Academic literature on the topic 'Nox enzymes in ASM cells'

Cancer cells can survive and maintain their high proliferation rate in spite of their hypoxic environment by deploying a variety of adaptative mechanisms, one of them being the reorientation of cellular metabolism. A key aspect of this metabolic rewiring is the promotion of the synthesis of antioxidant molecules in order to counter-balance the hypoxia-related elevation of reactive oxygen species (ROS) production and thus combat the onset of cellular oxidative stress. However, opposite to their negative role in the inception of oxidative stress, ROS are also key modulatory components of physiological cellular metabolism. One of the major physiological cellular ROS sources is the NADPH oxidase enzymes (NOX-es). Indeed, NOX-es produce ROS in a tightly regulated manner and control a variety of cellular processes. By contrast, pathologically elevated and unbridled NOX-derived ROS production is linked to diverse cancerogenic processes. In this respect, NOX4, one of the members of the NOX family enzymes, is of particular interest. In fact, NOX4 is closely linked to hypoxia-related signaling and is a regulator of diverse metabolic processes. Furthermore, NOX4 expression and function are altered in a variety of malignancies. The aim of this review is to provide a synopsis of our current knowledge concerning NOX4-related processes in the oncogenic metabolic adaptation of cancer cells.

NADPH oxidases (NOX) are commonly expressed ROS-producing enzymes that participate in the regulation of many signaling pathways, which influence cell metabolism, survival, and proliferation. Due to their high expression in several different types of cancer it was postulated that NOX promote tumor progression, growth, and survival. Thus, the inhibition of NOX activity was considered to have therapeutic potential. One of the possible outcomes of anticancer therapy, which has recently gained much interest, is cancer cell senescence. The induction of senescence leads to prolonged inhibition of proliferation and contributes to tumor growth restriction. The aim of our studies was to investigate the influence of low, non-toxic doses of diphenyleneiodonium chloride (DPI), a potent inhibitor of flavoenzymes including NADPH oxidases, on p53-proficient and p53-deficient HCT116 human colon cancer cells and MCF-7 breast cancer cells. We demonstrated that the temporal treatment of HCT116 and MCF-7 cancer cells (both p53 wild-type) with DPI caused induction of senescence, that was correlated with decreased level of ROS and upregulation of p53/p21 proteins. On the contrary, in the case of p53−/− HCT116 cells, apoptosis was shown to be the prevailing effect of DPI treatment. Thus, our studies provided a proof that inhibiting ROS production, and by this means influencing ROS sensitive pathways, remains an alternative strategy to facilitate so called therapy-induced senescence in cancers.

Diabetic kidney disease (DKD), a serious diabetic complication, results in podocyte loss and proteinuria through NADPH oxidases (NOX)-mediated ROS production. DUOX1 and 2 are NOX enzymes that require calcium for their activation which enters renal cells through the pivotal TRPC channels. Hypoglycemic drugs such as liraglutide can interfere with this deleterious mechanism imparting reno-protection. Herein, we aim to investigate the reno-protective effect of GLP1 receptor agonist (GLP1-RA), via its effect on TRPC6 and NADPH oxidases. To achieve our aim, control or STZ-induced T1DM Sprague–Dawley rats were used. Rats were treated with liraglutide, metformin, or their combination. Functional, histological, and molecular parameters of the kidneys were assessed. Our results show that treatment with liraglutide, metformin or their combination ameliorates DKD by rectifying renal function tests and protecting against fibrosis paralleled by restored mRNA levels of nephrin, DUOX1 and 2, and reduced ROS production. Treatment with liraglutide reduces TRPC6 expression, while metformin treatment shows no effect. Furthermore, TRPC6 was found to be directly interacting with nephrin, and indirectly interacting with DUOX1, DUOX2 and GLP1-R. Our findings suggest that treatment with liraglutide may prevent the progression of diabetic nephropathy by modulating the crosstalk between TRPC6 and NADPH oxidases.

Abstract Abstract 4801 Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic and leukemic cells. Reactive oxygen species (ROS) level is highly regulated. This regulation involves antioxydative enzymes and it has been recently described that leukemic stem cells (LSC) overexpress glutathione peroxydase 3 (Herault O et al, J. Exp. Med, 2012). This overexpression is associated with a decrease in ROS level and p38MAPK inactivation. ROS level in leukemic cells could be also regulated by the activity of ROS producers, such as NADPH oxidase, known to catalyze an electron transfer from NADPH to oxygen producing superoxides which could generate other downstream ROS. The expression of this enzymatic complex (NOX family, 6 isoforms) has been established in the plasma cell membrane of normal CD34+ hematopoietic progenitors (Piccoli C et al, Biochem. Biophys. Res. Commun., 2007). The aim of this study was to decipher the expression of NADPH oxydase components in various human acute myeloid leukemia (AML) Different leukemic cell lines were used according FAB classification: KG1a (MO/M1), KG1 (M1), HL60 (M2), Kasumi 1 (M2), NB4 (M3), ML2 (M4), THP1 (M5), U937 (M5), MV4–11 (M5), K562 (M6). The cells were cultured (2.105 cells/mL, 37°C in 95% humidified air and 5% CO2) in RPMI 1640 with 20mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100mg/mL streptomycin. The expression of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2, P22phox and P40phox, P47phox, P67phox, NOXO1, NOXA1 was quantified by RT-qPCR (Universal Probe Library, Roche). NOX2 and its regulatory subunits expression was quantified by SDS-PAGE and western-blot experiments. The effects of diphenylene iodonium (DPI), a specific NOX inhibitor, were evaluated by ROS quantification using dichlorodihydrofluorescein diacetate (DCF-DA) staining followed by fluorimetry and flow cytometry analyses. The cells were washed twice in the physiological saline buffer (PBS) without calcium and magnesium, then incubated in PBS complemented with 0.5M MgCl2, 0.9M CaCl2, 20mM glucose (Picciocchi A et al, J. Biol. Chem., 2011) with or without 20μM DPI for 1 hour. The cells were distributed at 106cells per 200μL well in 96 wells plates. DCF-DA (10μM) was added to quantify the ROS level (flow cytometry) and to monitor ROS production kinetic (fluorimetry). NOX family genes expression showed that phagocyte oxidase NOX2 is expressed in all leukemic cell lines. Conversely the NOX2 isoforms were not expressed, or very weakly expressed in leukemic cell lines (NOX3 in KG1a; NOX4 in K562; DUOX1 in KG1a, KG1; DUOX2 in KG1a, KG1, HL60). P22phox, the second cytochrome b558 component was also expressed in all cell lines, this expression being higher than NOX2. The cytochrome b558 components were more expressed in differentiated leukemic cells (granulocytic and monocytic) than in undifferentiated cells (KG1a, KG1). NOX2 regulatory subunits were expressed in all leukemic cell lines, the lower level (especially P40phox, P47phox) being observed in KG1a. Proteins quantification confirmed RNA results. Cytochrome b558 components and regulatory subunits were expressed in all cell lines with a higher level in differentiated leukemias. Interestingly, the regulatory subunits were not observed in KG1a cells. Functional flow cytometry and fluorimetry studies revealed a decrease in ROS production in DPI exposed leukemic cell lines. This effect was higher in monocytic cell lines than in granulocytic and undifferentiated leukemias. In conclusion, NADPH oxidases are present in the AML cell membrane, and NOX contribution to the ROS level is higher in differentiated cells than in immature leukemias. Altogether these results suggest that NADPH oxidase is constitutively active in leukemic cells and influences the ROS level, suggesting a role in the pathophysiology of AML. Disclosures: No relevant conflicts of interest to declare.

ABSTRACT Brachyspira (Serpulina)hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610 ) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmΔNOX and pKmΔNOX, were used to transform wild-type B. hyodysenteriae B204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-type nox genes in these strains had been replaced, through allelic exchange, by the inactivatednox gene containing cat or kan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that both nox mutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of both nox mutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204. In swine experimental infections, bothnox mutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. These results provide evidence that NADH oxidase serves to protect B. hyodysenteriae cells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete.

Thyroid hormone synthesis requires adequate hydrogen peroxide (H2O2) production that is utilized as an oxidative agent during the synthesis of thyroxin (T4) and triiodothyronine (T3). Thyroid H2O2 is generated by a member of the family of NADPH oxidase enzymes (NOX-es), termed dual oxidase 2 (DUOX2). NOX/DUOX enzymes produce reactive oxygen species (ROS) as their unique enzymatic activity in a timely and spatially regulated manner and therefore, are important regulators of diverse physiological processes. By contrast, dysfunctional NOX/DUOX-derived ROS production is associated with pathological conditions. Inappropriate DUOX2-generated H2O2 production results in thyroid hypofunction in rodent models. Recent studies also indicate that ROS improperly released by NOX4, another member of the NOX family, are involved in thyroid carcinogenesis. This review focuses on the current knowledge concerning the redox regulation of thyroid hormonogenesis and cancer development with a specific emphasis on the NOX and DUOX enzymes in these processes.

