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Biomedical subjects

M Sauter

Publications and source records attributed to M Sauter.

At least 55 records · Page 3Linked to original sources
PubMed1995

Gibberellin promotes histone H1 kinase activity and the expression of cdc2 and cyclin genes during the induction of rapid growth in deepwater rice internodes.

Partial submergence or treatment with either ethylene or gibberellin (GA) promotes rapid internodal growth in deepwater rice (Oryza sativa L.). Earlier work has shown that GA is the immediate hormonal signal for this growth response, which involves induction of the cell cycle at the G2/M phase transition and subsequent enhancement in the rate of DNA synthesis. In all eukaryotes, onset of mitosis is regulated by the p34cdc2/CDC28 protein kinase, whose activity is assayed by in vitro phosphorylation of histone H1. It was found that GA enhanced the activity of p34cdc2/CDC28-like histone H1 kinase in the intercalary meristem of rice internodes. The enzyme activity showed a sharp peak that correlated with a decrease in the population of cells in the G2 phase during the first 4 h of GA treatment but not with changes in DNA synthesis. The level of histone H1 kinase activity increased again when cell division activity in the intercalary meristem is known to be high. The expression of two cdc2 homologs was examined. The mRNA level of one of these, cdc2Os-2, was increased after 1 h of GA treatment, whereas the mRNA level of the other, cdc2Os-1, was not affected. Two cDNAs, cycOs1 and cycOs2, which show high homology to cyclin cDNAs, were cloned from rice. They share 75.1% sequence identity at the amino acid level, and both of them are encoded by mRNAs of 1.6 kb. Expression of the two corresponding cyclin genes was enhanced by GA, and the time course of the induction was compatible with a role for both cyclins in regulating the G2/M phase transition. The cyclins were expressed in the intercalary meristem and the elongation zone of the internode, but the GA-induced increase in transcript levels was restricted to the meristem only. The results support the hypothesis that induction of mitosis by GA is brought about by increased p34cdc2/CDC28 protein kinase activity, which may be the result of transcriptional activation of the cdc2Os-2, cycOs1 and cycOs2 genes.

Amino Acid Sequence↗
PubMed1995

Human endogenous retrovirus K10: expression of Gag protein and detection of antibodies in patients with seminomas.

The human endogenous retrovirus K10 (HERV-K10) has been identified in the human genome by its homology to retroviruses of other vertebrates (M. Ono, T. Yasunaga, T. Miyata, and H. Ushikubo, J. Virol. 60:589-598, 1986). Using PCR amplification, DNA cloning, sequencing, and procaryotic expression, we were able to demonstrate that HERV-K10 encodes a 73-kDa protein which was processed by a HERV-K10-encoded protease to yield proteins p22/p26, p30, and p15/16. Analysis of the teratocarcinoma cell line Tera 1 or tumor tissues by immunoblotting demonstrated that the 80-kDa polyprotein of HERV-K10 gag and a processed protein of 39 kDa were expressed. In addition, a major protein of 39 kDa and additional species of 30, 22, 19, and 17 kDa could be detected in the supernatant of Tera 1 cells, suggesting that HERV-K10 Gag proteins are either secreted or processed to probably incomplete viral particles. In addition, the gag gene of HERV-K10 was expressed in the baculovirus system. Using this recombinant system to test antisera from patients with different diseases and healthy individuals, we were able to detect antibodies against the N-terminal part of HERV-K10 Gag in 2 to 4% of groups of tumor patients with titers ranging between 1:80 and 1:640, while approximately 0.1 to 0.5% of healthy individuals exhibited antibodies with lower titers. In contrast, patients with seminoma had antibody titers in the range of 1:2,560 at the time when the tumor was detected. Immunohistochemistry using specific rabbit sera or monoclonal antibodies against HERV-K10 Gag revealed that the Gag protein is expressed in the cytoplasm of the tumor cells. Furthermore, an 80-kDa protein corresponding to the HERV-K10 Gag polyprotein could be detected in tumor biopsies. For the first time, these data indicate that HERV-K10 Gag proteins are synthesized in seminoma cells and tumors exhibit relatively high antibody titers against Gag. So far, no information on which role HERV-K10 plays in the development of this tumor exists.

Antibodies, Viral↗
PubMed1994

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C-terminal processing at Arg537.

Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel-containing polypeptide, which is subject to C-terminal proteolytic processing. This processing reaction could be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel. N-terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C-terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination. Nickel-containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (delta hycH) in the presence of the reducing agent dithiothreitol. Using 2-mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing. This conclusion was supported by the finding that HYCE precursor from strain HD708 (delta hycH) chromatographed with radioactivity from 83Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from strain BEF314 (delta hypB-E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.

