Effect of insulin on lipolysis in adipose tissue of rats of different ages

Several authors have not been able to find any antilipolytic effect of insulin in adipose tissue "in vitro". We investigated the possible role of cell size and/or age of donors on this phenomenon. The lipolytic rates (glycerol release per cell) were lower in the small cells of the 4-6 weeks old rats than in the larger cells of the 25-30 weeks old animals; however, the difference disappeared when the data were expressed per unit of cell surface area. Insulin (0.5-50 ng/ml) failed to inhibit both maximally and submaximally noradrenaline stimulated lipolysis in the adipocytes of the young rats, but its antilipolytic action was fully restored by using glucose-free medium. Therefore, at our experimental conditions, a glucose dependent factor, possibly involving the preferential hydrolysis of newly synthetized triglycerides, seems to blunt or to mask the insulin induced inhibition of glycerol release. Relatively higher rates of glucose metabolism and a lower lipolysis in small fat cells might explain the difference in the action of insulin on glycerol release in the adipose tissue of young rats as compared to the older ones.     

Variations of soil structure and microbial population in a compost amended soil

Changes in soil structure and in microbial population were recorded in a long term field experiment over the growing season of maize (June–November). Determinations were made on samples from plots which had received, for two years, the following treatments: mineral fertilizers, farmyard manure and three rates of compost. Seasonal variations were observed for the stability of soil aggregates, total porosity, pore size distribution, mycorrhizal infection and aerobic cellulolytic microorganisms. The stability of the soil aggregates changed in a similar way to that found for both mycorrhizal infection and the number of aerobic cellulolytic microorganisms. Physical characteristics were not affected in any instance by the organic dressings and microbiological populations were generally influenced only by the higher doses of compost.     

Neonatal Morphine Administration Leads to Changes in Hippocampal BDNF Levels and Antioxidant Enzyme Activity in the Adult Life of Rats

It is know that repeated exposure to opiates impairs spatial learning and memory and that the hippocampus has important neuromodulatory effects after drug exposure and withdrawal symptoms. Thus, the aim of this investigation was to assess hippocampal levels of BDNF, oxidative stress markers associated with cell viability, and TNF-α in the short, medium and long term after repeated morphine treatment in early life. Newborn male Wistar rats received subcutaneous injections of morphine (morphine group) or saline (control group), 5 μg in the mid-scapular area, starting on postnatal day 8 (P8), once daily for 7 days, and neurochemical parameters were assessed in the hippocampus on postnatal days 16 (P16), 30 (P30), and 60 (P60). For the first time, we observed that morphine treatment in early life modulates BDNF levels in the medium and long term and also modulates superoxide dismutase activity in the long term. In addition, it was observed effect of treatment and age in TNF-α levels, and no effects in lactate dehydrogenase levels, or cell viability. These findings show that repeated morphine treatment in the neonatal period can lead to long-lasting neurochemical changes in the hippocampus of male rats, and indicate the importance of cellular and intracellular adaptations in the hippocampus after early-life opioid exposure to tolerance, withdrawal and addiction.     

Green tea polyphenols: novel irreversible inhibitors of dopa decarboxylase

The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG), and (-)-epigallocatechin (EGC) were found to be inhibitors of Dopa decarboxylase (DDC). EGCG and EGC inactivate the enzyme in both a time- and concentration-dependent manner and exhibit saturation of the rate of inactivation at high concentrations, with efficiency of inactivation values (k(inact)/K(i)) of 868 and 1511 M(-1) min(-1), respectively. In contrast, gallic acid behaves as a weak inhibitor of DDC. Protection against inactivation by EGCG and EGC was observed in the presence of the active site-directed inhibitor D-Dopa. Either EGCG or EGC induce changes in the absorbance and CD bands of the visible spectrum of enzyme-bound PLP. Taken together, these findings indicate the active site nature of the interaction of DDC with both polyphenols. On the basis of the properties of the EGCG-inactivated enzyme, it can be suggested that inactivation could be ascribed to a covalent modification of not yet identified residue(s) of the active site of DDC.     

