Protein synthesis Research Papers - Academia.edu (original) (raw)

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate... more

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.

Oxidized LDL stimulates the expression of TGF-β and fibronectin in human glomerular epithelial cells. Abnormal lipid accumulation in glomeruli is a recognized early event in the development of glomerulosclerosis. The presence of LDL and... more

Oxidized LDL stimulates the expression of TGF-β and fibronectin in human glomerular epithelial cells. Abnormal lipid accumulation in glomeruli is a recognized early event in the development of glomerulosclerosis. The presence of LDL and scavenger receptors has recently been demonstrated in glomerular cells, including the visceral epithelial cells. To explore the possible molecular mechanisms of lipid-induced glomerular injury, the present

Muscle wasting and changes in muscle protein metabolism in chronic obstructive pulmonary disease. R.T. Jagoe, M.P.K.J. Engelen. #ERS Journals Ltd 2003. ABSTRACT: Loss of skeletal muscle mass is now recognised as an important feature of... more

Muscle wasting and changes in muscle protein metabolism in chronic obstructive pulmonary disease. R.T. Jagoe, M.P.K.J. Engelen. #ERS Journals Ltd 2003. ABSTRACT: Loss of skeletal muscle mass is now recognised as an important feature of chronic obstructive pulmonary disease (COPD) which contributes to symptoms and influences prognosis. The changes in skeletal muscle remain poorly understood, largely because only a few studies have been performed to define the adaptations in whole body and muscle protein metabolism in COPD.

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells.... more

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H 2 O 2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H 2 O 2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1. (Mol Cell Biochem 264: [169][170][171][172][173][174][175][176][177][178][179][180][181] 2004)

It has been increasingly recognized that cell death phenotypes and their molecular mechanisms are highly diverse. Necrosis is no longer considered a single entity, passively mediated by energy failure. Moreover, caspase-dependent... more

It has been increasingly recognized that cell death phenotypes and their molecular mechanisms are highly diverse. Necrosis is no longer considered a single entity, passively mediated by energy failure. Moreover, caspase-dependent apoptosis is not the only pathway involved in programmed cell death or even the only apoptotic mechanism. Recent experimental work emphasizes the diverse and interrelated nature of cell death mechanisms. Thus, there are both caspase-dependent and caspase-independent forms of apoptosis, which may differ morphologically as well as mechanistically. There are also necrotic-like phenotypes that requirede novo protein synthesis and are, therefore, forms of programmed cell death. In addition, forms of cell death showing certain morphological features of both necrosis and apoptosis have been identified, leading to the term aponecrosis. Considerable experimental evidence also shows that modulation of one form of cell death may lead to another. Together, these observations underscore the need to substantially revise our conceptions about neuroprotection strategies. Use of multiple treatments that target different cell death cascades, or single agents that moderate multiple cell death pathways, is likely to lead to more effective neuroprotection for clinical disorders.

We characterize the dynamics of primitive molecular synthesis machines operating in outer space on quasi-one-dimensional channels where polymers interact with ÿxed particles. We show that a generic property of particle=polymer... more

We characterize the dynamics of primitive molecular synthesis machines operating in outer space on quasi-one-dimensional channels where polymers interact with ÿxed particles. We show that a generic property of particle=polymer electrostatic interactions is an average three monomer spacing between consecutive interaction potential minima. We exhibit that this property translates into locomotion regularities with a slowing down every three monomers. We argue that this transport property may be at the origin of the three base codon composition of the genetic code. We relate these ÿndings to present day protein synthesis mechanisms.

In the present study, we tested the hypothesis of the indirect (via the sympathetic nervous system (SNS)) and direct (via AT1 receptors) contributions of Angiotensin II (Ang II) on the synthesis of collagen types I and III in the left... more

In the present study, we tested the hypothesis of the indirect (via the sympathetic nervous system (SNS)) and direct (via AT1 receptors) contributions of Angiotensin II (Ang II) on the synthesis of collagen types I and III in the left ventricle (LV) in vivo. Sympathectomy and blockade of the Ang II receptor AT1 were performed alone or in combination in normotensive rats. The mRNA and protein synthesis of collagen types I and III were examined by Q-RT-PCR and immunoblotting in the LV. Collagen types I and III mRNA were decreased respectively by 53% and 22% after sympathectomy and only collagen type I mRNA was increased by 52% after AT1 receptor blockade. mRNA was not changed for collagen type I but was decreased by 25% for collagen type III after double treatment. Only collagen protein type III was decreased after sympathectomy by 12%, but collagen proteins were increased respectively for types I and III by 145% and 52% after AT1 receptor blockade and by 45% and 60% after double treatment. Deducted interpretations from our experimental approach suggest that Ang II stimulates indirectly (via SNS) and inhibits directly (via AT1 receptors) the collagen type I at transcriptional and protein levels. For collagen type III, it stimulates indirectly the transcription and inhibited directly the protein level. Therefore, the Ang II regulates collagen synthesis differently through indirect and direct pathways.

