Failure of renal denervation to attenuate hypertension in Dahl NaCl-sensitive rats

In previous experiments we have demonstrated that the renal nerves play a significant role in all genetic and (or) induced models of hypertension that we have studied. The current experiments extended this research by investigating the contribution of the renal nerves to hypertension in the Dahl NaCl-sensitive rat. This was investigated by assessing the effect of bilateral phenol renal denervation carried out prior to initiation of a high NaCl (8% NaCl) diet. In two separate studies, renal denervation did not affect systolic blood pressure in either Dahl NaCl-sensitive rats or their normotensive counterparts, Dahl NaCl-resistant rats. Further, denervation did not increase absolute urinary sodium excretion, percent urinary sodium excretion, urinary volume output, or food or water intake; nor did it differentially alter creatinine clearance or body weight. Denervation was verified at the termination of each study by a greater than 80% depletion of renal noradrenaline stores. These results indicate that the renal nerves do not provide a major contribution to hypertension in the Dahl NaCl-sensitive rat.     

Characterization of autocatalytic conversion of precursor BACE1 by heteronuclear NMR spectroscopy

Accumulation of the cytotoxic 40- to 42-residue beta-amyloid peptide represents the primary pathological process in Alzheimer's disease (AD). BACE1 (beta-site APP cleaving enzyme 1) is responsible for the initial required step in the neuronal amyloidogenic processing of beta-amyloid precursor protein and is a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initially synthesized as an immature precursor protein containing part of the pre domain and the entire pro domain, and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme. To understand the mechanism of the conversion and the role of the pro domain, we monitored the autocatalytic conversion of BACE1 by heteronuclear NMR spectroscopy and used chemical shift perturbations as a probe to study the structural changes accompanying the autocatalytic conversion. NMR data revealed local conformational changes from a partially disordered to a well-folded conformation associated with the conversion. The conformational changes are largely concentrated in the NH(2)-terminal lobe. Conversely, the active site conformations are conserved during the autocatalytic conversion. The precursor and mature BACE1 proteins were further characterized for their ability to interact with a substrate-based transition state BACE1 peptide inhibitor. The precursor BACE1 rapidly adopted the bound conformation in the presence of the inhibitor, which is identical to the bound conformation of the mature protein. The interaction of the inhibitor with both the precursor BACE1 and the fully processed BACE1 is in slow exchange on the NMR time scale, indicating a tight binding interaction. Overall, the NMR data demonstrated that the pro domain does not hinder inhibitor binding and may assist in the proper folding of the protein. The fully processed BACE1 represents a high quality well-folded protein which is highly stable over a long period of time, and is suitable for evaluation of inhibitor binding by NMR for drug intervention.     

Regulation of human immunodeficiency virus type 1 envelope glycoprotein fusion by a membrane-interactive domain in the gp41 cytoplasmic tail

Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this "inside-out" regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.     

Comparative gene finding in chicken indicates that we are closing in on the set of multi-exonic widely expressed human genes

The recent availability of the chicken genome sequence poses the question of whether there are human protein-coding genes conserved in chicken that are currently not included in the human gene catalog. Here, we show, using comparative gene finding followed by experimental verification of exon pairs by RT–PCR, that the addition to the multi-exonic subset of this catalog could be as little as 0.2%, suggesting that we may be closing in on the human gene set. Our protocol, however, has two shortcomings: (i) the bioinformatic screening of the predicted genes, applied to filter out false positives, cannot handle intronless genes; and (ii) the experimental verification could fail to identify expression at a specific developmental time. This highlights the importance of developing methods that could provide a reliable estimate of the number of these two types of genes.     

Antihypertensive and cognitive effects of grape polyphenols in estrogen-depleted, female, spontaneously hypertensive rats

Both endogenous and dietary estrogens reduce hypertension and enhance cognitive abilities in estrogen-depleted female spontaneously hypertensive rats (SHR). Many of the beneficial effects of estrogens/phytoestrogens also appear to be provided by other polyphenols (e.g., proanthocyanidins) in grape seed, which lack appreciable estrogenic receptor binding. The present study tested the hypothesis that similar to phytoestrogens, proanthrocyanidins in grape seed polyphenols reduce salt-sensitive hypertension in young, estrogen-depleted SHR. SHR were ovariectomized at 4 wk of age and placed on phytoestrogen-free diets with or without 0.5% grape seed extract added and with high (8.0%) or basal (0.6%) NaCl. After 10 wk on the diets, grape proanthrocyanidin supplementation significantly reduced arterial pressure in the rats fed the basal (10 mmHg) and high (26 mmHg)-NaCl diet, compared with the nonsupplemented controls. In vitro superoxide production was significantly reduced (23%) by the grape seed polyphenols. Spatial learning (8-arm-radial maze) in the SHR on the basal NaCl diets was improved by dietary grape seed polyphenols. These results indicate that grape seed polyphenols decrease arterial pressure in SHR, probably via an antioxidant mechanism.     