The aim of this study was to examine the role of nitric oxide (NO) in the control of cardiac metabolism at 60 days of pregnancy (P60) in the dog. There was a basal increase in diastolic coronary blood flow during pregnancy and a statistically significant increase in cardiac output (55 ± 4%) and in cardiac NOx production (44 ± 4 to 59 ± 3 nmol/min, P < 0.05). Immunohistochemistry of the left ventricle showed an increase in endothelial nitric oxide synthase staining in the endothelial cells at P60. NO-dependent coronary vasodilation (Bezold-Jarisch reflex) was increased by 20% and blocked by NG-nitro-l-arginine methyl ester (l-NAME). Isotopically labeled substrates were infused to measure oleate, glucose uptake, and oxidation. Glucose oxidation was not significantly different in P60 hearts (5.4 ± 0.5 vs. 6.2 ± 0.4 μmol/min) but greatly increased in response to l-NAME injection (to 19.9 ± 0.9 μmol/min, P < 0.05). Free fatty acid (FFA) oxidation was increased in P60 (from 5.3 ± 0.6 to 10.4 ± 0.5 μmol/min, P < 0.05) and decreased in response to l-NAME (to 4.5 ± 0.5 μmol/min, P < 0.05). There was an increased oxidation of FFA for ATP production but no change in the respiratory quotient during pregnancy. Genes associated with glucose and glycogen metabolism were downregulated, whereas genes involved in FFA oxidation were elevated. The acute inhibition of NO shifts the heart away from FFA and toward glucose metabolism despite the downregulation of the carbohydrate oxidative pathway. The increase in endothelium-derived NO during pregnancy results in a tonic inhibition of glucose oxidation and reliance on FFA uptake and oxidation to support ATP synthesis in conjunction with upregulation of FFA metabolic enzymes.

ROS (reactive oxygen species; including superoxide and H2O2) are conventionally thought of as being broadly reactive and cytotoxic. Phagocytes utilize an NADPH oxidase to generate large amounts of ROS, and exploit their toxic properties as a host-defence mechanism to kill invading microbes. However, the recent discovery of the Nox and Duox enzymes that are expressed in many non-phagocytic cells implies that the 'deliberate' generation of ROS has additional cellular roles, which are currently incompletely understood. Functions of ROS in mammals have been inferred primarily from cell-culture experiments, and include signalling for mitogenic growth, apoptosis and angiogenesis. Nox/Duox enzymes may also provide H2O2 as a substrate for peroxidase enzymes (or, in the case of Duox, for its own peroxidase domain), thereby supporting peroxidative reactions. A broad comparison of biological functions of ROS and Nox enzymes across species and kingdoms provides insights into possible functions in mammals. To further understand novel biological roles for Nox/Duox enzymes, we are manipulating the expression of Nox/Duox enzymes in model organisms including Caenorhabditis elegans, Drosophila melanogaster and mouse. This chapter focuses on new insights into the roles of Nox enzymes gained from these approaches.

To investigate the effects of repeated exposure to nitrogen dioxide (NO2) on antioxidant enzymes in lung tissue and isolated lung cells, rats were continuously exposed to 20 mg/m3 NO2 (10.6 ppm) for 4 days. The activities of glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and glutathione peroxidase (GSHPx) were measured in the cytosolic fraction of lung tissue of both control and NO2-exposed rats as well as in isolated alveolar macrophages (AMs) and type II cells. Qualitative and quantitative changes in AM and type II cells were studied by electron microscopy and by morphometric analyses using enzyme and immunohistochemistry. NO2 exposure resulted in significantly increased pulmonary activities of G6PDH, GR, and GSHPx, both expressed per lung and per gram of lung weight. Morphometric data show that NO2 exposure significantly increased the number of type II cells, predominantly in the centriacinar region, indicating proliferation of epithelium following cellular injury. Type II cells in lungs of NO2-exposed rats had a squamous, less cuboidal appearance with more lamellar bodies compared to type II cells in lungs of control rats. Compared to control lungs, a higher number of macrophages could be isolated from NO2-exposed lungs, while numbers of type II cells isolated from lungs of control and NO2-exposed rats were the same. Isolated type II cells from control and NO2-exposed rats were polymorphic, with a small number of lamellar bodies and without polarity. Isolated macro-phages were rounded and contained many filopodia. NO2 exposure caused increases in the activities of G6PDH and GSHPx in isolated type II cells and of GSHPx in isolated macrophages, when expressed per number of cells. Macrophages and type II cells isolated from control and NO2-exposed rats and re-exposed in vitro to NO2, showed no differences in phagocytosis and viability features. Our results indicate that NO2-induced increases in pulmonary antioxidant enzymes are also reflected in isolated AM and type II cells. Since these lung cells do not display a decreased sensitivities toward an in vitro NO2 exposure, overall increase in antioxidant enzyme activities do not seem to play the most pivotal role in controlling cellular NO2 sensitivity and oxidant defence. Combined data from biochemical, morphological, and morphometric analyses of lungs and lung cells suggest that lung cell and tissue oxidant sensitivity and defence largely depends on the cell and tissue organisation, i.e., cell numbers and morphology as well as the ratio of surface area to cytoplasmic volume.

Radiation nephropathy usually arises after inadvertent exposure of kidneys to radiotherapy. It may manifest as acute nephropathy as early as 6 months after exposure, or later as chronic nephropathy, hypertension, or asymptomatic proteinuria. Glomerular and peritubular endothelium and renal tubular cells are especially radiosensitive. There are no pathognomonic histological features, but renal pathology may be similar to that of haemolytic uraemic syndrome. Radiation nephropathy may be prevented by renal shielding and mitigated by radiation dose fractionation. Control of hypertension is important and angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists appear to have protective effects beyond those of blood pressure control.

This chapter reviews the functions of the immune system, which has evolved to provide a defence mechanism against microbial challenges, and is divided into two main branches, innate and adaptive. In addition, there are physical and chemical barriers, including skin, mucous membrane, mucous secretions, saliva, and various enzymes, and these contribute to the first line of defence against pathogens. The innate immune system provides the initial quick response for rapid recognition and elimination of pathogens, as opposed to the adaptive immune system, which has evolved to provide a more definitive and finely tuned response. The common central feature of both of these systems is the ability to distinguish between self and non-self. The recognition of non-self or ‘foreign’ pathogens and the subsequent immune response is orchestrated by a whole range of cells and soluble (humoral) factors in both innate and adaptive immune systems.

In sickle cell anaemia (SCA) a single mutation in the haemoglobin beta-globin gene is responsible for a pleomorphic phenotype leading to acute and chronic life-threatening complications. Healthcare management programmes, patient and family education, infection prophylaxis (especially in childhood), and long-term treatment for some patients (such as hydroxyurea) have significantly improved survival, giving rise to some new long-term issues.Sickle cell-associated nephropathy (SCAN) leads in some cases to chronic renal failure with a significant impact on survival. SCAN is characterized by an increased effective plasma renal flow and glomerular filtration rate, glomerular hypertrophy, and damaged vasa recta system leading to albuminuria and impaired urinary concentration.Early onset of hyperfiltration occurs in 60% of SCA patients often associated with microalbuminuria. SCAN risk factors are still under investigation, but may be related to chronic haemolysis at an early time point. Other lesions in patients with sickle cell anaemia include papillary necrosis, and recurrent acute kidney injury in association with crises or infections.ACEI are recommended if there is proteinuria. There is no current agreement on whether angiotensin-converting enzyme inhibitors (ACEI) should be introduced earlier, but systematic screening for microalbuminuria and hypertension, and avoidance of nephrotoxic agents are strongly advised.Patients with sickle cell trait (carriers for sickle cell anaemia) are prone to microscopic haematuria and abnormalities of the vasa recta have been described. A very rare tumour, renal medullary carcinoma, is largely restricted to this group (in whom it is still extremely rare). Increased risk of other renal problems is still largely hypothetical rather than proven.The prevalence of nephropathies in other sickle cell diseases (in particular haemoglobin SC disease) is much lower.

The aerobic oxidation of organic material by microbes is the focus of this chapter. Microbes account for about 50% of primary production in the biosphere, but they probably account for more than 50% of organic material oxidization and respiration (oxygen use). The traditional role of microbes is to degrade organic material and to release plant nutrients such as phosphate and ammonium as well as carbon dioxide. Microbes are responsible for more than half of soil respiration, while size fractionation experiments show that bacteria are also responsible for about half of respiration in aquatic habitats. In soils, both fungi and bacteria are important, with relative abundances and activity varying with soil type. In contrast, fungi are not common in the oceans and lakes, where they are out-competed by bacteria with their small cell size. Dead organic material, detritus, used by microbes, comes from dead plants and waste products from herbivores. It and associated microbes can be eaten by many eukaryotic organisms, forming a detritus food web. These large organisms also break up detritus into small pieces, creating more surface area on which microbes can act. Microbes in turn need to use extracellular enzymes to hydrolyze large molecular weight compounds, which releases small compounds that can be transported into cells. Fungi and bacteria use a different mechanism, “oxidative decomposition,” to degrade lignin. Organic compounds that are otherwise easily degraded (“labile”) may resist decomposition if absorbed to surfaces or surrounded by refractory organic material. Addition of labile compounds can stimulate or “prime” the degradation of other organic material. Microbes also produce organic compounds, some eventually resisting degradation for thousands of years, and contributing substantially to soil organic material in terrestrial environments and dissolved organic material in aquatic ones. The relationship between community diversity and a biochemical process depends on the metabolic redundancy among members of the microbial community. This redundancy may provide “ecological insurance” and ensure the continuation of key biogeochemical processes when environmental conditions change.

Calcium pyrophosphate (CPP) dihydrate crystals form extracellularly. Their formation requires sufficient extracellular inorganic pyrophosphate (ePPi), calcium, and pro-nucleating factors. As inorganic pyrophosphate (PPi) cannot cross cell membranes passively due to its large size, ePPi results either from hydrolysis of extracellular ATP by the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (also known as plasma cell membrane glycoprotein 1) or from the transcellular transport of PPi by ANKH. ePPi is hydrolyzed to phosphate (Pi) by tissue non-specific alkaline phosphatase. The level of extracellular PPi and Pi is tightly regulated by several interlinked feedback mechanisms and growth factors. The relative concentration of Pi and PPi determines whether CPP or hydroxyapatite crystal is formed, with low Pi/PPi ratio resulting in CPP crystal formation, while a high Pi/PPi ratio promotes basic calcium phosphate crystal formation. CPP crystals are deposited in the cartilage matrix (preferentially in the middle layer) or in areas of chondroid metaplasia. Hypertrophic chondrocytes and specific cartilage matrix changes (e.g. high levels of dermatan sulfate and S-100 protein) are related to CPP crystal deposition and growth. CPP crystals cause inflammation by engaging with the NALP3 inflammasome, and with other components of the innate immune system, and is marked with a prolonged neutrophilic inflitrate. The pathogenesis of resolution of CPP crystal-induced inflammation is not well understood.

