Search PubMed⌕ Search

Biomedical subjects

M Bachmann

Publications and source records attributed to M Bachmann.

At least 73 records · Page 4Linked to original sources
PubMed1996

Different La/SS-B mRNA isoforms are expressed in salivary gland tissue of patients with primary Sjögren's syndrome.

Recently we isolated a La/SS-B mRNA isoform from a cDNA library made from peripheral blood lymphocytes of a patient with primary Sjögren's Syndrome. In the La/SS-B mRNA isoform the exon 1 was replaced. The alternative exon was termed exon 1'. Genomic analysis showed that the exon 1' La mRNA was the result of a promoter-switch in combination with alternative splicing. Due to the unusual structure of the exon 1' La/SS-B mRNA, the function and the behaviour under physiological and pathophysiological conditions in tissue of patients with primary Sjögren's syndrome or Systemic Lupus Erythematosus remained obscure. Therefore assays were established allowing a qualitative and quantitative estimation of expression of the exon 1 and 1' La mRNA form, including in situ and dot blot hybridization as well as reversed PCR. Both mRNA forms were found to represent finally processed cytoplasmic mRNAs belonging to the abundant class of mRNAs. They were expressed and regulated in parallel. A ratio exon 1 to 1' between 1:1 and 5:1 was determined. Both mRNA forms were downregulated in quiescent cells and upregulated in activated and proliferating cells including non-keratized stratified squamous epithelial, endothelial, salivary gland as well as infiltrating cells.

Autoantigens↗
PubMed1996

The autoantigen La/SS-B: analysis of the expression of alternatively spliced La mRNA isoforms.

The gene for the nuclear autoantigen La/SS-B encodes two La mRNA isoforms. In order to study the function and expression of both La mRNA forms, an in situ hybridization procedure was developed allowing the selective identification of either exon 1 or exon 1'. For this purpose, digoxigenin-labeled exon-specific sense and anti-sense probes were prepared by in vitro transcription from plasmids that contained the respective exon sequence. Detection of the probes was carried out by using rhodamine-conjugated anti-digoxigenin antibody and confocal laser scanning microscopy. Both La mRNAs were found in the cytoplasm of endothelial cells but not in smooth muscle cells. In addition to the in situ technique, an assay system was established allowing the expression ratio of the two mRNA forms to be determined. The estimation was based on the amplification of exon 1 and 1' La cDNAs in parallel by using a three primer polymerase chain reaction. The ratio of the exon 1 to exon 1' La mRNA forms was determined to be about 5:1 in liver tissue and endothelial cells. The data support the conclusion that both La mRNA forms represent finally processed cytoplasmic mRNAs that are up- or downregulated in parallel.

Adult↗
PubMed1996

Design and analysis of studies evaluating smoking cessation interventions where effects vary between practices or practitioners.

BACKGROUND: Patients grouped together in practices may share characteristics that cause them to have similar responses to an intervention. Sampling from such groups means that the power of a trial is less than when subjects are selected from the population at random. Knowledge of likely variation in outcome at the practice level is necessary to calculate the extent to which sample size would need to be inflated to maintain statistical power in the face of 'cluster effects'. OBJECTIVES: To plan sample size and precision requirements of a clinical trial, we examined reports of primary care smoking cessation trials for information on outcomes at the level of clusters, and found them unhelpful. We therefore constructed hypothetical scenarios to quantity the potential importance of this effect. METHOD: Scenarios of moderate and large inter cluster variation were compared with a sample where there was no difference in effect size at the level of practices. RESULTS: A study with 80% power to detect a difference of 20% versus 10% at a 5% significance level would need 200 patients in each arm in the absence of cluster effects. With moderate variation in outcome between clusters, over a thousand patients would be needed in the study to maintain this precision. With larger inter-cluster variation, close to 4000 subjects would be required. CONCLUSIONS: In the absence of detailed data from previous studies, hypothetical models can give insight into the statistical implications of possible cluster effects on study design and analysis. With even moderate inter-cluster variation, sample size will have to be inflated considerably to maintain the same statistical precision. Workers in this field will greatly assist those planning future research if they publish details of variation in outcome at the level of clusters.

