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M Wiedmann

Publications and source records attributed to M Wiedmann.

At least 91 records · Page 5Linked to original sources
PubMed1994

Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants.

Two outbreaks of epizootic listerial encephalitis, one in sheep and one in goats, were investigated through pathology, microbiology, and DNA amplification-based techniques. Efforts were made to survey the diversity of Listeria monocytogenes strains in the silage consumed by affected animals and to verify the causal relationship between silage and disease outbreak. In both outbreaks, L. monocytogenes was isolated from silage and brain tissue samples. Random amplified polymorphic DNA patterns revealed two distinct L. monocytogenes strains, one of which was identical to the sheep brain isolate, in the silage associated with the outbreak in sheep. Three brain isolates and one silage isolate, all of which had different random amplified polymorphic DNA patterns, were found in the outbreak involving goats. All isolates from both outbreaks were indistinguishable in an in vitro assay for cell-to-cell spread and growth in macrophages. All brain isolates from the goat outbreak had identical intracellular ActA patterns, which were different from the pattern for the silage isolate. While the sheep brain isolate had an ActA pattern different from that of the corresponding silage isolate, the patterns for the brain isolates from the two outbreaks were not identical. This survey demonstrates the diversity of L. monocytogenes in silage and suggests the existence of one or more selective processes by which certain strains are more prone to give rise to disease.

Animals↗
PubMed1994

Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction.

A nonisotopic ligase chain reaction (LCR) assay was developed to detect the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovine leukocyte adhesion deficiency (BLAD). Two sets of diagonally opposed discriminating LCR primers that differentiate the normal and BLAD allele were designed so that the 3' end of each primer overlapped the D128G mutation. These discriminating primers were synthesized with a 5' biotin and could be captured using streptavidin-coated microtitre wells. A common set of primers that abut these discriminating primers were also synthesized and 3'-tailed with digoxigenin-ddUTP. Captured LCR products were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos 530 and the entire assay including DNA isolation can be completed within 8 h.

Animals↗
PubMed1993

Overexpression of the ER-membrane protein P-450 CYP52A3 mimics sec mutant characteristics in Saccharomyces cerevisiae.

High expression of microsomal cytochrome P-450 CYP52A3 from Candida maltosa induces the formation of membrane stacks in Saccharomyces cerevisiae. Membrane proliferation is accompanied by coinduction of the ER proteins KAR2p and SEC61p and accumulation of precursor forms of proteins that have to translocate across the ER membrane (KAR2p, alpha factor). Cytosolic proteins (SSA1p and 2p) and mitochondrial proteins (CYT c1p and F1 beta p) are not affected. N-terminal truncated P-450 proteins remain in the cytoplasm and fail to induce membrane proliferation, KAR2p/SEC61p expression, and precursor accumulation. Membrane and precursor protein accumulation are typical features of sec mutants. We assume that the high amounts of P-450p block one or more factor(s) of the transport machinery and thereby cause the observed phenomena.

Amino Acid Sequence↗
PubMed1993

Control of folding and membrane translocation by binding of the chaperone DnaJ to nascent polypeptides.

Recent evidence supports the view that cellular protein folding may be mediated by molecular chaperones. A fundamental question concerns the stage in its biogenesis at which the folding protein makes first contact with these components. We show here by crosslinking that the chaperone DnaJ binds nascent ribosome-bound polypeptide chains as short as 55 residues. Cotranslational binding of DnaJ to firefly luciferase and chloramphenicol acetyltransferase resulted in an arrest of folding as long as the functional partners of DnaJ in Escherichia coli, DnaK and GrpE, were missing. Protein uptake into microsomes and mitochondria was also interrupted by DnaJ. Both folding and post-translational translocation recommenced upon addition of DnaK and GrpE. We propose that DnaJ protects nascent polypeptide chains against aggregation and, in cooperation with Hsp70, controls their productive folding once a complete polypeptide or a polypeptide domain has been synthesized.

Animals↗
PubMed1993

Non-radioactive detection of Mycobacterium tuberculosis LCR products in a microtitre plate format.

