Academic literature on the topic 'Metabolism; Enzymatic; Regulatory functions'

Sleep has a regulatory role in maintaining metabolic homeostasis and cellular functions. Inadequate sleep time and sleep disorders have become more prevalent in the modern lifestyle. Fragmentation of sleep pattern alters critical intracellular second messengers and neurotransmitters which have key functions in brain development and behavioral functions. Tryptophan metabolism has also been found to get altered in SD and it is linked to various neurodegenerative diseases. The kynurenine pathway is a major regulator of the immune response. Adequate sleep alleviates neuroinflammation and facilitates the cellular clearance of metabolic toxins produced within the brain, while sleep deprivation activates the enzymatic degradation of tryptophan via the kynurenine pathway, which results in an increased accumulation of neurotoxic metabolites. SD causes increased production and accumulation of kynurenic acid in various regions of the brain. Higher levels of kynurenic acid have been found to trigger apoptosis, leads to cognitive decline, and inhibit neurogenesis. This review aims to link the impact of sleep deprivation on tryptophan metabolism and associated complication in the brain.

Survival of the pathogenic yeast Candida albicans depends upon assimilation of fermentable and non-fermentable carbon sources detected in host microenvironments. Among the various carbon sources encountered in a human body, glucose is the primary source of energy. Its effective detection, metabolism and prioritization via glucose repression are primordial for the metabolic adaptation of the pathogen. In C. albicans, glucose phosphorylation is mainly performed by the hexokinase 2 (CaHxk2). In addition, in the presence of glucose, CaHxK2 migrates in the nucleus and contributes to the glucose repression signaling pathway. Based on the known dual function of the Saccharomyces cerevisiae hexokinase 2 (ScHxk2), we intended to explore the impact of both enzymatic and regulatory functions of CaHxk2 on virulence, using a site-directed mutagenesis approach. We show that the conserved aspartate residue at position 210, implicated in the interaction with glucose, is essential for enzymatic and glucose repression functions but also for filamentation and virulence in macrophages. Point mutations and deletion into the N-terminal region known to specifically affect glucose repression in ScHxk2 proved to be ineffective in CaHxk2. These results clearly show that enzymatic and regulatory functions of the hexokinase 2 cannot be unlinked in C. albicans.

Glycerol kinase (GYK) plays a critical role in hepatic metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. GYK isoform b is the only glycerol kinase present in whole cells, and has a non-enzymatic moonlighting function in the nucleus. GYK isoform b acts as a co-regulator of nuclear receptor subfamily 4 group A1 (NR4A1) and participates in the regulation of hepatic glucose metabolism by protein–protein interaction with NR4A1. Herein, GYK expression was found to upregulate the expression of NR4A1-mediated lipid metabolism-related genes (SREBP1C, FASN, ACACA, and GPAM) in HEK293T and L02 cells, and in mouse in vivo studies. GYK expression increased blood levels of cholesterol, triglyceride, and high-density lipoprotein cholesterol, but not low-density lipoprotein cholesterol levels. It enhanced the transcriptional activity of Nr4a1 target genes by negatively cooperating with NR4A1 and its enzymatic activity or by other undefined moonlighting functions. This enhancement was observed in both normal and diabetic mice. We also found a feed-forward regulation loop between GYK and NR4A1, serving as part of a GYK-NR4A1 regulatory mechanism in hepatic metabolism. Thus, GYK regulates the effect of NR4A1 on hepatic lipid metabolism in normal and diabetic mice, partially through the cooperation of GYK and NR4A1.

Current knowledge of the uptake and metabolism of the major energy yielding and nitrogenous nutrients that are naturally available to ruminal bacteria is reviewed. The potential use of metabolic engineering to manipulate these metabolic pathways and improve nutrient utilization in ruminant animals is briefly discussed. Metabolic engineering is the use of recombinant DNA techniques to enhance microbial function by manipulating enzymatic, transport and regulatory functions of the cell. Examples of the use of metabolic engineering in industrial fermentation are also given.

Mammalian patatin-like phospholipase domain–containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure–function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes.