Chi-Square Distribution↗
PubMed1996

Infant mortality rate inequalities in the Western Cape Province of South Africa.

BACKGROUND: Cape Town is undergoing rapid urbanization. South African vital statistics have routinely been stratified by racial categories but intra-urban and peri-urban geographical variations have been neglected. METHODS: To examine variations in infant mortality rates (IMR) and proportional infant mortality between urban, rural and informally settled areas, stratified by racial category, birth notifications and infant death certifications recorded by a large health authority were analysed. RESULTS: The IMR per 1000 livebirths was as high for coloureds on rural farms (34, 95% confidence interval 29-40) as for blacks in informal settlements (35, 95% CI: 32-37) and an exceptionally high IMR (60, 95% CI: 43-82) was found for coloureds in informal settlements. Inequalities between racial categories (11 (95% CI: 9-14) for whites, 19 (95% CI: 18-21) for coloureds and 33 (95% CI: 31-35) for blacks) were as expected from other South African studies. Of rural farm deaths, 22% were ascribed to ill-defined causes. Low birthweight was the most common defined cause of death in all areas except rural farm areas (14% ascribed to pneumonia), and gastro-enteritis was important in informally settled areas (18%). CONCLUSIONS: Routine mortality data are more informative if stratified by robust and readily available indicators of socio-economic status such as residential area and racial category. Place of residence may distinguish risk strata as well as racial category, but the latter is helpful within socioeconomically heterogeneous residential areas.

Black or African American↗
PubMed1996

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

Amino Acid Sequence↗
PubMed1996

[Lead poisoning: clinical picture, diagnosis and related environmental factors].

BACKGROUND: The predisposing environmental factors and clinical picture of lead poisoning, are not well known. AIM: To describe the clinical picture, diagnosis and treatment of lead poisoning in individuals under industry exposure. SUBJECTS AND METHODS: Twenty of 38 workers of a printing press were studied. Their medical and labor histories were recorded. Lead in air in the working area and the dose received by the workers was measured. RESULTS: The environmental lead in the linotype room was 25% over the accepted values. Twelve of the 20 workers were poisoned and three were highly exposed. Poisoned workers were working in areas with high environmental lead concentrations, were in direct contact with the metal, had plasma lead concentrations over 70 micrograms/dl and an average exposure time of over 17 years. Their clinical picture was not specific and related to plasma and environmental lead concentrations, length of exposure and type of contact. CONCLUSIONS: Lead poisoning must be suspected among workers exposed to high environmental concentrations and its treatment consists in withdrawing poisoned subjects from polluted areas.

Adult↗
PubMed1995

Recognition molecules myelin-associated glycoprotein and tenascin-C inhibit integrin-mediated adhesion of neural cells to collagen.

Because of the importance of collagens in mediating cell-substrate interactions and the association of collagens with neural recognition molecules in the peripheral nervous system, the ability of neural recognition molecules to modify the substrate properties of collagens, in particular collagen type I, for cell adhesion was determined. Two cell lines, the N2A neuroblastoma and PC12 pheochromocytoma, were investigated for their capacity to adhere to different collagen types in the absence or presence of several neural recognition molecules. Adhesion of N2A or PC12 cells and membrane vesicles from PC12 cells to collagen type I was reduced when the collagen had been preincubated prior to its application as substrate with the extracellular domain of myelin-associated glycoprotein (s-MAG) or, as control, fibroblast tenascin-C (F-tenascin). In mixture with other collagen types, s-MAG was only able to reduce the adhesiveness of collagen types III and V, but not of collagen types II and IV. F-tenascin reduced the adhesiveness of all collagen types tested. In contrast to F-tenascin, s-MAG had to be present during fibrillogenesis to exert its effect, indicating that it must be coassembled into the collagen fibril to block the binding site. Cell adhesion to collagen type I was dependent on Mg2+ or Mn2+ and inhibited by a monoclonal antibody to the alpha 1 integrin subunit. The combined observations indicate that s-MAG and F-tenascin interfere with cell binding, most probably by modifying the integrin binding site, and that the two molecules act by different mechanisms, both leading to reduction of adhesion.

