Effect of β-alanine administration on carbon tetrachloride-induced acute hepatotoxicity

Summary. Mice were supplemented with β-alanine (3%) in drinking water for one week. β-Alanine intake reduced hepatic taurine levels, but elevated cysteine levels significantly. Hepatotoxicity of CCl₄ in mice fed with β-alanine was decreased as determined by changes in serum enzyme activities. Hepatic glutathione and taurine concentrations after CCl₄ challenge were increased markedly by β-alanine intake. The enhanced availability of cysteine for synthesis of glutathione and/or taurine appears to account for the hepatoprotective effects of β-alanine against CCl₄-induced liver injury.     

Carbon isotope ratio (δC) values of urinary steroids for doping control in sport

The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (δ¹³C) profiling study (n = 1262) of urinary ketosteroids is reported that demonstrates the inter-individual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This δ¹³C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to δ¹³C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n = 100; consisting of three countries) is also presented with comparison to δ¹³C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.     

Detection of endogenous β-glucuronidase activity in Aspergillus niger

 An endogenous β-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous β-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uidAβ-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7–8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous β-glucuronidase activity, was achieved by staining at this pH. Received : 22 March 1995/Received last revision : 17 August 1995/Accepted : 22 August 1995     

Effect of carbon source on the β-glucosidase system of the thermophilic fungus Humicola grisea

H. grisea produced an extracellular -glucosidase (EC 3.2.1.21) at high activity in media supplemented with carboxymethyl cellulose (CMC) or cellobiose. Cellobiose-induced -glucosidase was insensitive to glucose repression whereas that of CMC-supplemented cultures was partially repressed. Molecular sieving revealed three main active components (M_r 50, 128 and 240 kDa). Glucose competitively inhibited -glucosidase activities with K_i values of 0.9mM and 3.3mM (extracellular) and 10.2mM and 22.6mM (cytosolic), induced in the presence of CMC or cellobiose respectively.The authors are with the Departamento de Biologia, Faculdade de Filosofia. Ciências e Letras de Ribeirão Preto, Universidade de São Paulo-14040-901 Ribeirão Preto, São Paulo, Brasil;     

Further studies on carbon catabolite regulation of β-lactam antibiotic synthesis inCephalosporium acremonium

A high glucose concentration (6%) interfered with production of -lactam antibiotics byCephalosporium acremonium. Production rate of the pathway intermediate, penicillin N, by resting cells harvested from a high glucose fermentation, peaked and declined early in the fermentation. When cells were grown in the standard medium (2.7% glucose + 3.6% sucrose), penicillin N productivity was prolonged, showing two peaks, the first during trophophase and the second afterwards. The decline in productivity was not prevented by addition of the amino acid precursors of -lactam antibiotics. The addition of glucose to resting cells drastically decreased formation of the end product, cephalosporin C, but had only a moderate effect on penicillin N production. Glucose markedly repressed the ring-expansion enzyme (deacetoxy-cephalosporin C synthetase) but had a lesser effect on the tripeptide cyclization enzyme (isopenicillin N synthetase). We conclude that the major effect of a low (2%) or a high (6%) concentration of a rapidly used carbon source (e.g., glucose, glycerol, maltose) onC. acremonium fermentations is repression of the metabolically unstable ring-expansion enzyme and hence of formation of cephalosporins. On the other hand, the lesser degree of repression of the cyclization enzyme and itsin vivo stability allow penicillin N to accumulate normally or even at increased rates except at high carbon source concentrations.     

Biocatalytic characterization of Aspergillus oryzae β-galactosidase immobilized on functionalized multi-walled carbon nanotubes

The objectives of this work were to immobilize commercial Aspergillus oryzae β-galactosidase on functionalized multi-walled carbon nanotubes (MWCNTs) using different treatments and to characterize the products. Treatments were performed with glutaraldehyde, ethylenediamine and a mixture of concentrated H₂SO₄:HNO₃. The MWCNTs and their derivatives were characterized by thermogravimetric analysis. The immobilized enzymes were evaluated using inactivation kinetics, operating conditions, that is pH and temperature, kinetic parameters and lactose hydrolysis reusability. Immobilization yield and efficiency were significantly higher for β-galactosidase immobilized on MWCNTs functionalized by the acid mixture (Ac-Gal-MWCNTs). These values were 97% and 82%, respectively, after 3?h of immobilization. The activity of the Ac-Gal-MWCNTs was maintained at ～51% of their initial activity after being stored for 90 days at 4?°C. The Ac-Gal-MWCNTs retained more than 90% of their initial activity up to the fourth recycle. As the acid functionalization was the most efficient method tested for immobilizing A. oryzae β-galactosidase on MWCNTs, this method shows promise for industrial applications.     