Amino Acid Sequence↗
PubMed1993

Evidence that HERV-K is the endogenous retrovirus sequence that codes for the human teratocarcinoma-derived retrovirus HTDV.

Human teratocarcinoma-derived viruses (HTDV) are retrovirus-like particles that are regularly observed by electron microscopy at low frequency in cell lines established from human teratocarcinomas. Over the last years, one of our teratocarcinoma cell lines spontaneously began to produce high amounts of HTDV. This cell line is stained in immunofluorescence tests by an antiserum raised against recombinant gag protein of HERV-K, an expressed human endogenous retrovirus sequence. In immunoelectron microscopy of ultrathin frozen sections, this anti HERV-Kgag-specific antiserum reacts specifically with HTDV particles. In Western blots, the antiserum recognizes predominantly a protein with an apparent molecular weight of 30 kDa, presumably the major core protein of HTDV particles. Taken together, these results provide evidence that HERV-K codes for HTDV.

Fluorescent Antibody Technique↗
PubMed1993

Molecular and biological characteristics of a novel HIV-2 isolate, HIV-2HOM.

In 1991 a new HIV-2 isolate (HIV-2HOM) was isolated first from a German individual most likely infected in West Africa in the beginning of the 1970s. The virus was isolated from both, the plasma and the peripheral blood lymphocytes of the patient by using OKT-3-stimulated cord blood lymphocytes. The recovered viruses could be further propagated on Jurkat cells and exhibited a broad cell tropism. Biochemical and antigenic properties of HIV-2HOM were examined by radioimmunoprecipitations. For a more detailed molecular characterization, a 1520 bp DNA fragment from the env gene and a 722 bp DNA fragment from the pol gene were amplified by polymerase chain reactions, cloned and sequenced. A comparison of both sequences to prototypic HIV-2 and SIV isolates revealed a close relationship to HIV-2ST. This strain originated from an asymptomatic Senegalese individual and is supposed to be of reduced pathogenicity. Taking into account genetic data, it may be assumed that HIV-2HOM and HIV-2ST are closely related strains with different growth characteristics and pathogenic features.

Base Sequence↗
PubMed1993

Genotoxicity studies of benzofuran dioxetanes and epoxides with isolated DNA, bacteria and mammalian cells.

1,2-Dioxetanes, very reactive and high energy molecules, are involved as labile intermediates in dioxygenase-activated aerobic metabolism and in physiological processes. Various toxicological tests reveal that dioxetanes are indeed genotoxic. In supercoiled DNA of bacteriophage PM2 they induce endonuclease-sensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, formamidopyrimidines). Pyrimidine dimers and sites of base loss (AP sites) which were probed by UV endonuclease and exonuclease III are minor lesions in this system. While the alkyl-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetranes such any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S. typhimurium strain TA100. DNA adducts formed with an intermediary alkylating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane. We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter, since benzofuran epoxides are highly mutagenic in the S. typhimurium strain TA100 and they form DNA adducts, as detected by the 32P-postlabelling technique. Our results imply that the type of DNA damage promoted by dioxetanes is dependent on the structural feature of dioxetanes. Furthermore, the direct photochemical DNA damage by energy transfer, i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes. Instead, photooxidation dominates in isolated DNA, while radical damage and alkylation prevail in the cellular system.

Alkylating Agents↗
PubMed1993

Human endogenous retroviral element K10 (HERV-K10) encodes a full-length gag homologous 73-kDa protein and a functional protease.

The gag-homologous region of the human endogenous retrovirus K10 (HERV-K10) was amplified by PCR from human genomic DNA and was analyzed by DNA cloning, sequencing, and expression of open reading frames in the prokaryotic pATH expression system. The analysis of genomic DNA of three donors provided evidence that HERV-K10 genes contain an open reading frame of 1966 bp spanning the entire gag-homologous region. In the prokaryotic system the entire reading frame of the HERV-K10 gag gene could be expressed as a fusion protein exhibiting a molecular weight of about 110,000. In addition, when the gag-homologous region and the adjacent HERV-K10 protease gene were prokaryotically expressed, we observed a Gag-protease fusion protein that exhibited specific autoproteolytic activities and processing of HERV-K10 Gag protein. By introducing deletions on the right end of the putative protease gene an autocatalytic site could be localized within 300 bp of the putative HERV-K10 protease gene. For the first time, these results provide evidence that the HERV-K10 encodes a full-length Gag protein and a functional protease.

