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Biomedical subjects

M Rohde

Publications and source records attributed to M Rohde.

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

Direct expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli and Salmonella typhimurium aroA.

Nonfused (i.e., nonhybrid) filamentous hemagglutinin (FHA) of Bordetella pertussis was efficiently expressed in Escherichia coli K-12 and Salmonella typhimurium aroA at levels higher than those found in wild-type B. pertussis when the upstream signals of the gene were replaced and the translation initiation region was engineered to optimize translational efficiency. Inclusion of part of the C-terminal FHA open reading frame, whose translation product does not appear to be part of the major secreted species of FHA, was shown to be important in achieving protein expression in both E. coli and S. typhimurium aroA; removal of the downstream gene sequence abolished recombinant FHA production. The levels of expression observed varied widely according to the construct and host bacterium used.

Adhesins, Bacterial↗
PubMed1991

Construction of minitransposons for constitutive and inducible expression of pertussis toxin in bvg-negative Bordetella bronchiseptica.

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations.

Bordetella bronchiseptica↗
PubMed1991

Production of recombinant Bordetella pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica: utility of Escherichia coli gene expression signals.

Serotype-specific fimbriae of Bordetella pertussis are considered potential components of new-generation vaccines against whooping cough. Attempts to characterize fimbriae, and indeed other virulence determinants, produced by B. pertussis have been frustrated on one hand by low yields from B. pertussis itself and on the other by an inability to produce native recombinant products in Escherichia coli. In order to try to circumvent this problem, we have examined the expression of B. pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica from native as well as E. coli expression signals. These studies revealed that the fimbrial gene product was expressed from the original B. pertussis promoter and Shine-Dalgarno sequence in both B. parapertussis and B. bronchiseptica. The transcriptional start site of the gene was located 146 nucleotides upstream of its ATG start codon. A recombinant fimbrial subunit gene containing PLAC and the atpE translation initiation region of E. coli was also expressed in B. bronchiseptica. In all cases in which gene expression was detected the gene product was expressed as serotype 2-specific fimbriae as determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopic investigation of the bacterial cell surface.

Base Sequence↗
PubMed1990

Immunocytochemical studies on the localization of 5-aminolevulinate synthase in rat liver.

The localization of 5-aminolevulinate synthase (ALAS) in hepatocytes of untreated and porphyrinogenic drug-treated rats has been examined by an immunocytochemical approach using a monoclonal antibody and protein A-gold labeling. Gold particles representing antigenic sites for ALAS were observed in the mitochondria and cytoplasm of untreated and drug-treated cells. Quantitative analysis of the labeling density showed that levels of ALAS increased significantly in both of these cellular compartments following drug treatment. Evidence that the detected cytoplasmic form of ALAS represents the precursor of the enzyme was obtained from immunoblotting experiments. The direct detection of cytosolic ALAS in vivo rules out the possibility that enzyme activity previously detected in the cytosol fraction resulted from mitochondrial leakage during cell fractionation. The results indicate that the cytosolic accumulation of ALAS is not a consequence of the inability of mitochondria to accommodate more enzyme. However, the molecular basis for this cytosolic accumulation is not known. The studies also established that the mitochondrial enzyme is predominantly, if not exclusively, associated with the matrix side of the inner mitochondrial membrane.

5-Aminolevulinate Synthetase↗
PubMed1990

Localization of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris using immunoelectron microscopic procedures.

The intracellular location of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris was determined using pre-embedding and post-embedding immunoelectron microscopic procedures. Polyclonal antisera directed against the purified (NiFe) and (NiFeSe) hydrogenases were raised in rabbits. One-day-old cultures of D. vulgaris, grown on a lactate/sulfate medium, were used for all experiments in these studies. For post-embedding labeling studies cells were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde, dehydrated with methanol, and embedded in the low-temperature resin Lowicryl K4M. Our post-embedding studies using antibody-gold or protein-A-gold as electron-dense markers revealed the location of the two hydrogenases exclusively at the cell periphery; the precise membrane location was then demonstrated by pre-embedding labeling. Spheroplasts were incubated with the polyclonal antisera against (NiFe) and (NiFeSe) hydrogenase followed by ferritin-linked secondary antibodies prior to embedding and sectioning. The observed labeling pattern unequivocally revealed that the antigenic reactive sites of the (NiFe) hydrogenase are located in the near vicinity of the cytoplasmic membrane facing into the periplasmic space, whereas the (NiFeSe) hydrogenase is associated with the cytoplasmic side of the cytoplasmic membrane.

Binding Sites↗
PubMed1990

Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures.

Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole-cell extracts. To engineer plasmids capable of directing the expression in E. coli of high levels of this product, both as a pre-protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda P(L) and P(R) promoters and the E. coli atpE translational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in several E. coli strains including DH5. However, they did in E. coli CAG629, which is lon protease and heat shock protein deficient. Both pre-protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface of E. coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionylated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with whole B. pertussis serotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild-type form of the fimbrial subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Bacterial↗
PubMed1990

[Evaluation of preoperative admission and anesthesia consultation in surgery].

