Biomedical subjects

M Bachmann

Publications and source records attributed to M Bachmann.

At least 55 records · Page 3Linked to original sources

PubMed1997

The nuclear autoantigen La/SS-B: mapping and sequencing of the gene and the three retropseudogenes.

One target of autoantibodies in sera of patients with systemic lupus erythematosus or primary Sjögren's syndrome is the nuclear autoantigen La/SS-B. Lambda clones and cosmids were isolated, which contained the sequences of the La gene and the three La pseudogenes. They were used for preparation of a physical map. Finally, the La gene and pseudogenes were sequenced. The pseudogenes were characterized as retropseudogenes. Their evolutionary ages were estimated to be approx. 4, 4.5 and 5 million years. Inserts of 4, 16 and 24 nucleotides, which were mostly A-residues, were found in exon 7 of the respective pseudogene. The oldest pseudogene contained the longest insert, the youngest pseudogene contained the smallest insert. The oligonucleotides seem to be the result of repeated inserts of A-residues in a hot spot region of the La genes. Two La cDNAs were isolated which contained either a deletion or an insert of an A-residue at the same position.

Autoantigens↗

PubMed1997

Transfection analysis of expression of mRNA isoforms encoding the nuclear autoantigen La/SS-B.

Transcription of the gene encoding for the nuclear autoantigen La resulted in La mRNA isoforms. A promoter switching combined with an alternative splicing pathway replaced the exon 1 with the exon 1'. The exon 1' contained GC-rich regions and an oligo(U) tail of 23 uridine residues. Moreover, it encoded for three open reading frames upstream of the La protein reading frame. Despite this unusual structure, when exon 1' La mRNAs were expressed in transfected cells, both exon 1 and 1' La mRNAs were translated to La protein, whereas the upstream open reading frames of the exon 1' were not translated. In addition to full-length exon 1' La mRNAs 5'-shortened exon 1' La mRNAs were detected. The exon 1' 5'-starts varied in dependence on the analyzed tissues. Like the full-length exon 1' La mRNA a 5'-shortened exon 1' construct starting downstream of the oligo(U) tail but upstream of the open reading frames 2 and 3 was also well translated when transfected in mouse cells. Thus all La mRNA forms represent functional La mRNAs.

Alternative Splicing↗

PubMed1997

The nuclear autoantigen La/SS-associated antigen B: one gene, three functional mRNAs.

Transcription of the gene encoding for the nuclear autoantigen La resulted in three mRNA forms. A promoter switching combined with an alternative splicing pathway replaced exon 1 with either exon 1' or exon 1'. The exon 1' donor splice site was located 4 nts downstream of the exon 1' donor splice site. All three La mRNA forms were expressed in all the tissues analysed including peripheral blood lymphocytes, liver, fetal spleen, cultured primary endothelial cells, and mouse LTA cell lines permanently transfected with the human La gene. Both the exons 1' and 1' had unusual structures. They contained GC-rich regions and an oligo(U)-tail of 23 uridine residues. Moreover, they encoded for three open reading frames upstream of the La protein reading frame. In spite of this unusual structure, when exon 1' or exon 1' La mRNAs were expressed in transfected mouse LTA cells, both La mRNAs were translated to nuclear La protein, indicating that all La mRNA forms are functional mRNAs.

Adenosine Triphosphatases↗

PubMed1997

Analysis of expression of an alternative La (SS-B) cDNA and localization of the encoded N- and C-terminal peptides.

A deletion of an (A)-residue was detected in a cDNA encoding for the nuclear autoantigen La/SS-B. The cDNA was recently isolated from a cDNA library made from peripheral blood lymphocytes of a patient with primary Sjögren's Syndrome. The region, where the deletion occurred, represents a hot spot region in the La gene(s). It leads to a frame shift mutation and a premature stop codon eleven amino acids downstream of the deletion site within one of the protease sensitive regions of the La protein. In spite of the frame shift mutation expression of full length La protein occurred efficiently in E. coli. Full length La protein was also made in SF9 cells infected with recombinant baculoviruses, although the efficiency of full length protein production was less. Two major peptides with molecular weights of 29 kDa and 25 kDa were made. The size of these peptides was similar to the known proteolytic degradation products of La protein. The N-terminal 29 kDa fragment containing the RNP consensus sequence located in the cytoplasm. The 25 kDa C-terminal fragment containing the nuclear location signal entered in the nucleus and associated with nuclear speckles. In conclusion, the ability to (i) enter, (ii) remain in the nucleus and (iii) assemble with nuclear speckles resides in the C-terminal domain of La protein and does not depend on the N-terminal RNP-consensus motif.

