Urotensin II in the central nervous system of the frog Rana ridibunda: Immunohistochemical localization and biochemical characterization

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians. (C) 1996 Wiley-Liss, Inc.

Biochemical and functional characterization of the tumor suppressors BRCA1 and BAP1

L’ubiquitination est une modification post-traductionnelle qui joue un rôle majeur dans la régulation d’une multitude de processus cellulaires. Dans cette thèse, je discuterai de la caractérisation de deux protéines, BRCA1 et BAP1, soit deux suppresseurs de tumeurs fonctionnellement reliés. BRCA1, une ubiquitine ligase qui catalyse la liaison de l’ubiquitine à une protéine cible, est mutée dans les cancers du sein et de l'ovaire. Il est bien établi que cette protéine aide à maintenir la stabilité génomique suite à un bris double brin de l’ADN (BDB), et ce, à l’aide d’un mécanisme de réparation bien caractérisé appelé recombinaison homologue. Cependant, les mécanismes de régulation de BRCA1 suite à des stresses génotoxiques n’impliquant pas directement un BDB ne sont pas pleinement élucidés. Nous avons démontré que BRCA1 est régulée par dégradation protéasomale suite à une exposition des cellules à deux agents génotoxiques reconnus pour ne pas directement générer des BDBs, soit les rayons UV, qui provoquent la distorsion de l’hélice d’ADN, et le méthyle méthanesulfonate (MMS), qui entraîne l’alkylation de l’ADN. La dégradation de BRCA1 est réversible et indépendante des kinases associées à la voie des PI3 kinase, soit ATM, ATR et DNA-PK, protéines qui sont rapidement activées par les dommages à l’ADN. Nous proposons que la dégradation de BRCA1 prévienne son recrutement intempestif, ainsi que celui des facteurs qui lui sont associés, à des sites de dommages d’ADN qui ne sont pas des BDBs, et que cette régulation coordonne la réparation de l’ADN. L’enzyme de déubiquitination BAP1 a initialement été identifiée comme une protéine capable d’interagir avec BRCA1 et de réguler sa fonction. Elle est également connue pour sa capacité à se lier avec les protéines du groupe Polycomb, ASXL1 et ASXL2. Cependant, l’importance de ces interactions n’a toujours pas été établie. Nous avons démontré que BAP1 forme deux complexes protéiques mutuellement exclusifs avec ASXL1 et ASXL2. Ces interactions sont critiques pour la liaison de BAP1 à l’ubiquitine ainsi que pour la stimulation de son activité enzymatique envers l’histone H2A. Nous avons également identifié des mutations de BAP1 dérivées de cancers qui empêchent à la fois son interaction avec ASXL1 et AXSL2, et son activité de déubiquitinase, ce qui fournit un lien mécanistique direct entre la déubiquitination de H2A et la tumorigenèse. Élucider les mécanismes de régulation de BRCA1 et BAP1 menera à une meilleure compréhension de leurs rôles de suppresseurs de tumeurs, permettant ainsi d’établir de nouvelles stratégies de diagnostic et traitement du cancer.

Biochemical Investigations on the Stability of Biological Membranes

In this project, an attempt has been made to study the stability of erythrocyte and lysosomal membranes biochemically. Erythrocytes were chosen for the study because of their ready availability and relative simplicity. Biological membranes forming closed boundaries between compartments of varying composition consist mainly of proteins and lipids. They are asymmetric, fluid structures that are thermodynamically stable and metabolically active. Normal cellular function begins with normal membrane structure and any variation in it may upset the normal functions. The degree of fluidity of a membrane depends on the chain length of its lipids and degree of unsaturation of constituent fatty acids. In response to environmental changes, many cells can regulate composition of their membranes to maintain the overall semi fluid environment necessary for many membrane associated functions. The assembly and Maintenance of membrane structures in cells is a dynamic process. The components are not only synthesized and inserted into a growing membrane but are also continuously degraded at a slower rate. This turnover process varies with each individual molecule.Lysosomes are important in the catabolic processes occurring in the cell. Lysosomes contain hydrolytic enzymes and are stable under normal conditions. In certain pathological conditions, the lysosomal membrane may rupture, releasing the hydrolytic enzymes into the cell and digestion of cell takes place as a whole. This is very dangerous. In normal life processes of multi cellular organisms, lysosomes rupture following the death of a cell and it may have some value as a built in mechanism for selfremoval of dead cells.An attempt has also been made in this project towards developing lysosome membrane stability as an index of fish spoilage during storage. Different membranes within the cell and between cells have different compositions as reflected in the ratio of protein to lipid. The difference is not surprising given the very different functions of membranes