Animals↗
PubMed1995

Efficient immune responses in mice lacking N-region diversity.

Mice with a null mutation in the terminal deoxynucleotidyl transferase (TdT) gene harbor immunoglobulin and T cell receptor repertoires essentially devoid of N-region diversity. Consequently, the CDR3 loops important for antigen recognition are shorter and considerably less diverse than those of wild-type controls. We find surprisingly normal immune responses in TdT0 mice, as regards both efficiency and specificity. This provokes a reconsideration of the assumption that N-region diversity is required for an effective T and B cell repertoire.

Amino Acid Sequence↗
PubMed1995

Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells.

The levels and subcellular distribution of the interferon-inducible double-stranded RNA-dependent protein kinase PKR have been measured in human Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase. Immunofluorescence of intact cells and quantitative immunoblotting of cell extracts indicate that PKR occurs in both the cytoplasm and the cell nucleus, with staining specifically in the nucleolus. The ratio of cytoplasmic to nuclear PKR is approximately 5:1 in control cells; in response to interferon treatment the protein kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly. Analysis of individual transfected cells by confocal microscopy reveals a pattern of distribution of PKR similar to that in Daudi cells, with immunostaining of cytoplasm and nucleoli. Similar results are observed whether cells expressing wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the antibody used. Two-dimensional isoelectric focusing analysis of PKR in whole cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pI. These results suggest that PKR may have a role in the cell nucleus as well as the cytoplasm and that the subcellular distribution of the protein kinase may be related to post-translational modifications.

3T3 Cells↗
PubMed1995

Activation of a murine autoreactive B cell by immunization with human recombinant autoantigen La/SS-B: characterization of the autoepitope.

Immunization of Balb/c mice with a homogeneously purified recombinant human La/SS-B protein resulted in activation of an autoreactive B cell secreting a novel monoclonal anti-La antibody termed La4B6. La4B6 reacted with La protein from a variety of sources including human, bovine, rat and mouse. ATP blocked the binding of La4B6 to recombinant La protein. The human epitope was identified as consisting of the amino acid sequence SKGRRFKGKGKGN, which includes the proposed ATP-binding site of the La protein. In the human and bovine La protein, the epitope exists as a continuous amino acid sequence. In rat and mouse the epitope was found to consist of the amino acid sequence SKG interrupted by a species-specific insert of 16 amino acids, and followed by the second half of the epitope, the amino acid sequence RRFKGKGKGN. Our data suggest that in the case of the rat and mouse La proteins the two separated parts of the epitope are able to form a conformational epitope which looks similar to the continuous human epitope.

3T3 Cells↗
PubMed1994

One gene, two transcripts: isolation of an alternative transcript encoding for the autoantigen La/SS-B from a cDNA library of a patient with primary Sjögrens' syndrome.

A cDNA library was prepared from peripheral blood lymphocytes of an autoimmune patient with primary Sjögrens' syndrome. The cDNA library was screened with the patients own autoimmune serum being monospecific for the nuclear autoantigen La/SS-B. Thereby an alternative type of La mRNA was identified that differed from the known La mRNA due to an exchange of the exon 1. Sequencing of the genomic region between the exons 1 and 2 showed that the alternative 5'-end is a part of the intron. In addition, the presence of an alternative promoter site, which exists within the intron downstream of the exon 1, became evident. In consequence, the alternative La mRNA is the result of a promoter switching combined with an alternative splicing mechanism. In the intron, further transcription factor binding sites, including a NF-kappa B element, were identified leading to the suggestion that the expression of the gene encoding for the nuclear autoantigen La/SS-B alters in dependence on disease conditions.

Alternative Splicing↗
PubMed1994

Expression of M-cadherin protein in myogenic cells during prenatal mouse development and differentiation of embryonic stem cells in culture.

