Six-month outcome of critically ill patients given glutamine-supplemented parenteral nutrition

Tetrahydrobiopterin is an essential cofactor required for activity of nitric oxide synthases. Existing evidence suggests that, during activation of constitutive and inducible isoforms of nitric oxide synthase, tetrahydrobiopterin is needed for allosteric and redox activation of enzymatic activity. However, precise mechanisms underlying the role of tetrahydrobiopterin in regulation of nitric oxide formation is not fully understood. In cerebral and peripheral arteries, increased availability of tetrahydrobiopterin can augment production of nitric oxide. In contrast, in arteries depleted of tetrahydrobiopterin, production of nitric oxide is impaired. Proinflammatory cytokines enhance mRNA expression of the rate-limiting enzyme of tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I and stimulate production of tetrahydrobiopterin. The ability of vascular tissues to synthesize tetrahydrobiopterin plays an important role in regulation of nitric oxide synthase under physiological conditions as well as during inflammation and sepsis. More recent studies concerning expression and function of recombinant nitric oxide synthases suggest that availability of tetrahydrobiopterin is important for production of nitric oxide in genetically engineered blood vessels. In this review, mechanisms regulating availability of intracellular tetrahydrobiopterin and its role in control of vascular tone under physiological and pathological conditions will be discussed.     

Metabolic and exercise endurance effects of coffee and caffeine ingestion

Caffeine (Caf) ingestion increases plasma epinephrine (Epi) and exercise endurance; these results are frequently transferred to coffee (Cof) consumption. We examined the impact of ingestion of the same dose of Caf in Cof or in water. Nine healthy, fit, young adults performed five trials after ingesting (double blind) either a capsule (Caf or placebo) with water or Cof (decaffeinated Cof, decaffeinated with Caf added, or regular Cof). In all three Caf trials, the Caf dose was 4.45 mg/kg body wt and the volume of liquid was 7.15 ml/kg. After 1 h of rest, the subject ran at 85% of maximal O2 consumption until voluntary exhaustion (approximately 32 min in the placebo and decaffeinated Cof tests). In the three Caf trials, the plasma Caf and paraxanthine concentrations were very similar. After 1 h of rest, the plasma Epi was increased (P < 0.05) by Caf ingestion, but the increase was greater (P < 0.05) with Caf capsules than with Cof. During the exercise there were no differences in Epi among the three Caf trials, and the Epi values were all greater (P < 0.05) than in the other tests. Endurance was only increased (P < 0. 05) in the Caf capsule trial; there were no differences among the other four tests. One cannot extrapolate the effects of Caf to Cof; there must be a component(s) of Cof that moderates the actions of Caf.     

Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.     

Dietary sodium restriction impairs insulin sensitivity in noninsulin-dependent diabetes mellitus

Dietary sodium restriction has a variety of effects on metabolism, including activation of the renin-angiotensin system. Angiotensin II has complex metabolic and cardiovascular effects, and these may be relevant to the effects of both nonpharmacological and pharmacological interventions in noninsulin-dependent diabetes mellitus (NIDDM). We have assessed the effect of dietary sodium restriction on insulin sensitivity and endogenous glucose production in eight normotensive patients with diet-controlled NIDDM who underwent hyperinsulinemic clamp studies in a randomized, double-blind, placebo-controlled cross-over protocol after two 4-day periods on sodium replete (160 mmol/day) and sodium deplete (40 mmol/day) diets. Mean +/- SD 24-h urinary sodium was 197 +/- 76.0 mmol (replete) and 67 +/- 19.5 mmol (deplete), P = 0.03. Insulin sensitivity was 42.0 +/- 11.3 mumol/kg.min (replete) and 37.0 +/- 11.6 mumol/kg.min (deplete), P = 0.04 (a reduction of 12%). Blood pressure was 130 +/- 21/78 +/- 11 mmHg (replete) and 128 +/- 12/73 +/- 10 mmHg (deplete). Dietary sodium restriction may result in a decrease in peripheral insulin sensitivity in normotensive patients with NIDDM, possibly via an elevation in prevailing angiotensin II concentrations.     

