Biomedical subjects

F Götz

Publications and source records attributed to F Götz.

At least 109 records · Page 6Linked to original sources

PubMed1994

Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine.

The Staphylococcus hyicus lipase is an acyl hydrolase with broad substrate specificity including neutral glycerides and phospholipids. To obtain further insight into the mechanism of action of this enzyme, we tested several sulfonyl fluorides as active site-directed inhibitors. The enzyme is resistant to the well-known serine protease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF), but is rapidly inactivated by hexadecylsulfonyl fluoride. The kinetics of inactivation were studied in Triton X-100 micelles. Inactivation is fast and the rate of inactivation is constant over the pH range where this lipase is active. Metal ions like Ca2+ and Sr2+ do not appreciably influence the rate of inactivation, although the enzymatic activity is significantly increased, suggesting a structural role for these ions. The S. hyicus lipase contains a consensus sequence G-H/Y-S-X-G. Substitution by site-directed mutagenesis of this serine (Ser369) by a cysteine resulted in a mutant with only 0.2% residual activity. The activity of this mutant could not be inhibited with water-soluble sulfhydryl reagents either in the presence or absence of Triton X-100 micelles. In the presence of Triton X-100 micelles, inactivation of the mutant occurred with 4-nitrophenylhexadecyl disulfide (t1/2 = 125 min) while the wild-type enzyme does not react at all. We conclude that Ser369 is the active site residue and that in water this residue is inaccessible. Only after interfacial activation Ser369 (or Cys369) becomes exposed and reacts with irreversible inhibitors.

Bacterial Proteins↗

PubMed1994

Biochemical properties of a novel metalloprotease from Staphylococcus hyicus subsp. hyicus involved in extracellular lipase processing.

Two extracellular proteases from Staphylococcus hyicus subsp. hyicus, ShpI and ShpII, have been characterized. ShpI is a neutral metalloprotease with broad substrate specificity; the gene has been cloned and sequenced. ShpII, characterized here, is mainly produced in the late logarithmic growth phase in contrast to ShpI, which is mainly produced in the late stationary growth phase. ShpII was purified from culture medium of S. hyicus by ammonium sulfate precipitation and DEAE-Sepharose chromatography. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 34 kDa. The temperature optimum of ShpII was 55 degrees C, and the pH optimum was 7.4. ShpII, a neutral metalloprotease, was strongly inhibited by zinc and calcium chelators. The amino-terminal sequence of the active enzyme was similar to the corresponding region of a Staphylococcus epidermidis metalloprotease. The substrate specificity of ShpII was similar to that of thermolysin-like proteases, with the exception that ShpII also recognized aromatic amino acids. We demonstrated in vitro that ShpII, but not ShpI, cleaved the 86-kDa S. hyicus subsp. hyicus prolipase between Thr-245 and Val-246 to generate the mature 46-kDa lipase. Results of additional in vivo experiments supported the model that ShpII is necessary for the extracellular processing and maturation of S. hyicus subsp. hyicus lipase.

Amino Acid Sequence↗

PubMed1994

Genetic and biochemical analyses of the biosynthesis of the yellow carotenoid 4,4'-diaponeurosporene of Staphylococcus aureus.