Several enzymes that were originally characterized to have one defined function in intermediatory metabolism are now shown to participate in a number of other cellular processes. Multifunctional proteins may be crucial for building of the highly complex networks that maintain the function and structure in the eukaryotic cell possessing a relatively low number of protein-encoding genes. One facet of this phenomenon, on which I will focus in this review, is the interaction of metabolic enzymes with RNA. The list of such enzymes known to be associated with RNA is constantly expanding, but the most intriguing question remains unanswered: are the metabolic enzyme-RNA interactions relevant in the regulation of cell metabolism? It has been proposed that metabolic RNA-binding enzymes participate in general regulatory circuits linking a metabolic function to a regulatory mechanism, similar to the situation of the metabolic enzyme aconitase, which also functions as iron-responsive RNA-binding regulatory element. However, some authors have cautioned that some of such enzymes may merely represent "molecular fossils" of the transition from an RNA to a protein world and that the RNA-binding properties may not have a functional significance. Here I will describe enzymes that have been shown to interact with RNA (in several cases a newly discovered RNA-binding protein has been identified as a well-known metabolic enzyme) and particularly point out those whose ability to interact with RNA seems to have a proven physiological significance. I will also try to depict the molecular switch between an enzyme's metabolic and regulatory functions in cases where such a mechanism has been elucidated. For most of these enzymes relations between their enzymatic functions and RNA metabolism are unclear or seem not to exist. All these enzymes are ancient, as judged by their wide distribution, and participate in fundamental biochemical pathways.

Hyaluronan is a matrix polymer prominent in tissues undergoing rapid growth, development, and repair, in embryology and during malignant progression. It reaches 107Daltons in size but also exists in fragmented forms with size-specific actions. It has intracellular forms whose functions are less well known. Hyaluronan occurs in all vertebrate tissues with 50% present in skin. Hyaluronan provides a scaffold on which sulfated proteoglycans and matrix proteins are organized. These supramolecular structures are able to entrap water and ions to provide tissues with hydration and turgor. Hyaluronan is recognized by membrane receptors that trigger intracellular signaling pathways regulating proliferation, migration, and differentiation. Cell responses are often dependent on polymer size. Catabolic turnover occurs by hyaluronidases and by free radicals, though proportions between these have not been determined. New aspects of hyaluronan biology have recently become realized: involvement in autophagy, in the pathology of diabetes., the ability to modulate immune responses through effects on T regulatory cells and, in its fragmented forms, by being able to engage several toll-like receptors. It is also apparent that hyaluronan synthases and hyaluronidases are regulated at many more levels than previously realized, and that the several hyaluronidases have functions in addition to their enzymatic activities.

Enzyme promiscuity toward substrates has been discussed in evolutionary terms as providing the flexibility to adapt to novel environments. In the present work, we describe an approach toward exploring such enzyme promiscuity in the space of a metabolic network. This approach leverages genome-scale models, which have been widely used for predicting growth phenotypes in various environments or following a genetic perturbation; however, these predictions occasionally fail. Failed predictions of gene essentiality offer an opportunity for targeting biological discovery, suggesting the presence of unknown underground pathways stemming from enzymatic cross-reactivity. We demonstrate a workflow that couples constraint-based modeling and bioinformatic tools with KO strain analysis and adaptive laboratory evolution for the purpose of predicting promiscuity at the genome scale. Three cases of genes that are incorrectly predicted as essential inEscherichia coli—aspC,argD, andgltA—are examined, and isozyme functions are uncovered for each to a different extent. Seven isozyme functions based on genetic and transcriptional evidence are suggested between the genesaspCandtyrB,argDandastC,gabTandpuuE, andgltAandprpC. This study demonstrates how a targeted model-driven approach to discovery can systematically fill knowledge gaps, characterize underground metabolism, and elucidate regulatory mechanisms of adaptation in response to gene KO perturbations.

AbstractPlants are redox systems and redox-active compounds control and regulate all aspects of their life. Recent studies have shown that changes in reactive oxygen species (ROS) concentration mediated by enzymatic and non-enzymatic antioxidants are transferred into redox signals used by plants to activate various physiological responses. An overview of the main antioxidants and redox signaling in plant cells is presented. In this review, the biological effects of ROS and related redox signals are discussed in the context of acclimation to changing environmental conditions. Special attention is paid to the role of thiol/disulfide exchange via thioredoxins (Trxs), glutaredoxins (Grxs) and peroxiredoxins (Prxs) in the redox regulatory network. In plants, chloroplasts and mitochondria occupying a chloroplasts and mitochondria play key roles in cellular metabolism as well as in redox regulation and signaling. The integrated redox functions of these organelles are discussed with emphasis on the importance of the chloroplast and mitochondrion to the nucleus retrograde signaling in acclimatory and stress response.

