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Biomedical subjects

P Hübner

Publications and source records attributed to P Hübner.

At least 19 recordsLinked to original sources
PubMed2000

Outbreak of viral gastroenteritis due to sewage-contaminated drinking water.

In August 1998, a large outbreak of gastroenteritis occurred in a Swiss village of 3500 inhabitants whereof more than 50% were affected. A high contamination of drinking water with faecal coliforms revealed a defect in the waste water system. The objective of the present study was to investigate the outbreak in respect of the presence of human pathogenic viruses. Drinking water and clinical samples from patients were examined for the presence of 'Norwalk-like viruses' (NLVs) and enteroviruses. NLVs and enteroviruses were detected by reverse transcription-polymerase chain reaction (RT-PCR) in one of two drinking water samples. Five of seven stool samples from ill persons were positive for NLVs. Typing of NLV-specific RT-PCR products by DNA sequencing revealed the presence of an identical genogroup-1 strain closely related to Southampton virus in both the water and one of the stool samples. A genogroup-2 NLV strain was identified in all positive stool samples. The enteroviral amplicon showed high sequence similarity with swine vesicular disease virus. These results demonstrate that the drinking water was highly contaminated with enteric viruses and that at least two NLV strains were involved in this outbreak of gastroenteritis.

Disease Outbreaks↗
PubMed1999

Study of the presence of Campylobacter jejuni and C. coli in sand samples from four Swiss chicken farms.

Chicken farms are frequently infected with Campylobacter jejuni and Campylobacter coli. The objective of the present study was to investigate environmental samples from chicken farms for the presence of C. jejuni and C. coli. Every week between July and November 1997, three sand samples from the runs of four chicken farms were analyzed by culture and directly by polymerase chain reaction (PCR). These two detection methods were compared to each other. A total of 231 samples were tested. Eleven samples (4%) were found to contain Campylobacter cells by culture, whereas 157 samples (68%) were positive by PCR. All samples which were positive by culture were also positive by PCR. All direct PCR products were further typed by restriction fragment length polymorphism (RFLP). Three different RFLP types and mixtures of these types were observed. Direct PCR products of one chicken farm were further typed by direct sequencing and two temporally separated sequence types could be distinguished. Campylobacter strains isolated by culture were also typed by RFLP and direct sequencing revealing close accordance with the corresponding direct PCR products.

Animal Husbandry↗
PubMed1999

PCR-RFLP analysis of mitochondrial DNA: a reliable method for species identification.

A method for identification of game species has been developed on the basis of the amplification of a specific part of the mitochondrial genome (tRNA(Glu)/cytochrome b) using the polymerase chain reaction (PCR). To distinguish between several game species, the obtained 464-bp-long PCR products were cut with different restriction endonucleases (RE) resulting in species-specific restriction fragment length polymorphism (RFLP). Even closely related deer species could be distinguished by application of one or two RE. Natural polymorphisms of the target sequence within one species were examined for red deer (Cervus elaphus), and base pair substitutions were identified affecting the RFLP pattern.

Animals↗
PubMed1998

PCR-based detection of verotoxin-producing Escherichia coli (VTEC) in ground beef.

Pathogenic strains of Escherichia coli producing verotoxins (VTs) have been recognized as a cause of human disease, and rapid and sensitive detection tests are urgently needed to ensure the safety of food, especially ground beef. We applied two nested polymerase chain reaction (PCR) assays to detect the genes encoding VT1 and VT2 irrespective of the bacterial serotype. In combination with a direct sample preparation protocol, we were able to uncover the presence of about 110 CFU of verotoxinogenic E. coli (VTEC) in 10 g of ground beef. When a six-hour enrichment was included, we found the detection limit to be in the range of 1 to 10 bacterial cells per 10 g of ground beef. To evaluate our detection system, we tested 30 ground beef samples originating from butcher shops in Berne, Switzerland. One sample yielded positive PCR results for both the VT1 and VT2 genes, indicating the presence of verotoxinogenic E. coli. Finally, 20 food homogenates, shown to contain E. coli strains by standard culture, were analysed with our method, and the gene encoding VT2 was detected in one cheese sample. The results suggest that the described PCR method can serve as a valuable tool for the surveillance of VTEC contamination of foods.