The major pigment produced by Staphylococcus aureus Newman is the deep-yellow carotenoid 4,4'-diaponeurosporene; after prolonged cultivation, this pigment is in part converted to the orange end product staphyloxanthin. From this strain a 3.5-kb DNA fragment was identified which after being cloned into Escherichia coli and Staphylococcus carnosus conferred the ability to produce 4,4'-diaponeurosporene. DNA sequencing of this fragment revealed two open reading frames (ORFs) which are very likely cotranscribed. ORF1 encodes a 254-amino-acid hydrophobic protein, CrtM (M(r), 30,121). The deduced sequence of CrtM exhibits in three domains similarities to the sequences of Saccharomyces cerevisiae and human squalene synthases and phytoene synthases of various bacteria. ORF2 encodes a 448-amino-acid hydrophobic protein, CrtN, with an M(r) of 50,853 whose deduced sequence is similar to those of phytoene desaturases of other bacteria. At the N terminus of CrtN a classical FAD-, NAD(P)-binding domain is found. Spectrophotometry and gas chromatography-mass spectrometry analyses of the carotenoid production of E. coli and S. carnosus clones containing either ORF1 or both ORFs together suggest that ORF1 and ORF2 represent the dehydrosqualene synthase gene (crtM) and the dehydrosqualene desaturase gene (crtN), respectively. The results furthermore suggest that the biosynthesis of 4,4'-diaponeurosporene starts with the condensation of two molecules of farnesyl diphosphate by dehydrosqualene synthase (CrtM); it is shown that the reaction product of this enzyme is dehydrosqualene and not squalene. Dehydrosqualene (4,4'-diapophytoene) is successively dehydrogenated by a desaturase (CrtN) to form the yellow main intermediate 4,4'-diaponeurosporene.

Amino Acid Sequence↗

PubMed1994

[Laparoscopic suture and knot techniques].

Laparoscopic suture and knot techniques are not yet considered standard laparoscopic procedures. In conventional surgery, it would be unthinkable that a procedure would be attempted without a good working knowledge of suture and knot techniques. In laparoscopic operations, limited mobility, the lever arm through the instruments and especially the conversion of 2D vision to 3D actions and the lack of a direct tactile sense all have limiting effect. The needle holders available leave room for improvement. The initially protracted operating times become shorter with training and a period of adjustment.

Humans↗

PubMed1993

Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis.

For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R-1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A(-4)-E-P-R(-1)- to -A(-4)-E-P-Q(-1)- by site-directed mutagenesis.

Amino Acid Sequence↗

PubMed1993

Expression of bacteriophage PhiX174 lysis gene E in Staphylococcus carnosus TM300.

Expression of the cloned PhiX174 gene E causes lysis of the Gram-negative bacterium Escherichia coli, which led to the proposal that a two-membrane system is necessary for the protein E lysis function. Gene E was cloned in an E. coli/Bacillus subtilis shuttle vector and expressed in the Gram-positive bacterium Staphylococcus carnosus TM300. Regulated gene E expression had a lethal effect on S. carnosus; however, no lysis was detected, lending support to the hypothesis.

Bacteriophage phi X 174↗

PubMed1993

Cloning and characterization of the scrA gene encoding the sucrose-specific Enzyme II of the phosphotransferase system from Staphylococcus xylosus.

By insertional mutagenesis with the staphylococcal transposon Tn551, mutants of Staphylococcus xylosus were isolated that were unable to utilize sucrose. One of these was found to be deficient in sucrose uptake. The genomic region containing this sucrose uptake gene of Staphylococcus xylosus (scrA) was cloned in Staphylococcus carnosus. The scrA gene was further localized to a 4.4 kb DNA fragment by complementation of the sucrose transport-deficient S. xylosus mutant. The DNA sequence analysis of the scrA region revealed three open reading frames, one of which encodes a protein of 480 amino acids (51.335 kDa) with significant similarity to sucrose-specific Enzymes II of phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems (PTS). A protein with an apparent molecular weight of 50 kDa was obtained in Escherichia coli by expression of scrA with the bacteriophage T7 RNA polymerase promoter system. Transcriptional start sites of the scrA gene were localized by primer extension analysis to positions 46 and 49 nucleotides upstream of the scrA start codon. No additional sucrose utilization genes are encoded close to scrA on the S. xylosus chromosome.

Amino Acid Sequence↗

PubMed1993

Regulation of epidermin biosynthetic genes by EpiQ.