Dissecting the order of bacteriophage T4 DNA polymerase holoenzyme assembly

Most biological organisms rely upon a DNA polymerase holoenzyme for processive DNA replication. The bacteriophage T4 DNA polymerase holoenzyme is composed of the polymerase enzyme and a clamp protein (the 45 protein), which functions as a processivity factor by strengthening the interaction between DNA and the holoenzyme. The 45 protein must be loaded onto DNA by a clamp loader ATPase complex (the 44/62 complex). In this paper, the order of events leading to holoenzyme formation is investigated using a combination of rapid-quench and stopped-flow fluorescence spectroscopy kinetic methods. A rapid-quench strand displacement assay in which the order of holoenzyme component addition is varied provided data indicating that the rate-limiting step in holoenzyme assembly is associated with the clamp loading process. Pre-steady-state analysis of the clamp loader ATPase activity demonstrated that the four bound ATP molecules are hydrolyzed stepwise during the clamp loading process in groups of two. Clamp loading was examined with stopped-flow fluorescence spectroscopy from the perspective of the clamp itself, using a site-specific, fluorescently labeled 45 protein. A mechanism for T4 DNA polymerase holoenzyme assembly is proposed in which the 45 protein interacts with the 44/62 complex leading to the hydrolysis of 2 equiv of ATP, and upon contacting DNA, the remaining two ATP molecules bound to the 44/62 complex are hydrolyzed. Once all four ATP molecules are hydrolyzed, the 45 protein is poised on DNA for association with the polymerase to form the holoenzyme.     

Contribution to the problem of tensile and bending-test method for SiC-fibre-reinforced glass

The Young's moduli of SiC-fibre-reinforced DURAN glass composites with different fibre concentrations obtained from tensile and three-point-bending tests were shown to be in good agreement, while the values of strength and bendover stress and strain were larger in the bending test than in the tensile test. It is assumed that this difference is mainly a consequence of the larger effectively stressed sample volume in the tensile-stress test, compared to the bending test, and it is a consequence of the shift of the neutral line in the bending test towards the compressive-stress region during the fracture process. The second consequence is the reason why the samples behave in a much more ductile way in the bending test and are brittle in the tensile test. The advantages and disadvantages of the two methods are discussed and demonstrated with special regard to the composites mentioned.     

Towards the cloning of genes underlying murine QTLs

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of bis azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to bis azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.     

Amphetamine-induced behavior, dopamine release, and c-fos mRNA expression: modulation by environmental novelty

We have shown recently that the psychomotor activating effects of amphetamine in the rat are much greater when this drug is administered in association with environmental novelty than when it is given in a home environment. The main purpose of the present study was to explore the neural basis of this phenomenon. We found, using in situ hybridization of c-fos mRNA, that the pattern of neuronal activation in the cortex, in the caudate, in the shell and core of the nucleus accumbens, and in other subcortical structures was markedly different when amphetamine (2.0 mg/kg, i.p.) was given in association with exposure to environmental novelty relative to when it was given at home. In most brain regions the magnitude of c-fos expression was over two times greater in rats given amphetamine plus novelty than in rats given amphetamine alone. In contrast, an in vivo microdialysis study indicated that environmental novelty did not affect amphetamine-induced dopamine release in either caudate or nucleus accumbens. Furthermore, a unilateral 6-hydroxydopamine lesion of the mesostriatal dopamine system reduced amphetamine- but not novelty-induced c-fos expression. Finally, we found no differences in the amount of corticosterone secreted after exposure to novelty, amphetamine, or both, suggesting that corticosterone does not play a critical role in the ability of novelty to modulate amphetamine-induced psychomotor activation. In conclusion, it seems that environmental novelty alters the neurobiological effects of amphetamine independently of the primary neuropharmacological actions of this drug in the striatum.