Animals↗
PubMed1997

Three-step isolation method for sensitive detection of enterovirus, rotavirus, hepatitis A virus, and small round structured viruses in water samples.

Control of drinking or bathing water quality in respect to viral contamination remains an unsolved problem. A highly sensitive isolation protocol was developed for concentration and detection of different enteric viruses from water samples. The three-step isolation procedure combines filtration with a positively charged nylon membrane, ultrafiltration and clean-up of the viral RNA with a silica based membrane. Detection of the viral RNA is accomplished by reverse-transcription polymerase chain reaction (RT-PCR). Detection limits were determined to be one 50% tissue culture infective dose (TCID50) of seeded coxsackievirus B2 or hepatitis A Virus per litre of tap water by RT-PCR compared to two orders of magnitude lower sensitivity for culture in the case of coxsackievirus B2. The isolation procedure is highly sensitive, easy to perform and allows the detection of different human pathogenic virus groups in one water sample. The application of the isolation procedure to six river water samples and subsequent detection with nested or semi-nested PCR revealed enterovirus in 6/6 (100%), rotavirus in 6/6 (100%), hepatitis A virus in 0/6 (0%), small round structured virus genotype I in 6/6 (100%) and small round structured virus genotype II in 2/6 (33%) of the samples. These findings suggest that first, we have developed a very sensitive detection procedure and second, that river water in Switzerland-where most of the wastewater is handled by sewage treatment plants-shows a high contamination rate with enteric viruses.

Base Sequence↗
PubMed1997

Seminested RT-PCR systems for small round structured viruses and detection of enteric viruses in seafood.

Highly sensitive seminested RT-PCR systems for the specific detection of genotype I and II small round structured viruses (SRSVs) were developed based on the nucleic acid information deposited in the databanks. SRSVs could be detected in 10(7)-fold dilutions of three different stool samples. In addition, a rapid and simple purification protocol for enteric viruses from seafood tissues was elaborated using poliovirus (PV) as model. The virus isolation and viral RNA purification include the following steps: elution of the viruses from the seafood tissue with glycine buffer, their concentration by PEG-precipitation, lysis of viral particles with guanidine hydrochloride and viral RNA isolation using a silica based membrane. The detection limit was 3 to 30 TCID50 of poliovirus in 1.25 g of seeded seafood tissues without marked food matrix differences, whereas SRSV viruses were 10- and 100-fold better detected in mussels than in shrimps and oysters, respectively. The newly developed purification method, which was shown to remove potential RT-PCR inhibitors present in mussel tissue samples, was applied in a small market survey. 15 mussels, 15 oysters and 12 shrimps were examined for the presence of Hepatitis A virus (HAV), Enterovirus (EV), Rotavirus (RV) and SRSV using specific RT-PCR detection systems. The finding of three oyster samples positive for Rotavirus demonstrated the successful application of our method for the detection of enteric viruses in naturally contaminated seafood samples. The rapid isolation method might be suitable for application in routine testing laboratories and will help to improve public health controls for seafood.

Base Sequence↗
PubMed1997

Genotoxicity of ethyl carbamate (urethane) in Salmonella, yeast and human lymphoblastoid cells.

Ethyl carbamate is a known carcinogen occurring in fermented food and beverages and is therefore of interest for food safety assurance. We studied the genotoxicity of ethyl carbamate in Salmonella typhimurium, in Saccharomyces cerevisiae and in human lymphoblastoid TK6 cells. In absence of cytochrome P450 enzymes, no ethyl carbamate-mediated genotoxicity was observed in any of the three test systems in the non-cytotoxic range. In the presence of an activating system, ethyl carbamate was found to be mutagenic in Salmonella typhimurium strain TA100 but not in strains TA98 and TA102, indicating base-pair substitutions at G-C base pairs. In contrast, no significant mutagenicity of ethyl carbamate could be detected in human lymphoblastoid TK6 cells. However, applied in cytotoxic concentrations, ethyl carbamate was genotoxic for Saccharomyces cerevisiae in the absence of P450-mediated metabolic activation. Inhibitors of P450IIE1 (DMSO, ethanol and dithiodiethylcarbamate) diminished ethyl carbamate-mediated mutagenicity in Salmonella typhimurium strain TA100 in a dose dependent manner, suggesting that P450IIE1 is the activating enzyme.