Base Sequence↗
PubMed1993

The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein.

The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 microM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-1. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 microM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.

Amino Acid Sequence↗
PubMed1993

Microbial metabolism of quinoline and related compounds. XX. Quinaldic acid 4-oxidoreductase from Pseudomonas sp. AK-2 compared to other procaryotic molybdenum-containing hydroxylases.

Quinaldic acid 4-oxidoreductase from Pseudomonas sp. AK-2 catalyses the hydroxylation of quinoline 2-carboxylic (quinaldic acid) to 4-hydroxyquinoline 2-carboxylic acid (kynurenic acid) with concomitant reduction of a suitable electron acceptor. An analogous hydroxylation in para-position relative to the N-heteroatom was only recently described for quinaldine 4-oxidoreductase (de Beyer & Lingens, 1993, Biol. Chem. Hoppe-Seyler 374, 101-110) and for quinaldic acid 4-oxidoreductase from Serratia marcescens 2CC-1 (Fetzner & Lingens, 1993, Biol. Chem. Hoppe-Seyler 374, 363-376). Quinaldic acid 4-oxidoreductase from Pseudomonas putida AK-2 was purified 78-fold to electrophoretic homogeneity with a recovery of 22%. The native enzyme (300 kDa) was composed of three subunits with molecular masses of 90, 34 and 20 kDa, indicating an alpha 2 beta 2 gamma 2 structure. Quinaldic acid 4-oxidoreductase contained FAD, molybdenum, iron and acid-labile sulfur in a ratio of 2:2:8:8. Molybdenum is probably associated with molybdopterin cytosine dinucleotide as organic part of the pterin molybdenum cofactor. The absorption spectrum of quinaldic acid 4-oxido-reductase exhibited the typical features of a molybdo-iron/sulfur-flavoprotein, namely, maxima at 274 nm, 340 nm and 450 nm, a shoulder at 550 nm, a ratio A280/A450 of 4.7 and a ratio A450/A550 of 3.5. The enzyme was susceptible to inactivation by methanol, sodium m-arsenite, p-hydroxymercuribenzoate, and potassium cyanide. Cyanide caused an alteration at 320 nm in the absorption spectrum, typical for the change in the coordination sphere of the molybdenum. Enzyme inactivated with cyanide was reactivated to 74% by incubation with sulfide. Thus, quinaldic acid 4-oxidoreductase possesses a monooxo-monosulfido-type molybdenum center.

Catalysis↗
PubMed1992

Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli.

In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.

Amino Acid Sequence↗
PubMed1991

Immunological characterization of the Epstein-Barr virus phosphoprotein PP58 and deoxyribonuclease expressed in the baculovirus expression system.

The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells. The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E. coli. DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities. Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58. The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E. coli. The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, B95-8, M-ABA and BL74 induced by TPA and n-butyrate. The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0). Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera. Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC. In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.

Animals↗
PubMed1990

Flow cytometric analysis of Epstein-Barr virus (EBV) latent membrane protein expression in EBV-infected Raji cells.

In cultures of the latently Epstein-Barr virus (EBV)-infected Burkitt's lymphoma cell line Raji, the detectable amount of the EBV-encoded latent membrane protein (LMP) is transiently increased after addition of fresh nutrient medium containing foetal calf serum. In the current study, the relative amount of LMP and DNA in Raji cells was determined by biparametric flow cytometry analysis at different times after the addition of fresh medium with 10% foetal calf serum to a dense Raji culture. A transient increase in the proportion of LMP-positive cells was observed during the lag phase of the culture. Subsequently, a subpopulation of cells, which had been arrested in the G0 or G1 phase, simultaneously started to progress through the cell cycle. Neither the amount of LMP in the cells, nor the enhanced expression of LMP, was restricted to a certain phase of the cell cycle. Further analysis revealed that the number of LMP-positive cells proceeding simultaneously from the G1 to the S phase of the cell cycle is about the same as the total number of cells changing phases. These results suggest that LMP expression might be one step in the pathway leading to growth activation of resting cells in cultures of the immortalized Raji cell line.

Antigens, Viral↗
PubMed1990

Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenlyase components.

