Letteratura scientifica selezionata sul tema "Cell wall deficiency"

Phosphorus is one of the mineral nutrient elements essential for plant growth and development. Low phosphate (Pi) availability in soils adversely affects crop production. To cope with low P stress, remodeling of root morphology and architecture is generally observed in plants, which must be accompanied by root cell wall modifications. It has been documented that cell wall proteins (CWPs) play critical roles in shaping cell walls, transmitting signals, and protecting cells against environmental stresses. However, understanding of the functions of CWPs involved in plant adaptation to P deficiency remains fragmentary. The aim of this review was to summarize advances in identification and functional characterization of CWPs in responses to P deficiency, and to highlight the critical roles of CWPs in mediating root growth, P reutilization, and mobilization in plants.

Intraperitoneal administration of group A streptococcal cell walls to Lewis rats induces a chronic arthritis, whereas the Fischer strain is resistant to the development of the lesion. Spleen cells from cell wall-treated rats (Lewis and Fischer) are deficient in the synthesis of IL-2. Using an mAb directed against the rat IL-2-R, the present studies indicate that the expression of IL-2-R on spleens of cell wall-treated rats is normal. However, the addition of exogenous IL-2 to spleen cells cultured with Con A does not stimulate the mitogenic response.

Bacillus subtilis Ni15 is deficient in cell wall turnover. This deficiency is removed if the medium contains 0.2 M NaCl, which does not affect growth. The levels of amidase and glucosaminidase, the most likely enzymes involved in turnover, were, in stationary phase Nil5 cells, similar to those in late-exponential phase cells of a standard strain. The Nil5 enzymes were not salt sensitive. However, the Nil5 walls contained 4.7-fold less phosphorus than the walls of the standard strain. Since the phosphorus content of B. subtilis walls reflects the level of teichoic acid, it is proposed that the turnover deficiency of this strain is due to a decrease in wall teichoic acid.

The cell wall plays a central role in protecting bacteria from some environmental stresses, but not against all. In fact, in some cases, an elaborate cell envelope may even render the cell more vulnerable. For example, it contains molecules or complexes that bacteriophages recognize as the first step of host invasion, such as proteins and sugars, or cell appendages such as pili or flagella. In order to counteract phages, bacteria have evolved multiple escape mechanisms, such as restriction-modification, abortive infection, CRISPR/Cas systems or phage inhibitors. In this perspective review, we present the hypothesis that bacteria may have additional means to escape phage attack. Some bacteria are known to be able to shed their cell wall in response to environmental stresses, yielding cells that transiently lack a cell wall. In this wall-less state, the bacteria may be temporarily protected against phages, since they lack the essential entities that are necessary for phage binding and infection. Given that cell wall deficiency can be triggered by clinically administered antibiotics, phage escape could be an unwanted consequence that limits the use of phage therapy for treating stubborn infections.

TCH4 is a xyloglucan endotransglucosylase/hydrolase (XTH) family member. Extensive studies have shown that XTHs are very important in cell wall homeostasis for plant growth and development. Boron (B), as an essential micronutrient for plants, plays an essential role in the cross-linking of cell wall pectin. However, the effect of B on cell wall organization is unclear. This study aimed to explore the mechanism of plant adaption to B stress by investigating the role of TCH4 in cell wall homeostasis. We conducted both plate and hydroponic cultures of wild-type Col-0 and overexpression and gene knockout lines of XTH22/TCH4 to analyze the phenotype, components, and characteristics of the cell wall using immunofluorescence, atomic force microscopy (AFM), and transmission electron microscopy (TEM). B deficiency induces the expression of TCH4. The overexpression lines of TCH4 presented more sensitivity to B deficiency than the wild-type Col-0, while the knockout lines of TCH4 were more resistant to low B stress. Up-regulation of TCH4 influenced the ratio of chelator-soluble pectin to alkali-soluble pectin and decreased the degree of methylesterification of pectin under B-deficient conditions. Moreover, we found that B deficiency disturbed the arrangement of cellulose, enlarged the gap between cellulose microfibrils, and decreased the mechanical strength of the cell wall, leading to the formation of a thickened and deformed triangular region of the cell wall. These symptoms were more profound in the TCH4 overexpression lines. Consistently, compared with Col-0, the O2− and MDA contents in the TCH4 overexpression lines increased under B-deficient conditions. This study identified the B-deficiency-induced TCH4 gene, which regulates cell wall homeostasis to influence plant growth under B-deficient conditions.

Abnormal sterols disrupt cellular functions through yet unclear mechanisms. In Saccharomyces cerevisiae, accumulation of Δ8-sterols, the same type of sterols observed in patients of Conradi–Hünermann–Happle syndrome or in fungi after amine fungicide treatment, leads to cell wall weakness. We have studied the influence of Δ8-sterols on the activity of glucan synthase I, the protein synthetizing the main polymer in fungal cell walls, its regulation by the Cell Wall Integrity (CWI) pathway, and its transport from the endoplasmic reticulum to the plasma membrane. We ascertained that the catalytic characteristics were mostly unaffected by the presence of abnormal sterols but the enzyme was partially retained in the endoplasmic reticulum, leading to glucan deficit at the cell wall. Furthermore, we observed that glucan synthase I traveled through an unconventional exocytic route to the plasma membrane that is associated with low density intracellular membranes. Also, we found out that the CWI pathway remained inactive despite low glucan levels at the cell wall. Taken together, these data suggest that Δ8-sterols affect cell walls by inhibiting unconventional secretion of proteins leading to retention and degradation of glucan synthase I, while the compensatory CWI pathway is unable to activate. These results could be instrumental to understand defects of bone development in cholesterol biosynthesis disorders and fungicide mechanisms of action.