Fungal respiratory infections are important causes of morbidity and mortality in immunocompromised patients. Invasive aspergillosis remains the most common invasive fungal infection whereas other filamentous fungi, such as Fusarium spp., Mucorales, and Scedosporium spp., are increasing in frequency, particularly in neutropenic hosts. Endemic mycoses, including those due to Histoplasma capsulatum, Coccidioides spp., and Talaromyces marneffei, are increasingly prevalent in patients with cell-mediated immunodeficiencies in respective geographic regions. Culture remains the gold standard of diagnosis but has limited sensitivity and often requires invasive procedures. Non-invasive diagnostic tests, including the serum sandwich enzyme immunoassay for the detection of galactomannan, the (1→3)-β‎-D-glucan assay, and molecular amplification methods have been developed to facilitate early and accurate diagnosis. Successful therapy depends upon early initiation of antifungal agents and reversal of immunosuppression. Lipid formulations of amphotericin B and newer generation triazoles including voriconazole, posaconazole, and isavuconazole have expanded the ability to treat multi-drug resistant pathogens more effectively and with less toxicity.

Anthracycline-induced cardiotoxicity is of considerable concern, as it may compromise the clinical effectiveness of treatment, affecting both quality of life and overall survival in cancer patients, independently of the oncological prognosis. It is probable that anthracycline-induced cardiotoxicity is a unique and continuous phenomenon starting with myocardial cell injury, followed by progressive left ventricular ejection fraction (LVEF) decline that, if disregarded and not treated progressively leads to overt heart failure. The main strategy for minimizing anthracycline-induced cardiotoxicity is early detection of high-risk patients and prompt prophylactic treatment. According to the current standard for monitoring cardiac function, cardiotoxicity is usually detected only when a functional impairment has already occurred, precluding any chance of its prevention. At present, anthracycline-induced cardiotoxicity can be detected at a preclinical phase, very much before the occurrence of heart failure symptoms, and before the LVEF drops by measurement of cardiospecific biochemical markers or by Doppler myocardial and deformation imaging. The role of troponins in identifying subclinical cardiotoxicity and treatment with angiotensin-converting enzyme inhibitors, in order to prevent LVEF reduction is an effective strategy that has emerged in the last 15 years. If cardiac dysfunction has already occurred, partial or complete LVEF recovery may still be achieved if cardiac dysfunction is detected early after the end of chemotherapy and heart failure treatment is promptly initiated.

Bones are multifunctional passive organs of movement that supports soft tissue and directly attached muscles. They also protect internal organs and are a reserve of calcium, phosphorus and magnesium. Each bone is covered with periosteum, and the adjacent bone surfaces are covered by articular cartilage. Histologically, the bone is an organ composed of many different tissues. The main component is bone tissue (cortical and spongy) composed of a set of bone cells and intercellular substance (mineral and organic), it also contains fat, hematopoietic (bone marrow) and cartilaginous tissue. Bones are a tissue that even in adult life retains the ability to change shape and structure depending on changes in their mechanical and hormonal environment, as well as self-renewal and repair capabilities. This process is called bone turnover. The basic processes of bone turnover are: • bone modeling (incessantly changes in bone shape during individual growth) following resorption and tissue formation at various locations (e.g. bone marrow formation) to increase mass and skeletal morphology. This process occurs in the bones of growing individuals and stops after reaching puberty • bone remodeling (processes involve in maintaining bone tissue by resorbing and replacing old bone tissue with new tissue in the same place, e.g. repairing micro fractures). It is a process involving the removal and internal remodeling of existing bone and is responsible for maintaining tissue mass and architecture of mature bones. Bone turnover is regulated by two types of transformation: • osteoclastogenesis, i.e. formation of cells responsible for bone resorption • osteoblastogenesis, i.e. formation of cells responsible for bone formation (bone matrix synthesis and mineralization) Bone maturity can be defined as the completion of basic structural development and mineralization leading to maximum mass and optimal mechanical strength. The highest rate of increase in pig bone mass is observed in the first twelve weeks after birth. This period of growth is considered crucial for optimizing the growth of the skeleton of pigs, because the degree of bone mineralization in later life stages (adulthood) depends largely on the amount of bone minerals accumulated in the early stages of their growth. The development of the technique allows to determine the condition of the skeletal system (or individual bones) in living animals by methods used in human medicine, or after their slaughter. For in vivo determination of bone properties, Abstract 10 double energy X-ray absorptiometry or computed tomography scanning techniques are used. Both methods allow the quantification of mineral content and bone mineral density. The most important property from a practical point of view is the bone’s bending strength, which is directly determined by the maximum bending force. The most important factors affecting bone strength are: • age (growth period), • gender and the associated hormonal balance, • genotype and modification of genes responsible for bone growth • chemical composition of the body (protein and fat content, and the proportion between these components), • physical activity and related bone load, • nutritional factors: – protein intake influencing synthesis of organic matrix of bone, – content of minerals in the feed (CA, P, Zn, Ca/P, Mg, Mn, Na, Cl, K, Cu ratio) influencing synthesis of the inorganic matrix of bone, – mineral/protein ratio in the diet (Ca/protein, P/protein, Zn/protein) – feed energy concentration, – energy source (content of saturated fatty acids - SFA, content of polyun saturated fatty acids - PUFA, in particular ALA, EPA, DPA, DHA), – feed additives, in particular: enzymes (e.g. phytase releasing of minerals bounded in phytin complexes), probiotics and prebiotics (e.g. inulin improving the function of the digestive tract by increasing absorption of nutrients), – vitamin content that regulate metabolism and biochemical changes occurring in bone tissue (e.g. vitamin D3, B6, C and K). This study was based on the results of research experiments from available literature, and studies on growing pigs carried out at the Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences. The tests were performed in total on 300 pigs of Duroc, Pietrain, Puławska breeds, line 990 and hybrids (Great White × Duroc, Great White × Landrace), PIC pigs, slaughtered at different body weight during the growth period from 15 to 130 kg. Bones for biomechanical tests were collected after slaughter from each pig. Their length, mass and volume were determined. Based on these measurements, the specific weight (density, g/cm3) was calculated. Then each bone was cut in the middle of the shaft and the outer and inner diameters were measured both horizontally and vertically. Based on these measurements, the following indicators were calculated: • cortical thickness, • cortical surface, • cortical index. Abstract 11 Bone strength was tested by a three-point bending test. The obtained data enabled the determination of: • bending force (the magnitude of the maximum force at which disintegration and disruption of bone structure occurs), • strength (the amount of maximum force needed to break/crack of bone), • stiffness (quotient of the force acting on the bone and the amount of displacement occurring under the influence of this force). Investigation of changes in physical and biomechanical features of bones during growth was performed on pigs of the synthetic 990 line growing from 15 to 130 kg body weight. The animals were slaughtered successively at a body weight of 15, 30, 40, 50, 70, 90, 110 and 130 kg. After slaughter, the following bones were separated from the right half-carcass: humerus, 3rd and 4th metatarsal bone, femur, tibia and fibula as well as 3rd and 4th metatarsal bone. The features of bones were determined using methods described in the methodology. Describing bone growth with the Gompertz equation, it was found that the earliest slowdown of bone growth curve was observed for metacarpal and metatarsal bones. This means that these bones matured the most quickly. The established data also indicate that the rib is the slowest maturing bone. The femur, humerus, tibia and fibula were between the values of these features for the metatarsal, metacarpal and rib bones. The rate of increase in bone mass and length differed significantly between the examined bones, but in all cases it was lower (coefficient b <1) than the growth rate of the whole body of the animal. The fastest growth rate was estimated for the rib mass (coefficient b = 0.93). Among the long bones, the humerus (coefficient b = 0.81) was characterized by the fastest rate of weight gain, however femur the smallest (coefficient b = 0.71). The lowest rate of bone mass increase was observed in the foot bones, with the metacarpal bones having a slightly higher value of coefficient b than the metatarsal bones (0.67 vs 0.62). The third bone had a lower growth rate than the fourth bone, regardless of whether they were metatarsal or metacarpal. The value of the bending force increased as the animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. The rate of change in the value of this indicator increased at a similar rate as the body weight changes of the animals in the case of the fibula and the fourth metacarpal bone (b value = 0.98), and more slowly in the case of the metatarsal bone, the third metacarpal bone, and the tibia bone (values of the b ratio 0.81–0.85), and the slowest femur, humerus and rib (value of b = 0.60–0.66). Bone stiffness increased as animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. Abstract 12 The rate of change in the value of this indicator changed at a faster rate than the increase in weight of pigs in the case of metacarpal and metatarsal bones (coefficient b = 1.01–1.22), slightly slower in the case of fibula (coefficient b = 0.92), definitely slower in the case of the tibia (b = 0.73), ribs (b = 0.66), femur (b = 0.59) and humerus (b = 0.50). Bone strength increased as animals grew. Regardless of the growth point tested, bone strength was as follows femur > tibia > humerus > 4 metacarpal> 3 metacarpal> 3 metatarsal > 4 metatarsal > rib> fibula. The rate of increase in strength of all examined bones was greater than the rate of weight gain of pigs (value of the coefficient b = 2.04–3.26). As the animals grew, the bone density increased. However, the growth rate of this indicator for the majority of bones was slower than the rate of weight gain (the value of the coefficient b ranged from 0.37 – humerus to 0.84 – fibula). The exception was the rib, whose density increased at a similar pace increasing the body weight of animals (value of the coefficient b = 0.97). The study on the influence of the breed and the feeding intensity on bone characteristics (physical and biomechanical) was performed on pigs of the breeds Duroc, Pietrain, and synthetic 990 during a growth period of 15 to 70 kg body weight. Animals were fed ad libitum or dosed system. After slaughter at a body weight of 70 kg, three bones were taken from the right half-carcass: femur, three metatarsal, and three metacarpal and subjected to the determinations described in the methodology. The weight of bones of animals fed aa libitum was significantly lower than in pigs fed restrictively All bones of Duroc breed were significantly heavier and longer than Pietrain and 990 pig bones. The average values of bending force for the examined bones took the following order: III metatarsal bone (63.5 kg) <III metacarpal bone (77.9 kg) <femur (271.5 kg). The feeding system and breed of pigs had no significant effect on the value of this indicator. The average values of the bones strength took the following order: III metatarsal bone (92.6 kg) <III metacarpal (107.2 kg) <femur (353.1 kg). Feeding intensity and breed of animals had no significant effect on the value of this feature of the bones tested. The average bone density took the following order: femur (1.23 g/cm3) <III metatarsal bone (1.26 g/cm3) <III metacarpal bone (1.34 g / cm3). The density of bones of animals fed aa libitum was higher (P<0.01) than in animals fed with a dosing system. The density of examined bones within the breeds took the following order: Pietrain race> line 990> Duroc race. The differences between the “extreme” breeds were: 7.2% (III metatarsal bone), 8.3% (III metacarpal bone), 8.4% (femur). Abstract 13 The average bone stiffness took the following order: III metatarsal bone (35.1 kg/mm) <III metacarpus (41.5 kg/mm) <femur (60.5 kg/mm). This indicator did not differ between the groups of pigs fed at different intensity, except for the metacarpal bone, which was more stiffer in pigs fed aa libitum (P<0.05). The femur of animals fed ad libitum showed a tendency (P<0.09) to be more stiffer and a force of 4.5 kg required for its displacement by 1 mm. Breed differences in stiffness were found for the femur (P <0.05) and III metacarpal bone (P <0.05). For femur, the highest value of this indicator was found in Pietrain pigs (64.5 kg/mm), lower in pigs of 990 line (61.6 kg/mm) and the lowest in Duroc pigs (55.3 kg/mm). In turn, the 3rd metacarpal bone of Duroc and Pietrain pigs had similar stiffness (39.0 and 40.0 kg/mm respectively) and was smaller than that of line 990 pigs (45.4 kg/mm). The thickness of the cortical bone layer took the following order: III metatarsal bone (2.25 mm) <III metacarpal bone (2.41 mm) <femur (5.12 mm). The feeding system did not affect this indicator. Breed differences (P <0.05) for this trait were found only for the femur bone: Duroc (5.42 mm)> line 990 (5.13 mm)> Pietrain (4.81 mm). The cross sectional area of the examined bones was arranged in the following order: III metatarsal bone (84 mm2) <III metacarpal bone (90 mm2) <femur (286 mm2). The feeding system had no effect on the value of this bone trait, with the exception of the femur, which in animals fed the dosing system was 4.7% higher (P<0.05) than in pigs fed ad libitum. Breed differences (P<0.01) in the coross sectional area were found only in femur and III metatarsal bone. The value of this indicator was the highest in Duroc pigs, lower in 990 animals and the lowest in Pietrain pigs. The cortical index of individual bones was in the following order: III metatarsal bone (31.86) <III metacarpal bone (33.86) <femur (44.75). However, its value did not significantly depend on the intensity of feeding or the breed of pigs.