Positioning of the mRNA codon towards the 18S ribosomal RNA in the A site of human 80S ribosomes has been studied applying short mRNA analogs containing either the stop codon UAA or the sense codon UCA with a per£uoroaryl azide group at... more

Positioning of the mRNA codon towards the 18S ribosomal RNA in the A site of human 80S ribosomes has been studied applying short mRNA analogs containing either the stop codon UAA or the sense codon UCA with a per£uoroaryl azide group at the uridine residue. Bound to the ribosomal A site, a modi¢ed codon crosslinks exclusively to the 40S subunits under mild UV irradiation. This result is inconsistent with the hypothesis RNA 7, 1683^1692] which requires direct contact between the large rRNA and the stop codon of the mRNA as recognition step at translation termination. Both sense and stop codons crosslink to the same A1823/ A1824 invariant dinucleotide in helix 44 of 18S rRNA. The data point to the resemblance between the ternary complexes formed at elongation (sense codonW Waminoacyl-tRNAW WAA dinucleotide of 18S rRNA) and termination (stop codonW WeRF1W WAA dinucleotide of 18S rRNA) steps of protein synthesis and support the view that eRF1 may be considered as a functional mimic of aminoacyl-tRNA. ß

Mapping regional brain development in terms of protein synthesis (PS) activity yields insight on specific spatio-temporal ontogenetic patterns. The biosynthetic activity of an individual brain nucleus is represented as a time-series... more

Mapping regional brain development in terms of protein synthesis (PS) activity yields insight on specific spatio-temporal ontogenetic patterns. The biosynthetic activity of an individual brain nucleus is represented as a time-series object, and clustering of time-series contributes to the problem of inducing indicative patterns of brain developmental events and forming respective PS chronological maps. Clustering analysis of PS chronological maps, in comparison with epigenetic influences of ␣ 2 adrenoceptors treatment, reveals relationships between distantly located brain structures. Clustering is performed with a novel graph theoretic clustering approach (GTC). The approach is based on the weighted graph arrangement of the input objects and the iterative partitioning of the corresponding minimum spanning tree. The final result is a hierarchical clustering-tree organization of the input objects. Application of GTC on the PS patterns in developing brain revealed five main clusters that correspond to respective brain development indicative profiles. The induced profiles confirm experimental findings, and provide evidence for further experimental studies.

Spatial learning and memory related morphological changes in the argyrophilic nucleolar organizer region (AgNOR) of telencephalic neurons in goldfish were quantitatively evaluated by means of AgNOR neurohistochemical stain. The AgNORs and... more

Spatial learning and memory related morphological changes in the argyrophilic nucleolar organizer region (AgNOR) of telencephalic neurons in goldfish were quantitatively evaluated by means of AgNOR neurohistochemical stain. The AgNORs and nuclei of nerve cells of two different telencephalic regions of goldfish trained in a spatial task or submitted to a similar non-contingent behavioral procedure (control group) were morphometrically evaluated. Results show that the area of AgNORs in goldfish dorsolateral telencephalic neurons increased significantly in the spatial learning group but not in control group. This effect seems to be highly specific as it did not appear in the dorsolateral area of the control group neither in the dorsomedial area of both groups. As the size of AgNORs in the nerve cell nuclei reflect the level of transcriptive activity, these morphological changes could be revealing increased protein synthesis in goldfish dorsolateral telencephalic neurons related with learning and memory. These findings could contribute to determining the subregions of the teleost telencephalon implicated in spatial learning and could indicate that the AgNOR staining technique would be a useful tool in assesing learning and memory related neuronal activity.

In T47D breast cancer cell line, progestin (R5020) induces de novo synthesis of an alkaline phosphatase enzyme. Based on inhibitor profiles and antigenic specificity, it is apparent that this enzyme belongs to the class of... more

In T47D breast cancer cell line, progestin (R5020) induces de novo synthesis of an alkaline phosphatase enzyme. Based on inhibitor profiles and antigenic specificity, it is apparent that this enzyme belongs to the class of membrane-associated tissue-unspecific alkaline phosphatases. Enzyme induction was uniquely specific to progestins and not altered by other steroid hormones or synthetic analogues. The progestin induction of the tissue-unspecific alkaline phosphatase was time and dose dependent. The protein synthesis inhibitor cycloheximide blocks the enzyme synthesis and tunicamycin blocks the enzyme activity, showing that the induction was new synthesis of protein in its complete glycosylated form and not activation of a preexisting enzyme. To our knowledge this is the first report of progesterone-induced expression of a tissue-unspecific alkaline phosphatase gene of such magnitude (about 30- to 100-fold) in a progesterone-responsive tissue.

With an objective to determine the period of persistence of the metal-induced adaptive response to chemical mutagens and heavy metals, growing root meristems of Allium cepa were conditioned by cadmium sulfate (CdSO4), 4×10−7 and 4×10−6 M... more

With an objective to determine the period of persistence of the metal-induced adaptive response to chemical mutagens and heavy metals, growing root meristems of Allium cepa were conditioned by cadmium sulfate (CdSO4), 4×10−7 and 4×10−6 M for 1 h and subsequently challenged by maleic hydrazide (MH), 5×10−3 M or methyl mercuric chloride (MMCl), 1.26×10−6 M for 3 h at different time intervals ranging from a few minutes to several hours following the conditioning dose. Root meristems, fixed at regular intervals during recovery from 6 to 48 h, were cytologically analysed for cells with micronuclei (MNC). The adaptive responses to MH and MMCl were observed as early as 5 min after the Cd-conditioning that persisted for at least 48 h. Metabolic inhibitors, cycloheximide (CH), 10−7 M and buthionine sulfoximine (BSO), 10−4 M administered either prior to or simultaneous with Cd-conditioning effectively prevented the adaptive response to MH. Whereas BSO, an inhibitor of phytochelatin synthesis, prevented the adaptive responses from 15 min to 8 h after the conditioning dose, CH an inhibitor of cytoplasmic protein synthesis prevented the same from 6 to 48 h. The findings underscored the differential roles of phytochelatins and proteins underlying the foregone metallo-adaptive response.