Antineoplastic Agents↗
PubMed1996

Polymerase chain reaction (PCR) in the quality and safety assurance of food: detection of soya in processed meat products.

A new method for the specific and sensitive detection of soya (Glycine max) in processed meat products has been developed using polymerase chain reaction (PCR) technology. The presence of soya deoxyribonucleic acid (DNA) from several soya protein concentrates was determined with two pairs of specific oligonucleotides yielding a 414-bp (bp = base pair) fragment and an internal 118-bp fragment amplified from the soya lectin Le1 gene. The test detected DNA from textured soya protein concentrates in meat products at a level of 1% and was confirmed by a commercially available enzyme-linked immunosorbent assay (ELISA).

Animals↗
PubMed1996

Molecular analysis of the Rhodobacter capsulatus chaperonin (groESL) operon: purification and characterization of Cpn60.

The heat-shock protein Cpn60 (chaperonin, GroEL homologue) from the phototrophic bacterium Rhodobacter capsulatus B10 was purified to homogeneity and biochemically characterized. Native Cpn60 from R. capsulatus was shown to be a tetradecamer of 840 kDa similar to that of homologous chaperones characterized so far. Cpn60 possesses ATPase activity and promotes refolding of chaotropically denatured citrate synthase. The groESL operon of R. capsulatus was cloned using a degenerate oligonucleotide and sequenced. Two open reading frames (285 and 1,635 bp) were found; they encode Cpn10 and Cpn60, with corresponding deduced molecular masses of 10.6 and 57.6 kDa. The deduced amino acid sequences coincided perfectly with those of the amino terminus and of three tryptic peptides of purified Cpn60 from R. capsulatus. Strong evidence that R. capsulatus encodes only one copy of the groESL operon was obtained. Primer-extension analysis revealed that the groESL operon is transcribed by a -35/-10-type promoter, and that transcription was initiated from the same positions before and after heat-shock under both chemotrophic and phototrophic conditions. The major initiation site is immediately followed by the inverted repeat structure CIRCE, which has been found upstream of many bacterial heat-shock operons. A second minor transcript starts just after the CIRCE element. Although heat-shock induction of a groEL-lacZ fusion failed because of thermal inactivation of the fusion protein, Western blot analysis revealed a two- to threefold induction of cellular Cpn60 levels 45-75 min after shifting from 28 degrees C to 39 degrees C. Deletion mapping of the groESL promoter identified upstream of the promoter a 19-bp element that enhances groESL transcription eightfold and contains the AT-rich sequence dAAATTTTT, which is found at similar positions in heat-shock operons of other gram-negative bacteria.

Adenosine Triphosphatases↗
PubMed1996

Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus.

The Rhodobacter capsulatus nifF gene and upstream sequence were cloned by using a probe based on the N-terminal sequence of NifF. nifF was found to not be contained in the previously described nif regions I, II, and III. Comparison of the deduced amino acid sequence showed that it is highly similar to NifF from Azotobacter vinelandii and NifF from Klebsiella pneumoniae. Analysis of translational fusions demonstrated that the regulation of transcription was the same as previously reported at the protein level. Insertional mutagen esis showed that NifF contributes significantly to nitrogenase activity under normal nitrogen-fixing conditions and that it is absolutely required for nitrogen fixation under iron limitation.

Amino Acid Sequence↗
PubMed1993

Sequence and transcript analysis of the nitrogenase structural gene operon (nifHDK) of Rhodobacter capsulatus: evidence for intramolecular processing of nifHDK mRNA.

