Relationship between postabsorptive respiratory exchange ratio and plasma free fatty acid concentrations

The relationship between overnight postabsorptive (fasting) respiratory exchange ratio (RER) and plasma FFA concentrations was addressed using data from three separate protocols, each of which involved careful control of the antecedent diet. Protocol 1 examined the relationship between fasting RER and the previous daytime RER. In Protocol 2 fasting, RER and plasma palmitate concentrations were measured in 29 women and 31 men (body mass index <30 kg·m⁻²). Protocol 3 analyzed data from Nielsen et al. (Nielsen, S., Z. K. Guo, J. B. Albu, S. Klein, P. C. O''Brien, M. D. Jensen. 2003. Energy expenditure, sex and endogenous fuel availability in humans. J. Clin. Invest. 111: 981-988.) to understand how fasting RER and palmitate concentrations relate within individuals during four consecutive measurements. The results were as follows: 1) Fasting RER was correlated (r = 0.74, P < 0.001) with the previous day''s average RER, and less so with RER variability. 2) Fasting RER was correlated (r = −0.39, P = 0.007) with fasting plasma palmitate concentrations. 3) The pattern of the RER/palmitate relationship was similar within individuals and between individuals; a negative slope was observed significantly more often than a positive slope (χ² test; P < 0.001). Our findings suggest that, despite a fixed food quotient, the slight departures from energy equilibrium in a controlled General Clinical Research Center environment can effect plasma FFA concentrations. We suggest that including indirect calorimetry as part of FFA metabolism studies may aid in data interpretation.     

A pertussis toxin-sensitive G-protein mediates some aspects of insulin action in BC3H-1 murine myocytes.

The involvement of G-proteins in the insulin signal transduction system has been studied in detail using the murine BC3H-1 myocyte system. Pertussis toxin (PT) treatment, previously shown to attenuate some of the metabolic effects of insulin in this cell line (Luttrell, L.M., Hewlett, E.L., Romero, G., and Rogol, A.D. (1988) J. Biol. Chem. 263, 6134-6141), abolished insulin-induced generation of diacylglycerol and inositolglycan mediators with no effects on either the autophosphorylation of the insulin receptor or the phosphorylation of the major endogenous substrates for insulin-stimulated tyrosine kinase activity (pp185 and pp42-45). In vitro ADP-ribosylation and immunoblotting studies suggest that the major PT substrate is a 40-kDa protein of the G alpha family. This protein band did not exhibit detectable tyrosine phosphorylation upon stimulation of either intact cells or cell membranes with insulin. In the presence of low concentrations of GTP, insulin treatment of isolated myocyte plasma membranes resulted in a small (30-40%) but significant stimulation of GTP hydrolysis. This effect was best observed in the presence of small concentrations of sodium dodecyl sulfate. The rate of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to BC3H-1 membranes was also significantly increased in the presence of insulin. The effects of insulin on GTP hydrolysis and GTP gamma S binding were found to be dependent on the concentration of insulin. These effects were not detected in plasma membranes prepared from PT-pretreated BC3H-1 myocytes. In contrast, pretreatment with the B (inactive) subunit of PT did not alter the response of myocyte membranes to insulin. High affinity binding of [125I]iodoinsulin to myocyte plasma membranes was reduced by 60-70% in the presence of guanine nucleotides. Similar effects on insulin binding were produced by PT pretreatment of the cells. In contrast, adenine nucleotides had no effect on insulin binding. Scatchard analysis of the binding data showed that the observed effects of guanine nucleotides and PT on insulin binding resulted either from a reduction in the number of high affinity insulin binding sites or from a significant reduction of the affinity of insulin for its receptor. Low affinity binding sites did not appear to be affected by either guanine nucleotides nor PT pretreatment. These results provide substantial evidence suggestive of a noncovalent interaction between the insulin receptor and a regulatory G-protein system during the process of insulin signaling.     

Peptides and multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase: preparation, immunogenicity and immunoprotective capacity in C57BL/6 mice.

Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail.     

External factors involved in the regulation of an extracellular proteinase synthesis in Bacillus megaterium

Summary A mathematical model was formulated to describe the kinetics and stoichiometry of growth and proteinase production in Bacillus megaterium. Synthesis of the extracellular proteinase in a batch culture is repressed by amino acids. The specific rate of formation of the enzyme (r _E) can be described by the formula {ie373-1}, where k ₂ and k ₃ stand for the non-repressible and repressible part of enzyme synthesis respectively, k _{S 2} is a repression coefficient and S ₂ indicates the concentration of amono acids; the values of k ₂ and k _{S 2} depend on the composition of the mixture of amino acids. Even in a high concentration, a single amino acid is less effective than a mixture of amino acids. The dependence of the proteinase repression on the concentration of an external amino acid (leucine) follows the same course as its rate of incorporation into proteins, approaching saturation at concentrations higher than 50 M (half saturation approximately 10 M). However, the total uptake of leucine did not exhibit any saturation even at 500 M external concentration.Symbols X biomass concentration, g/l - E proteinase concentration, unit/l - t time, h - S ₁ concentration of glucose, g/l - S ₂ concentration of amino acids, g/l - specific growth rate, l/h - rE specific rate of enzyme production, unit/g/h - k ₁ growth kinetic constant, l/h - k ₂ product formation kinetic constant (for non-repressible part of enzyme synthesis), unit/g - k ₃ product formation kinetic constant (for repressible portion of enzyme synthesis), unit/g - k _{S 1} saturation constant, g/l - k _{S 2} repression coefficient for a certain amino acid or amino acids mixture, g/l     

Physiological activity of immobilized cells of Claviceps fusiformis during long-term semicontinuous cultivation

Summary The 550-day semicontinuous cultivation of Claviceps fusiformis immobilized in calcium alginate is documented. The vegetative mycelium from seed or from early-production submerged culture is the best choice for immobilization. No extracellular glucans are produced by immobilized cells. Immobilized spores give low yields of clavine alkaloids. Alginate concentrations in a range of 2%–4% do not influence yield and spectrum of alkaloids. The cytoplasm of the immobilized cells becomes condensed (after 3 days), polysaccharides disappear, and centres of lipid synthesis are formed in the cytoplasm. After 60 days the cells harbour a great number of lipid particles, mitochondria are diminishing and their cristae partly disappear, indicating a decreased respiration capacity. After 350–500 days the volume of most cells is increased many times and the cells are filled with large oval bodies of electrondense material. Chloramphenicol protects immobilized cultures against bacterial contamination.     

Regulated nitrate transport in the cyanobacterium Anacystis nidulans.

Intracellular accumulation of nitrate, indicative of the operation of an active nitrate transport system, has been measured in intact cells of the cyanobacterium Anacystis nidulans. The ability of the cells to accumulate nitrate was effectively hindered by either ammonium addition or selective inhibition of CO2 fixation by DL-glyceraldehyde, with the effect of either compound being prevented by previously blocking ammonium assimilation. The results support the contention that nitrate utilization in cyanobacteria is regulated at the level of nitrate transport through the concerted action of ammonium assimilation and CO2 fixation.     

Glutathione content and GSH S-transferase activity in midgut gland of Procambarus clarkii. Sex differences, the effect of fasting, and their implications in cadmium toxicity

1. Glutathione content and GSH S-transferase activity in the midgut gland of Procambarus clarkii (P. c.) of different sex and body weight are presented. 2. Procambarus clarkii females' GSH concentration in the midgut gland decreases to a higher extent upon fasting, compared with males. 3. Procambarus clarkii females, both in control and fasting conditions, have a slightly higher GSH S-transferase activity than males. 4. Cadmium present in water only affects GSH content and GSH S-transferase activity (after 96 hr) in midgut gland, with cadmium chloride concentrations higher than 100 micrograms/l.     

Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase

Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.