Amino Acid Sequence↗

PubMed1997

Characterization of the autoantigen La (SS-B) as a dsRNA unwinding enzyme.

During the analysis of the La (SS-B) autoantigen for catalytic activities an ATP-dependent double-stranded RNA unwinding activity was detected. Both native and recombinant La proteins from different species displayed this activity, which could be inhibited by monospecific anti-La antibodies. La protein was able to melt dsRNA substrates with either two 3'-overhangs or a single 3'- and a 5'-overhang. Double-stranded RNAs with two 5'-overhangs were not unwound, indicating that at least one 3'-overhang is required for unwinding. Sequence elements of the La protein that might be involved in dsRNA unwinding, such as an evolutionarily conserved putative ATP-binding motif and an element that is homologous to the double-stranded RNA binding protein kinase PKR, are discussed.

Adenosine Triphosphate↗

PubMed1997

Antibodies protect mice against challenge with tick-borne encephalitis virus (TBEV)-infected macrophages.

TBEV is a flavivirus highly pathogenic for humans. By transfer of antibodies directed to the TBEV surface glycoprotein E into mice, immune protection against subsequent inoculation with free TBEV particles could be achieved. After natural TBEV infection via the skin, however, cells of the monocyte/macrophage lineage were recently demonstrated to represent an important source of local virus replication before viraemia occurs. Whether antibodies can protect against virus challenge when contracted in the form of infected cells, however, is still unclear. In the current study, TBEV antibodies protected mice against challenge with either free virus or TBEV-infected macrophages equally well. This observation may be of more general significance.

Animals↗

PubMed1997

Characterization of murine monoclonal antibodies against the Ro52 autoantigen.

Immunization of BALB/c mice with purified recombinant human Ro52 protein resulted in three anti-Ro52 MoAbs termed 2E7, 4C6 and 4F11. All anti-Ro52 MoAbs specifically reacted with recombinant human Ro52 protein, and also with Ro52 protein in total extracts of all human cell lines analysed, including the epithelial cell line HeLa, the B cell line Raji, the bladder carcinoma cell line RT112, and a fibroblast cell line derived from patients with xeroderma pigmentosum. The anti-Ro52 MoAbs were able to immunoprecipitate the recombinant human Ro52 protein expressed in wheat germ extract, but failed to precipitate hY RNAs from cell extracts. The staining pattern of the MoAbs strongly differed between the RT112 cells and the fibroblast cell line. RT112 cells displayed an intense cytoplasmic staining and in addition distinct fine nuclear speckles. In contrast, in the fibroblast cell line no cytoplasmic staining but only staining of distinct nuclear speckles was observed. Using deletion mutants of Ro52 the epitopes recognized by the anti-Ro52 MoAbs 2E7, 4C6 and 4F11 were partially mapped. All three MoAbs appeared to recognize distinct epitopes, that are located in the regions of Ro52 bordered by amino acids 136-164, 208-363 and 136-190, respectively. These MoAbs can be of great use in studying the cellular processes in which the Ro52 protein is involved.

Amino Acid Sequence↗

PubMed1997

Effect of cleaning dentine with soap and pumice on shear bond strength of dentine-bonding agents.

This in vitro study reports on the cleaning effect of different soaps on the shear bond strength of various dentine-bonding agents. Human teeth were coated with provisional cements for 24 h or for 14 days. After removing the provisional cements with a scaler, the dentinal surface was cleaned with a cotton pellet and non-fluoridated flour of pumice and soap for 10 sec. Different dentine-bonding agents and a luting resin were bonded to the dentinal surface according to manufacturers' instructions with the bonding agent and the composite material being light-cured at the same time. The bonding agents were tested under intrapulpal pressure and with thermal cycling to imitate physiological conditions. Compared with cleaning the dentine with water and pumice, all soaps investigated in this study decreased the shear bond strength values of the tested dentine-bonding agents considerably.