Trichoderma is one of the most studied genera of ascomycetous fungi due to the beneficial effects it has on plants. Trichoderma spp. are involved in the production of cell wall-degrading enzymes and metabolites with antimicrobial activity. It also produces volatile compounds that act together as direct biocontrol agents to protect plants against phytopathogenic fungi, oomycetes, nematodes and bacteria. Trichoderma spp. can also compete in the rhizosphere for space and nutrients while it can also protect plants by activating systemic immune responses that result in a faster and stronger induction of plant basal resistance mechanisms against biotic and abiotic stresses. The possibility that Trichoderma can also promote plant growth opens new opportunities to register strains as biostimulants. Adequate registration procedures are urgently needed as there is no appropriate legal framework for registering Trichoderma as both plant protection products and as biofertilizers.

Glycosylation substantially contributes to the physicochemical properties of proteins, and hence also cell walls. Moreover, they are key factors for the recognition of free or cell-bound glycoproteins by internal and external interaction partners. Green plants get by with a highly conserved, limited number of modifications of the pan-eukaryotic basic N-glycan structure. In detail, these are fucosylation of the innermost N-acetylglucosamine residue in 3-position, which renders plant glycoproteins immunogenic to mammals; xylosylation of the branching mannose; frequent occurrence of small N-glycans terminating with mannose or decoration of the antennae with Lewis A determinants. Bryophytes share all these features, but some mosses additionally display two peculiarities not seen in vascular plants. Many mosses exhibit 2,6-di-O-methylated mannose on the 6-arm and some mosses contain modified Lewis A termini with an as yet unspecified methyl pentose. Neither the responsible enzymes nor the function of these novel glycan features is currently known. Targeted glycoengineering of the moss Physcomitrella patens (Hedw.) Bruch & Schimp can allow the production of biopharmaceutical glycoproteins that are difficult to express in more established systems.

Benzo[a]pyrene (B[a]P) is a major polycyclic aromatic hydrocarbon (PAH), it can bind the DNA to produce DNA-adducts, which has major carcinogenic potential. Enzyme-linked immunosorbet assay (ELISA) is the method used to detect these DNA adducts of B[a] P diolepoxide (BPDE) within the living cells. The aim of this study is to evaluate exposure to bitumen fumes, and to B[a]P in asphalt plant workers by measuring the BPDE-DNA adducts in their peripheral white blood cells (WBC), which are considered biological markers for exposure risk assessment.In this study, Hemostatic blood (CBC, AST) were measured, and the levels of BPDE-DNA adducts were measured in DNA samples of WBC obtained from asphalt plant workers in Syria and compared to those measured from a control group. The measurement was performed using BPDE-DNA Adducts ELISA.kit. The sample size was determined to be 50 with 25 asphalt plant workers and 25 healthy volunteers with no occupational exposure to PAHs. The results showed some diseases associated with exposure to asphalt fumes among the workers in the study group and a statistically significant difference in the values of (CBC; WBC, leukocytes, HCT, MCHC and AST) between the study group and the control group. BPDE-DNA adducts were detected in WBC of 11 asphalt plant workers with concentrations ranging between 0 and 2.75 ng/ml and only one individual in the control group with concentration of 0.75 ng/ml. These results indicate significant positive relationship between exposure to the bitumen fumes and formation of BPDE-DNA adducts. BPDE-DNA adducts is potential biomarker for PAHs exposure and likely helpful indicator of PAH-induced DNA damage and possibly carcinogenesis.

It seems inescapable that at some point primitive cells incorporated chemical reactions related to what we now call metabolism. In all life today, metabolic reactions are driven by sources of chemical or photochemical energy, and each step is catalyzed by enzymes and regulated by feedback systems. There have been multiple proposals for the kinds of reactions that could have been incorporated into early life, but so far there is little consensus about a plausible way for metabolism to begin. This chapter will briefly review the main ideas that are familiar to chemists as solution chemistry but then ask a new question from the epigraph: how can reactions in bulk aqueous solutions be captured in membranous compartments? This question is still virtually unexplored, but I can offer some ideas in the hope of guiding potentially fruitful approaches. Because metabolism is such a complex process, it is helpful to use bullet points to help clarify the discussion. The first is a list of questions that guide the discussion, the second is list of facts to keep in mind, and the third is a list of assumptions that introduce the argument. Questions to be addressed: What are the primary metabolic reactions used by life today? What reactions can occur in prebiotic conditions that are related to metabolism? How can potential nutrient solutes cross membranes in order to support metabolism? How could metabolic systems become incorporated into primitive cellular life? Metabolism can be defined as the activity of catalyzed networks of intracellular chemical reactions that alter nutrient compounds available in the environment into a variety of compounds that are used by living systems. Most of the reactions are energetically downhill, so there is an intimate association between the energy sources available to life and the kinds of reactions that can occur. Here is a summary of energy sources used by life today: Light is by far the most abundant energy source, totaling 1360 watts per square meter as infrared and visible wavelengths. Chemical energy in the form of reduced carbon compounds is made available by photosynthesis.

A biofuel cell is an electrochemical device in which the energy stored in a fuel, such as ethanol, is converted to electrical energy by the means of the catalytic activity of enzymes. Biofuel cells have traditionally suffered from low power densities and short lifetimes due to the fragility of the enzyme catalyst. Utilizing a novel quaternary ammonium salt treated Nafion membrane for enzyme immobilization in a biofuel cell results in increases in power densities and enzyme lifetimes to commercially viable levels. Additionally, this method provides sufficient protection to develop a membrane electrode assembly style (MEA) biofuel cell, an important step for commercialization. Previously, it has not been possible to create a MEA-style biofuel cell due to the denaturing of the enzyme that would occur at the high temperatures experienced during the heat pressing step of fabrication. Quaternary ammonium salt treated Nafion membranes provide sufficient protection for the enzyme to retain activity after exposure to temperatures of 140°C. Thus, a MEA-style biofuel cell can be created. Preliminary results yield biofuel cell MEAs with power densities ranging from 0.15 to 1.49 mW/cm2 and open circuit potentials of 0.360 to 0.599 V.