Abstract Lignin is a complex phenylpropanoid polymer deposited in the secondary cell walls of vascular plants. Unlike most gymnosperm and eudicot lignins that are generated via the polymerization of monolignols, grass lignins additionally incorporate the flavonoid tricin as a natural lignin monomer. The biosynthesis and functions of tricin-integrated lignin (tricin-lignin) in grass cell walls and its effects on the utility of grass biomass remain largely unknown. We herein report a comparative analysis of rice (Oryza sativa) mutants deficient in the early flavonoid biosynthetic genes encoding CHALCONE SYNTHASE (CHS), CHALCONE ISOMERASE (CHI), and CHI-LIKE (CHIL), with an emphasis on the analyses of disrupted tricin-lignin formation and the concurrent changes in lignin profiles and cell wall digestibility. All examined CHS-, CHI-, and CHIL-deficient rice mutants were largely depleted of extractable flavones, including tricin, and nearly devoid of tricin-lignin in the cell walls, supporting the crucial roles of CHS and CHI as committed enzymes and CHIL as a noncatalytic enhancer in the conserved biosynthetic pathway leading to flavone and tricin-lignin formation. In-depth cell wall structural analyses further indicated that lignin content and composition, including the monolignol-derived units, were differentially altered in the mutants. However, regardless of the extent of the lignin alterations, cell wall saccharification efficiencies of all tested rice mutants were similar to that of the wild-type controls. Together with earlier studies on other tricin-depleted grass mutant and transgenic plants, our results reflect the complexity in the metabolic consequences of tricin pathway perturbations and the relationships between lignin profiles and cell wall properties.

Cell Wall-Deficiency in Staphylococcus aureus and its Role in Antibiotic Resistance. Elizabeth R. Fuller. Cell wall-deficient bacteria (CWDB) induced from Staphylococcus aureus ATCC 9144 (Oxford strain) were generated on medium with elevated osmolality in the presence of sublethal levels of penicillin G. On removal of antibiotic pressure the cell wall-competent (CWC) revertants along with these CWDB exhibited high-level penicillin and methicillin resistance, which was stable in the revertants. The revertants looked visually different, had an altered Gram stain and growth rate. Their matrixassisted laser desorption/ionisation time-of-flight (MALDI-TOF) `fingerprint' was also altered and they were more resistant to lysis by lysostaphin in comparison to the wild-type. Reversed-phaseh igh-performance liquid chromatography( RP-HPLC) showed that the revertants' cell walls had shorter glycan chains and more pentaglycine cross-bridges. A rapid,r eproduciblem ethodu sing liquid mediaw ase stablishedu singt he same medium and sublethal levels of penicillin G. The revertants produced using this method had the same characteristics as those cells produced from the original method. The high-level resistance seen in the revertants was homogenous and confirmed to be due to the transient CWD state, along with not being strain-specific. Transmission electron microscopy showed that the CWD cells and the revertant cells, when grown in penicillin, had a very disordered cell wall with areas where the cell wall appeared absent and were indistinguishable. The revertant cells were mecA-negative,ß -lactamase-negativea nd did not contain any mutations in the coding regions of pbp genes. The CWD cells and revertant cells, when grown in penicillin, were resistant to lysis by lysostaphin but were very sensitive to lysis with Triton X- 100. These data indicate that the resistant cells are not dependent upon an intact cell wall for osmotic stability and they are able to switch readily to this mode of growth in the presence of penicillin G.