Northern blot analysis of RNA prepared from cells of Rhodobacter capsulatus derepressed for nitrogenase (N2ase) synthesis, using a 6.0-kb DNA probe containing the entire nifHDK operon, revealed the presence of at least six hybridizing species of the estimated sizes, 4.4, 3.5, 2.7, 1.3, 0.9 and 0.38 kb. No hybridization was detected with RNA prepared from cells grown in the presence of an excess of NH4+, which represses N2ase synthesis. Hybridization with gene-specific probes revealed that the 4.4-kb species hybridized with all three probes, and presumably corresponded to the full-length nifHDK transcript, whereas the 3.5-kb species hybridized with nifD and nifK only, and the 2.7-kb transcript hybridized with nifH and nifD. The 1.3 and 0.9-kb species hybridized with all three probes, but appeared to hybridize most strongly with nifH. In contrast, the 0.38-kb species hybridized with none of the gene-specific probes, and was also detected in RNA from cells of strain RcM1, which contains a chromosomal deletion of the nifHDK operon. This species probably corresponds to the transcript of a gene, named fdxD, which was found to be located just upstream from the nifHDK operon. Nucleotide (nt) sequencing of the nifH-D and nifD-K intergenic regions revealed the presence of inverted repeat (IR) sequences potentially capable of forming stable stem-loop structures in mRNA. Primer extension analysis of the nifDK-homologous species showed that the 5' end was located one or two nt downstream from the IR sequence between nifH and nifD, suggesting that the putative stem-loop structure may be a target for intramolecular processing of the nifHDK mRNA.

Amino Acid Sequence↗
PubMed1993

Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5' half site of the wild type site is repeated in inverse orientation. The additional base pair in the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain which recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.

Base Sequence↗
PubMed1993

Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifUI SVW gene region: possible role of NifW in homocitrate processing.

DNA sequence analysis of a 3494-bp HindIII-BclI fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifUI and NifUII share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneumoniae. In contrast to nifA and nifB, which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifUISVW operon, which is preceded by a putative sigma 54-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifUI and nifW mutants as well as a nifUI/nifUII double mutant exhibited a Nif+ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV-NifW-), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase.

Amino Acid Sequence↗
PubMed1993

nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations.

The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif-specific alternative sigma factor of RNA polymerase and a nif-specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH-, rpoN-, nifA1- and nifA2- lacZ fusions were constructed and the expression of these different nif-lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifA1 expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of nif gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoN was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R. capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 over-expressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.

Bacterial Proteins↗
PubMed1992

Mapping and characterization of the promoter elements of the regulatory nif genes rpoN, nifA1 and nifA2 in Rhodobacter capsulatus.

The promoter elements responsible for the expression of the regulatory nif genes rpoN, nifA1 and nifA2 of Rhodobacter capsulatus were mapped by exonuclease-III-mediated deletions and by primer extension analysis. The rpoN promoter maps 600 bp upstream of rpoN and has the characteristic features of a -24/-12 promoter. The upstream activator sequence (UAS) displays two mismatches with the NIFA consensus sequence and is located 37 bp upstream of a perfect -24/-12 promoter element. The spacing and/or the helical phasing of these two promotor elements was found to be important for promoter function. In addition, an UAS half-site may contribute to optimal promoter function. The rpoN UAS can partially substitute for the UAS of the nifE promoter. An open reading frame with homology to Klebsiella pneumoniae NIFU was identified between the rpoN promoter and rpoN and termed nifU2 since another nifU-like gene (nifU1) is located in a conventional nifUSVW operon in nif region A. Thus, rpoN, encoding an alternative sigma factor for RNA polymerase, is cotranscribed with a nifU analogous gene from an rpoN-dependent promoter. Mapping of the promoter elements involved in the expression of nifA copy 1 and copy 2 identified a novel promoter type. A conserved distal promoter element is likely to represent the binding site of NTRC in R. capsulatus. The DNA region preceding the mapped 5' ends of the nifA transcripts displays much less homology. The distance between the distal and proximal elements is about 100 bp.

Amino Acid Sequence↗