Interferon-α (IFNα) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumours and melanoma. IFNα, the first cytokine to be produced by... more

Interferon-α (IFNα) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumours and melanoma. IFNα, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumour cell growth is directly suppressed by IFNα is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will discuss data obtained by us and others on the post-translational regulation of the expression of proteins involved in the occurrence of apoptotic process such as tissue transglutaminase (tTG) or in the modulation of cell cycle such as the cyclin-dependent kinase inhibitor p27. This new way of regulation of p27 and tTG occurs through the modulation of their proteasome-dependent degradation induced by the cytokine. We will also review the involvement of protein synthesis machinery in the induction of cell growth inhibition by IFNα. In details, we will describe the effects of IFNα on the expression and activity of the protein kinase dependent from dsRNA (PKR) and on the eukaryotic initiation factor of protein synthesis 5A (eIF-5A) and their correlations with the regulation of cancer cell growth. These data strongly suggest that the antitumour activity of IFNα against human tumours could involve still unexplored mechanisms based on post-translational and translational control of the expression of proteins that regulate cell proliferation and apoptosis.

Both light itself and excitation pressure have been implicated as the environmental signal that stimulates interchange of the two forms of the D1 protein of photosystem II (PS II) in Synechococcus sp. strain PCC 7942. We sought an... more

Both light itself and excitation pressure have been implicated as the environmental signal that stimulates interchange of the two forms of the D1 protein of photosystem II (PS II) in Synechococcus sp. strain PCC 7942. We sought an explanation for conflicting reports regarding the role of photosynthetic electron transport in regulation of psbA expression and D1 interchange. Inhibitors that block at different points in the photosynthetic electron transport chain were administered and the effect on expression of psbAII, which encodes the high-light-induced form II of D1, was examined by measuring changes in transcript levels and in the activities of reporter enzymes. Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of PSII, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), an inhibitor of the cytochrome b6/f complex, prevented high-light-induced increase in β-galactosidase activity from a psbAII::lacZ gene fusion when added at a concentration that completely inhibits photosynthetic electron transport (1 μM). The same effect was observed for luciferase activity from transcriptional and translational fusions of psbAII to the luxAB genes from Vibrio harveyi. DCMU (1 μM) arrested luciferase expression at low-light levels – thus eliminating the high light response – whereas a sublethal concentration (50 nM), which reduces electron transport by 50%, had intermediate effects on psbAII-driven luciferase activity. However, psbAII transcript levels, monitored by northern blot analysis, were not altered by electron transport inhibitors, either at low-light intensity or following a high-light exposure. The suppressive effect of DCMU on expression of reporter enzymes was not restricted to the high-light response of psbAII-driven reporter systems, but was also observed using an isopropyl-(-d)-thiogalactopyranoside (IPTG)-inducible trc promoter fused to luxAB. This construct only marginally responded to IPTG addition when DCMU was present. Thus, blocking electron transport in Synechococcus affects the translation machinery in a general way, and the use of electron transport inhibitors is of limited value when focusing on specific redox regulation of D1 protein synthesis or degradation.

The Bowman±Birk (BB) family of proteinase inhibitors (PI), initially reported from legume seeds, and thereafter also from wounded alfalfa and maize leaves appear to be regulated in similar ways as the extensively characterized PI I and PI... more

The Bowman±Birk (BB) family of proteinase inhibitors (PI), initially reported from legume seeds, and thereafter also from wounded alfalfa and maize leaves appear to be regulated in similar ways as the extensively characterized PI I and PI II family from dicots. Here, we report a ®rst characterization of the expression pro®les of a rice (Oryza sativa L. cv. Nipponbare) BBPI gene, OsBBPI, which is part of a multigene family as demonstrated by genomic Southern hybridization. OsBBPI was found to be rapidly induced in rice seedling leaf in response to cut, exogenous jasmonic acid (JA), and two potent protein phosphatase 2A (PP2A) inhibitors, cantharidin (CN) and endothall (EN), in a light/dark-, time-and dose-dependent manner; this induction was completely inhibited by cycloheximide (CHX), indicating a requirement for de novo protein synthesis in its induction. Surprisingly, dark strongly up regulated cut-, JA-, CN-, and EN-induced OsBBPI expression, with the strongest enhancement observed with JA. A simultaneous application of a serine/threonine protein kinase inhibitor staurosporine (ST) did not affect signi®cantly the JA-, CN-, and EN-induced OsBBPI transcript. Besides JA, it was found that the ethylene generator ethephon (ET) also had an enhancing effect on OsBBPI transcript, suggesting a direct effect of ethylene on OsBBPI expression. However, a simultaneous application of salicylic acid (SA) and abscisic acid (ABA), with JA, respectively, completely blocked OsBBPI gene expression, whereas kinetin (KN) was only partially effective. To the best of our knowledge, complete inhibition of JA-induced OsBBPI expression by SA is the ®rst report in monocots, and with ABA in plants. Taken together, these results suggest that among the phytohormones tested here, JA and ethylene play important role(s) in regulating OsBBPI expression, with an intimate interaction with light signals. Finally, that the induced OsBBPI expression follows a kinase-signaling cascade is implied by the use of PP2A inhibitors. q

A major component of consolidation theory holds that protein synthesis is required to produce the synaptic modification needed for long-term memory storage. Protein synthesis inhibitors have played a pivotal role in the development of... more

A major component of consolidation theory holds that protein synthesis is required to produce the synaptic modification needed for long-term memory storage. Protein synthesis inhibitors have played a pivotal role in the development of this theory. However, these commonly used drugs have unintended effects that have prompted some to reevaluate the role of protein synthesis in memory consolidation.

