NMR structure of C-terminally tagged gramicidin channels

A biotin group was covalently attached to the C terminus of gramicidin A (gA) through a linker arm comprising a glycine residue with either one (gAXB) or two caproyl groups (gAXXB). High-resolution two-dimensional NMR spectroscopy was used to determine the structure of these modified gA analogues and [Lys(16)]gramicidin A (gA-Lys) in sodium dodecyl-d(25) sulphate micelles. Gated gA ion channels based on linking a receptor group to these gA analogues have been used recently as a component in a sensing device. The conformations of the gA backbones and amino acid side chains of lysinated gA and biotinylated gA in detergent micelles were found to be almost identical to that of native gA, i.e. that of an N-terminal to N-terminal (head to head) dimer formed by two right-handed, single-stranded beta(6.3) helices. The biotin tail of the gAXB and gAXXB and the lysine extremity of gA-Lys appeared to lie outside the micelle. Thus it appears that the covalent attachment of functional groups to the C terminus of gA does not disrupt the peptide's helical configuration. Further, single channel measurements of all three gA analogues showed that functioning ion channels were preserved within a membrane environment. (C) 1999 Elsevier Science B.V. All rights reserved.

Algebraic Bethe ansatz for integrable Kondo impurities in the one-dimensional supersymmetric t-J model

An integrable Kondo problem in the one-dimensional supersymmetric t-J model is studied by means of the boundary supersymmetric quantum inverse scattering method. The boundary K matrices depending on the local moments of the impurities are presented as a nontrivial realization of the graded reflection equation algebras in a two-dimensional impurity Hilbert space. Further, the model is solved by using the algebraic Bethe ansatz method and the Bethe ansatz equations are obtained. (C) 1999 Elsevier Science B.V.

Optics of the developing fish eye: comparisons of Matthiessen's ratio and the focal length of the lens in the black bream Acanthopagrus butcheri (Sparidae, Teleostei)

Matthiessen's ratio (distance from centre of lens to retina: lens radius) was measured in developing black bream, Acanthopagrus butcheri (Sparidae, Teleostei). The value decreased over the first 10 days post-hatch from 3.6 to 2.3 along the nasal and from four to 2.6 along temporal axis. Coincidentally, there was a decrease in the focal ratio of the lens (focal length:lens radius). Morphologically, the accommodatory retractor lentis muscle appeared to become functional between 10-12 days post-hatch. The results suggest that a higher focal ratio compensates for the relatively high Matthiessen's ratio brought about by constraints of small eye size during early development. Combined with differences in axial length, this provides a means for larval fish to focus images from different distances prior to the ability to accommodate. (C) 1999 Elsevier Science Ltd. All rights reserved.

The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain

Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles, Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88, Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE(1-22) indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations, This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.

Molecular characterization of Drosophila C virus isolates

Reverse transcription coupled with polymerase chain reaction and restriction enzyme analysis was used to characterize 12 Drosophila C virus isolates from geographically different regions. A 1.2-kb fragment was amplified from cDNA and profiles from digestion with 20 restriction enzymes were generated. Analysis of the restriction fragment data gave estimates of nucleotide divergence of 0-10% between isolates. The isolates were grouped on the basis of genetic distance estimates derived from the restriction data. For the isolates from which a single genotype could be purified, a geographical pattern in the distribution of viral genotypes was identified. The 4 Moroccan isolates were very closely related to each other, differing in only 1 restriction profile. The 2 Australian isolates were each other's closest relatives, as were the 2 isolates first recovered in France. The PCR-RFLP technique used in this study has provided us with a simple procedure which can be used to characterize DCV isolates. A single enzyme, Tag I, generated 5 distinct and diagnostic restriction fragment patterns, which allowed easy assignment of isolates to one of the five viral genotypes identified in this study. (C) 1999 Academic Press.

Echinosulfonic acids A-C and echinosulfone A: Novel bromoindole sulfonic acids and a sulfone from a southern Australian marine sponge, Echinodictyum

The crude EtOH extract of an Echinodictyum sp. collected during trawling operations in the Great Australian Eight, Australia, displayed antibacterial and antiparasitic properties. Bioassay-directed fractionation yielded three novel sulfonic acids, the echinosulfonic acids A to C (1-3), and a new sulfone, echinosulfone A (4). Structures were assigned to these compounds on the basis of detailed spectroscopic analysis. It was determined that echinosulfonic acids A-C (1-3) and echinosulfone A(4) contributed to the antibacterial but not antiparasitic activity of the crude extract.

