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M Buck

Publications and source records attributed to M Buck.

At least 127 records · Page 7Linked to original sources
PubMed1991

Immunological study of the nef protein from HIV-1 by polyclonal and monoclonal antibodies.

We constructed and expressed different overlapping fusion proteins with the nef gene of HIV-1 and generated specific polyclonal rabbit and monoclonal mouse antibodies against these recombinant proteins. The rabbit antisera, one of the monoclonal antibodies as well as a serum from a HIV-1 infected patient recognized the nef protein with Mr 27 kDa in latently HIV-1 infected glioma cells in the immunoblot. In contrast, these antibodies could not detect nef in productively HIV-1 infected Molt-3 cells neither in immunoblot nor in indirect immunofluorescence assays. These results indicate the possible participation of nef in viral latency. The recombinant nef proteins were used as probes for anti-nef antibodies in human sera. We observed in 17 of 57 sera tested specific anti-nef antibodies. All of these anti-nef positive sera also contained antibodies directed against viral structural proteins. The NH2-terminal region of the recombinant nef was shown to be the major immunodominant antigenic site in the immunoblot assay.

Animals↗
PubMed1991

Preparation and properties of des-Tyr98 and des-Arg97-Tyr98 acylphosphatase (muscular isoenzyme).

Previous NMR reports indicated that Tyr98, the C-terminal residue of the muscular form of acylphosphatase, is likely to be part of the enzyme's active site. In addition, there is evidence that an arginine residue participates to the catalyzed reaction, possibly as phosphate binding site. Among all Arg residues present in the muscular forms of acylphosphatase, four, i.e. Arg23, Arg74, Arg77, and Arg97, appear to be conserved in all species checked thus far. We prepared the des-Tyr98 and des-Arg97-Tyr98 derivatives of the native acylphosphatase to investigate the properties of both modified enzymes. The enzyme lacking Tyr98 was found to be catalytically less effective than the native one, whereas the des-Arg97-Tyr98 acylphosphatase was completely inactive. This evidence suggests that Arg97 participates directly to the active site catalytic mechanism. Fluorescence and CD spectra revealed that the latter enzyme could have been undergone some conformational change that could account for the loss of activity; on the other hand, the one-dimensional NMR spectra of either native and des-Arg97-Tyr98 enzymes were strictly similar, thus demonstrating that the removal of the two C-terminal residues does not markedly affect the fold of the enzyme. The results reported are proof of a critical contribution of Arg97 to the acylphosphatase active site; however, we cannot exclude that the function of this residue is merely to stabilize the active site conformation and dynamics.

Acid Anhydride Hydrolases↗
PubMed1991

Biochemical characterization of phosphorylation site mutants of simian virus 40 large T antigen: evidence for interaction between amino- and carboxy-terminal domains.

The simian virus 40 large T antigen is phosphorylated at eight or more sites that are clustered in an amino-terminal region and a carboxy-terminal region of the protein. Mutants carrying exchanges at these phosphorylation sites have been generated in vitro by bisulfite or oligonucleotide-directed mutagenesis and analyzed for their phosphorylation patterns. Two-dimensional phosphopeptide analyses of the mutant large T antigens confirmed most of the previously identified phosphorylation sites, namely, serine residues 106, 112, 123, 639, 677, and 679 and threonine residues 124 and 701. In addition, serine residue 120 was identified as a new site, whereas serines residues 111 and 676 were excluded. Interestingly, several of the mutants exhibited secondary effects in that a mutation in the amino-terminal region affected phosphorylation at distant and even carboxy-terminal sites and vice versa. Thus, the amino- and carboxy-terminal domains appear to be in close proximity in the three-dimensional structure of large T antigen. The possible consequences of the above findings and the role of phosphorylation are discussed.

Amino Acid Sequence↗
PubMed1990

Activation of the Klebsiella pneumoniae nifU promoter: identification of multiple and overlapping upstream NifA binding sites.

The Klebsiella pneumoniae nifU promoter is positively controlled by the NifA protein and requires a form of RNA polymerase holoenzyme containing the rpoN encoded sigma factor, sigma 54. Occupancy of the K. pneumoniae nifU promoter by NifA was examined using in vivo dimethyl sulphate footprinting. Three binding sites for NifA (Upstream Activator Sequences, UASs 1, 2 and 3) located at -125, -116 and -72 were identified which conform to the UAS consensus sequence TGT-N10-ACA. An additional NifA binding site was identified at position -90. The UASs located at -125 (UAS1) and -116 (UAS2) overlap and do not appear to bind NifA as independent sites. They may represent a NifA binding site interacting with two NifA dimers. UAS3 is located at -72, and abuts a binding site for integration host factor (IHF) and is not normally highly occupied by NifA. In the absence of IHF UAS3 showed increased occupancy by NifA. Mutational and footprinting analysis of the three UASs indicates (1) IHF and NifA can compete for binding and that this competition influences the level of expression from the nifU promoter (2) that UAS2 is a principle sequence of the UAS 1,2 region required for activation and (3) that none of the NifA binding sites interacts with NifA independently. In vivo KMnO4 footprinting demonstrated that NifA catalyses open complex formation at the nifU promoter. IHF was required for maximal expression from the nifU and nifH promoters in Escherichia coli, and for the establishment of a Nif+ phenotype in E. coli from the nif plasmid pRD1.

Bacterial Proteins↗
PubMed1990

The influence of the Klebsiella pneumoniae regulatory gene nifL upon the transcriptional activator protein NifA.

The influence of the Klebsiella pneumoniae nifL gene product upon the interaction of the transcriptional activator protein NifA with the nifH promoter has been examined using in vivo dimethylsulphate 'footprinting'. Binding of NifA to the upstream activator sequence (UAS) of the nifH promoter in the presence of the NifL protein was observed under nitrogen-limiting growth conditions. Growth in the presence of NH4+ or addition of NH4+ to nitrogen-limited cells diminished the interaction of NifA with the UAS when NifL was present. Repression of nif transcription by NifL may therefore involve an interaction between NifL and NifA which reduces the affinity of NifA for the UAS.

Ammonia↗
PubMed1990

Tumor necrosis factor-alpha inhibits albumin gene expression in a murine model of cachexia.

The mechanisms responsible for decreased serum albumin levels in patients with cachexia-associated infection, inflammation, and cancer are unknown. Since tumor necrosis factor-alpha (TNF alpha) is elevated in cachexia-associated diseases, and chronic administration of TNF alpha induces cachexia in animal models, we assessed the regulation of albumin gene expression by TNF alpha in vivo. In this animal model of cachexia, Chinese hamster ovary cells transfected with the functional gene for human TNF alpha were inoculated into nude mice (TNF alpha mice). TNF alpha mice became cachectic and manifested decreased serum albumin levels, albumin synthesis, and albumin mRNA levels. However, even before the TNF alpha mice lost weight, their albumin mRNA steady-state levels were decreased approximately 90%, and in situ hybridization revealed a low level of albumin gene expression throughout the hepatic lobule. The mRNA levels of several other genes were unchanged. Hepatic nuclei from TNF alpha mice before the onset of weight loss were markedly less active in transcribing the albumin gene than hepatic nuclei from control mice. Therefore, TNF alpha selectively inhibits the genetic expression of albumin in this model before weight loss.

Animals↗
PubMed1990

Analysis of site-directed mutations in the alpha- and beta-subunits of Klebsiella pneumoniae nitrogenase.

Using directed mutagenesis, amino acid substitutions have been made in the alpha- and beta-subunits of the klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserved cysteine or tyrosine residues. Nif+, Nif- and intermediate phenotypes have been obtained. To extend our earlier biochemical characterization (Kent et al., 1989) the electrophoretic mobility of component 1 of the mutant and wild-type nitrogenases has been analysed by non-denaturing gel electrophoresis. The major and minor forms of component 1 separated by this methodology have been probed for by using both polyclonal and monoclonal antibodies. All Nif+ mutants exhibited a distribution of electrophoretic forms of component 1 comparable to the wild type, and the abundance of the major form found in purified nitrogenase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif- mutants exhibited either a major low-mobility form or a fast-moving form. Analysis of nitrogenase polypeptides synthesized in the absence of co-factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the alpha-subunit produces component 1 defective in its interaction with FeMoco. Substitution of other conserved cysteine residues by alanine appears to prevent early steps in nitrogenase assembly or to promote degradation. Two single mutations (cysteine 89 to alanine in the alpha-subunit and cysteine 94 to alanine in the beta-subunit) which are tightly Nif- can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the alpha- and beta-subunits.

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.

Acetylene↗
PubMed1989

In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex.