Compromise of the ubiquitin-proteasome system (UPS) is a potential basis for multiple physiological abnormalities and pathologies in the CNS. This could be because reduced protein turnover leads to bulk intracellular protein accumulation.... more

Compromise of the ubiquitin-proteasome system (UPS) is a potential basis for multiple physiological abnormalities and pathologies in the CNS. This could be because reduced protein turnover leads to bulk intracellular protein accumulation. However, conditions associated with compromised UPS function are also associated with impairments in protein synthesis, and impairment of UPS function is sufficient to inhibit protein synthesis. These data suggest that the toxicity of UPS inhibition need not depend on gross intracellular protein accumulation, and indicate the potential for crosstalk between the UPS and protein-synthesis pathways. In this review, we discuss evidence for interplay between the UPS and protein-synthesis machinery, and outline the implications of this crosstalk for physiological and pathological processes in the CNS.

Methionine is a dietary essential amino acid that plays unique roles, both in protein structure and in metabolism. Methionine serves as the initiating amino acid in eukaryotic protein synthesis. In globular proteins, most methionine... more

Methionine is a dietary essential amino acid that plays unique roles, both in protein structure and in metabolism. Methionine serves as the initiating amino acid in eukaryotic protein synthesis. In globular proteins, most methionine residues are buried inside the hydrophobic core. Some methionine residues, located on the surfaces of proteins are susceptible to oxidation to methionine sulfoxide. These may be reduced back to methionine by methionine sulfoxide reductase. Methionine's principal metabolic function lies in its conversion to S-adenosylmethionine which is the principal biological methylating agent. Methionine metabolism may be divided into transmethylation, remethylation and transsulfuration. S-adenosylmethionine, via allosteric mechanisms, exerts control over these processes. Creatine synthesis is a major user of S-adenosylmethionine-derived methyl groups. Piglets acquire considerable quantities of creatine during growth. About one third of this is provided in the milk; two thirds is synthesized within the piglet. This requires very high rates of creatine synthesis and has the potential to be a significant drain on dietary methionine.

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing... more

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis.

To establish a proteomic reference map of date palm leaves (Deglet Nour cultivar), we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, respectively. In total, 284 spots... more

To establish a proteomic reference map of date palm leaves (Deglet Nour cultivar), we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, respectively. In total, 284 spots were excised from gel and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among them, 158 were successfully identified (i.e, a success rate of 55.6%) conducting to the identification of 126 unique proteins. These proteins were then clustered according to their functional annotations. Identified proteins were involved in metabolism, electron transport, photosynthesis, protein synthesis, cell structure or defence. However, 29.4 % of the identifications gave unknown function. We then compared the proteome map of female and male trees. Only one discriminated spot was found to be specific of the gender. We identified the corresponding protein as an ABC superfamily ATP binding cassette transporter, ABC protein, a protein whose an ortholog in Arabidopsis thaliana was already reported as required for male fertility and pollen formation. The relevance of this protein as gender biomarker was then confirmed in four other cultivars, i.e., Aligue, Khouet Aligue, Kentichi and Kenta. Such biomarker should be helpful in rapidly distinguishing date palm gender of immature trees.

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A fundamental question in the field of circadian rhythms concerns the biochemical and molecular nature of the oscillator. There is strong evidence that circadian oscillators are cell autonomous and rely on periodic gene expression. In... more

A fundamental question in the field of circadian rhythms concerns the biochemical and molecular nature of the oscillator. There is strong evidence that circadian oscillators are cell autonomous and rely on periodic gene expression. In Drosophila, Neurospora, Aplysia, and vertebrates, circadian oscillators are thought to be based on molecular autoregulatory loops composed of transcription, translation, and negative feedback by proteins on nuclear transcription. By studying a mathematical model of molecular clocks based on this general concept, the authors sought to determine which features such clocks must have to generate robust and stable oscillations and to allow entrainment by external stimuli such as light. The model produced circadian oscillations as an emergent property even though a time delay in protein synthesis and rate constants of the feedback loop were much shorter than 24 h. Along with the delay in protein production, strong nonlinear interactions in macromolecular synthesis and nuclear feedback appeared to be required for the model to show well-behaved oscillatory behavior. Realistic phase-shifting patterns induced by external stimuli could be achieved by multiple mechanisms-namely, up-and downward perturbations of protein or mRNA synthesis or degradation rates. The model makes testable predictions about interactions between clock elements and mechanisms of entrainment and may help to understand the functions of the intricate molecular interactions governing circadian rhythmogenesis.

Enzyme analyses indicated that the metabolism of glycerol by Zygosaccharomyces rouxii occurred via either glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA). The route via DHA is significant in osmoregulation. The specific activities of... more

Enzyme analyses indicated that the metabolism of glycerol by Zygosaccharomyces rouxii occurred via either glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA). The route via DHA is significant in osmoregulation. The specific activities of glycerol dehydrogenase (GDHG) and DHA kinase, which metabolize glycerol via DHA, increased nine-and fourfold respectively during osmotic stress [0.960 water activity (aw) adjusted with NaCI] when compared to nonstressed conditions (0.998 aw). Both pathways are under metabolic regulation. Glycerol kinase, mitochondrial G3P dehydrogenase and DHA kinase are induced by glycerol while the latter is also repressed by glucose. Cells treated with cycloheximide prior to osmotic upshock showed significantly lower DHA kinase and GDHG levels and lower intracellular glycerol concentrations when compared to untreated control cells. Thus protein synthesis is essential for osmotic adaptation. pendent glycerol-3-phosphate dehydrogenase (G3PDHG; EC 1.1.1.8) and a phosphatase have been described in Z. rouxii (Spencer and Spencer 1978), Saccharomyces cerevisiae (Gancedo et al. 1968; Spencer and Spencer 1978) and Debaryomyces hansenii (Adler et al. 1985). Glycerol production via dihydroxyacetone (DHA) has not yet been reported in any yeasts. Glycerol dissimilation occurs via glycerol-3-phosphate (G3P) and involves a glycerol kinase (EC 2.7.1.30) and a mitochondrial G3PDHG (EC 1.1.99.5) in S. cerevisiae (Sprague and Cronan 1977), D. hansenii (Adler et al. 1985) and Candida utilis (Gancedo et al. 1968). An alternative dissimilatory pathway via DHA involving glycerol dehydrogenase (GDHG; EC 1.1.1.6) and DHA kinase occurs in Schizosaccharomyces pornbe (May and Sloan 1981; Gancedo et al. 1986). DHA kinase and GDHG activities have been reported in D. hansenii (Adler et al. 1985) and species of Candida,