Local or nonlocal? A research of strontium isotope ratios of teeth and bones on skeletal remains with artificial deformed skulls

The analysis of strontium isotopic composition of teeth and bone served as an approach to determine nonlocal individuals with artificially deformed skulls in Teuton and Gepid sites. A differing Sr-isotopic composition between tooth and bone from the same skeleton reveals a residence change between early childhood and the last ten years before death. The results show that most Teuton and Gepid individuals investigated are local habitants.     

Induction of xylanase and β-xylosidase in Cellulomonas flavigena growing on different carbon sources

When Cellulomonas flavigena CDBB-531 was grown on glucose, xylose, glycerol, solka floc, sugarcane bagasse or xylan, xylanase activity was found only in the fermentation broth, while -xylosidase activity was always associated with the cells. Both enzymes were inducible, sugar-cane bagasse was the best inducer, solka floc and avicel were moderately good, while xylan was poor. A synergistic effect on xylanase and -xylosidase synthesis was observed when cellulose and hemicellulose were used together as carbon sources. When this strain was grown on glucose, cellobiose, arabinose or xylose, only low levels of both enzymes were detected. These results indicate that xylanase and -xylosidase were carbon-source-repressed by readily metabolizable substrates. The effect of glycerol on enzymes that were already induced was studied. The addition of glycerol caused a significant decrease in the levels of xylanases, while -xylosidase activity remained unchanged.     

SHARPIN is an endogenous inhibitor of β1-integrin activation

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of β1-integrins in an RNAi screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.     

What sources of organic carbon drive food webs in billabongs? A study based on stable isotope analysis

We used the stable isotopes of carbon and nitrogen to examine the food webs of three small flood-plain lakes (billabongs) in south-eastern Australia. With few exceptions, stable carbon isotope analysis could not be used to discriminate among the conspicuous potential sources of fringing, emergent or floating vegetation or benthic detritus. These primary sources showed little spatial or temporal variation in ¹³C values, with means ranging from-28.5 to-26.8 in spring and-29.1 to-25.4 in late summer. Submerged vegetation had similar ¹³C values to the above sources in spring but showed greater spatial variation and were less ¹³C-depleted, considerably so in some species, in late summer. Epiphytes and algae were ¹³C-depleted in spring compared with the other primary sources but became more ¹³C-enriched in late summer. Mean ¹³C values for primary and secondary consumers were not only far more variable (-37.4 to-22.7) but in general were more negative than the potential food sources, particularly in spring. Using the combined information from stable carbon and nitrogen isotope analysis, we could narrow down the list of potential primary sources driving food webs in these billabongs. The freshwater crayfish (Cherax) was one of the few taxa that appeared to obtain its biomass carbon from detrital material. Gastropods and leptocerid caddis larvae on emergent or submerged vegetation obtained a mixture of carbon from epiphytes and macrophytes; in both taxa, epiphytes contributed more to biomass carbon than did the macrophytes. However, other common grazers and collector/gatherers sampled from macrophytes, e.g. baetid mayflies, chironomid larvae and atyid shrimps, were often too ¹³C-depleted even to have derived their biomass carbon solely from epiphytes. Many other primary consumers, including zooplankton, and mussels (Velesunio), and most of the secondary consumers, including water mites (Hydracarina), phantom midge larvae (Chaoborus) and fish, were also ¹³C-depleted. The enormous biomass of littoral and fringing vegetation could contribute to metazoan food webs in these billabongs only if an additional highly ¹³C-depleted source was consumed simultaneously. Methane released from billabong sediments could provide such a ¹³C-depleted carbon source that is re-introduced into metazoan food webs via the consumption of methanotrophic bacteria. Alternatively, food webs in these water bodies are largely driven by an unknown and inconspicuous ¹³C-depleted primary producer, such as planktonic Chlorophyta.     

The effect of environment on plasma cortisol and β-endorphin in the parturient pig and the involvement of endogenous opioids

Previous work has indicated that plasma cortisol increases during farrowing in the pig suggesting increasing physiological stress. The aim of this study was to determine changes in plasma cortisol and β-endorphin over farrowing in the pig to obtain a more detailed profile of pituitary and adrenal release at this time and also to investigate the involvement of endogenous opioids in the mediation of the HPA axis. Indwelling jugular catheters were implanted, under general anaesthesia, in 31 Large White×Landrace gilts approximately 15 days before the expected parturition day (EPD). Gilts were moved into either a farrowing crate, without straw (n=15), or a straw-bedded pen (n=16) 5 days before the EPD. Samples were taken during the pre-farrowing period and then during farrowing itself. At 7.5 min after the birth of the first piglet (BFP), gilts either received naloxone, an opioid antagonist, (1 mg kg⁻¹ body weight, i.v.) or a control dose of saline. Plasma β-endorphin increased following the BFP but remained fairly constant over the third and fourth hour of farrowing. Plasma cortisol continued to increase over the 4 h following the BFP. Changes seen in these hormones were generally insensitive to the environment and there was little evidence of opioid mediation of the HPA axis at parturition. From these results it is suggested that certain aspect(s) of parturition itself stimulate the HPA axis. However it is unknown if the rise in plasma cortisol is a result of some stress-inducing factor of the parturition process or whether it reflects a metabolic function. The study also demonstrates the lack of any inhibitory mediation of the HPA axis by endogenous opioids at parturition.     