Transcription from the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters requires the positive control protein NifA and the alternative sigma factor sigma 54, encoded by the rpoN gene. Transcription from the K. pneumoniae nifH promoter is fully dependent upon NifA bound at the upstream activator sequence (UAS) whereas the R. meliloti nifH promoter can be efficiently activated in the absence of this sequence and can also be activated by a mutant form of NifA unable to bind the UAS. The in vivo interaction of RNA polymerase-sigma 54 with these promoters was examined using dimethyl sulphate footprinting. The R. meliloti nifH promoter but not the K. pneumoniae nifH promoter showed sigma 54-dependent methylation protection of guanine residues at -14, -25 and -26, the most conserved nucleotides characteristic of sigma 54-dependent promoters. A mutant derivative of the K. pneumoniae nifH promoter bearing transitions at positions from -15 to -17 showed sigma 54-dependent methylation protection of guanines -13, -24 and -25. The enhanced interaction of the RNA polymerase-sigma 54 with this mutant promoter correlates with its increased level of activation by a form of NifA unable to bind the UAS. Use of in vivo KMnO4 footprinting to detect single-stranded pyrimidine residues and in vivo methylation protection demonstrated that the sigma 54-dependent protection observed in the R. meliloti and mutant K. pneumoniae nifH promoter results from the formation of a closed promoter complex. The isomerization of the pre-existing closed complex to an open promoter form, as judged by the local denaturation of promoter DNA which rendered sequences from +5 to -10 reactive towards KMnO4, was shown to be fully dependent on NifA. We propose a model in which the fidelity of activation of sigma 54-dependent promoters relies on a weak activator-independent interaction of RNA polymerase-sigma 54 with the promoter. A specific interaction of the appropriate activator with its respective UAS is then required for the positive control protein to facilitate open complex formation.

Bacterial Proteins↗
PubMed1989

Cloning and sequencing of the nifH gene of Desulfovibrio gigas.

The Desulfovibrio gigas nifH gene has been cloned and sequenced. It consists of an open-reading frame of 822 base pairs encoding a 274 amino acid polypeptide. A potential ntrA-dependent promotor sequence is present. The gene lacks an upstream activator sequence homologous to those often found in nif genes subject to activation by nifA.

Amino Acid Sequence↗
PubMed1989

Mutations in the RNA polymerase recognition sequence of the Klebsiella pneumoniae nifH promoter permitting transcriptional activation in the absence of NifA binding to upstream activator sequences.

Positive control of the wild-type Klebsiella pneumoniae nifH promoter by the NifA protein requires that NifA is bound at the upstream activator sequence (UAS). By introducing base substitutions at -15 to -17 in the RNA polymerase recognition sequence of the nifH promoter, positive control by a form of NifA unable to bind to the UAS was greatly increased when compared to the wild-type promoter. Transcriptional activation still required the rpoN encoded sigma factor and was initiated at the same nucleotide as in the wild-type promoter. Mutations at -15 to -17 suppressed the requirement that the UAS should be located on the correct face of the DNA helix with respect to the RNA polymerase recognition sequence in order that titration of NifA and efficient activation occur. This result supports the suggestion that upstream bound NifA interacts with the RNA polymerase-RpoN complex. To examine the minimal carboxy terminal sequences required for the positive control function of NifA a series of carboxy terminal deletions were constructed. Efficient positive control at a UAS-independent promoter was only observed in deletions which did not extend beyond the proposed boundary separating the carboxy terminal NifA DNA-binding domain from its central domain.

Amino Acid Sequence↗
PubMed1989

The effect of cotton bract extract on respiratory glycoconjugate secretion from human airways in vitro.

In vivo and in vitro studies with a water-soluble extract of cotton bracts (CBE) suggest that CBE may be responsible for some of the clinical manifestations of byssinosis. Since chronic bronchitis has been repeatedly documented as a major feature of byssinosis, we studied the effect of CBE on respiratory glycoconjugate (RGC) release from human airways (HAs) in vitro. HAs were incubated with [3H]glucosamine to label RGC molecules. CBE in increasing concentrations was added to radiolabeled HAs, and the release of 3H-RGC, histamine, and other mediators was measured. CBE in concentrations of 1 to 7 mg/ml caused a dose-related increase in RGC, as well as histamine release (RGC, 14% to 45% increase above control; histamine, 12 to 70 ng/ml released concurrently). Additionally, CBE in a dose of 5 mg/ml caused a more than threefold increase in peptidoleukotriene production above baseline. The effect of histamine H1 and H2 (pyrilamine and cimetidine), cyclooxygenase pathway inhibitor (indomethacin), leukotriene (LY 171883 and FPL 55712), and lipoxygenase pathway (BW nordihydroguaiarectic acid) blocking agents on CBE-induced RCG secretion was studied. In addition to histamine-H1 blockers, lipoxygenase inhibitors (nordihydroguaiarectic acid and BW 755C) and leukotriene blockers (FPL 55712 and LY 171883) are also potent inhibitors of CBE-induced RGC secretion. This suggests that CBE may act via the release of several mediators (histamine and leukotrienes), possibly from airway cells, such as mast cells, macrophages, or epithelial cells, to stimulate RGC secretion.