Listeria monocytogenes (Lm) est un pathogène intracellulaire facultatif responsable de la listériose, une infection alimentaire particulièrement dangereuse pour les femmes enceintes et les personnes immunodéprimées. Connue pour son adaptabilité, Lm persiste dans divers environnements, rendant son contrôle difficile. Lors d'infections prolongées de cellules épithéliales, telles que les hépatocytes et les trophoblastes, Lm passe d'un état réplicatif à un état quiescent au sein de vacuoles de type lysosomal, appelées Listeria-containing vacuoles (LisCVs). Cette transition est associée à la perte de ActA, protéine permettant la nucléation de l'actine, et à l'arrêt de la polymérisation de l'actine à la surface bactérienne. Au sein des LisCVs, la majorité des bactéries restent intactes et entrent dans un état peu voire non réplicatif jusqu'à atteindre un état viable mais non cultivable (VBNC), une forme dormante permettant la survie dans des conditions défavorables. L'infection prolongée des hépatocytes par Lm perturbe l'immunité de l'hôte, notamment en diminuant la sécrétion des protéines de phase aiguë (APP), essentielles à la réponse immunitaire. Ce processus pourrait empêcher l'élimination complète de Listeria du foie et favoriser ainsi l'établissement d'infection persistante. Lm montre également une capacité d'adaptation remarquable dans des environnements en dehors de l'hôte, notamment dans les milieux aquatiques, où il peut entrer dans un état VBNC. Les pathogènes VBNC posent un risque sanitaire majeur, car ils échappent aux méthodes de détection basées sur la croissance tout en conservant la capacité à se réactiver vers des formes virulentes. Une étude récente montre que lorsqu'elle est exposée à des conditions pauvres en nutriments, Lm perd sa forme en bâtonnet pour devenir ronde en raison de la perte de sa paroi cellulaire. Ces formes sans paroi cellulaire (cell wall-deficient, CWD) peuvent s'adapter aux déséquilibres physico-chimiques en modifiant leur membrane et en produisant des protéines spécifiques. La première partie de la thèse explore les interactions hôte-pathogène lors d'infections par Lm, en se focalisant sur les cellules trophoblastiques. À l'aide de la protéomique comparative par spectrométrie de masse (LC-MS/MS), cette étude compare les profils de sécrétome des cellules infectées et non infectées durant les phases réplicative (24h p.i.) et persistante (96h p.i) de l'infection. L'analyse des voies métaboliques mises en jeu dans le modèle trophoblastique indique que Lm module les réponses immunitaires par des processus intermédiaires tels que l'angiogenèse et les voies de signalisation, dont HIF-1α et MAPK, essentiels à la transduction du signal. Comme dans le foie, ces modulations pourraient être cruciales pour créer et maintenir une niche dans les cellules trophoblastiques ; cependant, les mécanismes restent à identifier. La seconde partie de la thèse étudie la persistance environnementale de Lm via un modèle in vitro d'eau minérale et une analyse de protéomique comparative. Les données montrent une régulation à la baisse des protéines liées à la paroi cellulaire, en cohérence avec l'établissement de forme CWD. L'analyse fonctionnelle a révélé également des réponses aux stress, notamment une diminution de la transduction des signaux, de la virulence et de la production d'énergie, cohérentes avec l'état VBNC. Ces résultats éclairent les stratégies de survie environnementale de Lm et ses mécanismes de persistance. Ce travail explore la persistance de Lm dans les trophoblastes et l'environnement, montrant son adaptation moléculaire à survivre dans des environnements variés. Dans les cellules hôtes, Lm réprime l'immunité, tandis que, dans des conditions pauvres en nutriments, elle privilégie l'acquisition de nutriments et la résistance aux stress. L'ensemble de ces données soulignent sa résilience et pourrait conduire à des applications potentielles pour détecter et traiter les infections persistantes
Listeria monocytogenes (Lm) is a facultative intracellular pathogen responsible for listeriosis, a severe foodborne illness in pregnant women and immunocompromised individuals. Known for its adaptability, Lm persists across varied environments, making it difficult to control. During long-term infection in epithelial cells, such as hepatocytes and trophoblasts, Lm shifts from a replicative to a quiescent state within lysosome-like vacuoles, termed Listeria-containing vacuoles (LisCVs). This transition is associated with the loss of the actin-nucleating protein ActA and the arrest of actin polymerisation at the bacterial surface. Within LisCVs, the majority of bacteria remain intact and enter a slow/non-replicative or a viable but non-culturable (VBNC) state, a dormant form enabling persistence under adverse conditions. Prolonged infection of hepatocytes by Listeria disrupts host immunity, particularly reducing the secretion of acute-phase proteins (APPs), key to the immune response. This process might prevent the complete elimination of Listeria from the liver, thereby favoring the establishment of persistent infection. Lm also displays notable adaptability outside host environments, particularly in water systems, where it can enter a VBNC dormant state. VBNC pathogens pose heightened health risks as they are undetectable by