Nutrition is the major intrauterine environ- mental factor that alters expression of the fetal genome and may have lifelong consequences. This phenomenon, termed "fetal programming," has led to the recent theory of "fetal... more

Nutrition is the major intrauterine environ- mental factor that alters expression of the fetal genome and may have lifelong consequences. This phenomenon, termed "fetal programming," has led to the recent theory of "fetal origins of adult disease." Namely, alterations in fetal nutrition and endocrine status may result in developmental adaptations that permanently change the structure, physi- ology, and metabolism of

We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this... more

We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO 2 )-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a twodimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO 2 -filled tips. To increase the trapping selectivity of the TiO 2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient.

We have previously quantified the extent of myofibrillar disruption which occurs following an acute bout of resistance exercise in untrained men, however the response of well-trained subjects is not known. We therefore recruited six... more

We have previously quantified the extent of myofibrillar disruption which occurs following an acute bout of resistance exercise in untrained men, however the response of well-trained subjects is not known. We therefore recruited six strength-trained men, who ceased training for 5 days and then performed 8 sets of 8 uni-lateral repetitions, using a load equivalent to 80% of their concentric (Con) 1-repetition maximum. One arm performed only Con actions by lifting the weight and the other arm performed only eccentric actions (Ecc) by lowering it. Needle biopsy samples were obtained from biceps brachii of each arm~21 h following exercise, and at baseline (i.e., after 5 days without training), and subsequently analyzed using electron microscopy to quantify myofibrillar disruption. A greater (P ≤ 0.05) proportion of disrupted fibres was found in the Ecc arm (45 ± 11%) compared with baseline values (4 ± 2%), whereas fibre disruption in the Con arm (27 ± 4%) was not different (P > 0.05) from baseline values. The proportion of disrupted fibres and the magnitude of disruption (quantified by sarcomere counting) was considerably less severe than previously observed in untrained subjects after an identical exercise bout. Mixed muscle protein synthesis, assessed from~21-29 h post-exercise, was not different between the Con-and Ecc-exercised arms. We conclude that the Ecc phase of resistance exercise is most disruptive to skeletal muscle and that training attenuates the severity of this effect. Moreover, it appears that fibre disruption induced by habitual weightlifting exercise is essentially repaired after 5 days of inactivity in trained men.

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size.... more

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size. Using improved patch-clamp techniques in brain slices, we recorded from mossy fiber terminals in the CA3 region of the hippocampus, which have a diameter of 2-5 lm. The major steps of improvement were the enhanced visibility provided by high-numerical aperture objectives and infrared illumination, the development of vibratomes with minimal vertical blade vibrations and the use of sucrose-based solutions for storage and cutting. Based on these improvements, we describe a protocol that allows us to routinely record from hippocampal mossy fiber boutons. Presynaptic recordings can be obtained in slices from both rats and mice. Presynaptic recordings can be also obtained in slices from transgenic mice in which terminals are labeled with enhanced green fluorescent protein.

Une caractéristique majeure du vieillissement est la sarcopénie liée à l'âge, à l'origine de conséquences négatives en terme de handicap et de morbidité mais faisant aussi le lit de la dénutrition et de la vulnérabilité. À côté d'ingesta... more

Une caractéristique majeure du vieillissement est la sarcopénie liée à l'âge, à l'origine de conséquences négatives en terme de handicap et de morbidité mais faisant aussi le lit de la dénutrition et de la vulnérabilité. À côté d'ingesta protéiques insuffisants, des anomalies du métabolisme protéique sont fréquentes, avec une plus forte extraction splanchnique des acides aminés et une diminution de leur action anabolisante au niveau musculaire, principalement du fait d'une moindre stimulation de la voie de signalisation mTOR. Une augmentation de la protéolyse (via le complexe ubiquitine-protéasome) est impliquée à un moindre degré. Les conséquences de ces anomalies sur les apports en acides aminés sont, en premier lieu, de ne jamais se situer sous le seuil d'apports de 1 g/kg par jour de protéines. L'administration des protéines en bolus ainsi que le recours à des protéines rapides sont des pistes prometteuses. Enfin, l'administration de quantités supraphysiologiques de certains acides aminés, tels la leucine surtout mais aussi la citrulline et l'arginine, permet de stimuler la synthèse protéique, principalement via l'activation de la voie mTOR, et représente une alternative thérapeutique intéressante à comparer aux pistes médicamenteuses.

In biology proteins are uniquely important. They are not to be classed with polysaccharides, for example, which by comparison play a very minor role. Their nearest rivals are the nucleic acids… The main function of proteins is to act as... more

In biology proteins are uniquely important. They are not to be classed with polysaccharides, for example, which by comparison play a very minor role. Their nearest rivals are the nucleic acids… The main function of proteins is to act as enzymes.