Magnetic iron oxide nanoparticles (NPs) have intrinsic advantages over other NPs for various biomedical applications. These advantages include visualization under Magnetic Resonance Imaging (MRI), heating with Radiofrequency (RF), and movement in a magnetic field. There are now numerous efforts to expand the applications of these particles for non-invasive drug and adjuvant delivery, cellular imaging and in vitro cell sorting and purification. In the present study, we describe methods to (i) assess and quantify NP cell association (ii) facilitate NP heat destruction of cells after association with RF and laser. First, we show that (i) the cell association of iron oxide NPs is dependent on the surface coating (surfactant greater than dextran), time, cell-type and extracellular NP concentrations (saturation with concentration and time). Furthermore, the association fits a simple enzyme Michealis-Menten model. Second, (ii) improved heat destruction of cells can be achieved after laser irradiation compared to traditional RF treatment for similar NP associations. These results and assays show promise for cell sorting and purification applications.

In the past decade the scientific community has showed considerable interest in the development of implantable medical devices. Such devices have low power requirements and can potentially be operated through fuel cells using reactants present in the body such as glucose and oxygen instead of non-rechargeable lithium batteries. In this paper we present a thin, enzyme-free fuel cell with high current density and good stability at a current density of 10μA cm−2. The fuel cell uses a stacked electrode design in order to achieve glucose and oxygen separation. In addition, it uses a porous carbon paper support for the anodic catalyst layer which reduces the amount of platinum or other noble metal catalysts required for fabricating high surface area electrodes with good reactivity. The peak power output of the fuel cell is approximately 2μW cm−2 and has a sustainable power density of 1.5μW cm−2 at 10μA cm−2. An analysis on the effects of electrode thickness and inter electrode gap on the maximum power output of the fuel cell is also performed.

Articular cartilage is a specialized avascular connective tissue found at the contact regions of diarthrodial joints. Cartilage has few cells (< 5% of the volume), though these cells can maintain the balance of turnover in healthy tissue, when the tissue is damaged, they are not able to repair the defects [1–3]. Extra cellular matrix (ECM) in cartilage comprises water, collagen (principally type II), proteoglycans and noncollagenous proteins. The type II collagen network, which is the dominant structural protein in cartilage ECM, constrains the expansion of the resident PGs and is generally held in mechanical tension. In osteoarthritis (OA), the balance of cartilage tissue production/degradation is thought to be affected by abnormal mechanical stimuli leading to net matrix resorption through production of excess degradative enzymes (e.g. matrix metalloproteinases (MMP) and aggrecanases) [4–8]. In OA tissue the amount of MMP-13 is thought to be increased relative to healthy tissue. OA typically occurs in older adults where, as cartilage ages, there is a marked decrease in the fixed charge density (FCD), the hydration and, consequently, mechanical tension on the collagen type II network [9–11]. We have hypothesized that loss of tension on the collagen network accelerates degradation by MMP. Detection of the effect of MMP on loaded, native cartilage could lead to insight about cartilage degradation kinetics in OA. However, it is quite difficult to controllably deliver MMP to cartilage, to activate the MMP during detensioning of the collagen network and to detect the effect on the cartilage mechanics (because cost limits the amount of MMP used). We have developed a transpirational enzyme loading method which is capable of precisely dosing bovine cartilage explants with a small, known quantity of MMP-13. Following enzyme insertion, we are able to detect the activity of the MMP on osmotically compressed cartilage (i.e. cartilage with a detensioned collagen network) via a simple hydration measurement.

It is well accepted that fluid flow is an important mechanical signal in regulating bone structure and function. Primary cilia, which are solitary, microtubule-based organelles that extend from the centrosome into extracellular space in many cell types, have been shown to mediate fluid flow-induced osteogenic responses in MLO-Y4 osteocyte-like cells [1], however, primary cilia did not mediate increases in intracellular Ca2+ concentration [1]. Recently, we identified cAMP as a novel early signaling molecule in primary cilia-dependent mechanotransduction of fluid flow in osteocytes. Specifically, we show that MLO-Y4 osteocyte-like cells respond to oscillatory flow with a rapid decrease in intracellular levels of cAMP that is dependent on the primary cilium [2]. Adenylyl cyclase 6 (AC6) is an enzyme responsible for the synthesis of cAMP from ATP. We found that AC 6 localizes to the primary cilium of bone cells (Fig. 1). In this study, our goal was to determine whether AC6 mediates the primary cilia-dependent, flow-induced decrease in cAMP.

The inhibition of thrombin as well as of factor Xa has been thought to occur primarily in plasma through the neutralizing action of the serine protease inhibitor antithrombin III (AT-III). However, inhibition of thrombin and Xa by this mechanism may not be sufficient for effective elimination of these clotting factors in states of increased coagulation activity. The potential role of the vascular endothelium in the inhibition of clotting factor activities has therefore received attention in recent years. The aim of the present investigation was to characterize the binding and inhibition of thrombin and factor Xa to the vascular endothelial cell (EC), smooth muscle cell (SMC) and rat hepatoma cell (RHC) in vitro, as well as to evaluate the effects of plasma constituents upon the inhibition of these factors. Purified bovine thrombin and factor Xa were used. The enzymatic activities of both factors were assayed using chromogenic substrates. The cells were exposed for 5 U/ml thrombin or 0.5 U/ml factor Xa. After 10 minutes incubation, the initial thrombin activity in the solution had decreased by about 20% in case of EC and SMC and about 11% when incubated with RHC. Thrombin activity recovered from the cell surface amounted to 0.02 U/cm2. When the cells with the surface bound enzyme were incubated with defibrinogenated plasma or AT-III for 30 seconds, only about 10% and 25-40%, respectively, of initial activity could be found. In similar experiments with factor Xa, after 10 minutes incubation, the initial activity in the solution had decreased by 10%. Factor Xa activity recovered from the cell surface was 0.001 U/cm2. After 30 seconds exposure to AT-III, no cell surface related factor Xa activity was recovered, whereas 10% of the cell surface activity was recovered after incubation with defibrinogenated plasma. It is concluded that thrombin and factor Xa are taken up and inhibited by EC, SMC and RHC cell surfaces in similar ratios suggesting that cell surface-mediated inactivation of activated clotting factors is not restricted to vascular wall cells. The inactivation of factor Xa was dependent on AT-III, however, the inactivation of thrombin was further promoted by an additional unidentified plasma constituent

An estimated 2.5 percent of the American population has heart valve (HV) disease and more than 100,000 US patients require a prosthetic valve replacement each year [1]. However, prosthetic valves can cause accelerated calcification leading to recurrence of HV disease in patients [2]. Thus, the development of a suitable tissue-engineered heart valve (TEHV) would greatly benefit patients with HV disease. Aortic valve interstitial cells (AVICs) play a crucial role in the progression of aortic valve disease as well as the maintenance of normal valve. Therefore, in order to design a suitable TEHV, these specialized cells need to be better understood. AVICs are known to synthesize ECM and express matrix degrading enzymes and their inhibitors that mediate and regulate remodeling of ECM components [3]. Interestingly, it was recently established that AVICs sense the stiffness of their surrounding ECM in vivo and are phenotypically responsive to mechanical cues with AVICs differentiating into myofibroblasts or osteoblasts, which are pathologic markers. Specifically, soft collagen gels (∼34kPa) caused less differentiation of AVICs than stiffer collagen gel (∼100kPa) [4]. However, for these experiments the AVICs were cultured on tissue culture polystyrene (TCPS) for at least one passage, and it is likely that AVICs cultured on TCPS might retain modified characteristics of AVICs in tissue prior to seed them on soft gels because of the memory to rigid substrate stiffness. Therefore, in this study, we examined the phenotype and function of AVICs on substrates that mimic ECM stiffness of adult leaflet as well as of developing embryo. In addition, we examine the effects of transforming growth factor-β1 (TGF-β1) which has been the most extensively studied cytokine initiator of fibrotic response of AVICs.

Vascular endothelial cells possess specific receptors for thrombin, and thrombin can interact with these receptors to activate the endothelial cells. However, the signal transduction mechanisms which mediate the cellular responses are not yet characterised. The aim of this study therefore, was to determine whether thrombin influenced the inositol phosphate transduction pathway in cultured human endothelial cells. Endothelial cells were isolated from both large and small vessels; these were human umbilical vein and the microvasculature of human omentum respectively. The endothelial cells stained positively with antibodies against Factor VIII antigen and another endothelial cell specific antigen (BMA 120). Pure human thrombin (0.1 - 10 units/ml) induced a dose-dependent formation of inositol phosphate, inositol biphosphate and inositol trisphosphate (IP3) in endothelial cells from large vessels prelabelled with tritiated inositol. The formation of IP3 was significantly increased after 15 sec., maximal after 1 min. and had returned almost to baseline levels after 4 min. This time course is consistent with its role as a second messenger. When the enzymic activity of thrombin was removed with phenylalanyl-prolyl-arginine chloromethyl ketone or d i i sopr opyIfluorophosphate, thrombin lost its ability to stimulate the accumulation of IP3. Thrombin at all concentrations tested was unable to stimulate the formation of IP3 in small vessel endothelial cells. However, IP3 formation could be stimulated by bradykinin (0.1-10 μM) in cells from both small and large vessels. The results demonstrate that active thrombin can induce the formation of IP3 in large vessel endothelium. But that there are differences in the way small vessel endothelium responds to thrombin.