growth-based methods and can reactivate into a virulent form. A recent study shows that when exposed to these nutrient-poor conditions, the bacteria lose their rod shape and become round due to their cell wall loss. These cell wall-deficient (CWD) forms can adapt to physicochemical imbalances by modifying their membrane and producing specific proteins. The first part of this thesis explores host-pathogen interactions during Lm infections, focusing on trophoblast cells. Using comparative proteomics via LC-MS/MS, this study analyses differences in secretome profiles between infected and uninfected cells across replicative (24h p.i.) and persistent (96h p.i.) infection phases. Pathway analysis in the trophoblast model indicates that Lm modulates immune responses through intermediary processes like angiogenesis and signalling pathways, including HIF-1α and MAPK, essential for signal transduction. Similar to the liver, these modulations may be crucial for creating and sustaining a niche in trophoblast cells; however, mechanisms remain to be identified. The second part of this thesis investigates Lm environmental persistence using an in vitro mineral water model and comparative proteomics. Proteomic data identified the downregulation of cell wall-related proteins, consistent with the establishment of CWD form. Functional analysis showed additional stress responses, including decreased signal transduction, virulence, and energy production, all consistent with the VBNC state. These findings provide insight into Lm environmental survival strategies, aiding in the understanding of its persistence mechanisms. This work examines Lm persistence in trophoblast cells and environmental conditions, demonstrating its molecular adaptation to survive in diverse environments. Within host cells, Lm emphasises immune repression, while in nutrient-poor conditions, it focuses on nutrient scavenging and stress resistance. These findings highlight its resilience and could lead to potential applications for detection and treatment of persistent infections
Το Listeria monocytogenes (Lm) είναι ένας προαιρετικά ενδοκυτταρικός παθογόνος μικροοργανισμός, υπεύθυνος για τη λιστερίωση, μια σοβαρή τροφιμογενή λοίμωξη που επηρεάζει κυρίως έγκυες γυναίκες και ανοσοκατασταλμένα άτομα. Γνωστό για την προσαρμοστικότητά του, το Lm επιμένει σε ποικίλα περιβάλλοντα, καθιστώντας τον έλεγχό του δύσκολο. Κατά τη διάρκεια χρόνιων λοιμώξεων σε επιθηλιακά κύτταρα, όπως ηπατοκύτταρα και τροφοβλάστες, το Lm μεταβαίνει από πολλαπλασιασμό σε λανθάνουσα κατάσταση σε Listeria-containing vacuoles (LisCVs). Αυτή η μετάβαση συνδέεται με την απώλεια της ActA και την αναστολή του πολυμερισμού της ακτίνης στην επιφάνειά του. Στα LisCVs, τα βακτήρια εισέρχονται σε μια αργή/μη πολλαπλασιαστική ή βιώσιμη αλλά μη καλλιεργήσιμη (VBNC) κατάσταση, ευνοώντας την επιμονή. Η παρατεταμένη λοίμωξη των ηπατοκυττάρων από τη Listeria διαταράσσει την ανοσία του ξενιστή, μειώνοντας την έκκριση πρωτεϊνών οξείας φάσης (APPs), κρίσιμων για την ανοσοαπόκριση. Αυτό μπορεί να εμποδίσει την πλήρη εξάλειψή της από το ήπαρ, προωθώντας χρόνια λοίμωξη. Το Lm εμφανίζει προσαρμοστικότητα και εκτός ξενιστών, ιδιαίτερα σε υδάτινα συστήματα, όπου εισέρχεται στη VBNC κατάσταση. Οι οργανισμοί VBNC συνιστούν κίνδυνο, καθώς δεν ανιχνεύονται με μεθόδους καλλιέργειας αλλά μπορούν να επανενεργοποιηθούν. Πρόσφατη μελέτη δείχνει ότι σε συνθήκες πτωχών θρεπτικών στοιχείων, τα βακτήρια χάνουν το ραβδοειδές σχήμα τους και γίνονται στρογγυλά λόγω απώλειας κυτταρικού τοιχώματος. Αυτές οι μορφές χωρίς κυτταρικό τοίχωμα (CWD) προσαρμόζονται σε φυσικοχημικές ανισορροπίες μέσω τροποποιήσεων στη μεμβράνη και παραγωγής ειδικών πρωτεϊνών. Το πρώτο μέρος αυτής της διατριβής εξετάζει τις αλληλεπιδράσεις ξενιστή-παθογόνου στις λοιμώξεις Lm, εστιάζοντας στα κύτταρα τροφοβλάστης. Με συγκριτική πρωτεομική (LC-MS/MS), αναλύονται οι διαφορές στο εκκρίτωμα μολυσμένων και μη μολυσμένων κυττάρων στις φάσεις πολλαπλασιασμού (24h) και επιμονής (96h). Η ανάλυση μονοπατιών δείχνει ότι το Lm τροποποιεί τις ανοσολογικές αποκρίσεις μέσω διαδικασιών όπως η αγγειογένεση και τα μονοπάτια σηματοδότησης HIF-1α και MAPK, κρίσιμα για τη μεταβίβαση σήματος. Όπως και στο ήπαρ, αυτές οι τροποποιήσεις μπορεί να συμβάλλουν στη διατήρηση μιας λοίμωξης στα κύτταρα τροφοβλάστης, αν και οι ακριβείς μηχανισμοί παραμένουν άγνωστοι. Το δεύτερο μέρος εξετάζει την περιβαλλοντική επιμονή του Lm χρησιμοποιώντας in vitro μοντέλο μεταλλικού νερού και συγκριτική πρωτεομική. Τα δεδομένα ανέδειξαν μείωση πρωτεϊνών κυτταρικού τοιχώματος, επιβεβαιώνοντας τη μορφή CWD. Επιπλέον, η λειτουργική ανάλυση έδειξε αποκρίσεις στο στρες, όπως μείωση μεταβίβασης σήματος, λοιμογόνου δράσης και παραγωγής ενέργειας, συμβατές με την κατάσταση VBNC. Αυτά τα ευρήματα προσφέρουν νέα δεδομένα για τις στρατηγικές περιβαλλοντικής επιβίωσης του Lm, συμβάλλοντας στην κατανόηση των μηχανισμών επιμονής του. Η εργασία αυτή εξετάζει την επιμονή του Lm σε κύτταρα τροφοβλάστης και περιβαλλοντικές συνθήκες, καταδεικνύοντας τη μοριακή του προσαρμογή. Στα κύτταρα ξενιστή, το Lm καταστέλλει το ανοσοποιητικό, ενώ σε συνθήκες πτωχών θρεπτικών στοιχείων εστιάζει στη συλλογή τους και την αντοχή στο στρες. Αυτά τα ευρήματα αναδεικνύουν την ανθεκτικότητά του και θα μπορούσαν να συμβάλουν στην ανίχνευση και θεραπεία επίμονων λοιμώξεων