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has... more

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process-from wild-type seeds to transgenic T 1 seeds-takes B8 months to complete.

Calotropis procera (Asclepiadaceae) is a well known plant in the Ayurvedic system of medicine. Based on its traditional use this plant was selected for evaluation of its wound healing potential. For this purpose four full thickness... more

Calotropis procera (Asclepiadaceae) is a well known plant in the Ayurvedic system of medicine. Based on its traditional use this plant was selected for evaluation of its wound healing potential. For this purpose four full thickness excisional wounds of 8.0 mm diameter were inflicted on the back of guinea pigs. Topical application of 20 μl of 1.0% sterile solution of the latex of C. procera twice daily was followed for 7 days. The latex significantly augmented the healing process by markedly increasing collagen, DNA and protein synthesis and epithelisation leading to reduction in wound area. Thus the present study provides a scientific rationale for the traditional use of this plant in the management of wound healing.

The regulation of crystal protein production in Bacillus thuringiensis 81 by sources of carbon and nitrogen was investigated. The highest titers of toxin were obtained on sucrose, lactose and inulin which also supported sporulation. Whey... more

The regulation of crystal protein production in Bacillus thuringiensis 81 by sources of carbon and nitrogen was investigated. The highest titers of toxin were obtained on sucrose, lactose and inulin which also supported sporulation. Whey and molasses were also potential carbon substrates for toxin production. Other carbohydrates including glucose, glycerol, maltose, starch and dextrin yielded lower amounts of toxin. Nitrogen sources were found to exert the most profound controls. Peptone was the best organic nitrogen source, supporting optimum production and sporulation as well as high cell density. The formation of CryI and CryII toxin proteins was found to be differentially regulated by the inorganic nitrogenous compounds incorporated into the medium.

Insulin Muscle Adipose tissue Non-esterified fatty acids Hexokinase 6-phosphofructokinase The major effects of insulin on muscle and adipose tissue are: (1) Carbohydrate metabolism: (a) it increases the rate of glucose transport across... more

Insulin Muscle Adipose tissue Non-esterified fatty acids Hexokinase 6-phosphofructokinase The major effects of insulin on muscle and adipose tissue are: (1) Carbohydrate metabolism: (a) it increases the rate of glucose transport across the cell membrane, (b) it increases the rate of glycolysis by increasing hexokinase and 6-phosphofructokinase activity, (c) it stimulates the rate of glycogen synthesis and decreases the rate of glycogen breakdown. (2) Lipid metabolism: (a) it decreases the rate of lipolysis in adipose tissue and hence lowers the plasma fatty acid level, (b) it stimulates fatty acid and triacylglycerol synthesis in tissues, (c) it increases the uptake of triglycerides from the blood into adipose tissue and muscle, (d) it decreases the rate of fatty acid oxidation in muscle and liver. (3) Protein metabolism: (a) it increases the rate of transport of some amino acids into tissues, (b) it increases the rate of protein synthesis in muscle, adipose tissue, liver, and other tissues, (c) it decreases the rate of protein degradation in muscle (and perhaps other tissues).

The possibility that c-phycocyanin serves as a nitrogen source in Spirutina platensis during nitrogen starvation was studied. The following evidence was obtained in support of this idea. 1. Under favourable conditions for growth,... more

The possibility that c-phycocyanin serves as a nitrogen source in Spirutina platensis during nitrogen starvation was studied. The following evidence was obtained in support of this idea. 1. Under favourable conditions for growth, c-phycocyanin existed in large excess in the algal cells. 2. When the supply of nitrogen was low, about 30-50 % of the c-phycocyanin disappeared without any effect on the maximal growth rate. 3. A culture which was deprived of nitrogen continued to grow unaffectedly for a period, the duration of which depended on the c-phycocyanin content in the cell before nitrogen starvation was initiated. 4. c-phycocyanin was the only nitrogenous compound that was depleted during the course of nitrogen starvation when growth was yet unaffected. 5. When protein synthesis was inhibited either by nitrogen starvation or by methionine sulfoximine (MSO), phycocyanin content began to decline immediately and growth contimled at normal rates as long as c-phycocyanin did not decline below 50 %. 6. The decrease in c-phycocyanin content during nitrogen starvation was accompanied by an increase in proteolytic activity.

Scientific studies support recommendations to increase dietary soluble fiber as part of hyperlipidemia treatment. Rice bran contains minimal soluble fiber, but rice bran oil has a hypolipidemic effect. Full-fat rice bran was compared with... more

Scientific studies support recommendations to increase dietary soluble fiber as part of hyperlipidemia treatment. Rice bran contains minimal soluble fiber, but rice bran oil has a hypolipidemic effect. Full-fat rice bran was compared with oat bran and a rice starch placebo in hyperlipidemic humans to see if it might have a role in the treatment of hyperlipidemia. Moderately hypercholesterolemic (5.95-8.02 mmol/L), nonsmoking, nonobese adults were studied in a 6-wk, randomized, double-blind, noncross-over trial. Three groups added 84 g/d of a heat-stabilized, full-fat, medium-grain rice bran product (nÅ 14), oat bran product (nÅ 13) or rice starch placebo (n Å 17) to their usual low-fat diet. Serum cholesterol, triglycerides, HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), apoA1 and apoB were measured before and at the end of the supplementation period. Serum cholesterol decreased significantly (P ¡ 0.05) by 8.3 { 2.4% and 13.0 { 1.8% in the rice bran and oat bran groups, respectiv...