Reaction and substrate specificity of recombinant pig kidney Dopa decarboxylase under aerobic and anaerobic conditions

Dopa decarboxylase (DDC) catalyzes as main reaction the stereospecific CO(2) abstraction from L-Dopa and L-5-hydroxytryptophan (5-HTP), generating the corresponding aromatic amines, dopamine and serotonin, respectively. Side reactions with turnover time of minutes are also catalyzed by the enzyme. In particular, DDC exhibits half-transaminase activity toward D-aromatic amino acids and oxidative deaminase activity toward aromatic amines. The latter reaction could represent a new activity for this class of enzymes. Studies on the effect exerted by O(2) on reaction specificity of DDC revealed that under anaerobic conditions decarboxylation of L-aromatic amino acids takes place with a k(cat) approximately half of that measured in the presence of O(2), and is accompanied by a decarboxylation-dependent transamination, whereas oxidative deamination of aromatic amines is replaced by half-transamination. Half-transamination of D-aromatic amino acids is unaffected by the presence or absence of O(2). Some structural elements relevant for the control of reaction and substrate specificity of DDC have been identified by means of limited tryptic digestion and site-directed mutagenesis studies. All together, the data indicate that the chemical nature of the substrate, the presence of O(2), the integrity of a mobile loop, the absence of perturbation in the coenzyme-binding cleft and pH are important requirements for the achievement of a closed conformational state where the highest level of reaction specificity is reached.     

Treponema denticola cystalysin catalyzes beta-desulfination of L-cysteine sulfinic acid and beta-decarboxylation of L-aspartate and oxalacetate

Pyridoxal 5'-phosphate-dependent cystalysin from Treponema denticola catalyzes the beta-displacement of the beta-substituent from both L-aspartate and L-cysteine sulfinic acid. The steady-state kinetic parameters for beta-desulfination of L-cysteine sulfinic acid, k(cat) and K(m), are 89+/-7 s(-1) and 49+/-9 mM, respectively, whereas those for beta-decarboxylation of L-aspartate are 0.8+/-0.1 s(-1) and 280+/-70 mM. Moreover, cystalysin in the pyridoxamine 5'-phosphate form has also been found to catalyze beta-decarboxylation of oxalacetate as shown by consumption of oxalacetate and a concomitant production of pyruvate. The k(cat) and K(m) of this reaction are 0.15+/-0.01 s(-1) and 13+/-2 mM, respectively. Possible mechanistic and physiological implications are discussed.     

Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine)

Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.     

Spectroscopic and kinetic analyses reveal the pyridoxal 5'-phosphate binding mode and the catalytic features of Treponema denticola cystalysin

To obtain insight into the functional properties of Treponema denticola cystalysin, we have analyzed the pH- and ligand-induced spectral transitions, the pH dependence of the kinetic parameters, and the substrate specificity of the purified enzyme. The absorption spectrum of cystalysin has maxima at 418 and 320 nm. The 320 nm band increases at high pH, while the 418 nm band decreases; the apparent pK(spec) of this spectral transition is about 8.4. Cystalysin emitted fluorescence at 367 and 504 nm upon excitation at 320 and 418 nm, respectively. The pH profile for the 367 nm emission intensity increases above a single pK of approximately 8.4. On this basis, the 418 and 320 nm absorbances have been attributed to the ketoenamine and substituted aldamine, respectively. The pH dependence of both log k(cat) and log k(cat)/K(m) for alpha,beta-elimination reaction indicates that a single ionizing group with a pK value of approximately 6.6 must be unprotonated to achieve maximum velocity. This implies that cystalysin is more catalytically competent in alkaline solution where a remarkable portion of its coenzyme exists as inactive aldamine structure. Binding of substrates or substrate analogues to the enzyme over the pH range 6-9.5 converts both the 418 and 320 nm bands into an absorbing band at 429 nm, assigned to the external aldimine in the ketoenamine form. All these data suggest that the equilibrium from the inactive aldamine form of the coenzyme shifts to the active ketoenamine form on substrate binding. In addition, reinvestigation of the substrate spectrum of alpha,beta-elimination indicates that cystalysin is a cyst(e)ine C-S lyase rather than a cysteine desulfhydrase as claimed previously.     

Recent decrease in chinstrap penguin (<Emphasis Type="Italic">Pygoscelis antarctica</Emphasis>) populations at two of Admiralty Bay’s islets on King George Island,South Shetland Islands,Antarctica

We examined the breeding populations of chinstrap penguins (Pygoscelis antarctica) on Chabrier Rock and Shag Island within Admiralty Bay, King George Island, South Shetland Islands, Antarctica from 2002 to 2004. When comparing our results to historic data from 1979, we found an overall decline of 57% in the last 25 years, mirroring the population trend of this species in other regions of the Antarctic Peninsula. Our results are discussed in relation to factors hypothesized to be driving the declines found at other sites, as well as the importance of consistent annual censuses to accurately determine population trends.