Abstract The patient (Fig. 17-1) was a 38-week-gestation product of a 22-year-old G,P1Abo (gravida 1, first pregnancy; paradelivery, first delivery; abortion, 0) woman and her 31-year-old firstcousin partner. Pregnancy was complicated by oligohydramnios (deficiency in the amount of amniotic fluid); an amniocentesis revealed a normal male karyotype. The infant was delivered by cesarean section because of maternal preeclampsia (development of hypertension due to pregnancy). Apgar scores were 8/9, weight was 2200 g, and length was 18.5 inches. On newborn examination, microcephaly (a broad nasal bridge with anteverted nostrils), micrognathia (smallness of the jaws, especially the underjaw), hypospadias (a defect in the wall of the urethra), and undescended testes were seen.A tentative diagnosis of Smith-Lemli- Opitz syndrome was postulated.

Abstract The patient (Fig. 17-1) was a 38-week-gestation product of a 22-year-old G,P1Abo (gravida 1, first pregnancy; paradelivery, first delivery; abortion, 0) woman and her 31-year-old first-cousin partner. Pregnancy was complicated by oligohydramnios (deficiency in the amount of amniotic fluid); an amniocentesis revealed a normal male karyotype. The infant was delivered by cesarean section because of maternal preeclampsia (development of hypertension due to pregnancy). Apgar scores were 8/9, weight was 2200 g, and length was 18.5 inches. On newborn examination, microcephaly (a broad nasal bridge with anteverted nostrils), micrognathia (smallness of the jaws, especially the underjaw), hypospadias (a defect in the wall of the urethra), and undescended testes were seen.A tentative diagnosis of Smith-LemliOpitz syndrome was postulated. By 9 months of age, marked developmental delay was noted. Repeat evaluation revealed bilateral inguinal hernias, dysmorphic features with synophrys (growing together of the eyebrows), and thick upper lip with gum hypertrophy; limitation of motion at the knees, hips, elbows, and wrists; camptodactyly (bending of fingers); rib flaring; hepatosplenomegaly; corneal clouding; and hypotonia in addition to those findings reported at birth.

Supravalvular aortic stenosis (SVAS) [1] is a disease of the cardiovascular system that leads to narrowing of the large arteries in humans. Studies have shown [2] that SVAS is caused by mutations or deletions in the elastin gene resulting in elastin haploinsufficiency. Elastin haploinsufficiency results in systemic hypertension [3], thinner and more numerous elastic lamellae [4], and altered arterial mechanics [5]. Genetically modified elastin deficient mice (ELN+/-) recapitulates the human phenotype including obstructive arterial disease and decreased arterial compliance [1,3]. Elastin deficiency in these mice is associated with changes in the mechanical microenvironment in the vascular wall [6], including enhanced wall thickness, increased smooth muscle cell (SMC) proliferation [7] and stiffening of arteries [8]. However, the molecular mechanisms for these changes are not fully understood. Also from a developmental perspective, no information is available regarding initiation and progression of aortic pathology in ELN+/− mice with time. The objectives of this study were to determine the temporal effects of elastin haploinsufficiency on the functional properties of aortic tissue and the aortic cell phenotype, using the elastin deficient mouse model (ELN+/-). We hypothesized that elastin haploinsufficiency will result in progressive abnormalities in aortic stiffness and dynamic alterations in aortic smooth muscle cell phenotype.

A thin liquid layer coating the airway can be unstable and forms a plug. Airway closure usually happens at the small airways near the end of expiration, often accompanied with hypersecretion or/and surfactant deficiency in the airway in a variety of lung diseases, such as chronic obstructive pulmonary disease (COPD) and acute respiratory distress syndrome (ARDS). Modeling work by Halpern and Grotberg [1] has shown that several forces could contribute to airway closure, such as the surface tension instability and the wall compliance. Experiments in a capillary tube were conducted by Cassidy et al. [2] who found that adding surfactant increased the airway closure time and the critical film thickness. In vitro studies [3] [4] illustrated that exposure of primary human airway epithelial cells to plug propagation and rupture led to significant cell injury. Experimental studies [5] [6] on excised lungs or in vivo animal models have shown that severe tissue damage was found in surfactant-deficient lungs due to the repetitive airway reopening. However, mechanical forces induced by airway closure have not been experimentally evaluated.

Immune thrombocytopenia has been recognized as a major hematologic manifestation associated with the acquired immune deficiency syndrome(AIDS) and AIDS-related complexes. The mechanism of thrombocytopenia in human immune deficiency virus infection is probably multifactorial.. The role of platelet-associated immuno-globulin(PAIgG)and circulating immune complexes(CIC) in mediating thrombocytopenia is controversial.. We experienced a case in which immune thrombocytopenia in AIDS-related complexes resolved during herpes zoster infection. A 37 y.o. white homosexual male with AIDS-related complexes and thrombocytopenia presented a 4-day history of painful, violaceous, non-pruritic vesicles which started on the right arm and hand, which then progressed to the chest wall, abdomen, back, and left arm. Peripheral lymphadenop-athy and splenomegaly was not noted. On admission WBG 13.5 Hct 40.8, platelet 46,000, Tzanck smear of vesicles revealed herpetic type giant cells. HTLV-III pos., Helper/suppressor T-cell ratio 0.3, Total protein 7.9 gm%, . Alb 3.95gm% IgG 1740 mg%, IgA 157 mg IgM 91.2 mg, Monospot neg., HBsAg Neg., HBsAb pos., HBcAb pos., VDRL neg. Before this admission, immune thrombocytopenia was documented by increased levels of PAIgG, CIC, bone marrow magakaryocytic hyperplasia, peripheral thrombocytopenia with giant platelets, absence of splenomegaly, and response to prednisone. He was treated with Acyclovir 250 mg/m2 1-hr infusion Q8h for 9 days ;to control the spread of his herpes infection. Recovery of thrombocytopenia wasobserved during herpes zo.ster infection. The platelet count rose to 158,000 and sustained over 4- weeks. Duringnormalization of platelet count thelevel of GIC (assayed by Raji cells, reference ranges, less than 12) droppedfrom 300 to less than 12 and PAIgG(fluorescence-activated flow cytometric assay, reference ranges, less than 1.5 RF) was markedly decreased from 90 to 2.9 When herpes infection had subsided the platelet count again decreased. These findings suggestthat PAIgG and GIG were contributing factors to immune thrombocytopenia and that herpes viral infection and Acyclovir altered this immunologically mediated thrombocytopenia in AIDS-related complexes.