A conditioned stimulus (CS) associated with a fearsome unconditioned stimulus (US) generates learned fear. Acquired fear is at the root of a variety of disorders, among which are phobias, generalized anxiety, and the posttraumatic stress... more

A conditioned stimulus (CS) associated with a fearsome unconditioned stimulus (US) generates learned fear. Acquired fear is at the root of a variety of disorders, among which are phobias, generalized anxiety, and the posttraumatic stress disorder (PTSD). The simplest way to inhibit learned fear is to extinguish it, which is usually done by repeatedly presenting the CS alone, so that a new association, CS-"no US", will eventually overcome the previously acquired CS-US association. Extinction was first described by Pavlov as a form of "ïnternal inhibition" and was recommended by Freud and Ferenczi in the 1920s (who called it "habituation") as the treatment of choice for phobic disorders. It is used with success till this day, often in association with anxiolytic drugs. Extinction has since then been applied, also successfully and also often in association with anxiolytics, to the treatment of panic, generalized anxiety disorders and, more recently, PTSD. Extinction of learned fear involves gene expression, protein synthesis, N-methyl-D-aspartate (NMDA) receptors and signaling pathways in the hippocampus and the amygdala at the time of the first CS-no US association. It can be enhanced by increasing the exposure to the "no US" component at the time of behavioral testing, to the point of causing the complete uninstallment of the original fear response. Some theorists have recently proposed that reiteration of the CS alone may induce a reconsolidation of the learned behavior instead of its extinction. Reconsolidation would preserve the original memory from the labilization induced by its retrieval. If true, this would of course be disastrous for the psychotherapy of fear-motivated disorders. Here we show that neither the CS nor retrieval cause anything remotely like reconsolida-tion, but just extinction. In fact, our findings indicate that the reconsolidation hypothesis is essentially incorrect, at least for the form of contextual fear most commonly studied in rodents. Therefore, it seems safe to continue using extinction-based forms of therapy for anxiety disorders secondary to acquired fear. Further, it is useful and desirable to devise procedures by which the "no US" component of the extinction is strengthened in order to alleviate the symptoms of victims of acquired fear.

Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of... more

Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of chloroplast-derived therapeutic proteins through plant cells eliminates expensive purification steps, low temperature storage, transportation and sterile injections for their delivery. Chloroplast transformation technology (CTT) has also been successfully used to engineer valuable agronomic traits and for the production of industrial enzymes and biomaterials. Here, we provide a detailed protocol for the construction of chloroplast expression and integration vectors, selection and regeneration of transformants, evaluation of transgene integration and inheritance, confirmation of transgene expression and extraction, and quantitation and purification of foreign proteins. Integration of appropriate transgenes into chloroplast genomes and the resulting high levels of functional protein expression can be achieved in B6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.

The most investigated Hsp70 chaperone system is the E. coli Hsp70 chaperone, DnaK, and its Hsp40 co-chaperone, DnaJ. DnaJ and DnaK were initially identified as being necessary for bacteriophage 8 DNA replication, hence the Dna... more

The most investigated Hsp70 chaperone system is the E. coli Hsp70 chaperone, DnaK, and its Hsp40 co-chaperone, DnaJ. DnaJ and DnaK were initially identified as being necessary for bacteriophage 8 DNA replication, hence the Dna nomenclature. 3 Hsp70s are found in all prokaryotes and eukaryotes, as well as in some, but not all, of the archaea. 14,15 Some methanogens, extreme halophiles and extreme thermophiles do not appear to synthesize, or even to contain, a genomic copy of an Hsp70 gene. There appear to be minimal differences between Hsc70 17 (also called Hsp73) and Hsp70 (also called Hsp72) in terms of function. The cognate form acts as a molecular chaperone under normal cellular conditions, whereas the heat shock protein will function under stress conditions. Hsp70s recognize unfolded proteins, binding and stabilizing them, thereby giving them enough time to fold correctly. They are also involved in the stabilization of precursor proteins, aiding in the translocation of newly synthesized proteins, the rearrangement of protein oligomers and the protection of the cell against the effects of cellular stress. 18 Hsp70s have also been implicated in protein degradation, 19 including the specific degradation of the heat shock transcription factor F 32 in E. coli, 20 and polyglutamine repeat containing proteins via the ubiquitination pathway in eukaryotes. These functions are all dependent on Hsp70 keeping polypeptides in an extended conformation, and stabilizing the exposed hydrophobic regions of the target polypeptide. 22 Hsp40-like proteins are involved in facilitating the folding of nascent polypeptides by regulating partner Hsp70 proteins, and have also been implicated in protein translocation, 24 protein degradation, 25 clathrin uncoating 26,27 and viral infection.

Like other more complex DNA viruses, it is believed that AAV interacts or modifies host cell proteins to carry out its infection cycle. To date, relatively little is known about the host proteins that interact with the viral Rep proteins,... more

Like other more complex DNA viruses, it is believed that AAV interacts or modifies host cell proteins to carry out its infection cycle. To date, relatively little is known about the host proteins that interact with the viral Rep proteins, which are known to be directly involved in DNA replication, control of viral and cellular transcription, splicing, and protein translation. In this study, we used affinity-tagged Rep protein to purify cellular protein complexes that were associated with Rep in cells that had been infected with Ad and AAV. In all, we identified 188 cellular proteins from 16 functional categories, including 14 transcription factors, 6 translation factors, 15 potential splicing proteins, 5 proteins involved in protein degradation, and 13 proteins involved in DNA replication or repair. This dramatically increases the number of potential interactions over the current number of approximately 26. Twelve of the novel proteins found were further tested by coimmunoprecipitation or colocalization using confocal immunomicroscopy. Of these, 10 were confirmed as proteins that formed complexes with Rep, including proteins of the MCM complex (DNA replication), RCN1 (membrane transport), SMC2 (

