Biomedical subjects

A S Weiss

Publications and source records attributed to A S Weiss.

At least 37 records · Page 2Linked to original sources

PubMed1998

Action of tropoelastin and synthetic elastin sequences on vascular tone and on free Ca2+ level in human vascular endothelial cells.

The elastic properties of extensible tissues such as arteries and skin are mainly due to the presence of elastic fibers whose major component is the extracellular matrix protein elastin. Pathophysiological degradation of this protein leads to the generation of elastin peptides that have been identified in the circulation in the ng/mL to microg/mL range. Similar concentrations of an elastin peptide preparation (kappa-elastin) were previously demonstrated to induce, among other biological actions, a dose- and endothelium-dependent vasorelaxation mediated by the elastin/laminin receptor and by endothelial NO production. To determine the elastin sequence(s) responsible for vasomotor activity and to learn more about possible signaling pathways, we have compared the action of different concentrations (10(-13) to 10(-7) mol/L) of recombinant human tropoelastin, eight synthetic elastin peptides, and a control peptide (VPVGGA) on both rat aortic ring tension and [Ca2+]i of cultured human umbilical vein endothelial cells. No vasoactivity could be detected for VPVGGA and for the elastin-related sequences VGVGVA, PGVGVA, and GVGVA. Tropoelastin, VGV, PGV, and VGVAPG were found to induce an endothelium- and dose-dependent vasorelaxation and to increase endothelial [Ca2+]i, whereas PVGV and VGVA produced these effects only at low concentration (10(-11) mol/L). A likely candidate for mediating the elastin peptide-related effects is the elastin/laminin receptor, since the presence of lactose strongly inhibited the vasoactivity associated with these compounds. Our results show that although the flanking amino acids modulate its activity, VGV seems to be the core sequence recognized by the elastin receptor.

Animals↗

PubMed1997

The nicotinic acetylcholine alpha-subunit gene tar-1 is located on the X chromosome but its coding sequence is not involved in levamisole resistance in an isolate of Trichostrongylus colubriformis.

The polymerase chain reaction was used to amplify fragments comprising the known reading frame of the nematode nicotinic acetylcholine alpha-subunit gene tar-1. Sequences were derived from DNA prepared from bulk collections of worms and from individual male and female Trichostrongylus colubriformis. In each case a levamisole-resistant (BCk) and a drug susceptible population were examined. Although several nucleotide transitions were detected no amino acid sequence variations were found between the isolates and between individual worms, indicating that the coding sequence of this gene is not responsible for levamisole-resistance in the isolate tested. However, an intronic allelic T/C variation at position 4955 was observed in both populations. It has been reported that levamisole-resistance in the BCk isolate of T. colubriformis is due to a sex-linked recessive gene or gene complex. A restriction fragment length polymorphism formed by the allelic variation was found and was detectable by digestion with the restriction endonuclease NlaIII. Statistical comparison of allele frequencies from individual male and female worms was consistent with sex-linkage of tar-1 (P < 0.05) but showed no correlation with levamisole resistance status. The polymorphism described will provide a useful X-chromosome marker and represents the first mapped genetic locus in this species.

Alleles↗

PubMed1997

Coacervation characteristics of recombinant human tropoelastin.

Coacervation of soluble tropoelastin molecules is characterized by thermodynamically reversible association as temperature is increased under appropriately juxtaposed ionic conditions, protein concentration and pH. Coacervation plays a critical role in the assembly of these elastin precursors in elastic fiber formation. To examine the effect of physiological parameters on the ability of tropoelastin molecules to associate, solutions of recombinant human tropoelastin were monitored spectrophotometrically by light scattering over a broad range of temperatures. Coacervation of recombinant human tropoelastin is strongly influenced by the concentration of protein and NaCl and to a lesser extent on pH. Trends towards maximal association are apparent when each of these parameters is varied. Remarkably, optimal coacervation is found at 37 degrees C, 150 mM NaCl and pH 7-8. Using the data generated by time courses, estimates of thermodynamic parameters were made. These estimates confirm that coacervation is endothermic and is marked by a strong entropic contribution. Circular dichroism of recombinant human tropoelastin revealed that, rather than being random, the structure is compatible with being largely that, of an all-beta protein (with secondary structure estimated to be 3% alpha-helix, 41% beta-sheet, 21% beta-turn and 33% other), exhibiting a spectrum as previously seen for tropoelastin populations and soluble elastin from naturally-derived sources.

