Inter-relationships between pituitary-adrenal hormones and catecholamines during a 6-day Nordic ski race

The aim of the study was to investigate the inter-relationships between pituitary-adrenal hormones and catecholamines during a prolonged competition over 6 days. Plasma adrenocorticotropic hormone (ACTH), cortisol (C), beta-endorphin (beta EP), free and sulphated adrenaline (A) and noradrenaline (NA) were measured in 11 volunteer male subjects during a national Nordic-ski race (323 km). Blood samples were obtained before the competition in the evening as control (D0), and before and after each day's racing (D1-D6). The mean daily heart rate (fc) was calculated from fc values recorded every minute during the race. The results showed the following: changes in mean fc [from 147 (SEM 3) to 156 (SEM 3) beats.min-1 according to the day] were not significant during the race. Diurnal variations in ACTH, beta EP and C were no longer apparent after the race: evening levels were higher than their respective D0 values during the race, except on D3 when there was a lack of response to exercise in the three hormones. Unlike ACTH and beta EP, pre- and postexercise C values on D1 and D2 were higher than those on the subsequent days (P less than 0.001). In contrast, there was a progressive accumulation of A and NA in pre- and postrace concentrations which reached a plateau in about 4 days. Positive correlations between exercise responses in ACTH, C and beta EP were found especially on D3 and D6 (P less than 0.001) but there were no significant correlations between catecholamines and the other three hormones. Thus, prolonged competition over 6 days evoked different control mechanisms for hormones of the pituitary-adrenal axis and catecholamines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Met-enkephalin, β-endorphin and cortisol responses to sub-maximal exercise after sleep disturbances

The present study compared the effects of partial sleep deprivation and the effects of an intake of a hypnotic compound (zolpidem) prior to bedtime, on sleep and on hormonal and metabolic adaptations to subsequent exercise. Sleep deprivation consisted of a delayed bedtime and an early getting-up time. Eight young subjects, who slept well and were highly trained athletes, were enrolled in this study. Sleep was recorded polygraphically and the following afternoon exercise was performed on a cycle ergometer for 30 min at 75% of maximal oxygen consumption (VO2max) after a 10-min warm up. Met-enkephalin, beta-endorphin, cortisol, and lactate concentrations were measured at rest and during exercise. The data obtained after experimental sleep, with and without medication were compared with those obtained in the reference condition with normal sleep. Both types of sleep reduction decreased the total sleep time, stage 2 sleep, and rapid eye movement sleep, whereas zolpidem administration did not modify either the duration of sleep or the sleep stages. After the reference night, plasma met-enkephalin did not show any significant change at the end of the submaximal exercise, whereas beta-endorphin, cortisol, and lactic acid concentrations increased significantly in all subjects. The changes in concentration in beta-endorphin were significantly related to the changes in cortisol (r = 0.78; P less than 0.01) and to the changes in plasma lactic acid (r = 0.58; P less than 0.05). Cortisol concentrations were also related to lactic acid values (r = 0.94; P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Activities ofβ-glucanases andβ-glucosidases during blastospore formation inSaccharomycopsis fibuligera

Summary The activities of three glycosidases, -glucosidase and (1,3)- and (1,6)-glucanases have been monitored during growth and blastospore formation inSaccharomycopsis fibuligera. The assays were carried out on the cell-free culture and in a cell-free extract and a wall autolysate preparation from the growing cells. In complex medium containing 1% glucose an increase in the level of all three enzymes was associated with the transition from mycelium to blastospores. When the level of glucose was increased to 5% blastospore formation was repressed and the level of -glucanases only increased at the end of the fermentation. The -glucosidase activity increased during the growth phase. In a defined medium in which slow growth in a wholly yeast-like form was observed, growth was not associated with a high level of -glucanase activity.     

Structural Changes of β-Lactoglobulin during Thermal Unfolding and Refolding – An FT-IR and Circular Dichroism Study

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of -lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly -sheet, still remained intact even at 90°C. Noticeable changes in secondary structure of -lactoglobulin were observed only above 70°C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded -lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded -lactoglobulin. The combined CD and FT-IR data indicated that refolded form of -lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

β-glucan degradation in malting sorghum

In all of four malting sorghum varieties, -glucan contents decreased by more than 50% 2 days after germination, due to enzymic digestion. The variety that had the least -glucan content in the malt gave the highest filterable volume of sweet wort while the variety with the highest -glucan content had the lowest volume of filterable wort.A.C. Ogbonna is with the Department of Brewing Science and Technology, University of Uyo, P.M.B. 1017, Uyo, Nigeria, A.L. Egunwu was with the University of Nigeria, Nsukka, Nigeria, and is now with Nigerian Breweries PLC, Aba, Abia State, Nigeria.     

Neurotoxic Potential of Three Structural Analogs of β-N-oxalyl-α,β-Diaminopropanoic Acid (β-ODAP)

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of -ODAP were synthesized. Carboxymethyl-,-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 M), but not by CNQX (10 M). N-acetyl-,-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three -ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2–3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [³H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for -ODAP. These data indicate that analogs of -ODAP may interact with glutamate receptors without producing neurotoxicity.     

Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.     

Exercise and recovery metabolism in the pacific spiny dogfish (<Emphasis Type="Italic">Squalus acanthias</Emphasis>)

We examined the effects of exhaustive exercise and post-exercise recovery on white muscle substrate depletion and metabolite distribution between white muscle and blood plasma in the Pacific spiny dogfish, both in vivo and in an electrically stimulated perfused tail-trunk preparation. Measurements of arterial-venous lactate, total ammonia, -hydroxybutyrate, glucose, and l-alanine concentrations in the perfused tail-trunk assessed white muscle metabolite fluxes. Exhaustive exercise was fuelled primarily by creatine phosphate hydrolysis and glycolysis as indicated by 62, 71, and 85% decreases in ATP, creatine phosphate, and glycogen, respectively. White muscle lactate production during exercise caused a sustained increase (~12 h post-exercise) in plasma lactate load and a short-lived increase (~4 h post-exercise) in plasma metabolic acid load during recovery. Exhaustive exercise and recovery did not affect arterial PO₂, PCO₂, or PNH₃ but the metabolic acidosis caused a decrease in arterial HCO₃^– immediately after exercise and during the first 8 h recovery. During recovery, lactate was retained in the white muscle at higher concentrations than in the plasma despite increased lactate efflux from the muscle. Pyruvate dehydrogenase activity was very low in dogfish white muscle at rest and during recovery (0.53±0.15 nmol g wet tissue^–1 min^–1; n=40) indicating that lactate oxidation is not the major fate of lactate during post-exercise recovery. The lack of change in white muscle free-carnitine and variable changes in short-chain fatty acyl-carnitine suggest that dogfish white muscle does not rely on lipid oxidation to fuel exhaustive exercise or recovery. These findings support the notion that extrahepatic tissues cannot utilize fatty acids as an oxidative fuel. Furthermore, our data strongly suggest that ketone body oxidation is important in fuelling recovery metabolism in dogfish white muscle and at least 20% of the ATP required for recovery could be supplied by uptake and oxidation of -hydroxybutyrate from the plasma.Abbreviations CoA-SH free coenzyme A - CPT-1 carnitine palmitoyltransferase-1 - CrP creatine phosphate - H⁺_m metabolic proton load - Lac lactate load - PDH pyruvate dehydrogenase - PVP polyvinylpyrrolidone - SCFA-carnitine short-chain fatty acyl-carnitine - TAG triacylglycerol - TENS trancutaneous electrical nerve stimulator Communicated by: L.C.-H. Wang     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.     

Estradiol Protects Against Oxygen and Glucose Deprivation in Rat Hippocampal Organotypic Cultures and Activates Akt and Inactivates GSK-3β

Here we investigated the neuroprotective effect of 17-estradiol in an in vitro model of ischemia. We used organotypic hippocampal slice cultures, acute or chronically treated with 17-estradiol (10 nM), and exposed to oxygen and glucose deprivation (OGD). Cellular death was quantified by measuring uptake of propidium iodide (PI), a marker of dead cells. In OGD exposed cultures, treated only with vehicle, about 70% of the CA1 area of hippocampus was labeled with PI, indicating a great percentage of cellular death. When cultures were treated with 17-estradiol (acute or chronically), this cellular death was reduced to 15%. This effect was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that both, chronic and acute, treatments with 17-estradiol induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of GSK-3. Our results show a clear neuroprotective effect of 17-estradiol and suggest that this effect could involve PI3-K pathway.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.     

Synthesis of Alkyl Glycosides Catalyzed by β-Glycosidases in a System of Reverse Micelles

A basic possibility of enzymic synthesis of alkyl glycosides in a system of the Aerosol-OT (AOT) reverse micelles was studied. Octyl -D-galactopyranoside and octyl -D-glucopyranoside were synthesized from the corresponding sugars (lactose or glucose) and octyl alcohol under catalysis with glycolytic enzymes, -galactosidase and -glucosidase, respectively. The transglycosylation/hydrolysis ratio was shifted toward transglycosylation by using octyl alcohol, one of the substrates, as an organic solvent. The alkyl glycosides were thus obtained in one step from a hydrophilic mono- or disaccharide and a hydrophobic aliphatic alcohol. The direction of the reaction was shown to depend on the pH of aqueous solution solubilized in reverse micelles. The maximum yields were 45% and 40% for octyl galactoside and octyl glucoside, respectively; they markedly exceeded the yields of enzymic syntheses in a two-phase system reported previously.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

A continuous process for the synthesis of hexyl β-D-glucoside in aqueous phase using immobilized β-glucosidase and extractive product recovery with 1-hexanol

Hexyl -d-glucoside was synthesized at 1 mM using immobilized -glucosidase with 2.0 M glucose solution with 1-hexanol. The continuous production of hexyl glucoside was achieved by its extractive recovery with 1-hexanol, and 4.9 g of product was obtained during a one week operation using a 26.5 ml reactor.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .