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Biomedical subjects

R Mertz

Publications and source records attributed to R Mertz.

17 recordsLinked to original sources
PubMed1996

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

Amino Acid Sequence↗
PubMed1994

Cloning, expression, and distribution of functionally distinct Ca(2+)-activated K+ channel isoforms from human brain.

We have cloned and expressed nine Ca(2+)-activated K+ channel isoforms from human brain. The open reading frames encode proteins ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse. All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 (hslo). RNA splicing occurs at four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1-hbr9). hslo mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in Xenopus oocytes produces robust voltage and Ca(2+)-activated K+ currents. Splice variants differ significantly in their Ca2+ sensitivity, suggesting a broad functional role for these channels in the regulation of neuronal excitability.

Alternative Splicing↗
PubMed1990

Interleukin-1 beta inhibits glucokinase activity in clonal HIT-T15 beta-cells.

Interleukin-1 beta (IL-1 beta) has been implicated in the pathogenesis of insulin-dependent diabetes mellitus. In the present study we have investigated the effects of IL-1 beta on glucose metabolism in clonal HIT-T15 beta cells. In the short-term (1 h), 25 U/ml IL-1 beta significantly increased the rates of insulin release and glucose utilisation, but not glucose oxidation. In contrast, after 48 h, IL-1 beta inhibited insulin release and glucose utilisation and oxidation. By assaying enzymes (hexokinase, glucokinase, pyruvate dehydrogenase, glucose 6-phosphatase) and nucleotides (ATP, ADP) associated with the regulation of glycolysis and glucose oxidation, we conclude that the inhibitory effects of IL-1 beta may be due to impaired glucokinase activity.

Adenine Nucleotides↗
PubMed1990

Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro.

Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with anti-L antibodies, the immunocomplex already showed protein kinase activity. In the presence of P protein, the NP protein was more highly phosphorylated. The results show that Sendai virus L protein possesses a protein kinase activity phosphorylating the other proteins of the viral nucleocapsid in vitro.

Amino Acid Sequence↗
PubMed1989

Carrier-bound synthetic peptides. Use as antigen in HIV-1 ELISA tests and in antiserum production.

Chemically synthesize carrier-bound peptides have been used as antigens in diagnostic test systems (ELISA) and for raising antipeptide-specific antisera. The method does not require prior cleavage of the peptides from the support used for the solid-phase synthesis. Using the same resin for both the synthesis and the subsequent applications it was possible to avoid expensive and time-consuming purification procedures and artificial recoupling to solid supports. A quick and specific ELISA-based diagnostic test system for HIV-specific antipeptide antibodies in human sera was established. In addition the carrier-bound peptides were shown to be potent antigens for raising antibodies in animals.

Animals↗
PubMed1989

Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter.

Transcription from one of the two initiation sites, P1 and P2, of the dual human MYC promoter seems to be essential in all proliferating cells. To identify proteins and target structures for MYC regulation, a DNA region was analyzed that is critical for P2 promoter activity. Here, we show that a nuclear factor binds to a DNA element within P2, which is conserved perfectly between mouse and man and displays a striking homology to the E1a-inducible E2 promoter of adenovirus type 5 (Ad5). We demonstrate that the same transcription factor, defined recently as E2F, which plays an essential role in the activation of adenovirus early promoters and enhancers, also interacts as a dominant nuclear factor with the MYC promoter. The presence of an intact E2F binding site is required for basic expression and for trans-activation of the P2 promoter by E1a proteins. The human MYC promoter is the first cellular target described for E2F. The results suggest that expression of MYC might be regulated via modulation of E2F by cellular 'E1a-like' factors.

Base Sequence↗
PubMed1989

[Ethically chosen studies with early-weaned piglets during their raising in pens with different applications of straw. 1. The effects of different applications of straw and different floor conditions in areas of uniform size].

In extension of earlier experiments in housing systems with perforated floors, utilization rates of solid floor with litter, straw in a rack and deep litter, as well as the behaviour and physical condition, especially of claws, were investigated. Each pen had an area of 0.45 m2/piglet. In further experiments the influences of race (DL instead of DL x Piétrain), kind of rearing (without straw instead of rearing with straw), lowering temperature and the effect of a preferred perforated floor in comparison with solid floor were investigated. Deep litter was preferred for activity and lying behaviour only in the case of low temperature (14-18 degrees C) whereas solid floor with litter was preferred in the case of higher temperature (19-25 degrees C). The perforated floor was equivalent to solid floor on the condition that the area dimension was sufficient, temperature 3 degrees C higher than in the other experiments and straw in a rack was offered. However, the utilization rates of solid floor increased in the case of lowering temperature by 4 degrees C. There was no significant influences of the different race or kind of rearing. In the experiments there were significant signs of a more harmonious condition of the animals and a shorter, respectively no period where the animals had to adapt to the housing conditions. These findings were in contrast to those of earlier investigations. The possibility of acting with straw led to an undisturbed circadian rhythm and to a higher rate of standing within the total activity which was as high as in earlier studies. Further social companions and environmental objects lost attractiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI)--generation of protein sequence information.

The paper describes a potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins. This procedure exploits the sequence-specific DNA-binding affinity of such proteins for their enrichment, comparable to recognition site DNA affinity chromatography. The method was employed to obtain a pure preparation of nuclear factor I (NFI) from porcine liver from which sequences of partial peptides could be obtained. Oligonucleotide probes derived from these amino-acid sequences were used to identify genomic and cDNA clones of NFI.

Amino Acid Sequence↗
PubMed1988

Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease.

Retroviruses code for a virus-specific protease which is essential for polyprotein processing and viral infectivity. The human immune deficiency virus-1 protease is an aspartic protease of 9 kDa which was synthesized by recombinant DNA technology and arises by autocatalytic processing from a polyprotein precursor which has recently been demonstrated by use of a protease-specific monoclonal antibody. The protease was shown to form dimers. Here we demonstrate that synthetic peptides can be used as both model substrates as well as inhibitors for investigation of the protease. 14 synthetic peptides, 7-18 amino acids in length, containing putative protease cleavage sites of the viral polyprotein gag and pol precursors, have been analyzed with the partially purified protease by the use of high performance liquid chromatography. In seven cases, where cleavage was observed, the length of the peptides did not significantly influence the cleavage efficiencies, heptapeptides being large enough as model substrates. No cleavage was observed with a protein preparation purified in parallel from control bacteria not expressing the human immune deficiency virus-1 protease. The protease was not only able to cut next to a proline but also between other peptides indicating that the proline is not a prerequisite. Three peptides with either reduced bonds at the cleavage site or a substitution by statin were inhibitory while another uncleaved substrate was not. The usefulness of small model substrates for characterization of the protease is further demonstrated by determination of a kinetic optimum pH (3.5-5.5) and incubation temperature (37 degrees C).

Escherichia coli↗
PubMed1988

Isolation and characterization of the porcine nuclear factor I (NFI) gene.

This study describes the isolation of a major portion of the gene for nuclear factor I (NFI) including its 5'-flanking region with transcriptional start sites. We screened a porcine liver, genomic DNA library in phage EMBL3A with synthetic oligonucleotides derived from tryptic and cyanogen-bromide peptide sequences obtained from purified NFI protein. The NFI gene is present as a single copy in porcine DNA.

Amino Acid Sequence↗
PubMed1988

Beta 3 tubulin expression characterizes the differentiating mesodermal germ layer during Drosophila embryogenesis.

During embryogenesis, the beta 3 tubulin gene of Drosophila is transcribed predominantly in the mesoderm. We have raised antibodies specific to the C-terminal domain of the beta 3 tubulin and analysed by immunostaining the distribution of this tubulin isotype during Drosophila embryogenesis. The protein is first detectable in the cephalic mesoderm at maximal germband extension. Shortly afterwards, beta 3 tubulin is expressed in single cells at identical positions of the thoracic and abdominal segments. We suggest that these cells represent muscle pioneer cells of Drosophila. During later embryonic development the somatic musclature, visceral musculature, dorsal vessel and macrophages contain beta 3 tubulin. In dorsalizing mutants dorsal, snail and twist, which do not form a ventral furrow during gastrulation, beta 3 expression is greatly reduced but not completely abolished. Our analysis shows that beta 3 tubulin immunostaining characterizes the differentiation of mesodermal derivatives during embryogenesis.

Animals↗
PubMed1987

Clinical and socio-professional fate of isocyanate-induced asthma.