Iron-sulfur clusters-containing proteins participate in many cellular processes, including crucial biological events like DNA synthesis and processing of dioxygen. In most iron-sulfur proteins, the clusters function as electron-transfer groups in mediating one-electron redox processes and as such they are integral components of respiratory and photosynthetic electron transfer chains and numerous redox enzymes involved in carbon, oxygen, hydrogen, sulfur and nitrogen metabolism. Recently, novel regulatory and enzymatic functions of these proteins have emerged. Iron-sulfur cluster proteins participate in the control of gene expression, oxygen/nitrogen sensing, control of labile iron pool and DNA damage recognition and repair. Their role in cellular response to oxidative stress and as a source of free iron ions is also discussed.

To cope with various endogenous toxin and xenobiotics nature has equipped the organisms with a proper protection system. Glutathione transferases (GSTs) are important components of the cellular defense against oxidative stress. These proteins appear to be suited for different tasks.

Based on catalytic activity of GSTs with monochlorobimane (MCB), a screening method was developed for identification of active GSTs in bacterial colonies and for characterization of combinatorial GST libraries.

Solvent viscosity effects on k_cat and k_cat/K_m on wild-type human GST A1-1 and phenylalanine-220 mutants indicate a physical step being the rate-limiting step in the catalytic mechanism.

Three residues that were under evolutionary selection pressure were identified in Mu class GSTs. By changing these residues in human GSTM2-2, a 1000-fold change of catalytic activity towards GSTM1-1 was accomplished.

Using peptide phage display, a peptide sequence was found that acts as non-substrate ligand for human GST M2-2. The peptide sequence was shown to be highly similar to the C-terminal region of c-Jun N-terminal kinase (JNK). JNK is a kinase linked to activating protein-1 (AP-1) transcriptional activity, which is part of the regulation of cell proliferation and apoptosis in response to cellular stress. Reporter gene assays in cell lines showed that human GST M2-2 coactivates the transcriptional activity of AP-1.

GSTs as part of the cellular defense against oxidative stress could be important in inflammatory processes. The distribution of GSTs in the intestine of both mice and human in abnormal inflammatory state was investigated immunohistochemically. Using an experimental mouse model, it was shown that mouse GST A4-4 is markedly induced while, the expression of Mu and Pi class GSTs is reduced in the colon of conventional and germ-free mice with extensive colitis. Moreover, the expression of mouse GST A4-4 was elevated with time when germ-free mice were exposed to normal bacteria flora. In contrast, Mu and Pi class GSTs showed decreased expression in the colon of germ-free mice associated with commensal flora. The Alpha, Mu and Pi class GST levels in mouse colon were increased when germ-free mice received Lactobacillus strain GG.

The distribution of Alpha, Mu and Pi class GST in the intestinal tissues of patients with Crohn’s disease was investigated using immunohistochemistry. All the three classes were consistently expressed in the intestinal epithelium as well as in macrophage-like cells and smooth muscle tissue. The mucus secreting goblet cells, however, did not express Alpha class GST.