Bronchi↗
PubMed1988

The DNA-binding domain of the transcriptional activator protein NifA resides in its carboxy terminus, recognises the upstream activator sequences of nif promoters and can be separated from the positive control function of NifA.

The positive control protein NifA activates transcription of nitrogen fixation promoters in Klebsiella pneumoniae. NifA is believed to bind to specific sites, the upstream activator sequences (UAS's), of the nif promoters which it activates. We have now shown by mutation of the carboxy terminus of NifA that this is the DNA-binding domain and that the DNA-binding and positive activator functions of NifA can be separated. Mutational analysis of the nifH UAS and in vivo methylation protection analysis of the interaction of NifA with the nifH promoter demonstrates that the UAS is recognised by the carboxy terminus of NifA. The UAS's of K. pneumoniae nif promoters are also required for activation by the Rhizobium meliloti NifA indicating that this activator also possesses DNA-binding activity.

Amino Acid Sequence↗
PubMed1988

NifA-dependent in vivo protection demonstrates that the upstream activator sequence of nif promoters is a protein binding site.

Primer-extension analysis of the Klebsiella pneumoniae nifH promoter was used to determine changes in the accessibility of the promoter DNA to methylation after exposure of growing cells to dimethyl sulfate. Four guanine residues present in the nifH upstream activator sequence (UAS), the proposed NifA binding site, were protected from methylation and two guanine residues were hypermethylated when the transcriptional activator protein NifA was present in the cells. The interaction detected at the nifH UAS was independent of the alternative sigma factor NtrA required for transcription of the nifH and other nif promoters. Mutations within the nifH UAS that diminish NifA-dependent transcriptional activation reduced the interaction at the UAS. It seems likely that the pattern of methylation protection observed in the nifH UAS is the result of NifA binding.

Bacterial Proteins↗
PubMed1988

Cardiac arrest--a manifestation of 5-fluorouracil cardiotoxicity.

We report a case of cardiac arrest occurring during an infusion of 5-fluorouracil where electrocardiograms suggested coronary artery spasm as the underlying event. Treatment with vasodilators and diltiazem, proved effective. This clinical presentation of 5-fluorouracil cardiotoxicity has not previously been described.

Carcinoma, Squamous Cell↗
PubMed1987

Mutational analysis of upstream sequences required for transcriptional activation of the Klebsiella pneumoniae nifH promoter.

Upstream sequences of the Klebsiella pneumoniae nifH promoter were mutagenised and activation of the mutated promoters by the nif-specific transcriptional activator protein NifA examined in vivo. Of the sixteen mutations analysed, only those within the nifH upstream activator sequence (UAS), characterised by a TGT-N10-ACA motif, influenced nifH promoter activity. Mutations altering the two-fold rotational symmetry of the UAS or the spacing between the TGT and ACA motifs reduced promoter activity, consistent with the UAS functioning as a NifA binding site. The bases flanking the TGT-ACA motif of the UAS also appear to influence activation by NifA. Substituting the nifH UAS with a binding site for the transcriptional activator NtrC resulted in improved NtrC-dependent activation of the nifH promoter demonstrating that the activator specificity of the nifH promoter is dependent upon the presence of the appropriate upstream sequences to which the activator binds.

Base Sequence↗
PubMed1987

Nucleotide sequence of the Klebsiella pneumoniae nifD gene and predicted amino acid sequence of the alpha-subunit of nitrogenase MoFe protein.

The nucleotide sequence of the Klebsiella pneumoniae nifD gene is presented and together with the accompanying paper [Holland, Zilberstein, Zamir & Sussman (1987) Biochem. J. 247, 277-285] completes the sequence of the nifHDK genes encoding the nitrogenase polypeptides. The K. pneumoniae nifD gene encodes the 483-amino acid-residue nitrogenase alpha-subunit polypeptide of Mr 54156. The alpha-subunit has five strongly conserved cysteine residues at positions 63, 89, 155, 184 and 275, some occurring in a region showing both primary sequence and potential structural homology to the K. pneumoniae nitrogenase beta-subunit. A comparison with six other alpha-subunit amino acid sequences has been made, which indicates a number of potentially important domains within alpha-subunits.

Amino Acid Sequence↗