Thirty-one patients with isocyanate-induced asthma were studied 6-54 months after diagnosis. Four had the same work conditions and unchanged or worse respiratory symptoms; seven had an alternative job or safer work conditions at the same work-place and suffered from mild to severe symptoms. The remaining twenty subjects were definitely removed from exposure; of these, ten (50%) remained symptomatic after being removed from exposure for an average of 19 months. Asymptomatic patients appeared to be younger and to have shorter durations of total and symptomatic exposures, while symptomatic patients were more reactive to acetylcholine at diagnosis. For patients removed from isocyanate exposure and for those re-employed at the same work-place, quality of the new work site seems to play a role in the evolution of isocyanate-induced asthma.

Adult↗
PubMed1986

Upstream regulatory sequences of immunoglobulin genes are recognized by nuclear proteins which also bind to other gene regions.

The decanucleotide sequence (dc) TNATTTGCAT is an upstream regulatory sequence of immunoglobulin genes and occurs also upstream of certain other eukaryotic and prokaryotic genes (compiled in the accompanying paper). We now investigated the binding of proteins from nuclear extracts of a number of cell types and organisms to the dc sequence using a sensitive gel electrophoretic DNA binding assay. Binding studies with specifically designed oligonucleotides led to the following conclusions: the central T of the dc sequence can be altered with only a slight decrease of protein binding activity: the sequences in the neighborhood of dc have a positive or negative effect on the efficiency of protein binding; C-rich sequences which occur in many K chain promoters have a protein binding activity independent of dc; the dc binding protein(s) of human lymphoid cells elute from a Sephadex column in the 30.000-60.000 molecular weight range; dc binding proteins were found in nuclear extracts of lymphoid as well as non-lymphoid human and murine cell lines, of Xenopus oocytes, and of yeast cells. The finding of dc binding proteins in a wide variety of different organisms and the occurrence of dc-related sequences in the regulatory regions of several gene families point to a general role in the transcriptional regulation of the respective genes.

Animals↗
PubMed1986

A sensitive and rapid gel retention assay for nuclear factor I and other DNA-binding proteins in crude nuclear extracts.

The paper describes a rapid and sensitive assay for DNA binding proteins which interact with specific and defined binding sites. It exploits the observation that complexes of proteins and small synthetic DNA fragments (40 bp) containing the protein/DNA binding site can enter native polyacrylamide gels and remain stably associated during electrophoresis under non-denaturing conditions. The assay was applied to nuclear factor I, to its identification and purification from porcine liver, to an analysis of its binding site on adenovirus type 5 DNA and to an exploration of other potential binding sites for DNA binding proteins within the inverted terminal repetition of adenovirus DNA. The extreme sensitivity of the assay which surpasses that of conventional footprint assays by at least two orders of magnitude permitted the identification of nuclear factor I-like activities in Saccharomyces cerevisiae.

Animals↗
PubMed1985

Seventeen base pairs of region I encode a novel tripartite binding signal for SV40 T antigen.

Three sequence components direct high affinity binding of dimeric SV40 T antigen to SV40 origin region I. Two signals are encoded by two directly repeated 5'-GAGGC-3' pentanucleotides. Approximately equal contributions to binding stability are made by each pentanucleotide, and both spacing and orientation of the pentanucleotides are important for binding affinity. The third vital component is contained in a 5'-TTTTTTG-3' spacer sequence that separates the pentanucleotides. Sequence-specific features of the spacer stabilize binding to the adjacent pentanucleotides. The asymmetry of the spacer suggests that a novel binding mechanism is involved. Because the alignment of T antigen on mutant and wild-type DNAs is similar, we propose that any two of the three sequence signals are sufficient to determine the unique arrangement of a bound protein dimer.

Antigens, Polyomavirus Transforming↗
PubMedDate not supplied

Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli.

This paper reports the expression of an artificial functional polypeptide in bacteria. The gene of a designed 24-residue DDT-binding polypeptide (DBP) was inserted between the BamHI and PstI cleavage sites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, was cloned in Escherichia coli JM109. After induction by isopropyl-beta-D-thiogalactopyranoside a fusion protein was expressed in which DBP was linked to the COOH-terminus of beta-galactosidase. DBP, which is stable to trypsin, was obtained by tryptic digestion of the fusion protein and subsequent fractionation of the tryptic peptides by reversed-phase h.p.l.c. Recombinant and chemically synthesized DBP showed identical chromatographic properties, amino acid composition, and chymotryptic digestion patterns. Both the beta-galactosidase-DBP fusion and isolated recombinant DBP bound DDT. The fusion protein was 25 times as potent as the designed 24-residue DBP in activating a cytochrome P-450 model system using equimolar catalytic amounts of the two proteins.

Amino Acid Sequence↗