Increased arterial stiffness is directly correlated with hypertension and cardiovascular disease. Stiffness of the conducting arteries is largely determined by the extracellular matrix (ECM) proteins in the wall, such as collagen and elastin, produced by the smooth muscle cells (SMCs) found in the medial layer. Elastin is deposited as soluble tropoelastin and is later crosslinked into elastin fibers. Newborn mice lacking the elastin protein ( Eln−/−) have increased arterial wall stiffness and SMCs with altered proliferation, migration and morphology [1]. Vessel elasticity is also mediated by other ECM proteins, such as fibulin-4. Elastic tissue, such as lung, skin, and arteries, from fibulin-4 deficient ( Fbln4−/−) mice show no decrease in elastin content, but have reduced elasticity due to disrupted elastin fibers [2]. Arteries from both elastin and fibulin-4 deficient mice have been previously studied, but the mechanical properties of their SMCs have not been investigated. Recent experiments comparing arterial SMCs from old and young animals suggest that mechanical properties of the SMCs themselves may contribute to changes in wall stiffness [3]. Hence, we investigated the stiffness of isolated arterial SMCs from elastin and fibulin-4 deficient mice using atomic force microscopy (AFM). In addition, we studied the effects of two elastin treatments on the mechanical properties of SMCs from Eln+/+ and Eln−/− mice. Differences between the treatments may elucidate the importance of soluble versus crosslinked elastin on single cell stiffness.

From Jan. 1982 to Jan. 1987, 1213 patients were investigated either because of thrombotic episode(s) or thrombosis heredity. Diagnosis of DVT was confirmed by phlebography in 567 cases. Patients were first examined at least three months after an acute episode.Methods. Anti thrombin (AT) and plasminogen were assayed, using chromogenic substrates S-2238 and S-2251 , respectively, followed if values were low by immunochemical assessment. Furthermore, fibrinogen, thrombin and reptilase times, APTT, P…P were assessed. Only in the latter study period was protein C activity determined (129 cases). Fibrinolysis was assayed on fibrin plates after 20 min venous occlusion of the arms. In 1984 only, plasminogen activator inhibitor of endothelial cell type (PAI 1) was measured in most DVT patients (n=75).Results. DVT first occurred by the age of 45 in 337 patients, of whom five had AT deficiency (four classic and one abnormal), one had low and another abnormal plasminogen), one had abnormal fibrinogen, seven had lupus anticoagulants (LA), and 72 (21%) had decreased lysis on fibrin plates. Defective fibrinolysis was re-investigated in 42 patients; at check-up 13 were found to have normalised. Of the 75 patients from 1984, fibrinolysis on fibrin plates was normal in 50 cases, of which PAI 1 was normal in 44 and increased in six; of the remaining 25 patients from 1984, fibrin plate activity was decreased, PAI 1 was normal in five cases and increased in 20.DVT first occurred after the age of 45 in 230 patients, of whom none had pathological AT or protein C, five had LA, and one had abnormal plasminogen; of the 51 (22%) patients found to have defective fibrinolysis, 29 were re-investigated at check-up and 14 of them found to have normalised.Conclusion. Alterations in coagulation inhibitors are rare in patients with DVT. A more frequent finding, although intraindividual fluctuations occur, is defective vessel wall fibrinolysis.

Platelet (P) Cyclo-oxygenase (CO) deficiency is characterized by bleeding tendency due to platelet release mechanism defect. Pareti et al. have reported a patient with PCO deficiency whose CO activity was defective not only in platelets but also in vessel walls. But it has not been demonstrated whether other cells than platelets do have this enzyme in other.patients with PCO deficiency. Recent data suggest taht hemorrhagic or thrombotic diathesis is dependent on the site(s) (platelet, vessel wall or others) or CO deficiency. In order to determine the CO activity in kidney in a patient with PCO deficiency (Scand. J. Haematol., 32; 167 – 174, 1984), We studied the urinary PGE2 excretion and renal function at basal state and before and after intravenous administration of angiotensin II (ANG II). Before ANG II infusion to the patient, 600 ml H20 was orally intaked, and then AGN II (5, 10, 18 ng/min. each dose for 10 min.) was infused intravenously. Urine samples were collected before (20 min.) AGN II infusion. Urinary PGE2 was measured by RIA. Urine osmorality was determined by Fiske osmometer. To measure basal daily excretion of urinary PGE2, daily total urine was collected into ice box for 2 days, and then stocked at −80°C until PGE2 measurement by RIA.A reduction of Uosm following the intravenous infusion of ANG II to normal humans has been reported by Usberti et al. (Am. J. Physiol. 248; 254-259, 1985), atK] aiso reported that Uosm increased after aspirin administration.It was concluded that CO activity in thd kidney of this patient was no al least defective, but endogenous PGE2 synthesis stumulated by ANG II ma be decreased.