Les thérapies anti-tumorales se sont considérablement améliorées au cours de la dernière décennie. Toutefois, les traitements utilisés actuellement rencontrent d'importantes limitations, notamment dans le cas de cancers métastatiques, révélant l'urgence de développer de nouvelles approches. Ainsi, l'immunothérapie par transfert adoptif de cellules T représente une approche innovante particulièrement prometteuse. Son principe s'appuie sur l'injection de cellules T autologues spécifiques d'antigènes tumoraux, préalablement manipulées et amplifiées ex vivo, chez des patients rendus lymphopéniques par chimiothérapie et/ou radiothérapie. Toutefois, même si l'état lymphopénique est induit par ces 2 protocoles de conditionnements, leurs effets sur l'environnement de l'hôte ainsi que sur le devenir des cellules T greffées étaient, jusqu'à nos travaux, mal connus. Par le biais de modèles murins, nous avons pu démontrer que le devenir des cellules T diffère après transfert dans des souris irradiées ou traitées par chimiothérapie (Bu/Cy). Ainsi, après transfert dans des animaux irradiés, on observe une prolifération préférentielle des cellules T CD8, dépendante de l'IL-7, est observée alors qu'un transfert chez des souris traitées Bu/Cy se traduit par une prolifération rapide, indépendante de l'IL-7, des cellules T CD4. De plus, ces comportements sont associés à d'importantes modifications de l'environnement généré chez l'hôte. Plus spécifiquement, nous avons démontré, dans les organes lymphoïdes secondaires, que la localisation et la représentation des différentes sous-populations de cellules dendritiques présentes étaient différentiellement modulées par le type de conditionnement utilisé. Par ailleurs, l'élimination spécifique des cellules CD11c+ chez des souris traitées Bu/Cy était accompagnée d'une inhibition importante de la prolifération rapide des cellules T CD4 greffées. L'ensemble de nos travaux montrent que les traitements lymphopéniques génèrent des environnements distincts capables de moduler le devenir des cellules T greffées.Durant ma thèse, nous avons également abordé de façon originale un aspect novateur de l'environnement en étudiant le rôle potentiel des nutriments comme régulateurs métaboliques des fonctions effectrices des cellules T. La glutamine est l'acide aminé le plus abondant du plasma, pouvant contribuer aux besoins bionénergétiques et biosynthétiques des cellules T en prolifération. Nous avons démontré dans nos travaux qu'une carence en glutamine lors de l'activation de cellules T CD4 par leur TCR entrainait un délai dans l'activation de la voie mTOR, une réduction de la production intracellulaire d'ATP aux temps précoces et se traduisait par une diminution de la prolifération. De plus, ces conditions étaient associées à une augmentation de la conversion de cellules CD4 T naïves, via TGFβ, en cellules régulatrices Foxp3+ , y compris en condition de polarization Th1. Par contre, la carence en glutamine n'a pas inhibé la différenciation Th2. Les cellules T Foxp3+ ainsi générées en condition limitante de glutamine présentaient in vivo des fonctions suppressives aussi efficaces que celles des cellules régulatrices nTregs. En effet, elles ont la capacité de bloquer l'induction de la colite provoquée par la greffe de cellules T effectrices dans des souris Rag2-/- . Nos travaux démontrent ainsi que l'environnement métabolique peut être un régulateur clé de la différenciation des cellules T CD4. L'ensemble de mes travaux de thèse ont mis en évidence de nouveaux paramètres capables de potentiellement modifier la survie et la réactivité des cellules T greffées
Anti-tumor therapies have improved significantly over the decade. However, the currently used treatments have important limitations, notably for metastatic cancers, and the development of new approaches is therefore a high priority. Adoptive T cell therapy (ACT) represents an innovative strategy that has shown much promise. This therapy is based on the infusion of tumor-specific T cells, which have been manipulated and expanded ex vivo, into patients who have been rendered lymphopenic by chemotherapy and/or irradiation. It is interesting to note that while lymphodepletion is attained by the vast majority of conditioning regimens, the effects of these protocols on the host environment and potentially, on the destiny of adoptively-transferred T cells had not been elucidated prior to the studies which we initiated. Using a murine model, we found that the fate of adoptively-transferred T cells differs markedly in mice rendered lymphopenic by sub-lethal irradiation as compared to a busulfan/cyclophosphamide (Bu/Cy) chemotherapy regimen. Irradiation-mediated lymphopenia resulted in a skewed IL-7-dependent proliferation of donor CD8+ T cells, whereas Bu/Cy treatment led to an increased IL-7-independent, rapid CD4+ T cell proliferation. These alterations in T cell proliferation were associated with striking changes in the host microenvironment. More specifically, we demonstrated that the proportion and localization of different dendritic cell (DC) subsets in lymphoid organs were differentially affected by the type of conditioning. Furthermore, we found that these DC controlled the rapid donor CD4+ T cell division detected in Bu/Cy-treated mice as depletion of CD11c+ DC inhibited this proliferation. Altogether, our studies demonstrate that lymphopenic regimens generate distinct host environments which modulate the fate of adoptively-transferred T cells. Durind my PhD, we also investigated an original and novel aspect of the microenvironement by studying the potential role of nutrients as metabolic regulators of T cell effector function. Glutamine is the most abundant amino acid in the plasma and contributes to the bioenergetic and biosynthetic requirements of proliferating T cells. Here, we demonstrated that activation of CD4+ T cells under glutamine-deprived conditions results in a delayed mTOR activation with reduced early ATP production and decreased proliferation. Moreover, these conditions resulted in the conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs). This de novo Treg differentiation occurred even under Th1-polarizing conditions and was TGFβ-dependent. Interestingly, glutamine deprivation did not inhibit Th2 differentiation. Importantly, these converted Foxp3+ T cells showed enhanced in vivo persistence and were highly suppressive, completely protecting Rag-deficient mice from the development of autoimmune inflammatory bowel disease as efficiently as natural-occuring Tregs. Thus, our data reveal the external metabolic environment to be a key regulator of a CD4 T lymphocyte's differentiation. Altogether, the data generated during my PhD provide new insights into the identification of parameters that can potentially alter the survival and reactivity of adoptively-transferred T cells