Circular Dichroism↗

PubMed1997

mle-1, a mariner-like transposable element in the nematode Trichostrongylus colubriformis.

A mariner-like element termed mle-1 was discovered in the parasitic nematode Trichostrongylus colubriformis. The mle-1 has features which support its assignment as a mariner-like transposable element. Cloned mle-1 was derived from an intron of the tar-1 gene. It comprises 893 bp, includes two 27 bp flanking perfect inverted repeats and is present at approximately 50 copies in the genome. The element contains a coding region which displays homology to transposases, with the greatest amino acid similarity to a Caenorhabditis elegans mariner-like transposase. The coding region contains two 12 bp repeats; these repeats flank an 11 bp segment which accounts for a frameshift in this region. As a candidate transposon, mle-1 provides potential for genetic manipulation of this and related organisms.

Amino Acid Sequence↗

PubMed1997

Characterization of an acetylcholine receptor gene of Haemonchus contortus in relation to levamisole resistance.

The anthelminitic drug levamisole is thought to bind to nicotinic acetylcholine receptors of nematodes. It is possible that resistance to this drug is associated with either a change in binding characteristics or a reduction in the number of nicotinic acetylcholine receptors. Therefore, the molecular mechanism of levamisole resistance in the parasitic nematode Haemonchus contortus was studied by isolating and characterising cDNA clones encoding a putative ligand binding nicotinic acetylcholine receptor subunit, HCAl, of two susceptible and one levamisole resistant population. Hcal is related to unc-38, a nicotinic acetylcholine receptor subunit gene associated with levamisole resistance in Caenorhabditis elegans. Although extensive sequence analyses of hcal sequences revealed polymorphism at amino acid level, no association with levamisole resistance could be detected. Restriction fragment length polymorphism analyses confirmed that, although polymorphism was detected, no selection of a specific allele of hcal has taken place during selection for levamisole resistance in various levamisole resistant populations.

Amino Acid Sequence↗

PubMed1996

Cloning and sequence analysis of the candidate nicotinic acetylcholine receptor alpha subunit gene tar-1 from Trichostrongylus colubriformis.

A T. colubriformis genomic library in lambda EMBL3 was screened for sequences homologous to the Caenorhabditis elegans unc-38 nicotinic acetylcholine receptor (nAChR) alpha-subunit gene. The candidate gene tar-1 (for Trichostrongylus acetylcholine receptor subunit gene 1) comprising 13704 base pairs was thus identified. BLAST comparison of the sequenced clone with GenBank, followed by comparison of translated regions in six reading frames with protein databases, identified clearly defined tracts corresponding to 12 putative exons sharing high sequence homology to other nAChR genes and able to code for sequential regions of a putative nAChR alpha-subunit protein (tar-1). Tar-1 shares sequence similarities with over 40 nAChR subunit proteins. The highest similarity (91.6%) is with unc-38, suggesting that nAChR sequences from nematodes are closely related. The sequence includes motifs typical of these molecules including adjacent cysteine residues at the ACh binding site and four transmembrane regions. The DNA sequence presents the longest genomic tract described for this organism and should prove useful as a probe source in the search for nAChR genes from this and other nematodes and for studying the molecular mechanism of resistance to levamisole, a drug which is known to act on nAChRs of worms and which is widely used for parasite control.

Amino Acid Sequence↗

PubMed1996

Development of a sensitive peptide-based immunoassay: application to detection of the Jun and Fos oncoproteins.

c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36 --> E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., & Easterbrook-Smith, S.B. (1993) Anal. Biochem. 209, 183-187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 degrees C was determined to be 0.99 +/- 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 +/- 0.13 microM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.

Amino Acid Sequence↗

PubMed1996

High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer.

The solution structure of the c-Jun leucine zipper domain has been determined to high resolution using a new calculation protocol designed to handle highly ambiguous sets of interproton distance restraints. The domain comprises a coiled coil of parallel alpha-helices in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface; this interface extends over 10 helical turns and is the most elongated protein domain solved to date using NMR methods. The backbone fold is very similar to that seen in crystal structures of the GCN4 and Jun-Fos leucine zippers; however, in contrast with these crystal structures, the Jun leucine zipper dimer appears to be devoid of favorable intermolecular electrostatic interactions. A polar asparagine residue, located at the dimer interface, forms the sole point of asymmetry in the structure; furthermore, the side chain of this residue is disordered due to motional averaging. This residue, which is highly conserved in the leucine zipper family of transcription factors, provides a destabilizing influence that is likely to facilitate the rapid exchange of zipper strands in vivo.