In the last years, encapsulation of functional biomolecules (such as enzymes, proteins and antibodies) in three-dimensional nanoporous silica matrices has received considerable attention for its potential applications in biocatalysis, biosensing, and biopreservation.1 A handful of studies conducted with bacteria, plant, and mammalian cells have shown that these complex microorganisms can also function when they are encapsulated in silica matrices.2 When encapsulated in rigid permeable mesopores, mobility and proliferation of the microorganisms are prevented, while nutrients and by-products diffuse freely through the porous matrix. However, it is not known what mechanism(s) enables the biomolecules to be stable, and the microorganisms to be active, while they are mechanically confined in a matrix in a “no-growth” state. It is known that the motions and properties of water change when confined in nanopores. Therefore, we envision that the altered properties of confined water play a role in increasing the stability of biomolecules. This study is focused on understanding the effects of confinement on the kinetic and thermodynamic transitions of water, and identifying its effects on the structure of confined biomolecules. We have utilized Fourier Transform Infrared Spectroscopy (FTIR) to identify the different states and phase transitions of nanopore confined water at cryogenic temperatures. We have extended our analysis to quantify the structural changes in nanopore confined isolated and endogenous proteins of intact organisms.

Articular cartilage lines the surfaces of joints and functions to absorb shock and distribute load. To date, few strategies exist for restoring damaged articular surfaces; therefore, cartilage tissue engineering (TE) has emerged as a means to generate functional replacement tissues. To optimize growth and maturation of TE constructs, various methodologies have been employed, including 3D culture, coupled with mechanical stimulation [1] and growth factor supplementation [2]. Recently, studies have shown that temporal application of TGF-β3 enhances the compressive properties and GAG content of chondrocyte-laden hydrogels to near-native levels after the removal of this morphogen [3, 4]. However, as chondrocytes may prove impractical for clinical use, mesenchymal stem cells (MSCs) have been increasingly utilized in cartilage TE. While MSCs are able to undergo chondrogenesis and deposit cartilaginous extracellular matrix (ECM), they have not demonstrated functional parity with chondrocytes [5]. One recent study showed MSCs were able to maintain a chondrocytic phenotype after brief exposure of TGF-β3, though mechanical and biochemical properties were not assessed [6]. In this study, we evaluated these properties in chondrocyte- and MSC-laden hydrogels with transient exposure of TGF-β3 in a chemically defined medium. In addition, we explored the effects of varying seeding density in MSC-laden constructs on functional properties. We hypothesized that transient application of TGF-β3 would improve functional properties of MSC-laden constructs in a seeding density-dependent manner, and that these changes would be marked by differences in cartilaginous gene expression, particularly of enzymes involved in proteoglycan synthesis.

The overall goal of the research was to understand the processes involved in the development of woolliness in peaches and nectarines. Four specific hypotheses were proposed and in the course of the research evidence was gathered t support two of them and to not support two others. The hypotheses and a summary of the evidence are outlined below. 1. That woolliness arises from an imbalance between the activities of the cell wall pectin degrading enzymes. Using 'Flavortop' nectarines and 'Hermoza' peaches as model systems, storage regimes were manipulated to induce or prevent woolliness. The expression (mRNA abundance), protein content (Western blotting), and activity of polygalacturonase (PG) and pectin esterase (PE) were followed. Expression of the enzymes was not different, but activity and the ratio between PG and PE activities were quite different in fruits developing woolliness or ripening normally. This was also examined by looking at the substrate, the pectin moiety of the cell wall, and i woolly fruit there were more high molecular weight pectins with regions of non-methylated galacturonic acid residues. Taking an in vitro approach it was found a) that PE activity was stable at 0oC while PG activity decreased; b) incubating the calcium pectate fraction of the cell wall with PE extracted from peaches caused the polymers to form a gel characteristic of the visual woolly symptoms in peaches. 2. That continued cell wall synthesis occurs during storage and contributes to structural changes i cell walls and improper dissolution and softening after storage. We tried to adapt our technique of adding 13C-glucose to fruit discs, which was used successfully to follow cell wall synthesis during tomato ripening. However, the difference in sugar content between the two fruits (4% in tomato and 12% in peach) meant that the 13C-glucose was much more diluted within the general metabolite pool. We were unable to see any cell wall synthesis which meant that either the dilution factor was too great, or that synthesis was not occurring. 3. That controlled atmosphere (CA) prevents woolliness by lowering all enzyme activities. CA was found to greatly reduce mRNA abundance of the cell wall enzymes compared to regular air storage. However, their synthesis and activity recovered during ripening after CA storage and did not after regular air storage. Therefore, CA prevented the inhibition of enzyme activation found in regular air storage. 4. That changes in cell wall turgor and membrane function are important events in the development of woolliness. Using a micro pressure probe, turgor was measured in cells of individual 'O'Henry' and 'CalRed' peaches which were woolly or healthy. The relationship between firmness and turgor was the same in both fruit conditions. These data indicate that the development and expression of woolliness are not associated with differences in membrane function, at least with regard to the factors that determine cell turgor pressure. In addition, during the period of the grant additional areas were explored. Encoglucanase, and enzyme metabolizing hemicellulose, was found to be highly expressed air stored, but not in unstored or CA stored fruit. Activity gels showed higher activity in air stored fruit as well. This is the first indication that other components of the cell wall may be involved in woolliness. The role of ethylene in woolliness development was also investigated at it was found a) that woolly fruits had decreased ability to produce ethylene, b) storing fruits in the presence of ethylene delayed the appearance of woolliness. This latter finding has implication for an inexpensive strategy for storing peaches and nectarines.

The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.

The goal of this work was to understand the role of the enzyme sucrose synthase (SuSy) in synthesis of cellulose and callose in plants. The work resulting from the this grant leads to a number of conclusions. SuSy clearly plays diverse roles in carbon metabolism. It can associate with the plasma membrane of cells undergoing rapid cellulose deposition, such as cotton fibers, developing maize endosperm, gravistimulated pulvini, and transfer cells of the cotton seed. It is also concentrated at sites of high callose deposition (tapetal cells; cell plates). When SuSy levels are lowered by mutation or by anti-sense technology, cell walls undergo degeneration (maize endosperm) and show reduced levels of cellulose (potato tubers). In sum, our evidence has very much strengthened the concept that SuSy does function in the plasma membrane to channel carbon from sucrose via UDP-glucose to glucan synthase complexes. Soluble SuSy also clearly plays a role in providing carbon for starch synthesis and respiration. Surprisingly, we found that the cotton seed is one unique case where SuSy apparently does not play a role in starch synthesis. Current evidence in sum suggests that no specific SuSy gene encodes the membrane-associated form, although in maize the SS 1 form of SuSy may be most important for cell wall synthesis in the early stages of endosperm development. Work is still in progress to determine what does control membrane localization - and the current evidence we have favors a role for Ca2+, and possibly also protein phosphorylation by differentially regulated protein kinases. Finally, we have discovered for the first time, a major new family of genes that encode the catalytic subunit of the cellulose synthase of plants - a result that has been widely cited and opens many new approaches for the study of this important plant function.

Citrus fruit quality standards have been determined empirically, depending on species and on the particular growing regions. In general, the TSS (total soluble solids) to total acidity (TA) ratio determines whether citrus fruit can be marketed. Soluble sugars account for most of the TSS during harvest while TA is determined almost solely by the citric acid content, which reaches levels of 1-5% by weight in many cultivated varieties. Acid and sugar homeostasis in the fruit is critical for the management of existing cultivars, the development of new cultivars, the improvement of pre- and post-harvest strategies and the control of fruit quality and disorders. The current proposal (a continuation of a previous proposal) aimed at: (1) completing the citrus fruit proteome and metabolome, and establish a citrus fruit functional database, (2) further characterization of the control of fruit acidity by studying the regulation of key steps affecting citrate metabolism, and determine the fate of citrate during acid decline stage, and (3) Studying acid and sugar homeostasis in citrus fruits by characterizing transport mechanisms across membranes. These aims were completed as the following: (1) Our initial efforts were aimed at the characterization and identification of citric acid transporters in citrus juice cells. The identification of citrate transporters at the vacuole of the citrus juice cell indicated that the steady-state citrate cytosolic concentration and the action of the cytosolic aconitase were key elements in establishing the pH homeostat in the cell that regulates the metabolic shift towards carbon usage in the fruit during the later stages of fruit development. We focused on the action of aconitase, the enzyme mediating the metabolic use of citric acid in the cells, and identified processes that control carbon fluxes in developing citrus fruits that control the fruit acid load; (2) The regulation of aconitase, catalyzing a key step in citrate metabolism, was further characterized by using two inhibitors, citramalte and oxalomalte. These compounds significantly increased citrate content and reduced the enzyme’s activity. Metabolite profiling and changes of amino-acid metabolizing enzymes in oxalomalate- treated cells suggested that the increase in citrate, caused by aconitase inhibition, induces amino acid synthesis and the GABA shunt, in accordance with the suggested fate of citrate during the acid decline stage in citrus fruit. (3) We have placed a considerable amount of time on the development of a citrus fruit proteome that will serve to identify all of the proteins in the juice cells and will also serve as an aid to the genomics efforts of the citrus research community (validating the annotation of the fruit genes and the different ESTs). Initially, we identified more than 2,500 specific fruit proteins and were able to assign a function to more than 2,100 proteins (Katz et al., 2007). We have now developed a novel Differential Quantitative LC-MS/MS Proteomics Methodology for the identification and quantitation of key biochemical pathways in fruits (Katz et al., 2010) and applied this methodology to identify determinants of key traits for fruit quality (Katz et al., 2011). We built “biosynthesis maps” that will aid in defining key pathways associated with the development of key fruit quality traits. In addition, we constructed iCitrus (http://wiki.bioinformatics.ucdavis.edu/index.php/ICitrus), a “functional database” that is essentially a web interface to a look-up table that allows users to use functional annotations in the web to identify poorly annotated citrus proteins. This resource will serve as a tool for growers and field extension specialists.