We investigated the role of epiQ in the biosynthesis of the lantibiotic epidermin. epiQ was essential for epidermin production. It was shown that EpiQ controls epidermin production by transcriptionally activating the epiA promoter, used for transcription of most of the epidermin biosynthetic genes. Additional copies of epiQ increased epidermin production in the epidermin-producing wild-type strain Staphylococcus epidermidis Tü3298. The epiA promoter region was characterized by primer extension analysis. Two inverted repeats, putative operator sites for EpiQ binding, are located upstream of the -35 region and one is localized downstream of the -10 region. Crude protein extracts from S. epidermidis Tü3298 and epiQ expressing Escherichia coli cells led to gel mobility shifts of a DNA fragment bearing the inverted repeat which is located immediately upstream of the -35 region. DNA fragments bearing the other two inverted repeats were not shifted. The epiQ gene product could be detected by overexpression in the E. coli T7 system using antiserum raised against synthetic peptides of EpiQ. Furthermore, EpiQ, like other DNA-binding proteins, was shown to bind strongly to heparin sepharose.

Amino Acid Sequence↗

PubMed1993

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis.

The gene sepA from Staphylococcus epidermidis TU3298-P, encoding the extracellular neutral metalloprotease SepP1, was cloned into pT181mcs. DNA sequencing revealed an open reading frame of 1,521 nucleotides encoding a 507-amino-acid protein with an M(r) of 55,819. The sepA-containing DNA fragment did not hybridize with Staphylococcus hyicus or Staphylococcus carnosus DNA. Expression of sepA in the protease-negative S. carnosus (pT181mcsP1) resulted in overproduction of a 33-kDa protease found in the culture medium. The first 15 N-terminal amino acids of the partially purified protease completely matched the deduced DNA sequence starting at GCA (Ala-208). This finding indicated that SepP1 is synthesized as a preproenzyme with a 28-amino-acid signal peptide, a 179-amino-acid hydrophilic pro region, and a 300-amino-acid extracellular mature form with a calculated M(r) of 32,739. In activity staining, the mature protease prepared from S. carnosus (pT181mcsP1) corresponded to the extracellular S. epidermidis Tü3298-P protease. The partially purified protease had a pH optimum between 5 and 7, and its activity could be inhibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, indicating that it is a neutral metalloprotease. The protease had a low substrate specificity. Glucagon was cleaved preferentially between aromatic (Phe) and hydrophobic (Val) amino acids. The protease hydrolyzed casein and elastin. The amino acid sequence of the mature form of SepP1 revealed pronounced similarities with the thermolabile and thermostable neutral proteases of various bacilli (44 to 55% identity) and a central part of the mature form of the Pseudomonas aeruginosa elastase (31% identity). From homology comparison with the Bacillus thermoproteolyticus thermolysin, we predict that mature SepP1 binds one zinc ion at a conserved zinc-binding site.

Amino Acid Sequence↗

PubMed1993

Cloning, sequencing, and characterization of the gene encoding the class I fructose-1,6-bisphosphate aldolase of Staphylococcus carnosus.

fda from Staphylococcus carnosus TM300, encoding the class I fructose-1,6-bisphosphate aldolase, was cloned in Escherichia coli and sequenced. The 888-nucleotide open reading frame encoding a protein with an M(r) of 32,855 had an E. coli-like promoter sequence. Plasmids containing fda complemented E. coli NP315 (Fda-). Expression of fda in S. carnosus led to a six- to eightfold increase in aldolase production and activity; low levels of glucose in the growth medium stimulated activity.

Amino Acid Sequence↗

PubMed1993

Characterization of a sucrase gene from Staphylococcus xylosus.

The Staphylococcus xylosus gene scrB, encoding a sucrase, has been isolated from a genomic library of S. xylosus constructed in Escherichia coli. The gene was detected by its ability to confer utilization of the glucose and fructose residues of raffinose in an E. coli strain that is not able to metabolize galactose. It was found to reside within a 1.8-kb DNA fragment, the nucleotide sequence of which was determined. One large open reading frame, which is preceded by a ribosome binding site, is encoded on the fragment. Its deduced amino acid sequence yields a protein with a molecular mass of 57.377 kDa which shows significant homology with bacterial sucrose-6-phosphate hydrolases and sucrases. Overexpression of scrB in E. coli by the bacteriophage T7 polymerase promoter system resulted in the production of a protein with an apparent molecular mass of 58 kDa. Disruption of the scrB gene in the S. xylosus genome rendered S. xylosus unable to utilize sucrose. Thus, the ScrB sucrase is essential for sucrose metabolism in S. xylosus.

