The disposition of citric acid cycle intermediates by isolated rat heart mitochondria

The mechanism of depletion of tricarboxylic acid cycle intermediates by isolated rat heart mitochondria was studied using hydroxymalonate (an inhibitor of malic enzymes) and mercaptopicolinate (an inhibitor of phosphoenolpyruvate carboxykinase) as tools. Hydroxymalonate inhibited the respiration rate of isolated mitochondria in state 3 by 40% when 2 mM malate was the only external substrate, but no inhibition was found with 2 mM malate plus 0.5 mM pyruvate as substrates. In the prescence od bicarbonate, arsenite and ATP, propionate was converted to pyruvate and malate at the rates of 14.0 ± 2.9 and 2.8 ± 1.8 nmol/mg protein in 5 min, respectively. Under these conditions, 0.1 mM mercaptopicolinate did not affect this conversion, but 2 mM hydroxymalonate inhibited pyruvate formation completely and resulted in an accumulation of malate up to 13.2 ± 2.9 nmol/mg protein. No accumulation of phosphoenolpyruvate was found under any condition tested. It is concluded that malic enzymes but not phosphoenolpyruvate carboxykinase, are involved in conversion of propionate to pyruvate in isolated rat heart mitochondria.     

Local Conformation and Dynamics of Isoleucine in the Collagenase Cleavage Site Provide a Recognition Signal for Matrix Metalloproteinases

The mechanism by which enzymes recognize the “uniform” collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Gly∼[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P₃ subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.     

Choline Administration Elevates Brain Phosphorylcholine Concentrations

Abstract: The phosphorylcholine concentration of rat brain rises and falls in response to parallel changes in the concentration of circulating choline. A single oral dose of choline chloride (20 mmol/kg) elevated whole-brain concentrations of both choline and phosphorylcholine 5 h after administration; a greater proportion of exogenously administered choline was retained by the brain in its phosphorylated form than as the free arnine. Striatal phosphorylcholine concentrations were elevated within 2 h of choline administration and continued to be significantly greater than control values for up to 34 h after treatment. The response of striatal choline levels to exogenous choline was of shorter duration than that of phosphorylcholine and was correlated with a significant increase in striatal acetylcholine concentrations. The consumption of a choline-free diet for 7 days lowered both serum choline and striatal phosphorylcholine concentrations, but had no effect on striatal choline or acetylcholine. These results suggest that choline kinase is unsaturated by its substrate in vivo and may thus serve to modulate the response of brain choline concentrations to alterations in the supply of circulating choline.     

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

Mathematical model of the activation of prothrombin by factor X a and factor V t

A mathematical model of prothrombin activation is being proposed which includes the feedback mechanism of thrombin and the alteration of factor V by thrombin. This model is in good agreement with experimental data for the dependence of the rate of thrombin formation on the concentrations of factors V and X _a . In particular, it correctly predicts the existence and location of a maximum in both of these cases.     

Performing arts medicine.

Arts medicine has come of age, resulting from 3 important developments over the past decade: improved methods of diagnosis and treatment, an awareness that artists suffer from special problems related to their occupation and lifestyle, and the establishment of health programs emphasizing an interdisciplinary approach to these patients. We focus on the patterns of illness afflicting performing artists, specifically dancers, singers, actors, and instrumental musicians, and explain some of the things a health care team can do in treating these patients. The conditions governing these patients'' lives--early exposure to high expectations of excellence, incessant demands for perfection, long periods of intense practicing, fierce competition, high levels of anxiety associated with performance, and uncertain careers--need to be understood. Levels of disease and disability are remarkably high, but artists often ignore symptoms. We discuss the musculoskeletal, neurologic, vocal, psychological, and other syndromes found among performers and some of the difficulties in treating them. The prevention of injury, conservative management, collaboration with teachers, and a psychotherapeutic approach are desirable. Arts medicine programs for professional consultation exist in several major cities of the United States and abroad. Although research is needed regarding the effectiveness of health care services for performing artists, the scientific literature devoted to this field is growing.     

Methylated poly(L-lysine): conformational effects and interactions with polynucleotides

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of epsilon-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally-induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO4)2. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from beta, to 50% alpha, to random coil at the maximum methylation. Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)n.(dAdT)n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)n.(dC)n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative psi-spectra are induced, which, in the case of (dGdC)n.(dGdC)n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration. It is suggested that the biological effects of methylation proteins may be evoke by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.     

Sympathetic and adrenocorticoid influences on diet-induced thermogenesis and brown fat activity in the rat

Bilateral adrenalectomy markedly reduced body weight and energy gain and energetic efficiency of adult cafeteria-fed rats but enhanced the thermogenic response to food and stimulated brown fat activity. These changes were totally prevented by replacement of the animals with corticosterone (1 mg/rat/day). Unilateral denervation of the sympathetic nerves supplying the interscapular brown adipose tissue abolished the enhanced activity resulting from adrenalectomy and inhibited thermogenic activity in brown fat from cafeteria rats with intact adrenals, but had no effect in adrenalectomised animals treated with a high dose of corticosterone.     

Interaction of the serum amyloid A proteins with phospholipid

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.