Seasonal ramification in the biochemical composition of organic matter in Chalakudy river-estuary

The present study which is the first of its kind in this region is an attempt to generate adequate information on the relative abundances, the seasonal and spatial variations as well as on the source and fate of organic compounds found associated with the dissolved, particulate and sedimentary compartments of Chalakudy river system. The study aimed at investigating variations, the relative proportion of dissolved, particulate and sedimentary fractions of these materials as well as the pollution extent so as to be able to comment on the present condition of this river-estuarine system. This thesis focuses attention on the role of biogeoorganics in modifying the ecological and environmental condition of the dissolved, particuIate and sediment compartments with their minute variability subjected to various physical, chemical and biogeochemical processes. A scheme of study encompassing all these objectives provides the frame work for the present investigation.

Biochemical Studies on the Protective Effect of Taurine on Experimentally Induced Fulminat Hepatic Failure in Rats

Fulminant hepatic failure (FHF) is a dramatic and challenging syndrome in clinical medicine. Although an uncommon disorder, it is usually fatal and occurs in previously healthy person. While the causes of FHF remain unclear, viral hepatitis and drug-induced liver injury account for the majority of cases. Hepatitis E causes large-scale epidemics of hepatitis in the Indian subcontinent, involving hundreds of thousands of cases with high mortality. FHF is associated with several clinical features like jaundice, shrunken liver, easy bruising, low levels of serum proteins, fatigue, multi-organ failure etc and metabolic derangements like hypoglycemia, hyperlipidemia, hyponatremia, defective protein synthesis, reduced energy production, decreased rate of urea production etc. These disturbances are predominantly attributed to oxidative stress, membrane destabilization and osmolytic imbalances. The options available for these patients are quite minimal with liver transplantation being one of them. But the procedure is ridden with issues causing it to find less favor among the patients and the caregivers. Use of hepatoprotective and cytoprotective drugs, is being considered to be a more acceptable alternative as a strategy to enhance liver regeneration. In this regard use of taurine a naturally occurring amino acid that plays a crucial role in many physiological processes would prove to be effective. In the present study, hepatoprotective effect of taurine on a rat model of induced FHF was studied. Taurine supplementation has effectively counteracted the metabolic and structural aberrations in the liver caused by D-galactosamine intoxication.

Physicochemical and Biochemical Characterization of Enzymes Immobilized on lnorganic Matrices

Biotechnology is currently considered as a useful altemative to conventional process technology in industrial and catalytic fields. The increasing awareness of the need to create green and sustainable production processes in all fields of chemistry has stimulated materials scientists to search for innovative catalysts supports. lmmobilization of enzymes in inorganic matrices is very useful in practical applications due to the preserved stability and catalytic activity of the immobilized enzymes under extreme conditions. Nanostructured inorganic, organic or hybrid organic-inorganic nanocomposites present paramount advantages to facilitate integration and miniaturization of the devices (nanotechnologies), thus affording a direct connection between the inorganic, organic and biological worlds. These properties, combined with good chemical stability, make them competent candidates for designed biocatalysts, protein-separation devices, drug delivery systems, and biosensors Aluininosilicate clays and layered double hydroxides, displaying, respectively, cation and anion exchange properties, were found to be attractive materials for immobilization because of their hydrophilic, swelling and porosity properties, as well as their mechanical and thermal stability.The aim of this study is the replacement of inorganic catalysts by immobilized lipases to obtain purer and healthier products.Mesocellular silica foams were synthesized by oil-in-water microemulsion templating route and were functionalized with silane and glutaraldehyde. " The experimental results from IR spectroscopy and elemental analysis demonstrated the presence of immobilized lipase and also functionalisation with silane and glutaraldehyde on the supports.The present work is a comprehensive study on enzymatic synthesis of butyl isobutyrate through esterification reaction using lipase immobilized onto mesocellular siliceous foams and montmorillonite K-10 via adsorption and covalent binding. Moreover, the irnrnobil-ization does not modify the nature of the kinetic mechanism proposed which is of the Bi-Bi Ping—Pong type with inhibition by n-butanol. The immobilized biocatalyst can be commercially exploited for the synthesis of other short chain flavor esters. Mesocellular silica foams (MCF) were synthesized by microemusion templating method via two different routes (hydrothermal and room temperature). and were functionalized with silane and glutaraldehyde. Candida rugosa lipase was adsorbed onto MCF silica and clay using heptane as the coupling medium for reactions in non-aqueous media. I From XRD results, a slight broadening and lowering of d spacing values after immobilization and modification was observed in the case of MCF 160 and MCF35 but there was no change in the d-spacing in the case of K-10 which showed that the enzymes are adsorbed only on the external surface. This was further confirmed from the nitrogen adsorption measurements