Effect of glucocorticoid administration on the rate of muscle protein breakdown in vivo in rats, as measured by urinary excretion of Nτ-methylhistidine

The role of glucocorticoids in regulating the rate of muscle protein breakdown was evaluated by measuring excretion of N(tau)-methylhistidine during administration of various doses of corticosterone to adrenalectomized rats. Groups of rats received daily subcutaneous injections of 0, 0.2, 0.5, 1.0, 5.0 or 10.0mg of corticosterone/day per 100g body wt. for 7 days, followed by 3 days without hormone treatment, after which they were killed. A group with intact adrenal glands served as an additional control. All animals were pair-fed with the untreated adrenalectomized group. No significant differences were noted in growth rate or N(tau)-methylhistidine excretion between the intact or adrenalectomized control groups, or those given 0.2, 0.5 and 1.0mg of corticosterone, whereas growth ceased and N(tau)-methylhistidine excretion rose markedly in the groups receiving 5 and 10mg of corticosterone. After these two high doses of corticosterone, but not after lower doses, there was a loss of weight of the gastrocnemius muscle per 100g of final body wt., but not of the soleus and extensor digitorum longus muscles. The two highest doses of corticosterone also resulted in an increase in liver weight per 100g of final body wt. Lower doses of corticosterone did not cause these changes. Plasma corticosterone concentrations, measured on the final day of injection and again at the time of killing, were decreased to near zero by adrenalectomy and were little raised by doses of 0.2 and 0.5mg daily, but were increased to within the normal range by the 1mg dose. At 5 and 10mg doses, plasma corticosterone concentrations were sustained at 2-3 times those of intact rats, and thus in the range reported for rats exposed to severe stress. Rats given 5 and 10mg doses of corticosterone had glycosuria, and showed considerably elevated concentrations of insulin in the plasma. It is concluded that plasma concentrations of glucocorticoids within the normal range do not regulate the rate of muscle protein breakdown, whereas excessive plasma concentrations of corticosteroids, equivalent to those observed in severe stress, can accelerate muscle protein breakdown.     

Electrochemical immunosensor for the milk allergen β-lactoglobulin based on electrografting of organic film on graphene modified screen-printed carbon electrodes

A novel label-free voltammetric immunosensor for sensitive detection of β-lactoglobulin using graphene modified screen printed electrodes has been developed. The derivatization of the graphene electrode surface was achieved by electrochemical reduction of in situ generated 4-nitrophenyl diazonium cations in aqueous acidic solution, followed by electrochemical reduction of the terminal nitro groups to amines. The electrochemical modification protocol was optimized in order to generate monolayer of nitrophenyl groups on the graphene surface without complete passivation of the electrode. Unlike the reported method for graphene functionalization, we demonstrated here the ability of the electrografting of aryl diazonium salt to attach an organic film to the graphene surface in a controlled manner by choosing the suitable grafting protocol. Next, the amine groups on the graphene surface were activated using glutaraldehyde and used for the covalent immobilization of β-lactoglobulin antibodies. Cyclic and differential pulse voltammetry carried out in an aqueous solution containing [Fe(CN)(6)](3-/4-) redox pair have been used for the immunosensor characterization. The results demonstrated that the DPV reduction peak current of [Fe(CN)(6)](3-/4-) decreased linearly with increasing the concentration of β-lactoglobulin due to the formation of antibody-antigen complex on the modified electrode surface. The immunosensor obtained using this novel approach enabled a detection limit of 0.85pgmL(-1) and a dynamic range from 1pgmL(-1) to 100ngmL(-1) of β-lactoglobulin in PBS buffer. In addition, the immunosensor evaluated in different samples including cake, cheese snacks, a sweet biscuit, showing excellent correlation with the results obtained from commercially enzyme-linked immunosorbent assay (ELISA) method.     

Distribution of endogenous and exogenous 5′-nucleotidase on bovine spermatozoa

A polyclonal rabbit antibody against 5-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.     