Effect of ion concentration on the rupture and survival of Azotobacter cysts

The ability of ethylenediaminetetraacetic acid (EDTA) and dipicolinic acid (DPA) to rupture Azotobacter cysts was directly related to the molarity of the reaction mixture. Cysts were not ruptured at concentrations above 0.15 m (NaCl or phosphate). These higher salt concentrations prevented the formation of metallo:EDTA (or DPA) chelate complexes. Sudden shifts in the tonicity of NaCl or phosphate mixtures induced cyst rupture in the absence of EDTA. The effect of these mechanisms on the survival and germination of Azotobacter cysts in the soil is discussed.     

Endogenous Encystment of Azotobacter vinelandii

When young cells of Azotobacter vinelandii are impinged on membrane filters, washed free of carbon substrate, and placed on a mineral salts basal medium, the culture will proceed to encyst although at a slower rate than if n-butanol were supplied as a substrate. The endogenous cysts are depleted in polyβ-hydroxybutyrate and have a narrower intine but show an increased resistance to desiccation and are susceptible to lysis by chelating agents. Membrane-supported cells reveal details of the encystment process such as the formation of a zone within the capsule prior to exine formation and the early deposition of exine structures.     

FLIPPERS AND PINNIPED PHYLOGENY: HAS THE PROBLEM OF CONVERGENCE BEEN OVERRATED?

The phylogenetic relationships of the major groups of pinnipeds are of longstanding controversy. According to prevailing paleontological opinion, pinnipeds comprise two distinct lineages of independent terrestrial carnivoran origin, A logical correlate of this view is that an enormous degree of convergence accounts for the striking resemblances of these groups.
It has been generally supposed that derived similarities common to pinnipeds are independently acquired adaptations to a highly constraining environment. This has not been adequately substantiated. Moreover, considerable evidence favors a contrary conclusion. Acceptance of convergence necessarily rests on demonstration that taxa possessing characters suspected to be convergent are in fact not mutal nearest allies. In the absence of such phylogenetic information, shared derived characters are indicative of common ancestry irrespective of whether they are deemed "functionally important." Given meager support for the proposition that pinnipeds are related to different terrestrial lineages, it is currently more judicious to regard the group's origin as monophyletic. In addition to the presumed prevalence of convergence, biogeography, the high degree of morphological divergence of phocids, and the aberrant nature of phocines have improperly contributed to acceptance of independent pinniped origins.     

delta-Aminolevulinic acid dehydratase: effects of succinylacetone in rat liver and kidney in an in vivo model of the renal Fanconi syndrome.

Succinylacetone (SA) is a known inhibitor of the heme biosynthetic pathway in liver. We have demonstrated previously the SA enhancement of delta-aminolevulinic acid dehydratase (ALAD) in renal tubules, while this enzyme is known to be impaired by SA in the liver. The present studies, based on in vivo treatment of animals with SA, show equivalent degree of inhibition of specific ALAD activity in liver and kidney. Both tissues evidenced an ability to restore enzyme activity with time, the recovery occurring much more slowly in kidney than in liver. The discrepant in vitro and in vivo effect of SA on renal ALAD may be due to differences between a direct inhibitor-enzyme interaction and inhibitor actions in the living cell, respectively. Persistent tissue levels of SA, consistent with demonstrated SA in plasma and urine, might account for continuing inhibition, with the greatest tissue accumulation in kidney where the substance must be cleared for excretion.     

A high accuracy linear rate meter.

By implementing analog computer techniques using digital circuits, an instantaneous rate meter was built with approximately 100 times the accuracy of commercially available rate meters. The circuit is accurate to within +/- 0.2 events/min over a range of rates of 0.2-900 epm. Modifications can be made to provide a digital display of rate. The circuit design techniques used in developing the rate meter may be used to generate a wide variety of functions of time with very high accuracy.