Sclerotinia sclerotiorum is a filamentous fungus (mold) that causes plant disease. It has an extremely wide range of hosts (>400 species) and causes considerable damage (annual multimillion dollar losses) in economically important crops. It has proven difficult to control (culturally or chemically) and host resistance to this fungus has generally been inadequate. It is believed that this fungus occurs in almost every country. Virulence of this aggressive pathogen is bolstered by a wide array of plant cell wall degrading enzymes and various compounds (secondary metabolites) produced by the fungus. It is well established that plant pathogenic fungi secrete proteins and small molecules that interact with host cells and play a critical role in disease development. Such secreted proteins have been collectively designated as “effectors”. Plant resistance against some pathogens can be mediated by recognition of such effectors. Alternatively, effectors can interfere with plant defense. Some such effectors are recognized by the host plant and can culminate in a programmed cell death (PCD) resistant response. During the course of this study, we analyzed an effector in Sclerotiniasclerotiorum. This specific effector, SsCM1 is the protein chorismatemutase, which is an enzyme involved in a pathway which is important in the production of important amino acids, such a Tryptophan. We have characterized the Sclerotiniaeffector, SsCM1, and have shown that inactivation of Sscm1 does not affect fungal vegetative growth, development or production of oxalic acid (one of this fungus’ secondary metabolites associated with disease) production. However, yhis does result in reduced fungal virulence. We show that, unexpectedly, the SsCM1 protein translocates to the host chloroplast, and demonstrated that this process is required for full fungal virulence. We have also determined that the fungal SsCM1 protein can interact with similar proteins produced by the host. In addition, we have shown that the fungal SsCM1 is able to suppress at least some of the effects imposed by reactive oxygen species which are produced as a defense mechanism by the host. Last, but not least, the results of our studies have provided evidence contradicting the current dogma on at least some of the mechanist aspects of how this pathogen infects the host. Contrary to previousons, indicating that this pathogen kills its host by use of metabolites and enzymes that degrade the host tissue (a process called necrotrophy), we now know that at least in the early phases of infection, the fungus interacts with live host tissue (a phenomenon known as biotrophy). Taken together, the results of our studies provide novel insights concerning the mechanistic aspects of Sclerotinia-host interactions. We hope this information will be used to interfere with the disease cycle in a manner that will protect plants from this devastating fungus.

Research objectives 1) Analyze the combined effects of hexose phosphorylation and P level in tomato and Arabidopsis plants 2) Analyze the combined effects of hexose phosphorylation and P level in pho1 and pho2 Arabidopsis mutants 3) Clone and analyze the PHO2 gene 4) Select Arabidopsis mutants resistant to high and low P 5) Analyze the Arabidopsis mutants and clone the corresponding genes 6) Survey wild tomato species for growth characteristics at various P levels Background to the topic Hexose phosphorylating enzymes, the first enzymes of sugar metabolism, regulate key processes in plants such as photosynthesis, growth, senescence and vascular transport. We have previously discovered that hexose phosphorylating enzymes might regulate these processes as a function of phosphorous (P) concentration, and might accelerate acquisition of P, one of the most limiting nutrients in the soil. These discoveries have opened new avenues to gain fundamental knowledge about the relationship between P, sugar phosphorylation and plant development. Since both hexose phosphorylating enzymes and P levels affect plant development, their interaction is of major importance for agriculture. Due to the acceleration of senescence caused by the combined effects of hexose phosphorylation and P concentration, traits affecting P uptake may have been lost in the course of cultivation in which fertilization with relatively high P (30 mg/L) are commonly used. We therefore intended to survey wild tomato species for high P-acquisition at low P soil levels. Genetic resources with high P-acquisition will serve not only to generate a segregating population to map the trait and clone the gene, but will also provide a means to follow the trait in classical breeding programs. This approach could potentially be applicable for other crops as well. Major conclusions, solutions, achievements Our results confirm the mutual effect of hexose phosphorylating enzymes and P level on plant development. Two major aspects of this mutual effect arose. One is related to P toxicity in which HXK seems to play a major role, and the second is related to the effect of HXK on P concentration in the plant. Using tomato plants we demonstrated that high HXK activity increased leaf P concentration, and induced P toxicity when leaf P concentration increases above a certain high level. These results further support our prediction that the desired trait of high-P acquisition might have been lost in the course of cultivation and might exist in wild species. Indeed, in a survey of wild species we identified tomato species that acquired P and performed better at low P (in the irrigation water) compared to the cultivated Lycopersicon esculentum species. The connection between hexose phosphorylation and P toxicity has also been shown with the P sensitive species VerticordiaplumosaL . in which P toxicity is manifested by accelerated senescence (Silber et al., 2003). In a previous work we uncovered the phenomenon of sugar induced cell death (SICD) in yeast cells. Subsequently we showed that SICD is dependent on the rate of hexose phosphorylation as determined by Arabidopsis thaliana hexokinase. In this study we have shown that hexokinase dependent SICD has many characteristics of programmed cell death (PCD) (Granot et al., 2003). High hexokinase activity accelerates senescence (a PCD process) of tomato plants, which is further enhanced by high P. Hence, hexokinase mediated PCD might be a general phenomena. Botrytis cinerea is a non-specific, necrotrophic pathogen that attacks many plant species, including tomato. Senescing leaves are particularly susceptible to B. cinerea infection and delaying leaf senescence might reduce this susceptibility. It has been suggested that B. cinerea’s mode of action may be based on induction of precocious senescence. Using tomato plants developed in the course of the preceding BARD grant (IS 2894-97) and characterized throughout this research (Swartzberg et al., 2006), we have shown that B. cinerea indeed induces senescence and is inhibited by autoregulated production of cytokinin (Swartzberg et al., submitted). To further determine how hexokinase mediates sugar effects we have analyzed tomato plants that express Arabidopsis HXK1 (AtHXK1) grown at different P levels in the irrigation water. We found that Arabidopsis hexokinase mediates sugar signalling in tomato plants independently of hexose phosphate (Kandel-Kfir et al., submitted). To study which hexokinase is involved in sugar sensing we searched and identified two additional HXK genes in tomato plants (Kandel-Kfir et al., 2006). Tomato plants have two different hexose phosphorylating enzymes; hexokinases (HXKs) that can phosphorylate either glucose or fructose, and fructokinases (FRKs) that specifically phosphorylate fructose. To complete the search for genes encoding hexose phosphorylating enzymes we identified a forth fructokinase gene (FRK) (German et al., 2004). The intracellular localization of the four tomato HXK and four FRK enzymes has been determined using GFP fusion analysis in tobacco protoplasts (Kandel-Kfir et al., 2006; Hilla-Weissler et al., 2006). One of the HXK isozymes and one of the FRK isozymes are located within plastids. The other three HXK isozymes are associated with the mitochondria while the other three FRK isozymes are dispersed in the cytosol. We concluded that HXK and FRK are spatially separated in plant cytoplasm and accordingly might play different metabolic and perhaps signalling roles. We have started to analyze the role of the various HXK and FRK genes in plant development. So far we found that LeFRK2 is required for xylem development (German et al., 2003). Irrigation with different P levels had no effect on the phenotype of LeFRK2 antisense plants. In the course of this research we developed a rapid method for the analysis of zygosity in transgenic plants (German et al., 2003).

Our study was designed to elucidate the chemical determinants of pectin cross-linking in developing fruits of apple and peach and to evaluate the role of breakage cross-linkages in swelling, softening, and cell separation during the ripening. Peaches cell walls soften and swell considerably during the ripening, whereas apples fruit cells maintain wall firmness but cells separate during late stages of ripening. We used a "double-reduction" technique to show that levels of non-methyl esters of polyuronic acid molecules were constant during the development and ripening and decreased only in overripe fruit. In peach, methyl and non-methyl esters increased during the development and decreased markedly during the ripening. Non-methyl ester linkages in both fruit decreased accompanied fruit softening. The identity of the second component of the linkage and its definitive role in the fruit softening remain elusive. In preliminary examination of isolated apples cell walls, we found that phenolic compounds accumulate early in wall development but decrease markedly during ripening. Quantitative texture analysis was used to correlate with changes to wall chemistry from the fresh-picked ripe stage to the stage during storage when the cell separation occurs. Cell wall composition is similar in all cultivars, with arabinose as the principal neutral sugar. Extensive de-branching of these highly branched arabinans pre-stages softening and cell-cell separation during over-ripening of apple. The longer 5-arabinans remain attached to the major pectic polymer rhamnogalacturonan I (RG I) backbone. The degree of RG I branching, as judged from the ratios of 2-Rha:2,4-Rha, also decreases, specially after an extensive arabinan de-branching. Loss of the 4-Rham linkages correlated strongly with the softening of the fruit. Loss of the monomer or polymer linked to the RG I produce directly or indirectly the softening of the fruit. This result will help to understand the fruit softening and to have better control of the textural changes in fruit during the ripening and especially during the storage. 'Wooliness', an undesirable mealy texture that is induced during chilling of some peach cultivars, greatly reduces the fruit storage possibilities. In order to examine the hypothesis that the basis for this disorder is related to abnormality in the cell wall softening process we have carried out a comparative analysis using the resistant cultivar, Sunsnow, and a sensitive one, Hermosa. We investigated the activity of several pectin- and glycan-modifying enzymes and the expression of their genes during ripening, chilling, and subsequent shelf-life. The changes in carbohydrate status and in methyl vs. non-methyl uronate ester levels in the walls of these cultivars were examined as well to provide a basis for comparison of the relevant gene expression that may impact appearance of the wooly character. The activities of the specific polygalacturonase (PGase) and a CMC-cellulase activities are significantly elevated in walls of peaches that have become wooly. Cellulase activities correlated well with increased level of the transcript, but differential expression of PGase did not correspond with the observed pattern of mRNA accumulation. When expression of ethylene biosynthesis related genes was followed no significant differences in ACC synthase gene expression was observed in the wooly fruit while the normal activation of the ACC oxidase was partially repressed in the Hermosa wooly fruits. Normal ripening-related loss of the uronic acid-rich polymers was stalled in the wooly Hermosa inconsistent with the observed elevation in a specific PGase activity but consistent with PG gene expression. In general, analysis of the level of total esterification, degree of methyl esterification and level of non-methyl esters did not reveal any major alterations between the different fruit varieties or between normal and abnormal ripening. Some decrease in the level of uronic acids methyl esterification was observed for both Hermosa and Sunsnow undergoing ripening following storage at low temperature but not in fruits ripening after harvest. Our results support a role for imbalanced cell wall degradation as a basis for the chilling disorder. While these results do not support a role for the imbalance between PG and pectin methyl esterase (PME) activities as the basis for the disorder they suggest a possible role for imbalance between cellulose and other cell wall polymer degradation during the softening process.