Molecules regulating morphogenesis by cell-cell interactions are the cadherins, a class of calcium-dependent adhesion molecules. One of its members, M-cadherin, has been isolated from a myoblast cell line (Donalies et al. [1991] Proc. Natl. Acad. Sci. U.S.A. 88:8024-8028). In mouse development, expression of M-cadherin mRNA first appears at day 8.5 of gestation (E8.5) in somites and has been postulated to be down-regulated in developing muscle masses (Moore and Walsh [1993] Development 117:1409-1420). Affinity-purified polyclonal M-cadherin antibodies, detecting a protein of approximately 120 kDa, were used to study the cell expression pattern of M-cadherin protein. It was first visualized in somites at E10 1/3 and could be confined to desmin positive, myotomal cells. At all subsequent prenatal stages, M-cadherin was only found in myogenic cells of somitic origin. The detection of the protein at E10 1/3 suggests a translational delay of M-cadherin mRNA of 1 to 2 days (E8.5 vs. E10 1/3). This was further supported by the finding that during differentiation of ES cell line BLC6 into skeletal muscle cells in culture, expression of M-cadherin mRNA can be detected 2 days prior to M-cadherin protein. During prenatal development, the pattern of M-cadherin expression changes: In E10 1/3 embryos and also in myotomal cells of later stages, M-cadherin is evenly distributed on the cell surface. In developing muscle masses (tested at E16 to E18), however, M-cadherin protein becomes clustered most likely at sites of cell-cell contact as indicated by double-labelling experiments: M-cadherin-staining is the positive image of laminin negative areas excluding the presence of a basal lamina at M-cadherin positive sites. Furthermore, M-cadherin is coexpressed with the neuronal cell adhesion molecule N-CAM which has been shown to mediate cell-cell contact in myogenic cells. In summary, our results are in line with the idea that M-cadherin might play a central role in myogenic morphogenesis.

Amino Acid Sequence↗
PubMed1994

Localization of antigens PwA33 and La on lampbrush chromosomes and on nucleoplasmic structures in the oocyte of the urodele Pleurodeles waltl: light and electron microscopic immunocytochemical studies.

Monoclonal antibodies A33/22 and La11G7 have been used to study the distribution of the corresponding antigens, PwA33 and La, on the lampbrush chromosome loops and nucleoplasmic structures of P. waltl oocytes, using immunofluorescence, confocal laser scanning microscopy and immunogold labeling. The results obtained with these antibodies have been compared with those obtained with the Sm-antigen-specific monoclonal antibody Y12. All these monoclonal antibodies (mAbs) labeled the matrices of the majority of normal loops along their whole length. Nucleoplasmic RNP granules showed a strong staining with the mAbs La11G7 and Y12 throughout their mass, but with the mAb A33/22, they showed only a weak peripheral labeling in the form of patches on their surface. This patchy labeling was confirmed by confocal laser scanning microscopy. Electron microscopy revealed that this patchy labeling might be due to a hitherto undescribed type of submicroscopic granular structure, around 100 nm in either dimension, formed by 10-nm particles. Such granules were observed either attached to the RNP granules or free in the nucleoplasm, but rarely in relation with the normal loop matrices. These 100-nm granules may have a role in the movement of proteins and snRNPs inside the oocyte nuclei for storage, recycling, and/or degradation. Our results also suggest that all the microscopically visible free RNP granules of the nucleoplasm of P. waltl oocytes correspond to B snurposomes. The granules forming the B (globular) loops showed a labeling pattern similar to that of B snurposomes; their possible relationship is discussed.

Animals↗
PubMed1993

Isolation of rat cDNA clones coding for the autoantigen SS-B/La: detection of species-specific variations.

Clones of cDNA coding for the autoantigen La (or SS-B) were isolated from a library made from rat liver. A comparison of the rat La cDNA (encoding from nt 38 to 1281 for rat La protein) with the sequences known for human and bovine La protein resulted in the identification of species-specific inserts. The inserts seem to be the result of multiplication of flanking sequences during evolution. In addition to these variations, we observed that rat La cDNAs exhibit non-canonical polyadenylation sites. Finally, a databank search resulted in the identification of a DNA sequence originally termed as TAG or TSG20X (GenBank accession No. X61893) which represents the C terminus of mouse La/SS-B protein.

