Acid Denaturation of alpha1-antitrypsin: characterization of a novel mechanism of serpin polymerization

The native serpin architecture is extremely sensitive to mutation and environmental factors. These factors induce the formation of a partially folded species that results in the production of inactive loop-sheet polymers. The deposition of these aggregates in tissue, results in diseases such as liver cirrhosis, thrombosis, angioedema and dementia. In this study, we characterize the kinetics and conformational changes of alpha(1)-antitrypsin polymerization at pH 4 using tryptophan fluorescence, circular dichroism, turbidity changes and thioflavin T binding. These biophysical techniques have demonstrated that polymerization begins with a reversible conformational change that results in partial loss of secondary structure and distortion at the top of beta-sheet A. This is followed by two bimolecular processes. First, protodimers are formed, which can be dissociated by changing the pH back to 8. Then, an irreversible conformational change occurs, resulting in the stabilization of the dimers with a concomitant increase in beta-sheet structure, allowing for subsequent polymer extension. Electron microscopy analysis of the polymers, coupled with the far-UV CD and thioflavin T properties of the pH 4 polymers suggest they do not form via the classical loop-beta-sheet A linkage. However, they more closely resemble those formed by the pathological variant M(malton). Taken together, these data describe a novel kinetic mechanism of serine proteinase inhibitor polymerization.     

A Spiking Neuron Model for Binocular Rivalry

We present a biologically plausible model of binocular rivalry consisting of a network of Hodgkin-Huxley type neurons. Our model accounts for the experimentally and psychophysically observed phenomena: (1) it reproduces the distribution of dominance durations seen in both humans and primates, (2) it exhibits a lack of correlation between lengths of successive dominance durations, (3) variation of stimulus strength to one eye influences only the mean dominance duration of the contralateral eye, not the mean dominance duration of the ipsilateral eye, (4) increasing both stimuli strengths in parallel decreases the mean dominance durations. We have also derived a reduced population rate model from our spiking model from which explicit expressions for the dependence of the dominance durations on input strengths are analytically calculated. We also use this reduced model to derive an expression for the distribution of dominance durations seen within an individual.     

Voltage-dependent calcium channels in the corpora allata of the adult male loreyi leafworm, Mythimna loreyi

In the corpora allata (CA) of the adult male loreyi leafworm, Mythimna loreyi, juvenile hormone acid (JHA) biosynthesis and release show a dose dependence on extracellular Ca(2+) concentration. Maxima are obtained with Ca(2+) concentrations of 2-10 mM, and synthesis and release are significantly inhibited under a Ca(2+)-free condition. The Ca(2+)-free inhibition of JHA release can be reversed by returning the glands to medium at 5 mM Ca(2+). The cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which was measured with fura-2, in individual CA cells also shows a dose dependence on extracellular Ca(2+) concentration, with significant [Ca(2+)](i) depression being observed in the absence of extracellular Ca(2+).High K(+) significantly increases the JHA release and causes a transient [Ca(2+)](i) increase within seconds in CA cells. High-K(+)-stimulated JHA release is partially inhibited by the benzothiazepine (BTZ)-, dihydropyridine (DHP)- and phenylalkylamine (PAA)-sensitive L-type voltage-dependent calcium channel (VDCC) antagonists diltiazem, nifedipine and verapamil, respectively; by the N- and P/Q-type VDCC antagonist omega-conotoxin (omega-CgTx) MVIIC; and by the T-type VDCC antagonist amiloride. The N-type antagonist omega-CgTx GVIA is the most potent in inhibiting the high-K(+)-stimulated JHA release. No inhibitory effect is shown by the P-type antagonist omega-agatoxin TK (omega-Aga TK). The high-K(+)-induced transient [Ca(2+)](i) increase is largely inhibited by the L-type antagonists (diltiazem, nifedipine, verapamil), by the N- and P/Q-type antagonist omega-CgTx MVIIC and by the T-type antagonist amiloride, and is totally inhibited by the N-type antagonist omega-CgTx GVIA. No inhibitory effect is shown by the P-type antagonist omega-Aga TK.We hypothesize that L-type, N-type and T-type VDCCs may be involved to different degrees in the high-K(+)-stimulated JHA release and transient [Ca(2+)](i) increase in the individual CA cells of the adult male M. loreyi, and that the N-type VDCCs may play important roles in these cellular events.     