A suite of biomarkers was measured in caged mussels at areas impacted by different anthropogenic activities along the Greek coastline to assess biological effects of environmental pollution. Mussels were caged at coastal sites in the... more

A suite of biomarkers was measured in caged mussels at areas impacted by different anthropogenic activities along the Greek coastline to assess biological effects of environmental pollution. Mussels were caged at coastal sites in the vicinity of major cities, in areas influenced by major industries, agricultural practices and in islands away from known sources of pollution. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AchE), oxidative stress (catalase, CAT), phase II biotransformation of xenobiotics (glutathione Stransferase, GST), metal exposure (metallothioneins, MTs) and protein synthesis (RNA:DNA ratio) were measured to assess effects of various types of pollutants. AchE activity proved to be the most responsive biomarker with decreased values at sites influenced by agricultural, urban and industrial activities. Decreased CAT and GST activities and increased MTs levels were recorded at a number of anthropogenicimpacted sites. RNA:DNA ratio showed a biphasic response as both high and low values were found at impacted sites. Principal component analysis clearly distinguished sites receiving pollution inputs from nonpolluted sites. The combination of the selected biomarkers used in caged mussels resulted useful in the assessment of the effects of environmental pollution.

Patients with end-stage liver disease (ESLD) frequently have diverse abnormalities of carbohydrate, lipid, and protein metabolism that cause progressive deterioration of their clinical condition and lead to malnutrition. Malnutrition is... more

Patients with end-stage liver disease (ESLD) frequently have diverse abnormalities of carbohydrate, lipid, and protein metabolism that cause progressive deterioration of their clinical condition and lead to malnutrition. Malnutrition is almost universally present in patients with ESLD undergoing liver transplantation and has been associated with increased morbidity and mortality. It is essential to identify and correct nutritional deficiencies in this population and provide an adequate nutritional support during all phases of liver transplantation. In conclusion, this article reviews the etiologic factors, prevalence, assessment and management guidelines of nutritional disorders seen in patients with ESLD undergoing liver transplantation.

The hPepT1-mediated transport properties of a series of 11 synthesized -and γ-peptides have been studied in Caco-2 cells. The results show that several of the compounds interact with the peptide transporter, but only two -dipeptides act... more

The hPepT1-mediated transport properties of a series of 11 synthesized -and γ-peptides have been studied in Caco-2 cells. The results show that several of the compounds interact with the peptide transporter, but only two -dipeptides act as substrates and are transported across the cell monolayers. These two are lessefficient substrates than R-peptides. Larger derivatives than -dipeptides do not act as hPepT1 substrates, but instead, they appear to be substrates for P-glycoprotein efflux. ionization; HOBt, 1-hydroxy-1H-benzotriazole hydrate; TFA, trifluoroacetic acid. Three-letter amino-acid abbreviations are used for homologated amino acid derivatives: hXaa ( n , homoamino acid) 1 and γhhXaa (γ m,n , doubly homologated amino acid), where n indicates the position of the proteinogenic side chain.

We report here the successful establishment of callus, cell and root cultures from explants of in-vitrogrown plantlets of the soapwort Saponaria officinalis L. The production of saporin in the different tissue systems was evaluated by... more

We report here the successful establishment of callus, cell and root cultures from explants of in-vitrogrown plantlets of the soapwort Saponaria officinalis L. The production of saporin in the different tissue systems was evaluated by determining the capability of crude extracts to inactivate protein synthesis and by Western blotting analysis. Protein synthesis inhibition varied in callus and derived cell suspensions and in cultured roots, the latter, in particular, showing the lowest specific activity. The ribosome-inactivating principle from root cultures was purified to homogeneity by cation exchange chromatography. Key words Ribosome-inactivating protein • Roots • Saponaria officinalis • Saporin Abbreviations BAP 6-Benzylaminopurine • 2,4-D 2,4-Dichlorophenoxyacetic acid • IAA Indole-3-acetic acid • KIN Kinetin • MS Murashige and Skoog medium • NAA α-Naphthaleneacetic acid • PBS Phosphate buffer saline • RIP Ribosome-inactivating protein

The eutardigrade Adorybiotus (Richtersius) coronifer survives cryptobiosis for years. During entrance into anhydrobiosis this species accumulates the disaccharide trehalose reaching a maximum content of 2.3% d.w. In the present study we... more

The eutardigrade Adorybiotus (Richtersius) coronifer survives cryptobiosis for years. During entrance into anhydrobiosis this species accumulates the disaccharide trehalose reaching a maximum content of 2.3% d.w. In the present study we examined the survival of anhydrobiotic A. (R) coronifer during exposure to alcohols of various polarity, and to high temperatures, as well as qualitative changes in protein synthesis during entrance into anhydrobiosis. Results showed that A. (R) coronifer in anhydrobiosis survived exposure to ethanol for less than 10 minutes whereas exposure to 1-butanol only decreased survival to 40% after the first 7 days and 1-hexanol did not change survival from the controls after the first 7 days. A. (R) coronifer survived temperatures up to approximately 70 °C for 60 minutes without any decrease in survival. However, survival decreased rapidly when the exposure temperature was increased to above 70 °C and no animals survived exposure to 100 °C. During the entrance into anhydrobiosis a protein with a molecular weight of approximately 71 kDa appeared on acryl amide gels showing protein bands after the animals had been incubated with 3 H-Leucine. This protein may belong to the Heat-shock protein (Hsp) 70 family. The results on the survival of A. (R) coronifer during exposure to alcohols and high temperature are discussed in light of the trehalose content earlier described in this animal during anhydrobiosis.