Amino Acid Sequence↗
PubMed1993

Disruption of the murine IL-4 gene blocks Th2 cytokine responses.

Murine T-helper clones are classified into two distinct subsets (Th1 and Th2) on the basis of their patterns of lymphokine secretion. Th1 clones secrete interleukin-2 (IL-2), tumour necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), whereas Th2 clones secrete IL-4, IL-5 and IL-10 (ref. 1). These subsets are reciprocally regulated by IL-4, IL-10 and IFN-gamma and differentially promote antibody or delayed-type hypersensitivity responses. To evaluate whether IL-4 is required for mounting Th2 responses, we generated IL-4-mutant mice (IL-4-/-) and assessed the cytokine secretion pattern of T cells both from naive and Nippostrongylus brasiliensis infected mice. CD4+ T cells from naive IL-4-/- mice failed to produce Th2-derived cytokines after in vitro stimulation. The levels of Th2 cytokines IL-5, IL-9 and IL-10 from CD4+ T cells obtained after nematode infection were significantly reduced. The reduced IL-5 production in IL-4-/- mice correlated with reduced helminth-induced eosinophilia, which has been shown to be dependent on IL-5 in vivo. We conclude that IL-4 is required for the generation of the Th2-derived cytokines and that immune responses dependent on these cytokines are impaired.

Alleles↗
PubMed1993

Kinetics of expression of prion protein in uninfected and scrapie-infected N2a mouse neuroblastoma cells.

The scrapie prion protein, PrPSc, is formed from its isoform, the cellular PrPc. There is evidence available indicating that PrPSc is a necessary component of the infectious prion particle to cause a series of transmissible spongiform encephalopathies. We have used immunocytochemistry and RNA blotting techniques to investigate if infection with prions results in an increased PrP gene expression. For the experiments we used N2a cells which had been infected with prions (ScN2a cells). We demonstrated by confocal laser scanning microscopy that PrP-protein was present in the nucleus (predominantly in the nucleoli) of ScN2a cells. Analysis of the PrP-mRNA levels both in N2a- and in ScN2a cells using cDNA encoding PrPc revealed no marked alteration of the mRNA steady state level between the two cell strains. Likewise, in run-off experiments no changes in either PrP-specific transcription or in general transcriptional activity were found. The half-life of PrP-mRNA was found to be identical in both cell strains (7h). Taken together, these results show that PrPSc and/or PrPc is present in the nucleus (nucleoli) of ScN2a cells but does not display an effect on the expression of the PrP gene.

Animals↗
PubMed1993

Nuclear localization of the protein encoded by the Wilms' tumor gene WT1 in embryonic and adult tissues.

The human Wilms' tumor gene WT1 encodes a putative transcription factor implicated in tumorigenesis and in specifying normal urogenital development. We have studied the distribution of WT1 protein and mRNA using immunohistochemistry and in situ hybridization. Monoclonal antibodies were raised against a peptide specific to the first alternative splice site of WT1. Two antibodies specifically reacted on Western blot to this WT1 isoform. Immunofluorescence localized WT1 protein to podocytes during mesonephric and metanephric development. In situ hybridization revealed a similar pattern of expression except that WT1 mRNA was also present in metanephric blastema and renal vesicles. Messenger RNA expression was most pronounced in the kidneys during early fetal development and declined thereafter. In contrast, WT1 protein was readily detectable in glomerular podocytes throughout adulthood. WT1 protein in Wilms' tumor was present in blastema and glomeruloid structures. Expression in the female gonad was linked to the different stages of granulosa cell development. In the male gonad, expression was restricted to Sertoli cells and their precursors, the embryonic tunica albuginea and the rete testis. The intracellular distribution of the WT1 protein was investigated by confocal laser microscopy and was demonstrated to be exclusively nuclear. The nuclear distribution and the selective pattern of expression support the proposed role of WT1 as a transcription factor active during urogenital development. The persistence of WT1 expression in the adult kidney suggests a role in homeostasis of the podocyte.

Blotting, Western↗