Malevolence, stigma, and social distance: Maximizing intergroup differences in HIV/AIDS discourse

This study examined the intergroup language used by young heterosexual Australians in conversations about HIV/AIDS and safe sex. Sixty male and 72 female heterosexuals participated in four-person facilitated conversations (same-sex or mixed-sex) about HIV/AIDS and safe sex, which were recorded and transcribed. We focused on extracts concerning strangers or malevolent individuals who appear to be group members, along with extracts involving foreign national groups. Discourse analysis showed that groups at lower levels of social distance were constructed mainly in terms of individual responsibility. At moderate social distance, stereotypes were more negative, but sub-typing was common, whereas at the highest levels, people were constructed entirely in intergroup terms. The findings of this study suggest that HN prevention programs should make reference to all salient outgroups, so as to neutralize communicative strategies that strengthen intergroup boundaries as a means of reducing perceived personal threat of HIV infection.

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

Cell death and immunohistochemistry of p53, c-Fos and c-Jun after spermine infection into the rat striatum

Administration of polyamines into the central nervous system results in tissue damage, possibly through the excitotoxic actions of the NMDA receptor. Direct injection of 100 nmol of spermine into the rat striatum produced a lesion equivalent to approximately 50% of the striatum. Analysis of the DNA in this region revealed the distinct ladder-like pattern of degradation often associated with apoptosis. This DNA fragmentation was confirmed in vivo using terminal deoxynucleotidyl-transferase-mediated biotinylated deoxyuridine triphosphate nick end labelling (TUNEL). The morphology of the TUNEL-positive cells showed marked differences at the needle tract when compared with cells in damaged areas away from the needle tract, suggesting a differential mechanism of cell death in these two regions. The patterns of p53, c-Fos and c-Jun protein expression were determined using immunohistochemistry. The number of p53-immunoreactive cells increased up to 14 h and returned to basal levels by 24 h. c-Fos protein expression transiently increased, peaking at 8 h after injection, c-Jun exhibited a protracted pattern of expression, remaining elevated up to 24 h. p53 protein expression was colocalised with TUNEL staining in areas away from the needle tract, but not in cells at the needle tract, suggesting once again a differential mechanism of cell death. At 14 h, c-Fos and c-Jun were not colocalised with TUNEL staining, suggesting that they are either not involved with the cell death process or that the time course of protein expression and the onset of DNA fragmentation do not overlap. This work represents the first characterisation of processes associated with cell death induced by spermine in vivo.

Clathrins A-C: Metabolites from a southern Australian marine sponge, Clathria species

A Clathria sp. collected in the Great Australian Bight has yielded the novel metabolites clathrins A (6), B (7), and C (8). Structures were assigned to clathrins A-C on the basis of spectroscopic analysis. Clathrin A (6) represents a plausible biosynthetic intermediate that provides an inferred link between marine sesquiterpene/benzenoids and mixed terpene/shikimate biosynthesis.

The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-alpha

It is now well recognized that cervical cancer is caused by infection with certain human papillomavirus (HPV) subtypes and while interferon-alpha (IFN-alpha) is used to treat HPV-infected lesions, HPV appears to have developed a means to avoid the effects of IFN-alpha. Clinically, resistance appears to be associated with the expression of the E7 oncoprotein. Here we investigated the effects of expression in cells of the E7 protein from high- and low-risk papillomavirus subtypes on a range of responses to IFN-alpha. 2fTGH, a cell line dependent on IFN-alpha for growth in selection medium, grew significantly less well in the presence of E7, and the antiproliferative effects of IFN-alpha upon epithelial cells was lost upon E7 expression. The antiviral effects of IFN-alpha were abrogated in E7-expressing cells. Loss of response to IFN-alpha was found to occur in both high- and low-risk papillomaviruses. Finally, deletion of amino acids 21-24 of HPV type 16 E7 protein partially reversed repression. We conclude that E7 inhibits the functional effects of IFN-alpha and that this property is shared by all HPV subtypes tested. (C) 2000 Academic Press.