The use of transgenic mice is an incredibly powerful tool in understanding the underlying etiology of disease. To understand the usefulness of specific transgenic mice, the systems of interest should be characterized. We have created TGFβ2-deficient mouse fetuses that develop widespread aortic and coronary artery aneurysms [1]. Several studies have pointed to a strong connection between elevated TGFβ signaling and aortic aneurysm [2]. In situ hybridization has shown that Tgfb2 and Tgfb3 are major ligands expressed in the aortic medial wall. Further reduction of TGFβ signaling by combining TGFβ2- and TGFβ3-deficient mice exacerbated cardiovascular aneurysms in TGFβ2/TGFβ3-doubly deficient embryos. In vitro cell culture experiments demonstrated an impaired ability of TGFβ2-deficient mouse embryonic fibroblasts to reorganize collagen. Previous data indicate reduced levels of TGFβ2 leading to a higher susceptibility to aortic aneurysm. We present here the macroscopic biomechanical characterization of the aorta of a transgenic mouse line showing this susceptibility and compare it to wild-type mice. We also present results comparing the microstructure between mouse lines.

PCC is used empirically for its factor VIII (FVIII) bypassing activity in hemophiliacs with inhibitors to FVIII. The effect of of PCC on plasma coagulation tests is variable and is not predictive of clinical response. We are testing the hypothesis that bypass activity is expressed locally at the vessel wall, which functions as a procoagulant surface at times of hemostatic stress. Monolayer cultures of umbilical vein endothelial cells (EC) were incubated for 1 hour with commerical PCC (1 u/ml), and then extensively washed to remove fluid phase PCC components. Ellagic acid-activated PTT determinations were then done over the treated monolayers using plasma deficient in FVIII (<1 u/ml). PTTs over EC pre-treated with PCC were shorter than PTTs with control EC or EC pre-treated with commercial FVIII concentrate (control 101 s, PCC treated 57.7 s, FVIII treated 84 s, means of n=3). Similar results were obtained using non-activated PTTs and with adult saphenous vein EC. The bypass effect of PCC was dose-related:non-activated PTTs were 172 s (control),134 s (PCC 0.1 u/ml),and 107 s (PCC 1 u/ml). Pre-treatment of EC with cycloheximide shortened the PTT of FVIII deficient plasma, presumably due to impaired expression of endogenous anticoagulant activity.Subsequent incubation of cycloheximide pre-treated EC with PCC still provided FVIII bypass activity. In contrast, FVIII consistently failed to shorten the PTT using FVIII deficient plasma added to cycloheximide pre-treated EC. Thfe ability of PCC to bypass the FVIII defect was not impaired using paraformaldehyde 1fixed in place of live EC, but was reduced or abolished if the ECwere incubated with PCC at 4° instead of 37°.The putative FVIII bypassing component present in PCC may therefore express its activity only upon interacting with EC at sites of vascular interruption. Further studies using purified reagents will be necessary to identify the responsible substance(s). These observations may help explain the discrepancy between the clinical. efficacy of PCC and its inconsistent effect on plasma coagulation tests.

For many years it is known that activation of the factor XII (FXII) -prekallikrein (PKK)- kininogen system of coagulation (contact activation) also may be involved in activation of fibrinolysis. Despite the numerous efforts over the past two decades to clarify this process, our current insights in this matter are far from complete. Also the physiological meaning of this possible interlinkage of coagulation and fibrinolysis is still uncertain; clearcut clinical manifestations in patients deficient in FXII or PKK are not found.No doubt, activation of fibrinolysis is a much more complicated process than it originally was thought to be, and it is only recently that the importance of urokinase for fibrinolysis in the circulation became clear. Two pathways of plasminogen (Pig) activation may be distinguished: 1. the extrinsic system, catalysed by t-PA, which upon stimulus is increasingly released from the endothelial cells of the vessel wall and 2. the intrinsic system, catalysed via Pig proactivators which circulate in the blood at a fairly constant level of concentration. The discovery that the virgin 55 kD urokinase molecule in fact is a single-chain proenzym (now denoted by scu-PA, single-chain urokinase-type PA), the notion that 55 kD scu-PA occurs in the blood and that its concentration even among individuals is fairly constant (2.1+/-0.4 ng/ml, n=52), and the observation that the efficacy of scu-PA is fibrin selective, all are recent findings pointing to the involvement of scu-PA in the intrinsic system.Still the relation between contact activation and the activation of scu-PA is obscure. Active KK, for instance, is an effective activator of 55 kD scu-PA, but proteolytic cleavage of scu-PA resulting in an active molecule, is readily achieved in plasma’s deficient in FXII or PKK. In addition, a portion of Pig activator activity which is dependent for its activation on FXII and PKK, is fully recovered in plasma’s artificially depleted in 55 kD scu-PA. Yet, both portions are activated by negatively charged surfaces or dextransulphate (DXS) as a substitute! These observations have led to the concept of two co-ordinative pathways of Pig activation for the intrinsic system: one containing scu-PA, the other containing FXII, PKK and a postulated Pig proactivator (note that the Pig activator activities of FXIIa and KK per se do not account for the latter portion of activity). Until recently in both pathways was a missing link: in the former it was the step between the negatively charged surface and scu-PA, in the latter it was the postulated Pig proactivator between active KK and Pig. This year, however, it became clear that in plasma artificially depleted in u-PA, still a substantial amount of protein immunochemically related to u-PA, can be detected (at least 35 ng/ml), but only after SDS PAGE. Part of this protein is a single-chain 110 kD molecule which in plasma can be converted to a cleaved molecule with Pig activator activity provided the plasma contains FXII and PKK. Although the relation with the 55 kD scu-PA remained unclear, the discovery of this 110 kD PA with latent urokinase antigen, undoubtedly, explains the missing link between KK and Pig. The other missing link still remains unexplained. It could be an in vitro artefact by DXS causing scu-PA catalysed activation of Pig as fibrin clots do. Since subsequently generated plasmin is capable of activation of both scu-PA and FXII, the two intrinsic pathways are thus interlinked via feed-back activation and consequently may be co-operative in function.