Amino Acid Sequence↗

PubMed1993

Short- and long-term effects of perinatal interleukin-1 beta-application in rats.

Nervous, endocrine and immune systems are regarded as a complex functional unit, interacting by their specific chemical messengers-neurotransmitters, systemic hormones and hormone-like mediators of immune cells (cytokines). Cytokines are known to affect several endocrine axes. Interleukin-1 beta (IL) was administered in rats intraperitoneally twice daily from day 17 to 21 of pregnancy. Some of the IL-treated mothers were rapidly decapitated 1.5 h after the last injection. The fetuses were delivered by cesarean section. Maternal plasma CRF, ACTH and corticosterone were found to be significantly elevated. Fetal adrenal and thymus weights were lower, and plasma corticosterone did not differ from controls. Fetal plasma testosterone was decreased in males, androstenedione was increased in females. Open-field testing revealed a higher total locomotor activity of IL offspring than of controls. IL offspring showed worse results in Skinner box learning than controls. Sexual behavior was only affected in males, showing a higher percentage of female-type lordosis behavior after castration and estrogen treatment compared to controls. At the age of 6 months responsiveness to 'novel-environment stress' of IL groups was significantly lower than that of controls in terms of plasma corticosterone. These results indicate that prenatal treatment with IL-1 beta results in long-lasting alterations in psychomotor development, behavior as well as in the neuroendocrine system.

Adrenal Glands↗

PubMed1992

Lipase of Staphylococcus hyicus: analysis of the catalytic triad by site-directed mutagenesis.

In this study the putative catalytic triad Ser-His-Asp of the Staphylococcus hyicus ssp. hyicus lipase was investigated. Putative catalytic sites determined by homology comparisons of three staphylococcal and other non-staphylococcal lipases were altered by site-directed mutagenesis. Since the mutations did not influence the secretion of the lipase, the decrease in lipase activity of the mutants strongly supports the proposed involvement of Ser369 and His600 in catalysis. Asp559 is postulated to be the third amino acid of the triad.

Amino Acid Sequence↗

PubMed1992

Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis.

Epidermin is produced by Staphylococcus epidermidis Tü3298 which harbors the 54-kb plasmid, pTü32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C, D, Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., Götz, F. & Entian, K.-D. (1992) Eur. J. Biochem. 204, 57-68]. We isolated a number of stable mutants from strain Tü3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT181mcs and pCU1 led to their classification as epiA, epiB, epiC or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co-transcribed from the epiA promoter. There is evidence that epiC and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S. carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D, and the DNA region comprising epiQ and P, host-encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8-kb DNA fragment of pTü32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of Gly10 by Glu.

Amino Acid Sequence↗

PubMed1992

Analysis of genes involved in the biosynthesis of lantibiotic epidermin.

The structural gene of the lanthionine-containing peptide antibiotic epidermin is located on a 54-kb plasmid of Staphylococcus epidermidis [Schnell et al. (1988) Nature 333, 276-278]. A 13.5-kb DNA region neighbouring the epidermin structural gene (epiA) was subcloned and its sequencing revealed five additional open reading frames. Three of these reading frames, epiB, epiC and epiD shared no homology with previously described proteins stored in data bases. They were located 3' adjacent to epiA. Using epiB as a probe, a 5-kb mRNA was identified indicating that three or all four reading frames are transcribed as an operon. Additionally, a 0.3-kb mRNA specific for epiA was identified. Two open reading frames (epiP and epiQ) were located 3' to epiA, epiB, epiC and epiD, but in the reverse orientation. The epiQ gene product shows similarity to the positive regulatory factor PhoB. This might indicate a regulatory function of epiQ in epidermin biosynthesis. The epiP gene product shows striking similarity to several serine proteases which makes epiP a likely candidate for processing the epidermin prepeptide. Heterologous epidermin synthesis in the non-producing organism Staphylococcus carnosus finally proved that these reading frames are necessary for epidermin biosynthesis.