Immunohematology is an area dedicated to the study of the interactions of the immune system and blood cells in transfusion practice. Blood transfusion is a therapeutic technique that has been widely used since the 17th century. The transfusion medicine aims to repair the pathological needs of blood components in the living organism, be it red blood cells, plasma, platelets, clotting factors, among others. Despite being a therapeutic means, transfusion of blood components can be considered at risk because it is a biological material and due to the transfusion immunological reactions that can be caused during or after the moment of transfusion. In the surface structure of red blood cells, numerous molecules of a protein, glycoprotein or glycolipid nature are found, which are also called membrane antigens that make up structures and perform transport functions, as receptors, as adhesion, enzymatic and / or complement regulatory molecules. The formation of these antigens occurs by an approximate amount of 39 genes involved in their production, of which 282 different antigens are organized in more than 30 blood group systems. This antigenic diversity is a major cause of the formation of irregular anti-erythrocyte antibodies. Therefore, with the increase in blood transfusions in surgeries, transplants and clinical treatment of cancer and other chronic diseases, a significant increase in the occurrence of alloimmunizations in polytransfused patients began to be observed. Such biological phenomena motivated us to carry out this study and the antigenic diversity motivated us to elaborate this small compendium where we also describe the main blood groups.

The action of most xenobiotics ends in either excretion or metabolic inactivation. Some compounds, on the other hand, require metabolic activation before they can exert any biological action. In most cases these biotransformations, activations as well as inactivations, are carried out by specialized enzyme systems. The essential role of these enzymes is to facilitate elimination of xenobiotics. Water-soluble compounds usually do not need to be metabolized, as they can be excreted in their original forms. Lipophilic compounds can be disposed of through biliary excretion, or they may undergo metabolism to become more polar and thus more water-soluble so that they can be disposed of through the kidneys. The metabolism of xenobiotics is usually carried out in two phases. Phase 1 involves oxidative reactions in most cases, whereas phase 2 involves conjugation (combination) with highly water-soluble moieties. Occasionally the products of biotransformation are unstable and decompose to release highly reactive compounds such as free radicals, strong electrophiles, or highly stressed three-member rings (epoxides, azaridines, episulfides, and diazomethane; Figure 3.1) that have a tendency toward nucleophilic ring opening. For order to be retained within the cells, the chemical reactions have to occur through enzymatic processes in which the substrate is activated while bound to the enzyme. Only after the desired reaction takes place is a stable product released. Freely roaming reactive compounds are not welcome in a living organism because they react randomly with macromolecules such as DNA, RNA, and proteins. Alteration of DNA leads to faulty replication and transcription. Alteration of RNA causes faulty messages that, in turn, lead to the synthesis of abnormal proteins and thus alter enzymatic and regulatory activity. Phase 1 processes are carried out by a series of similar enzymes (commonly designated as mixed-function monooxidases) or cytochrome P-450. The basic reactions catalyzed by cytochrome P-450 enzymes involve introduction of oxygen into a molecule. In most cases the oxygen is retained, but sometimes it is removed from the end product. The oxygen carrier is a prosthetic group containing porphyrin-bound iron. The overall reaction catalyzed by these enzymes is hydroxylation.