The objectives of the research in this proposal were to (A) identify synergy among proteins that provide enhanced activity over single proteins for control of plant pathogenic fungi, (B) clone and characterize genetic sequences coding for proteins with ability to control pathogenic fungi, (C) produce transgenic organisms with enhanced biocontrol ability using genes and gene combinations and determine their efficiency in protecting plants against plant pathogenic fungi. A related objective was to produce disease-resistant plants. Fungal cell wall degrading enzymes from any source are strongly synergistic with any membrane active compound and, further, different classes of cell wall degrading enzymes are also strongly synergistic. We have cloned and sequenced a number of genes from bacterial and fungal sources including five that are structurally unrelated. We have prepared transgenic fungi that are deficient in production of enzymes and useful in mechanistic studies. Others are hyperproducers of specific enzymes that permit us, for the first time, to produce enzymes from T. harzianum in sufficient quantity to conduct tests of their potential use in commercial agriculture. Finally, genes from these studies have been inserted into several species of crop plants were they produce a high level of resistance to several plant pathogenic fungi.

The objectives of the project were: a) to construct the site-specific integrative expression cassettes carrying: (i) the chiA gene for a 58-kDa endochitinase, (ii) the pyrrolnitrin biosynthesis operon, and (iii) the acdS gene encoding ACC deaminase; b) to employ these constructs to engineer stable recombinant strains with an expanded repertoire of beneficial activities; c) to evaluate the rhizosphere competence and antifungal activity of the WT and modified strains against pathogenic fungi under laboratory and greenhouse conditions; and d) to monitor the persistence and impact of the introduced strains on culturable and nonculturable rhizosphere microbial populations in the greenhouse and the field. The research generally support our concepts that combining strategically selected genes conferring diverse modes of action against plant pathogens into one organism can improve the efficacy of biological control agents. We hypothesized that biocontrol agents (BCAs) engineered to expand their repertoire of beneficial activities will more effectively control soilborne plant pathogens. In this work, we demonstrated that biocontrol activity of Pseudomonas fluorescens Q8r1-96 and Q2-87, both producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) effective against the plant pathogenic fungus Rhizoctonia solani, can be improved significantly by introducing and expressing either the 1.6-kb gene chiA, encoding the 58-kDa endochitinase ChiA from the rhizosphere strain SerratiaplymuthicaIC1270, or the 5.8-kb prnABCDoperon encoding the broad-range antibiotic pyrrolnitrin (Prn) from another rhizosphere strain, P. fluorescens Pf-5. The PₜₐcchiAandPₜₐcprnABCDcassettes were cloned into the integrative pBK-miniTn7-ΩGm plasmid, and inserted into the genomic DNA of the recipient bacteria. Recombinant derivatives of strains Q8r1-96 and Q2-87 expressing the PₜₐcchiA or PₜₐcprnABCD cassettes produced endochitinase ChiA, or Prn, respectively, in addition to 2,4-DAPG, and the recombinants gave significantly better biocontrol of R. solani on beans under greenhouse conditions. The disease reduction index increased in comparison to the parental strains Q8r1-96 and Q2-87 to 17.5 and 39.0% from 3.2 and 12.4%, respectively, in the case of derivatives carrying the PₜₐcchiAcassette and to 63.1 and 70% vs. 2.8 and 12,4%, respectively, in the case of derivatives carrying the PₜₐcprnABCDcassette. The genetically modified strains exhibited persistence and non-target effects comparable to those of the parental strains in greenhouse soil. Three integrative cassettes carrying the acdS gene encoding ACC deaminase cloned under the control of different promoters were constructed and tested for enhancement of plant growth promotion by biocontrol strains of P. fluorescens and S. plymuthica. The integrative cassettes constructed in this work are already being used as a simple and efficient tool to improve biocontrol activity of various PGPR bacteria against fungi containing chitin in the cell walls or highly sensitive to Prn. Some parts of the work (e. g., construction of integrative cassettes) was collaborative while other parts e.g., (enzyme and antibiotic activity analyses) were fully synergistic. The US partners isolated and provided to the Israeli collaborators the original biocontrol strains P. fluorescens strains Q8r1-96 and Q2-87 and their mutants deficient in 2,4-DAPG production, which were used to evaluate the relative importance of introduction of Prn, chitinase or ACC deaminase genes for improvement of the biocontrol activity of the parental strains. The recombinant strains obtained at HUJI were supplied to the US collaborators for further analysis.