Original objectives (revisions from original proposal are highlighted) 1. Elucidate the direct effects O3 and H2O2 on guard cell function, utilizing assays of stomatal response in isolated epidermal peels and whole cell gas exchange. 2. Determine the mechanistic basis of O3 and H2O2 effects on the plasma membrane through application of the electrophysiological technique of patch clamping to isolated guard cells. 3. Determine the relative sensitivity of Israeli cultivars of economically important crops to O3 and determine whether differential leaf conductance responses to O3 can explain relative sensitivity to the air pollutant: transfer of technological expertise to Israel. Background to the topic For a long time O3 has been known to reduce gas exchange in plants; it has however been unclear if O3 can affect the stomatal complex directly. Ion channels are essential in stomatal regulation, but O3 has never before been shown to affect these directly. Major conclusions, solution, achievements 1. Ozone inhibits light-induced stomatal opening in epidermal peels isolated from Vicia faba, Arabidopsis thaliana and Nicotiana tabacum in V. faba plants this leads to reduced assimilation without a direct effect on the photosynthetic apparatus. Stomatal opening is more sensitive to O3 than stomatal closure. 2. Ozone causes inhibition of inward K+ channels (involved in stomatal opening) while no detectable effect is observed o the outward K+ channels (stomatal closure). 3. Hydrogen peroxide inhibits stomatal opening and induces stomatal closure in epidermal peels isolated from Vicia faba. 4. Hydrogen peroxide enhances stomatal closure by increasing K+ efflux from guard cells via outward rectifying K+ channels. 5. Based on epidermal peel experiments we have indirectly shown that Ca2+ may play a role in the guard cell response to O3. However, direct measurement of the guard cell [Ca2+]cyt did not show a response to O3. 6. Three Israeli cultivars of zucchini, Clarita, Yarden and Bareqet, were shown to be relatively sensitive to O3 (0.12 ml1-1 ). 7. Two environmentally important Israeli pine species are adversely affected by O3, even at 0.050 ml1-1 , a level frequently exceeded under local tropospheric conditions. P. brutia may be better equipped than P. halepensis to tolerate O3 stress. 8. Ozone directly affects pigment biosynthesis in pine seedlings, as well as the metabolism of O5 precursors, thus affecting the allocation of resources among various metabolic pathways. 9. Ozone induces activity of antioxidant enzymes, and of ascorbate content i the mesophyll and epidermis cells of Commelina communis L. Implications, both scientific and agricultural We have improved the understanding of how O3 and H2O2 do affect guard cell and stomatal function. We have shown that economical important Israeli species like zucchini and pine are relatively sensitive to O3.

Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato plants are largely unknown. The goal of the project was to elucidate the molecular interactions between Cmmand tomato. The first objective was to analyze gene expression profiles of susceptible tomato plants infected with pathogenic and endophytic Cmmstrains. Microarray analysis identified 122 genes that were differentially expressed during early stages of infection. Cmm activated typical basal defense responses in the host including induction of defense-related genes, production of scavenging of free oxygen radicals, enhanced protein turnover and hormone synthesis. Proteomic investigation of the Cmm-tomato interaction was performed with Multi-Dimensional Protein Identification Technology (MudPIT) and mass spectroscopy. A wide range of enzymes secreted by Cmm382, including cell-wall degrading enzymes and a large group of serine proteases from different families were identified in the xylem sap of infected tomato. Based on proteomic results, the expression pattern of selected bacterial virulence genes and plant defense genes were examined by qRT-PCR. Expression of the plasmid-borne cellulase (celA), serine protease (pat-1) and serine proteases residing on the chp/tomA pathogenicity island (chpCandppaA), were significantly induced within 96 hr after inoculation. Transcription of chromosomal genes involved in cell wall degradation (i.e., pelA1, celB, xysA and xysB) was also induced in early infection stages. The second objective was to identify by VIGS technology host genes affecting Cmm multiplication and appearance of disease symptoms in plant. VIGS screening showed that out of 160 tomato genes, which could be involved in defense-related signaling, suppression of 14 genes led to increase host susceptibility. Noteworthy are the genes Snakin-2 (inhibitor of Cmm growth) and extensin-like protein (ELP) involved in cell wall fortification. To further test the significance of Snakin -2 and ELP in resistance towards Cmm, transgenic tomato plants over-expressing the two genes were generated. These plants showed partial resistance to Cmm resulting in a significant delay of the wilt symptoms and reduction in size of canker lesion compared to control. Furthermore, colonization of the transgenic plants was significantly lower. The third objective was to assess the involvement of ethylene (ET), jasmonate (JA) and salicylic acid (SA) in Cmm infection. Microarray and proteomic studies showed the induction of enzymes involved in ET and JA biosynthesis. Cmm promoted ET production 8 days after inoculation and SIACO, a key enzyme of ET biosynthesis, was upregulated. Inoculation of the tomato mutants Never ripe (Nr) impaired in ET perception and transgenic plants with reduced ET synthesis significantly delayed wilt symptoms as compared to the wild-type plants. The retarded wilting in Nr plants was shown to be a specific effect of ET insensitivity and was not due to altered expression of defense related genes, reduced bacterial population or decrease in ethylene biosynthesis . In contrast, infection of various tomato mutants impaired in JA biosynthesis (e.g., def1, acx1) and JA insensitive mutant (jai1) yielded unequivocal results. The fourth objective was to determine the role of cell wall degrading enzymes produced by Cmm in xylem colonization and symptoms development. A significance increase (2 to 7 fold) in expression of cellulases (CelA, CelB), pectate lyases (PelA1, PelA2), polygalacturonase and xylanases (XylA, XylB) was detected by qRT-PCR and by proteomic analysis of the xylem sap. However, with the exception of CelA, whose inactivation led to reduced wilt symptoms, inactivation of any of the other cell wall degrading enzymes did not lead to reduced virulence. Results achieved emphasized the complexity involved in Cmm-tomato interactions. Nevertheless they provide the basis for additional research which will unravel the mechanism of Cmm pathogenicity and formulating disease control measures.

There is a need for renewable antimicrobial surface treatments that are semi- permanent, can eradicate both biofilms and planktonic pathogens over long periods of time and that do not select for resistant strains. This proposal describes a dopamine binding technology that is inexpensive, bio-friendly, non-toxic, and uses straight-forward commercially available products. The antimicrobial agents are peptidoglycanhydrolase enzymes that are non-toxic and highly refractory to resistance development. The goal of this project is to create a treatment that will be applicable to a wide variety of surfaces and will convey long-lasting antimicrobial activity. Although the immediate goal is to create staphylolytic surfaces, the technology should be applicable to any pathogen and will thus contribute to no less than 3 BARD priorities: 1) increased animal production by protecting animals from invasive and emerging diseases, 2) Antimicrobial food packaging will improve food safety and security and 3) sustainable bio- energy systems will be supported by coating fermentation vats with antimicrobials that could protect ethanolic fermentations from Lactobacillus contamination that reduces ethanol yields. The dopamine-based modification of surfaces is inspired by the strong adhesion of mussel adhesion proteins to virtually all types of surfaces, including metals, polymers, and inorganic materials. Peptidoglycanhydrolases (PGHs) meet the criteria of a surface bound antimicrobial with their site of action being extracellular peptidoglycan (the structural basis of the bacterial cell wall) that when breached causes osmotic lysis. As a proof of principle, we will develop technology using peptidoglycanhydrolase enzymes that target Staphylococcus aureus, a notoriously contagious and antimicrobial-resistant pathogen. We will test for susceptibility of the coating to a variety of environmental stresses including UV light, abrasive cleaning and dessication. In order to avoid resistance development, we intend to use three unique, synergistic, simultaneous staphylococcal enzyme activities. The hydrolases are modular such that we have created fusion proteins with three lytic activities that are highly refractory to resistance development. It is essential to use multiple simultaneous activities to avoid selecting for antimicrobial resistant strains. This strategy is applicable to both Gram positive and negative pathogens. We anticipate that upon completion of this award the technology will be available for commercialization within the time required to achieve a suitable high volume production scheme for the required enzymes (~1-2 years). We expect the modified surface will remain antimicrobial for several days, and when necessary, the protocol for renewal of the surface will be easily applied in a diverse array of environments, from food processing plants to barnyards.