There is enormous interest in the biology of complex reaction systems, be it in metabolism, signal transduction, gene regulatory networks, protein synthesis, and many others. The field of the interpretation of experiments on such systems by application of the methods of information science, computer science, and biostatistics is called bioinformatics (see for a presentation of this subject). Part of it is an extension of the chemical approaches that we have discussed for obtaining information on the reaction mechanisms of complex chemical systems to complex biological and genetic systems. We present here a very brief introduction to this field, which is exploding with scientific and technical activity. No review is intended, only an indication of several approaches on the subject of our book, with apologies for the omission of vast numbers of publications. A few reminders: The entire complement of DNA molecules constitute the genome, which consists of many genes. RNA is generated from DNA in a process called transcription; the RNA that codes for proteins is known as messenger RNA, abbreviated tomRNA. Other RNAs code for functional molecules such as transfer RNAs, ribosomal components, and regulatory molecules, or even have enzymatic function. Protein synthesis is regulated by many mechanisms, including that for transcription initiation, RNA splicing (in eukaryotes), mRNA transport, translation initiation, post-translational modifications, and degradation of mRNA. Proteins perform perhaps most cellular functions. Advances in microarray technology, with the use of cDNA or oligonucleotides immobilized in a predefined organization on a solid phase, have led to measurements of mRNA expression levels on a genome-wide scale (see chapter 3). The results of the measurements can be displayed on a plot on which a row represents one gene at various times, a column the whole set of genes, and the time of gene expression is plotted along the axis of rows. The changes in expression levels, as measured by fluorescence, are indicated by colors, for example green for decreased expression, black for no change in expression, and red for increased expression. Responses in expression levels have been measured for various biochemical and physiological conditions. We turn now to a few methods of obtaining information on genomic networks from microarray measurements.

Most enzymatic reactions proceed with chemical changes that cannot be brought about by the side chains of amino acid residues. These enzymes function in cooperation with coenzymes and cofactors, which lend physicochemical potentialities not found in amino acids. Many coenzymes are organic molecules incorporating functional groups with chemical properties that enable them to facilitate reactions of certain types. These molecules bind to active sites tailored for them through evolution and equipped to assist in their coenzymatic functions. Many of these coenzymes were derived from vitamins, and in early biochemistry investigations, vitamins and coenzymes were often regarded as closely linked and even synonymous. However, vitamins such as vitamin D are more akin to hormones than to coenzymes, and in modern biochemistry, the newly discovered coenzymes are not related to vitamins and have identities independent of any nutritional origin. More than 25 biological molecules may be regarded as coenzymatic in nature. In this book, the most common coenzymes and their functions are described in two chapters, the organic coenzymes in this chapter, and the metallo-coenzymes in chapter 4. Each coenzyme and cognate enzyme form a union that allows them to act as a single catalytic entity functioning in concert to bring about a difficult chemical transformation. Each coenzyme provides the chemistry required for a class of enzymatic processes, and the mechanisms of enzymatic catalysis are often revealed through the actions of the participating coenzymes. Nicotinamide adenine dinucleotide (NAD+) is the coenzymatic form of the vitamin niacin (vitamin B1). The structural formula for NAD+ is shown in fig. 3-1 along with the biochemically reactive bonds and their importance in metabolism. NADH is the reduced form of NAD+ and is produced in the dehydrogenation of substrates. The closely related forms NADP+ and NADPH are phosphorylated at the 2′-hydroxyl group of the adenosyl moiety. NADP+ and NADPH generally participate in biosynthesis (anabolism), whereas NAD+ and NADH generally participate in biodegradative metabolism (catabolism). NAD+ and NADH were formerly known as DPN+ and DPNH, for diphosphopyridine nucleotide, and TPN+ and TPNH, for triphosphopyridine nucleotide. The most frequent function of NAD+ is as an acceptor of a hydrogen atom and two electrons, a hydride equivalent, in reactions of oxidoreductases, commonly known as dehydrogenases.

Cell volume regulation has been observed in almost all cell types examined to date. When cells are exposed to hypotonic solutions a quick increase in volume is followed by a more gradual return, termed regulatory volume decrease (RVD). The mechanism associated with RVD depends upon cell type and species, but in bovine chondrocytes the non-selective osmolyte channels are mainly responsible [1]. In a chondrocyte, volume control is critical for the maintenance of metabolism, and biosynthesis. Volume fluctuations can be due to changes in hydrostatic pressure, fluid flows, deformation, and extracellular matrix (ECM) hydration. Alterations in hydration can occur during static loading of articular cartilage or during the early stages of osteoarthritis [1], which have been correlated with changes in cellular metabolism. The swelling behaviour of chondrocytes, and the mechanism by which they sense and respond to changes in their physico-chemical environment, are not well understood [1]. To investigate the effects of osmotic environment on chondrocyte behaviour it is often beneficial to isolate cells from the ECM, which can be achieved by a variety of techniques. To investigate the effect of isolation technique on the swelling behaviour of bovine chondrocytes, two enzymatic digestion techniques were chosen for this study.