‘Biochemical Responses to Heavy Metals in Oreochromis mossambicus with special reference to Metal Detoxifying Mechanism

The present work is focused on the organelle and biochemical responses to heavy metal exposure in the fish Oreochromis mossambicus giving particular importance to the metal detoxifying machinery of the organism. The thesis is an outcome of the effort aimed at developing practicable monitoring techniques to deliver guidelines for biological effect monitoring and the need for specific biochemical methods to detect biological effects of heavy metals that can be interpreted in terms of the health status of the individual organism and eventually alterations in vital processes as growth and reproduction. The efficiency of the metal detoxifying metallothioneins which is an attractive tool for biological monitoring, their role as scavengers of trace metal ions and thus in relieving the biological machinery from their toxicity effects are important themes of this study. Efforts have also been made to test the reliability of the spill over hypothesis of the action of metallothioneins (Winge et a1.,1973) and their use as a biological barometer of heavy metal stress.

Theoretical studies of systems of biochemical interest containing Fe and Cu transition metals

La presència de la química teòrica i computacional està augmentant en quasi tots els camps de la recerca en química. Els càlculs teòrics poden ajudar a entendre millor l'estructura, les propietats i la reactivitat de compostos metàl·lics d'àrees tan diferents com la química inorgànica, organometàl·lica i bioinorgànica. No obstant això, és imprescindible utilitzar la metodologia adequada per obtenir resultats teòrics fiables. Els estudis d'aquesta tesi es poden dividir en dos grups diferents. El primer grup inclou l'estudi teòric del mecanisme de reacció de diversos sistemes que contenen coure i tenen diferents estructures Cun-O2. Aquests estudies s'han dut a terme amb l'objectiu de profunditzar en la natura dels processos oxidants químics i biològics promoguts per sistemes que contenen coure. En la segona part de la tesi, s'estudia la fiabilitat de diferents tècniques utilitzades per estudiar l'estructura electrònica i la reactivitat de sistemes que contenen coure, ferro i altres metalls de transició.

Immersive Projection for biochemical pattern matching

In all biological processes, protein molecules and other small molecules interact to function and form transient macromolecular complexes. This interaction of two or more molecules can be described by a docking event. Docking is an important phase for structure-based drug design strategies, as it can be used as a method to simulate protein-ligand interactions. Various docking programs exist that allow automated docking, but most of them have limited visualization and user interaction. It would be advantageous if scientists could visualize the molecules participating in the docking process, manipulate their structures and manually dock them before submitting the new conformations to an automated docking process in an immersive environment, which can help stimulate the design/docking process. This also could greatly reduce docking time and resources. To achieve this, we propose a new virtual modelling/docking program, whereby the advantages of virtual modelling programs and the efficiency of the algorithms in existing docking programs will be merged.

Physiological, biochemical and transcriptional analysis of onion bulbs during storage