Preoperative consultation with patients on their imminent hospitalisation for surgery and on aspects of necessary anaesthesia was introduced at the Lübben District Hospital for planned operations in 1977. Five categories of diseases which required general surgery have now been evaluated, in this context. That consultation, based on outpatient practice, has proved helpful in reducing the time of hospitalisation, disability, and pre-admission waiting periods. Surgical and bed capacities were more effectively utilised, and duplicated examinations were avoided. Patients were given a say in surgical timing.

Cholecystectomy↗
PubMed1989

Immunoelectron microscopic localization of the F1F0 ATPase (ATP synthase) on the cytoplasmic membrane of alkalophilic Bacillus firmus RAB.

Evidence that the F1F0 ATPase (ATP synthase) of alkalophilic Bacillus firmus RAB is localized exclusively on the cytoplasmic membrane was obtained by immunogold electron microscopy using a highly specific polyclonal antibody against the beta subunit of Escherichia coli F1F0 ATPase. The energetic problem faced by cells of B. firmus RAB growing oxidatively at pH 10.5 despite a low protonmotive force across the cytoplasmic membrane cannot, therefore, be circumvented by localization of energy transducing functions on hypothetical internal membranes.

Bacillus↗
PubMed1988

Pyruvate carboxylase from Pseudomonas citronellolis: shape of the enzyme, and localization of its prosthetic biotin group by electron microscopic affinity labeling.

Pseudomonas citronellolis is known to contain a pyruvate carboxylase with an alpha 4 beta 4 composition. All the other pyruvate carboxylases investigated so far are made up of four seemingly identical subunits. Nevertheless, this exceptional pyruvate carboxylase exhibits a size and overall shape similar to other pyruvate carboxylases. Electron microscopic affinity labeling with avidin revealed that the prosthetic biotin groups (one per alpha beta unit, i.e. four per enzyme particle) are located close to the inter-unit junctions of pairs of alpha beta units making up the enzyme. This position of the prosthetic biotin groups is very similar to the location of the biotin in the other carboxylases.

Biotin↗
PubMed1988

The methanoreductosome: a high-molecular-weight enzyme complex in the methanogenic bacterium strain Gö1 that contains components of the methylreductase system.

The methanogenic bacterium strain Gö1 harbors a high-molecular-weight enzyme complex containing methyl coenzyme M methylreductase as revealed by immunoelectron microscopy. This complex consists of a spherelike, hollow head piece, in the wall of which a number of copies of the methyl coenzyme M methylreductase are located. It is named Rc (c indicates collector). Intimately bound to it is a group of additional subunits of unknown composition referred to as Rm (m indicates mediator). Electron microscopy of negatively stained samples indicated that Rm contains a functional pore or channel which connects the internal volume of Rc with the outside. The RcRm complex is named Rs (s indicates spherelike). This complex was often found detached from the inside of the cytoplasmic membrane when membrane vesicles were investigated. However, Rs was also seen attached to a third component of the complex located in the membrane, the attachment being mediated by Rm. This membrane part of the complex is designated Rt (t indicates translocator). It consists of subunits with unknown composition. When Rs is attached to the membrane, the pore in Rm appears to be plugged by Rt. This indicates that the internal volume in Rc is in contact, via the pore in Rm, with Rt. The RcRmRt complex is referred to as methanoreductosome. Functional implications of the structural organization of the methylreductase system are discussed in view of methane formation and the creation of a transmembrane proton gradient used by the cell for ATP synthesis.

Cell Membrane↗
PubMed1987

Pyruvate carboxylase in the yeast pyc mutant.

Pyruvate carboxylase deficiency was previously reported to be the biochemical lesion in a yeast mutant, designated pyc, which cannot utilize ethanol, acetate, pyruvate, aspartate, or oxaloacetate as the sole carbon source [C. Wills and T. Melham (1985) Arch. Biochem. Biophys. 236, 782-791; C. Wills et al. (1986) Arch. Biochem. Biophys. 246, 306-320]. We present evidence here that the level of pyruvate carboxylase activity as well as the native and subunit molecular weights of this enzyme are identical in the mutant and the wild type. In addition we have used immunocytochemical labeling to demonstrate the exclusively cytosolic localization of this enzyme in both the mutant and wild-type yeast.

Carbon Dioxide↗
PubMed1987

Immunoelectron microscopic demonstration of ATPase on the cytoplasmic membrane of the methanogenic bacterium strain Göl.

ATPase was shown to be present on the cytoplasmic membrane of the methanogenic bacterium strain Göl. The enzyme was identified by an immunoelectron microscopic technique by using polyclonal antiserum directed against the beta subunit of Escherichia coli F0F1-ATPase. Negatively stained membrane vesicles exhibited a dense population of stalked particles similar in dimensions and fine structure to typical F0F1-ATPase particles.

Adenosine Triphosphatases↗