As part of the development of the ligase chain reaction (LCR) into a tool which can be used by a wide variety of researchers, we have investigated several analytical detection systems for the products of this amplification reaction. While early work with this technology has used gel electrophoresis to separate the LCR probes from the ligated product, solid phase capture techniques are also applicable, particularly when one of the probes is modified with a 'hook' such as biotin, and the adjoining probe modified with a detectable label. In this study we report a comparison of eight different non-radioactive detection techniques and discuss the analytical sensitivity of each. Detection with laser scanning fluorescent gel electrophoresis remains the most sensitive, with the assay described herein capable of detecting 100 molecules of the Mycobacterium tuberculosis insertion element IS6110 in a background of 4 micrograms of unrelated DNA. This method was followed closely by solid-phase capture and chemiluminescence detection which gave a sensitivity of 1000 molecules of IS6110. Fluorescence detection was approximately 10-fold less sensitive than chemiluminescence detection, and absorbance detection was a further 10-fold less sensitive than fluorescence detection. However, absorbance detection even at this level can still be useful for systems where visual interpretation is desired.

Alkaline Phosphatase↗
PubMed1993

Detection of bovine herpesvirus-1 in bovine semen by a nested PCR assay.

A nested PCR assay targeting a portion of the glycoprotein IV gene has been developed for the detection of Bovine Herpesvirus-1 (BHV-1). Rapid and sensitive detection of the PCR products was achieved using a nonisotopic reverse dot-blot format with a visible color readout. Cross-reactivity of this PCR assay was not observed with the closely related BHV-3. The sensitivity of this assay when tested on a supernatant from a BHV-1 cell culture was approximately 4.5 TCID50 (50% tissue culture infectious dose). A procedure using the chelating resin Chelex 100 was used to prepare viral DNA from artificially inoculated samples of extended and raw semen for use in the PCR assay. In combination with nested PCR and reverse dot blot, this method allowed the detection of 5 x 10(3) TCID per 0.5 ml of semen, which is comparable to the detection in the Cornell Semen Test. The whole procedure can be completed in approximately 8 h. This assay has therefore the potential of replacing the currently available yet time consuming and costly detection methods for BHV-1 in bovine semen.

Animals↗
PubMed1993

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.

Base Sequence↗
PubMed1993

Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assay.

A polymerase chain reaction (PCR)-coupled ligase chain reaction (LCR) assay for the specific detection of Listeria monocytogenes (M. Wiedmann, J. Czajka, F. Barany, and C. A. Batt, Appl. Environ. Microbiol. 58:3443-3447, 1992) has been modified for detection of the LCR products with a nonisotopic readout. When a chemiluminescent or a colorimetric substrate for the nonisotopic detection of the LCR products was used, the PCR-coupled LCR gave a sensitivity of 10 CFU of L. monocytogenes. The detection method with the chemiluminescent substrate Lumi-Phos 530 permitted detection of the LCR products in less than 3 h, so that the whole assay can be completed within 10 h.

Base Sequence↗
PubMed1992

Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) bind to a multi-SNAP receptor complex in Golgi membranes.

Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) are required for the binding of N-ethylmaleimide-sensitive fusion protein (NSF) to Golgi membranes and are, therefore, required for intra-Golgi transport. We report the existence of distinct alpha/beta-SNAP and gamma-SNAP-binding sites in Golgi membranes that appear to be part of the same receptor complex. Cross-linking studies with alpha-SNAP demonstrate that an integral membrane protein of between 30-40 kDa is the alpha-SNAP binding component of the multi-SNAP receptor complex. These data suggest that SNAPs function by independently binding to a multi-SNAP membrane-receptor complex, thereby activating them to serve as adaptors for the targeting of NSF.

Amino Acid Sequence↗
PubMed1992

Yeast Sec proteins interact with polypeptides traversing the endoplasmic reticulum membrane.

We show by photocross-linking that nascent secretory proteins, during their passage through the endoplasmic reticulum membrane of S. cerevisiae, are in physical contact with Sec61p and Sec62p, two genetically identified membrane proteins that are essential for in vivo translocation. Sec61p seems to be in continuous contact, whereas Sec62p is involved only transiently. Translocation comprises both ATP-dependent and -independent phases of interaction with the Sec proteins. The results suggest a direct role of the Sec proteins in translocation.