Amino Acid Sequence↗

PubMed1992

Lifelong enhanced diabetes susceptibility and obesity after temporary intrahypothalamic hyperinsulinism during brain organization.

Newborn male Wistar-rats received bilateral intrahypothalamic insulin-agar-implants on the 2nd or 8th day of life. In male control animals only the insulin-free indifferent agar-vehicle was implanted at the same age. In both experimental groups with temporary intrahypothalamic hyperinsulinism during brain organization the following results were obtained: 1) Higher body weight gain starting at the end of the hypothalamic differentiation period and continuing during juvenile life until adulthood, resulting in increased relative body weight as a sign of obesity; 2) A tendency to basal hyperinsulinaemia in juvenile and adult age; 3) Impaired glucose tolerance in adulthood; 4) Increased diabetes susceptibility to a single "subdiabetogenic" dose of streptozotocin in adult age. In view of these and previous observations a teratogenetic role of high insulin concentrations during the organization of glucoregulatory hypothalamic structures is hypothesized and the possible relevance of such hyperinsulinism as a predisposing factor for a lifelong enhanced diabetes and/or obesity risk is suggested.

Animals↗

PubMed1992

Obesity and enhanced diabetes and cardiovascular risk in adult rats due to early postnatal overfeeding.

To investigate possible permanent consequences of an early postnatal overfeeding, the following experimental model was used: Male Wistar rats were divided into three groups after birth: (1) Small litters with 3-4 newborns (overnutrition), (2) normal litters with 12 animals (normonutrition), and (3) large litters with 20-24 newborn rats (undernutrition). After weaning all animals had free access to tap water and standard pellet diet. The serum insulin level of animals from small litters on day 15 of life was highly significantly increased as compared to the other groups. These overfed hyperinsulinaemic rats showed a higher body weight gain during the suckling period trough juvenile life until adulthood, associated with enhanced mean food intake and resulting in an increased relative body weight (per body length) as a sign of obesity. The obesity was found to be correlated with basal hyperinsulinaemia and increased systolic blood pressure in the small-litter-adults. Moreover, the early postnatally overnourished animals developed an increased type I-like diabetes susceptibility to a "subdiabetogenic" dose of streptozotocin in adulthood. These results suggest once more that hyperinsulinism during brain differentiation, in the present experiment induced by early postnatal overnutrition, may represent a predisposing factor for the development of obesity, of increased diabetes susceptibility and also of increased cardiovascular risk in later life.

Animals↗

PubMed1992

Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus xylosus plasmid pSX267.

The arsenate, arsenite, and antimonite resistance region of the Staphylococcus xylosus plasmid pSX267 was subcloned in Staphylococcus carnosus. The sequenced DNA region revealed three consecutive open reading frames, named arsR, arsB, and arsC. Expression studies in Escherichia coli with the bacteriophage T7 RNA polymerase-promoter system yielded three polypeptides with apparent molecular weights of 8,000, 35,000, and 15,000, which very likely correspond to ArsR, ArsB, and ArsC, respectively. ArsB was distinguished by its overall hydrophobic character, suggesting a membrane association. The arsenate, arsenite, and antimonite resistance was shown to be inducible by all three heavy metal ions. Inactivation of the first gene, arsR, resulted in constitutive expression of resistance. Similar results were obtained with transcriptional fusions of various portions of the ars genes with a lipase reporter gene, indicating a function of ArsR as a negative regulator of a putative promoter in front of arsR. The inactivation of arsR also resulted in reduction of resistance to arsenite and antimonite, while arsenate resistance was unaffected. The three ars genes conferred arsenite resistance in E. coli and arsenite as well as arsenate resistance in Bacillus subtilis.

Adenosine Triphosphatases↗