Amino Acid Sequence↗

PubMed1996

NOD mouse diabetes: the ubiquitous mouse hsp60 is a beta-cell target antigen of autoimmune T cells.

In the NOD mouse, the onset of beta-cell destruction is associated with spontaneous development of T-lymphocytes reactive to members of the 60 kDa heat shock protein (hsp60) family, including the Mycobacterial (MT) and the human (H) hsp60 molecules. Diabetes in the NOD mouse is a spontaneous tissue-specific autoimmune disease occurring without prior immunization. Therefore, it has been suggested that the anti-hsp60 T cells involved in the autoimmune diabetes of NOD mice might reflect molecular mimicry between MT-hsp60 and a beta-cell tissue specific molecule sharing similar T cell epitopes, the p277 peptide of hsp60 in particular. We cloned and expressed the mouse hsp60 cDNA from a beta-cell tumour. This mouse beta-cell hsp60 cDNA was found to be identical in sequence to the hsp60 of mouse fibroblasts. We further report that NOD spleen cells and an NOD diabetogenic T cell clone C9 responded to the recombinant mouse hsp60 and to its peptide M-p277 to the same extent as to H-hsp60 and H-p277. Splenocytes of mice of other strains did not respond to p277. Moreover, treatment of 3 month old NOD mice with the non-modified self M-p277 peptide was as efficient as H-p277, from which it differs in one amino acid, in halting progression of the disease. Thus, anti-H-p277 T cells modulating diabetes in the NOD mouse are autoreactive, and are targeted at the mouse beta-cell hsp60, which is not tissue specific. These findings raise the question of how a non-tissue specific molecule may be a target of a tissue-specific autoimmune disease.

Amino Acid Sequence↗

PubMed1996

Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module.

Human c-Jun and c-Fos leucine zipper domains were examined for their ability to serve as autonomous dimerization domains as part of a heterologous protein construct. Schistosoma japonicum glutathione S-transferase (GST) was fused to recombinant Jun leucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains. SDS-PAGE 'snapshot' analyses based on disulphide linkage of monomers demonstrated the ability of rJunLZ to function as a dimerization motif in a foreign protein environment. Steric hindrance prevented formation of rJunLZ-GST::rFosLZ-GST heterodimers whereas rJunLZ-GST::rFosLZ and rJunLZ:: rFosLZGST formed readily. Furthermore, rJunLZGST generated homodimers suggesting fusion protein heterodimers interact differently to homodimers. Gel filtration chromatography confirmed that GST is a dimer in solution and that attachment of a leucine zipper domain allows further interactions to take place. Sedimentation equilibrium analyses showed that GST is a stable dimer (K(a) > 10(6) M(-1)) with no higher multimeric forms. rFosLZ-GST weakly associates beyond a dimer (K(a) approximately 4 x 10(4) M(-1)) and rJunLZ-GST associates indefinitely (K(a) approximately 4 x 10(5) M(-1)) [corrected], consistent with an isodesmic model of association. The interaction of these leucine zippers independently of GST association demonstrates their utility in the modification of proteins when multimer formation is desired.

Animals↗

PubMed1995

Nuclear magnetic resonance characterization of the Jun leucine zipper domain: unusual properties of coiled-coil interfacial polar residues.