Adenosine Triphosphate↗
PubMed1992

Mapping of the protein import machinery in the mitochondrial outer membrane by crosslinking of translocation intermediates.

Mitochondria contain a complex machinery for the import of nuclear-encoded proteins. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein). Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were crosslinked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOM8, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.

Animals↗
PubMed1992

A multisubunit particle implicated in membrane fusion.

The N-ethylmaleimide sensitive fusion protein (NSF) is required for fusion of lipid bilayers at many locations within eukaryotic cells. Binding of NSF to Golgi membranes is known to require an integral membrane receptor and one or more members of a family of related soluble NSF attachment proteins (alpha-, beta-, and gamma-SNAPs). Here we demonstrate the direct interaction of NSF, SNAPs and an integral membrane component in a detergent solubilized system. We show that NSF only binds to SNAPs in the presence of the integral receptor, resulting in the formation of a multisubunit protein complex with a sedimentation coefficient of 20S. Particle assembly reveals striking differences between members of the SNAP protein family; gamma-SNAP associates with the complex via a binding site distinct from that used by alpha- and beta-SNAPs, which are themselves equivalent, alternative subunits of the particle. Once formed, the 20S particle is subsequently able to disassemble in a process coupled to the hydrolysis of ATP. We suggest how cycles of complex assembly and disassembly could help confer specificity to the generalized NSF-dependent fusion apparatus.

Adenosine Triphosphate↗
PubMed1992

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.

Base Sequence↗
PubMed1991

The identification of proteins in the proximity of signal-anchor sequences during their targeting to and insertion into the membrane of the ER.

Using a photocross-linking approach we have investigated the cytosolic and membrane components involved in the targeting and insertion of signal-anchor proteins into the membrane of the ER. The nascent chains of both type I and type II signal-anchor proteins can be cross-linked to the 54-kD subunit of the signal recognition particle. Upon addition of rough microsomes the type I and type II signal-anchor proteins interact with a number of components. Both types of protein interact with an integral membrane protein, the signal sequence receptor, previously identified by its proximity to preprolactin during its translocation (Wiedmann, M., T.V. Kurzchalia, E. Hartmann, and T.A. Rapoport. 1987. Nature [Lond.] 328:830-833). Three proteins, previously unidentified, were found to be cross-linked to the nascent chains of the signal-anchor proteins. Among them was a 37-kD protein that was found to be the main component interacting with the type I SA protein used. These proteins were not seen in the absence of membranes suggesting they are components of the ER. The ability of the nascent chains to be cross-linked to these identified proteins was shown to be abolished by prior treatment with agents known to disrupt translocation intermediates or ribosomes. We propose that the newly identified proteins function either in the membrane insertion of only a subset of proteins or only at a specific stage of insertion.

Amino Acid Sequence↗
PubMed1990

The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents.

Bifunctional cross-linking reagents were used to probe the protein environment in the ER membrane of the signal sequence receptor (SSR), a 24-kD integral membrane glycoprotein (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328:830-833). The proximity of several polypeptides was demonstrated. A 22-kD glycoprotein was identified tightly bound to the 34-kD SSR even after membrane solubilization. The 34-kD polypeptide, now termed alpha SSR, and the 22-kD polypeptide, the beta SSR, represent a heterodimer. We report on the sequence of the beta SSR, its membrane topology, and on the mechanism of its integration into the membrane. Cross-linking also produced dimers of the alpha-subunit of the SSR indicating that oligomers of the SSR exist in the ER membrane. Various bifunctional cross-linking reagents were used to study the relation to ER membrane proteins of nascent chains of preprolactin and beta-lactamase at different stages of their translocation through the membrane. The predominant cross-linked products obtained in high yields contained the alpha SSR, indicating in conjunction with previous results that it is a major membrane protein in the neighborhood of translocating nascent chains of secretory proteins. The results support the existence of a translocon, a translocation complex involving the SSR, which constitutes the specific site of protein translocation across the ER membrane.

Amino Acid Sequence↗