An 8kb segment of DNA from the 58/59 min region of the E. coli chromosome, which complements the defect of a mutant devoid of hydrogenase 3 activity, has been sequenced. Eight open reading frames were identified which are arranged in a transcriptional unit; all open reading frames were transcribed and translated in vivo in a T7 promoter/polymerase system. Analysis of the amino acid sequences derived from the nucleic acid sequences revealed that one of them, open reading frame 5 (ORF5), exhibits significant sequence similarity to conserved regions of the large subunit from Ni/Fe hydrogenases. Two of the open reading frames (orf2, orf6) code for proteins apparently carrying iron-sulphur clusters of the 4Fe/4S ferredoxin type. The product of one of the open reading frames, orf7, displays extensive sequence similarity with protein G from the chloroplast electron transport chain. ORF3 and ORF4, on the other hand, are extremely hydrophobic proteins with nine and six putative transmembrane helices, respectively. Over a limited hydrophilic sequence stretch, bordered by putative transmembrane areas, ORF3 and ORF4 exhibit homology with subunits 4 and 1 of mitochondrial and plastid NADH-ubiquinol oxidoreductases, respectively. The operon described, therefore, appears to comprise genes for redox carriers linking formate oxidation to proton reduction and for a hydrogenase of hitherto unique composition.

Aldehyde Oxidoreductases↗
PubMed1990

Transcriptional analysis of the gene encoding pyruvate formate-lyase-activating enzyme of Escherichia coli.

The act gene of Escherichia coli encodes the pyruvate formate-lyase-activating enzyme which is necessary for the post-translational modification of pyruvate formate-lyase. The gene is located 191 bp downstream from the pfl structural gene. Northern blot analysis revealed that the act transcript is monocistronic and that transcription is independent of pfl gene expression. Through mapping of the 5' and 3' ends of the act transcript, sequences could be identified showing similarity to both an Escherichia coli sigma 70 promoter and to a rho-independent transcription terminator. Expression of the act gene was analysed with the aid of chromosomally integrated transcriptional and translational lacZ fusions. The results verified that the act gene is transcribed from its own promoter and that expression of the gene is essentially constitutive. Anaerobiosis led only to a two-fold increase in expression over that observed in aerobically grown cells and this elevated expression was independent of the transcriptional regulator, Fnr. Moreover, effectors such as pyruvate and nitrate, which substantially influence anaerobic transcription of the pfl gene, did not affect act gene expression.

Acetyltransferases↗
PubMed1989

[In vitro studies on the quality of proximal margins of Cermet fillings in posterior teeth].

Since Cerment-cement has been recommended also for Black's Class II cavities in posterior teeth, it was the goal of this study to compare its marginal adaptation with that of amalgam after alternating thermal loads. Black's Class II cavities were prepared in extracted premolar teeth and filled with amalgam or Cermet. Three different application techniques were used for the Cermet fillings: 1. syringe only, 2. syringe and amalgam plugger, 3. individual occlusal plugger. Before and after alternating thermal loads the proximal margins were studied by SEM using replicas. The poorest results were found in the Cermet fillings applied with the syringe only. In the other three groups the results were comparable. These findings suggest that cermet cement should not be applied with exclusive use of the syringe, but should always be additionally adapted with a plugger.

Bicuspid↗
PubMed1988

Characterization of a latent protein encoded by the large internal repeats and the BamHI Y fragment of the Epstein-Barr virus (EBV) genome.

Analysis of EBV nuclear antigen 1 (EBNA 1) encoding transcripts by cDNA characterization revealed a potentially polycistronic message generated by long-range splicing of several exons (Speck, S., and Strominger, J., Proc. Natl. Acad. Sci. USA 82, 8305-8309, 1985). Besides the open reading frame encoding EBNA 1, two other open reading frames are found in the EBNA 1-specific cDNA. The first reading frame consists of several exons from BamHI W and Y viral genome fragments (W1, W2, Y1, and Y2). In our experiments, the W1 exon was expressed in the tryptophan-regulated expression vector pATH11. Rabbit sera, raised against the bacterial fusion protein, recognized one or two proteins of molecular weights between 30,000 and 100,000 in several EBV genome harboring Burkitt lymphoma and EBV immortalized peripheral blood cell lines. Although, in a few cell lines from both groups no specific protein could be detected. Immunofluorescence analysis and characterization of subcellular distribution demonstrated that this W/Y fragment encoded latent protein is located, in part, in the cytoskeleton fraction, and in the chromatin. In addition, 2-D immunoblot analysis revealed post-translational modifications of this latent protein, probably due to phosphorylation. In DNA-binding studies on DNA cellulose columns, this W/Y encoded latent protein exhibited specific DNA binding activities.

Amino Acid Sequence↗