Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.     

Low-flow vascular remodeling in the metabolic syndrome X

Peripheral microvascular dysfunction is a common affliction in patients with the metabolic syndrome X. Previous studies have described a number of vascular impairments in vasomotor control in both human patients and animal models of syndrome X, but the net effect of these impairments on microvascular structure has not been examined. The goal of the current study was to test the hypothesis that syndrome X reduces muscle perfusion and induces vascular remodeling. The obese Zucker rat was used as a model of syndrome X, and the microcirculation of the hindlimb and brain were examined. Obese Zucker rats were obese, hyperlipidemic, hyperinsulinemic, and hyperglycemic. Blood flow to the hindlimb was reduced by 59% in obese rats relative to lean rats. Skeletal muscle resistance arteries of the hindlimb microcirculation of obese rats had thinner walls, smaller lumens, and reduced distensibility. Hindlimb microvessels from obese rats also demonstrated reduced expression of vascular smooth muscle cell markers. Each of these traits is consistent with low-flow remodeling. In contrast, the cerebral microcirculation, where flow is vigorously autoregulated, showed no vascular remodeling nor were there changes in microvascular smooth muscle marker expression. Neither physical activity nor muscle mass were significantly different between lean and obese rats. Taken together, these findings suggest that syndrome X, by reducing hindlimb blood flow, induces a marked remodeling of microcirculation to favor smaller, less distensible vessels. This remodeling may result in an architectural limitation of maximum perfusion capacity and may be an important maladaption in the progression of peripheral microvascular disease.     

Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo

We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng. kg(-1). min(-1) ic) caused dose-dependent vasodilation similarly before and after administration of N(G)-monomethyl-L-arginine (L-NMMA) (3 micromol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK with L-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade with L-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with L-NMMA and Indo, sodium nitroprusside (SNP, 1-3 microgram. kg(-1). min(-1) ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP during L-NMMA and Indo (P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence of L-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.     

Adenosine preconditions against endothelin-induced constriction of coronary arterioles

Myocardial hypoperfusion is accompanied by concomitant increases in adenosine and endothelin-1 (ET-1) production, but the vasodilatory effect of adenosine prevails over that of ET-1. Therefore, we hypothesized that adenosine-induced or ischemic preconditioning reduces the vasoconstrictive effect of ET-1. Coronary arteriolar diameter in vivo was measured using fluorescence microangiography in anesthetized open-thorax dogs. ET-1 (5 ng. kg(-1). min(-1) administered intracoronary, n = 10) induced progressive constriction over 45 min [25 +/- 6% (SE)]. The constriction was blocked by preconditioning with adenosine (25 microgram. kg(-1). min(-1) administered intracoronary) for 20 min and 10 min of washout (n = 10) or attenuated by ischemic preconditioning (four 5-min periods of ischemia, 9 +/- 5% at 45 min). To investigate the receptor involved in this process, coronary arterioles (50-150 micrometer) were isolated and pressurized at 60 mmHg in vitro. The ET-1 dose-response curve (1 pM-5 nM) was rightward shifted after preconditioning with adenosine (1 microM) for 20 min and 10 min of washout (n = 11). Blockade of A(2) receptors [8-(3-chlorostyryl)caffeine, 1 microM, n = 9] but not A(1) receptors (8-cyclopentyl-1,3-dipropylxanthine, 100 nM, n = 7) prevented this shift. These results suggest that adenosine confers a vascular preconditioning effect, mediated via the A(2) receptor, against endothelin-induced constriction. This effect may offer a new protective function of adenosine in preventing excessive coronary constriction.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Synaptic Plasticity Enables Adaptive Self-Tuning Critical Networks

During rest, the mammalian cortex displays spontaneous neural activity. Spiking of single neurons during rest has been described as irregular and asynchronous. In contrast, recent in vivo and in vitro population measures of spontaneous activity, using the LFP, EEG, MEG or fMRI suggest that the default state of the cortex is critical, manifested by spontaneous, scale-invariant, cascades of activity known as neuronal avalanches. Criticality keeps a network poised for optimal information processing, but this view seems to be difficult to reconcile with apparently irregular single neuron spiking. Here, we simulate a 10,000 neuron, deterministic, plastic network of spiking neurons. We show that a combination of short- and long-term synaptic plasticity enables these networks to exhibit criticality in the face of intrinsic, i.e. self-sustained, asynchronous spiking. Brief external perturbations lead to adaptive, long-term modification of intrinsic network connectivity through long-term excitatory plasticity, whereas long-term inhibitory plasticity enables rapid self-tuning of the network back to a critical state. The critical state is characterized by a branching parameter oscillating around unity, a critical exponent close to -3/2 and a long tail distribution of a self-similarity parameter between 0.5 and 1.     

A high-potassium diet reduces infarct size and improves vascular structure in hypertensive rats

High-potassium diets can improve vascular function, yet the effects of potassium supplementation on ischemic stroke have not been studied. We hypothesized that dietary potassium supplementation would reduce ischemic cerebral infarct size by reversing cerebral artery hypertrophy. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were fed diets containing 0.79% potassium (LK) or 2.11% potassium (HK) for 6 wk; Wistar-Kyoto (WKY) rats were fed the LK diet. The HK diet did not reduce blood pressure, as measured by telemetry, in the SHRSP. Cerebral ischemia was induced by middle cerebral artery (MCA) occlusion. The resultant infarct was smaller in the HK-SHRSP than in the LK-SHRSP: 55.1 +/- 6.3 vs. 71.4 +/- 2.4% of the hemisphere infarcted (P < 0.05). Infarcts were smaller in WKY rats (33.5 +/- 4.8%) than in LK-SHRSP or HK-SHRSP. The vessel wall of MCAs from LK-SHRSP was hypertrophied compared with WKY rats; this was reversed in HK-SHRSP. RT-PCR analysis of the cerebral vessels showed that expression of platelet-derived growth factor receptors-alpha and -beta, epidermal growth factor receptor, and collagen I and III was increased in the vessels from LK-SHRSP compared with WKY rats and reduced in HK-SHRSP. These results suggest that potassium supplementation provides neuroprotection in a model of ischemic stroke independent of blood pressure and possibly through changes in vascular structure.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.