Abstract: During the transition from endo-dormancy to eco-dormancy and subsequent growth, the onion bulb undergoes the transition from sink organ to source, to sustain cell division in the meristematic tissue. The mechanisms controlling these processes are not fully understood. Here, a detailed analysis of whole onion bulb physiological, biochemical and transcriptional changes in response to sprouting is reported, enabling a better knowledge of the mechanisms regulating post-harvest onion sprout development. Biochemical and physiological analyses were conducted on different cultivars ('Wellington', 'Sherpa' and 'Red Baron') grown at different sites over 3 years, cured at different temperatures (20, 24 and 28 degrees C) and stored under different regimes (1, 3, 6 and 6 1 degrees C). In addition, the first onion oligonucleotide microarray was developed to determine differential gene expression in onion during curing and storage, so that transcriptional changes could support biochemical and physiological analyses. There were greater transcriptional differences between samples at harvest and before sprouting than between the samples taken before and after sprouting, with some significant changes occurring during the relatively short curing period. These changes are likely to represent the transition from endo-dormancy to sprout suppression, and suggest that endo-dormancy is a relatively short period ending just after curing. Principal component analysis of biochemical and physiological data identified the ratio of monosaccharides (fructose and glucose) to disaccharide (sucrose), along with the concentration of zeatin riboside, as important factors in discriminating between sprouting and pre-sprouting bulbs. These detailed analyses provide novel insights into key regulatory triggers for sprout dormancy release in onion bulbs and provide the potential for the development of biochemical or transcriptional markers for sprout initiation. Evidence presented herein also suggests there is no detrimental effect on bulb storage life and quality caused by curing at 20 degrees C, producing a considerable saving in energy and costs.

Determination of volatile organic compounds in recycled polyethylene terephthalate and high-density polyethylene by headspace solid phase microextraction gas chromatography mass spectrometry to evaluate the efficiency of recycling processes

A method for the determination of volatile organic compounds (VOCs) in recycled polyethylene terephthalate and high-density polyethylene using headspace sampling by solid-phase microextraction and gas chromatography coupled to mass spectrometry detection is presented. This method was used to evaluate the efficiency of cleaning processes for VOC removal from recycled PET. In addition, the method was also employed to evaluate the level of VOC contamination in multilayer packaging material containing recycled HDPE material. The optimisation of the extraction procedure for volatile compounds was performed and the best extraction conditions were found using a 75 mu m carboxen-polydimethylsiloxane (CAR-PDMS) fibre for 20 min at 60 degrees C. The validation parameters for the established method were linear range, linearity, sensitivity, precision (repeatability), accuracy (recovery) and detection and quantification limits. The results indicated that the method could easily be used in quality control for the production of recycled PET and HDPE. (C) 2011 Elsevier B.V. All rights reserved.

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.

Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum

P>Scedosporium apiospermum is an emerging agent of opportunistic mycoses in humans. Previously, we showed that mycelia of S. apiospermum secreted metallopeptidases which were directly linked to the destruction of key host proteins. In this study, we analysed the effect of metallopeptidase inhibitors on S. apiospermum development. As germination of inhaled conidia is a crucial event in the infectious process of S. apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 degrees C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 mu mol l-1, while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis (beta-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l-1. EGTA did not promote any significant reduction in the conidial growth, while PHEN and EDTA, both at 10 mmol l-1, inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum.

Phosphoproteomics Profiling Suggests a Role for Nuclear beta IPKC in Transcription Processes of Undifferentiated Murine Embryonic Stem Cells

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal and differentiation However, the function of specific PKC Isoenzymes have yet to be determined Of the PKCs expressed in undifferentiated ESCs, beta IPKC was the only isoenzyme abundantly expressed in the nuclei To investigate the role of beta IPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one beta IPKC-specific inhibitor peptide We identified 13 nuclear proteins that are direct or indirect beta IPKC substrates in undifferentiated ESCs These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation Inhibiting beta IPKC had no effect on DNA synthesis in undifferentiated ESCs However, upon differentiation many cells seized to express beta IPKC and beta IPKC was frequently found in the cytoplasm Taken together, our results suggest that beta IPKC takes part in the processes that maintain ESCs in their undifferentiated state

Psychological and biochemical mechanisms of schizoprenia

Schizophrenia is a disease whose physical cause is unknown despite the attempts of several research teams to discover a physical basis for it. Some success has been gained in genetic studies which indicate that schizophrenia is an inherited disability. However, since research tools are at present so sadly inadequate, the value of pursuing a genetic line of reasoning is questionable. To compensate for the lack of biochemical certainties in treating mental illness, psychological theories have been constructed to explain the schizophrenia syndrome. Normal personality is seen as the resultant of environmental and inherited influences. Involved in the formation of personality are the processes of differentiation and integration, maturation of inherited traits, and the learning processes. As personality develops. consciousness of the self, inferiority feelings, and compensatory mechanisms, and the transformation of interests into drives exert a decided influence upon personality growth. Finally, in the mature personality, an integrating philosophy of life, a large variety of interests, and the possibility of self-objectification become evident.