Exposure to oxidized low-density lipoprotein reduces activable Ras protein in vascular endothelial cells

Oxidized low-density lipoprotein (ox-LDL) has been shown to alter the migratory and proliferative activities of the vascular endothelial cells (EC) in response to serum and growth factors. The mechanism underlying the antiproliferative effect of ox-LDL on vascular EC has not been fully elucidated. In this report, we show that exposure of vascular EC to ox-LDL results in a marked reduction of the membrane-associated Ras protein. Further study shows that in ox-LDL-treated EC, reduction of the membrane-associated Ras protein is correlated with a reduced amount of active Ras (Ras-guanosine triphosphate), indicating that the Ras signaling pathway is attenuated. The attenuation of the Ras signaling pathway in ox-LDL-treated EC may thus be responsible for the retarded response to the mitogenic stimulation of serum and growth factors.     

Gastric H-K-ATPase and acid-resistant surface proteins

Despite the fact that mucus and bicarbonate are important macroscopic components of the gastric mucosal barrier, severe acidic and peptic conditions surely exist at the apical membrane of gastric glandular cells, and these membranes must have highly specialized adaptations to oppose external insults. Parietal cells abundantly express the heterodimeric, acid-pumping H-K-ATPase in their apical membranes. Its beta-subunit (HKbeta), a glycoprotein with >70% of its mass and all its oligosaccharides on the extracellular side, may play a protective role. Here, we show that the extracellular domain of HKbeta is highly resistant to trypsin in the native state (much more than that of the structurally related Na-K-ATPase beta-subunit) and requires denaturation to expose tryptic sites. Native HKbeta also resists other proteases, such as chymotrypsin and V8 protease, which hydrolyze at hydrophobic and anionic amino acids, respectively. Removal of terminal alpha-anomeric-linked galactose does not appreciably alter tryptic sensitivity of HKbeta. However, full deglycosylation makes HKbeta much more susceptible to all proteases tested, including pepsin at pH <2.0. We propose that 1) intrinsic folding of HKbeta, 2) bonding forces between subunits, and 3) oligosaccharides on HKbeta provide a luminal protein domain that resists gastric lytic conditions. Protein folding that protects susceptible charged amino acids and is maintained by disulfide bonding and hydrophilic oligosaccharides would provide a stable structure in the face of large pH changes. The H-K-ATPase is an obvious model, but other gastric luminally exposed proteins are likely to possess analogous protective specializations.     

Photoinduced cleavage by a rhodium complex at G.U mismatches and exposed guanines in large and small RNAs

Photoinduced cleavage reactions by the rhodium complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) [Rh(DIP)(3)(3+)] with three RNA hairpins, r(GGGGU UCGCUC CACCA) (16 nucleotide, tetraloop(Ala2)), r(GGGGCUAUAGCUCUAGCUC CACCA) (24 nucleotide, microhelix(Ala)), and r(GGCGGUUAGAUAUCGCC) (17 nucleotide, 790 loop), and full-length (1542 nucleotide) 16S rRNA from Escherichia coli were investigated. The cleavage reactions were monitored by gel electrophoresis and the sites of cleavage by Rh(DIP)(3)(3+) were determined by comparisons with chemical or enzymatic sequencing reactions. In general, RNA backbone scission by the metal complex was induced at G.U mismatches and at exposed G residues. The cleavage activity was observed on the three small RNA hairpins as well as on the isolated 1542-nucleotide ribosomal RNA.     

The tree shrews: adjuncts and alternatives to primates as models for biomedical research

The tree shrews are non-rodent, primate-like, small animals. There is increasing interest in using them to establish animal models for medical and biological research. This review focuses on the use of the tree shrews in in vivo studies on viral hepatitis, hepatocellular carcinoma (HCC), myopia, and psychosocial stress. Because of the susceptibility of the tree shrews (Tupaia belangeri) and their hepatocytes to infection with human hepatitis B virus (HBV) in vivo and in vitro, these animals have been used to establish human hepatitis virus-induced hepatitis and human HBV- and aflatoxin B1-associated HCC models. As these animals are phylogenetically close to primates in evolution and have a well-developed visual system and color vision in some species, they have been utilized to establish myopia models. Because dramatic behavioral, physiological, and neuroendocrine changes in subordinate male tree shrews are similar to those observed in depressed human patients, the tree shrews have been successfully employed to experimentally study psychosocial stress. However, the tree shrews holds significant promise as research models and great use could be made of these animals in biomedical research.