Leucine zippers constitute a widely observed structural motif which serves to promote both homo- and heterodimerization in a number of DNA-binding proteins. As part of our ongoing efforts to characterize both the structure and the dynamical properties of this dimerization domain as they relate to biological function, we report here the secondary structure in solution of a recombinant dimeric peptide (rJunLZ) comprising residues Arg276-Asn314 of the leucine zipper domain of c-Jun. Two- and three-dimensional homo- and heteronuclear NMR experiments have allowed definition of the secondary structure of rJunLZ and have provided a total of approximately 1500 interproton distance and 62 phi dihedral angle constraints for tertiary structure calculations. Amide proton protection factors, calculated from hydrogen-deuterium exchange experiments, have identified 62 hydrogen bonds in the rJunLZ dimer. We have also examined the role of Asn22, the only polar residue situated at the hydrophobic dimer interface. Virtually all leucine zipper sequences contain such a polar residue (usually Asn) near the center of the motif. X-ray crystallographic studies showed that, in the case of the GCN4 homodimer, the polar residue (Asn) adopts an asymmetric conformation in an otherwise essentially symmetric structure. In contrast, all NMR studies of leucine zipper homodimers to date have suggested that the dimers are completely symmetric in solution. We present evidence that the side-chain amide protons of Asn22 are hydrogen-bonded in solution and that this side chain exchanges rapidly between two distinct conformations. On the basis of these observations, we propose a dynamic model which can explain the apparent differences in symmetry observed in NMR and X-ray crystallographic studies of leucine zipper homodimers. We show that mutation of Asn22 to a hydrophobic Leu residue markedly increases the thermal stability of the rJunLZ homodimer, consistent with a destabilizing role for this residue. However, at temperatures below 30 degrees C, the Asn22-->Leu mutant rearranges to form oligomers larger than the dimer, as was previously observed for the corresponding Asn-->Val mutation in the GCN4 leucine zipper. These results are consistent with the hypothesis that the polar Asn residue commonly observed at the interface of leucine zippers imposes specificity for the dimer structure at the expense of stability [Harbury, P.B., Zhang, T., Kim, P.S., & Alber, T. (1993) Science 262, 1401-1407].

Amino Acid Sequence↗

PubMed1995

Hutchinson-Gilford progeria fibroblasts exhibit metabolically normal uridine uptake and RNA synthetic rates.

The accelerated aging disease Hutchinson-Gilford progeria syndrome displays altered messenger RNA levels in cultured fibroblasts, yet little is known of effects on transcription by RNA polymerases other than RNA polymerase II. Total RNA metabolism was examined by incubation of Hutchinson-Gilford progeria fibroblasts with [5-3H]uridine in asynchronous culture. Uptake of radiolabel was quantitative and was incorporated preferentially (99 +/- 0.3%) into newly synthesized RNA. Progeria and control cultures showed comparable rates of uptake of radiolabel, time courses of RNA synthesis, and relative intensity profiles of newly synthesized ribosomal RNA. These characteristics held over a greater than ten-fold range of cell densities (5 x 10(3) to 8 x 10(4) cells/cm2). Progeria and control fibroblasts thus have comparable metabolic capacities for uridine uptake and net RNA production, emphasizing the relative specificity of transcriptional changes previously identified in progeria cells.

Biological Transport↗

PubMed1995

Hutchinson-Gilford progeria types defined by differential binding of lectin DSA.

Hutchinson-Gilford Progeria Syndrome (progeria) is an extremely rare childhood disorder characterized by precocious senility which presents features similar to those seen in human aging. We have previously described a consistent increase of the glycoprotein gp200 in progeria skin fibroblasts in vitro. Here we extend these glycosylation studies and present evidence for the existence of two types of progeria skin fibroblasts. These two forms, called D- and D+, are distinguished by their response to the lectin DSA. In the D- group, DSA bound glycoproteins from progeria fibroblast strains AG03513B and AG10750 with markedly lower affinities compared with glycoproteins from three control fibroblast strains. In the D+ group, DSA binding to glycoproteins from four other progeria strains AG01972A, AG06297A, AG06917 and AG03198, was comparable to controls. Discrimination by DSA is the most distinctive feature of the D- and D+ groups, in contrast to binding of lectins Con A, GNA, PHA-L, RCA120, AAA and PNA which show no such selectivity. The data are consistent with a model of altered glycosylation in the D- type of progeria fibroblasts.

Adolescent↗

PubMed1995

Tandem integration of multiple ILV5 copies and elevated transcription in polyploid yeast.