Lower–middle Frasnian organic carbon isotope record of conodonts in East European Platform

The early–middle Frasnian boundary interval of the northern part of the East European Platform (northwest of Russia) corresponding to the transitans-punctata isotope event is revealed by biostratigraphically constrained conodont carbon stable isotope data (δ¹³C_con). The dynamics of δ¹³C_con demonstrate a three-fold pattern with positive peaks at the onset of the main phase of the transitans-punctata isotope event (upper part of Montagne Noire 4 conodont Zone, MN4; up to -22.5‰ VPDB), and during the late part of the event (lower and middle parts of MN6 Zone; up to -24.0‰ VPDB and -22.1‰ VPDB). The stratigraphic level near the MN5 and MN6 boundary is marked by a prominent negative excursion in δ¹³C_con (down to -31.8‰ VPDB) that resembles the negative excursion in the terminal phase of the transitans-punctata isotope event worldwide. The δ¹³C_con variations in different taxa are generally consistent but demonstrate some differences in values and amplitudes. It is assumed that variations in the carbon isotopic compositions of conodonts were mainly controlled by changes in the isotope composition of the planktons as the main food source for conodonts.     

Diversity,metabolic types and δ13C carbon isotope ratios in the grass flora of Namibia in relation to growth form,precipitation and habitat conditions

The grass flora of Namibia (374 species in 110 genera) shows surprisingly little variation in ¹³C values along a rainfall gradient (50–600 mm) and in different habitat conditions. However, there are significant differences in the ¹³C values between the metabolic types of the C4 photosynthetic pathway. NADP-ME-type C4 species exhibit the highest ¹³C values (–11.7 ) and occur mainly in regions with high rainfall. NAD-ME-type C4 species have significantly lower ¹³C values (–13.4 ) and dominate in the most arid part of the precipitation regime. PCK-type C4 species play an intermediate role (–12.5 ) and reach a maximum abundance in areas of intermediate precipitation. This pattern is also evident in genera containing species of different metabolic types. Within the same genus NAD species reach more negative ¹³C values than PCK species and ¹³C values decreased with rainfall. Also in Aristida, with NADP-ME-type photosynthesis, ¹³C values decreased from –11 in the inland region (600 mm precipitation) to –15 near the coast (150 mm precipitation), which is a change in discrimination which is otherwise associated by a change in metabolism. The exceptional C3 species Eragrostis walteri and Panicum heterostachyum are coastal species experiencing 50 mm precipitation only. Many of the rare species and monotypic genera grow in moist habitats rather than in the desert, and they are not different in their carbon isotope ratios from the more common flora. The role of species diversity with respect to habitat occupation and carbon metabolism is discussed.     

Effect of β-Propiolactone on Sendai Virus

The biological properties (infectivity, hemagglutination, hemolysis, cell fusion, neuraminidase) of Sendai virus were dissociated on the basis of sensitivity to beta-propiolactone, by freeze-thawing, by heating at different temperatures, and by adsorption-elution with formalinized chicken erythrocytes. Possible mechanisms whereby beta-propiolactone selectively destroys viral infectivity are discussed.     

Effect of protein-surfactant interactions on aggregation of β-lactoglobulin

The milk protein β-lactoglobulin (βLG) dominates the properties of whey aggregates in food products. Here we use spectroscopic and calorimetric techniques to elucidate how anionic, cationic and non-ionic surfactants interact with bovine βLG and modulate its heat-induced aggregation. Alkyl trimethyl ammonium chlorides (xTAC) strongly promote aggregation, while sodium alkyl sulfates (SxS) and alkyl maltopyranosides (xM) reduce aggregation. Sodium dodecyl sulfate (SDS) binds to non-aggregated βLG in several steps, but reduction of aggregation was associated with the first binding step, which occurs far below the critical micelle concentration. In contrast, micellar concentrations of xMs are required to reduce aggregation. The ranking order for reduction of aggregation (normalized to their tendency to self-associate) was C10-C12>C8>C14 for SxS and C8>C10>C12>C14>C16 for xM. xTAC promote aggregation in the same ranking order as xM reduce it. We conclude that SxS reduce aggregation by stabilizing the protein's ligand-bound state (the melting temperature t(m) increases by up to 10°C) and altering its charge potential. xM monomers also stabilize the protein's ligand-bound state (increasing t(m) up to 6°C) but in the absence of charged head groups this is not sufficient by itself to prevent aggregation. Although micelles of both anionic and non-ionic surfactants destabilize βLG, they also solubilize unfolded protein monomers, leaving them unavailable for protein-protein association and thus inhibiting aggregation. Cationic surfactants promote aggregation by a combination of destabilization and charge neutralization. The food compatible surfactant sodium dodecanoate also inhibited aggregation well below the cmc, suggesting that surfactants may be a practical way to modulate whey protein properties.