An industrial yeast strain was modified by introducing DNA into brewing yeast such that the derived cells contain only yeast DNA. Thus selectable markers and bacterial sequences are not present in the final strain, making this procedure attractive for the development of generally acceptable brewing yeast. Linear DNA containing the cloned ILV5 gene was introduced into lager yeast along with an unlinked circular bifunctional plasmid containing a dominant resistance marker. Resistant colonies were screened for site-directed integration of the ILV5 DNA. Candidates were examined by several methods including Southern transfer and polymerase chain reaction. In this way, a strain WM56 was identified containing three tandem copies of ILV5. The amplified ILV5 region is stable during repeated subculturing in the absence of selective pressure. Correspondingly elevated levels of ILV5 transcript in strain WM56 compared to the control (i.e. non-tandem) parental strain led to increased amounts of encoded acetohydroxyacid reductoisomerase as evidenced by significantly lower diacetyl production. WM56 appears to be identical to the parental strain judged by CHEF, total restriction digestion patterns, and probing, but differs in the ILV5 region of the chromosome. The method is generally applicable to other yeast strains, and if desired, is amenable to iterated cycles of integration to increase the number of copies.

Genes, Fungal↗

PubMed1995

Total synthesis and expression in Escherichia coli of a gene encoding human tropoelastin.

To elucidate the structural features and interactions of tropoelastin (TEL) molecules which assist in giving the elastic fibre its physical properties, a 2210-bp synthetic human TEL-encoding gene (SHEL) was constructed for expression in Escherichia coli. To this end, a model of codon adjustment was tested which better suits the polypeptide biosynthetic needs of E. coli than the human sequence, where over one-third of this natural sequence contains expression-limiting rare codons and 4 amino acids alone account for 75% of the resulting polypeptide. This large synthetic TEL gene was expressed at a high level as the recombinant counterpart of human TEL and as a C-terminal fusion with glutathione S-transferase. This demonstrates that a synthetic approach based upon matching codon usage to that of the host organism can support significant expression of recombinant sequences. The synthetic gene incorporates the facility for simple cassette replacement in future insertion, deletion and mutagenesis experiments, including the introduction and removal of exon homologues. The resulting soluble polypeptide is easily purified and displays properties expected for this protein.

Amino Acid Sequence↗

PubMed1994

Conservation of the 168 divIB gene in Bacillus subtilis W23 and B. licheniformis, and evidence for homology to ftsQ of Escherichia coli.

The chromosomal regions of Bacillus subtilis (Bs) W23 and Bacillus licheniformis (Bl), which span the sequence encoding the homolog of the division initiation gene, divIB, of Bs168 were cloned and sequenced. The high level of conservation of the amino acid (aa) sequence of the DivIB protein (99 and 68% identity for BsW23 and Bl, respectively) was consistent with a significant role for this protein in the cell cycle of the two species. The hydropathy profile for DivIB of Bl was almost identical to that of Bs168 and consistent with a membrane location, as previously established for the latter. The higher than average level of identity (87%) of the 31-aa N-terminal cytoplasmic domain of DivIB between Bs168 and Bl raised the possibility of a special role for this domain. Database analyses using the Bl DivIB sequence and similarity analyses also strongly suggested that DivIB, of Bl and Bs, is a homolog of FtsQ of Escherichia coli. The flanking sequences extending into the unidentified orfs both upstream and downstream from divIB were highly conserved between Bs168 and Bl at both the nucleotide and aa levels. It was confirmed that orf4 of Bs168 is dispensable.

Amino Acid Sequence↗

PubMed1994

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain.

Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of c-Jun plus a total of five engineered non-native residues at the N- and C-termini. The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (approximately 15% total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy-terminal portion of Schistosoma japonicum glutathione S-transferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel alpha-helices. We also present 15N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy.

Amino Acid Sequence↗

PubMed1993

The solution structure of the leucine zipper motif of the Jun oncoprotein homodimer.

Proton NMR studies have been performed on a 9.8-kDa synthetic fragment comprising the homodimeric leucine zipper domain of the human oncoprotein Jun to ascertain its conformation in aqueous solution. Analysis of two-dimensional scalar and dipolar-coupling experiments enabled almost all proton resonances to be sequence-specifically assigned and further revealed that the Jun leucine zipper forms a completely symmetric dimer in solution, consistent with the formation of a coiled-coil arrangement of parallel alpha-helical strands. The rates of exchange of individual amide protons with solvent, as well as hydrogen-bond lengths predicted from amide proton chemical shifts, are shown to correlate with residue position in the coiled-coil. A subset of 209 unambiguous distance constraints was compiled using rules recently formulated for interpreting the NOESY spectra of symmetric coiled-coils, and these were used in combination with experimentally determined hydrogen bond and dihedral angle constraints to compute a solution structure for the Jun leucine zipper domain.

Amino Acid Sequence↗