Dental Bonding↗

PubMed1997

Intrathecal infusion of bupivacaine with or without morphine for postoperative analgesia after hip and knee arthroplasty.

Postoperative pain after major orthopaedic operations can be controlled by continuous intrathecal administration of opioids or local anaesthetics. Effective intrathecal analgesia can be achieved through synergism of low doses of the two analgesic drugs and, possibly, less drug-related adverse effects. Therefore, we have evaluated the usefulness of a combined low-dose bupivacaine and morphine infusion in patients undergoing hip and knee arthroplasty. Spinal anaesthesia was induced in 55 ASA I-III patients with 0.5% bupivacaine 2 ml via a 28-gauge spinal catheter (L3-4 interspace) and 0.5-ml increments were given if needed. Intrathecal 24-h infusions consisted of bupivacaine 2 mg h-1 alone (n = 18), bupivacaine 1 mg h-1 alone (n = 18) or bupivacaine 1 mg h-1 combined with morphine 8 micrograms h-1 (n = 19). The interview after 3, 6, 12 and 24 h included assessment of pain at rest and on movement (VAS scale), occurrence of sensory and motor block and nausea/vomiting. Bupivacaine 1 mg h-1 combined with an infusion of morphine provided as good postoperative analgesia as bupivacaine 2 mg h-1, but motor block disappeared earlier (P = 0.01). Patients in the bupivacaine 1-mg h-1 group required more supplementary doses of oxycodone i.m. than the other groups (P = 0.04). Time to first oxycodone dose from the start of intrathecal infusion did not differ between groups. The frequency of nausea and vomiting was similar in all groups. In spite of this, antiemetic medication was required more often in the bupivacaine 1-mg h-1 group (possible because of opioid rescue medication). On the ward, one patient in the bupivacaine 2-mg h-1 group experienced a new increase in sensory block with concomitant hypotension. One patient in the same group had minor decubitus on the heel of the operated leg, probably because of prolonged motor block. We conclude that intrathecal infusion of a combination of bupivacaine 1 mg h-1 and morphine 8 micrograms h-1 produced adequate postoperative analgesia. Unfortunately, postoperative nausea and vomiting was a frequent disturbing adverse effect.

Adult↗

PubMed1997

Patients' experience of epilepsy and health care.

OBJECTIVE: The aim of this study was to assess the severity of epilepsy and its effect on patients lives, and to describe patients' use of and attitudes to health care. METHOD: A questionnaire was sent to 595 people with epilepsy identified from 14 general practices in north-west Bristol. All patients aged 16 years and over receiving anti-epileptic medication for their epilepsy were included in the study. Areas investigated included severity of epilepsy and its effect on quality of life, anti-epileptic medication and its perceived effect, health care utilization and preferences for health care. RESULTS: Seizure frequency was strongly associated with adverse effects of epilepsy. Attacks of epilepsy were experienced at least monthly by 20.4% (95% confidence intervals (Cl) 17.0-23.7%) of patients, 29.4% (25.4-33.4%) took more than one anti-epileptic drug, 56.1% (50.1-62.2%) reported drug side effects, 74.1% (70.3-77.8%) would prefer to receive all or most of their epilepsy care in a general practice setting, and 69.8% (63.5-76.2%) would like contact with a primary care-based epilepsy specialist nurse. During the previous year 42.4% (35.9-48.8%) of patients had not seen a doctor about their epilepsy. Of patients who had attended the general practice only 13.4% (9.6-17.2%) had regular arrangements to see their GP about epilepsy. Patients receiving both primary and secondary care had the greatest needs and wants for improved care. CONCLUSIONS: Structured care, including regular appointments, co-ordination of primary and secondary care, and increased monitoring and discussion, may improve the quality of life of people with epilepsy, but requires evaluation.

Adolescent↗

PubMed1997

De novo acquisition of neuronal polarity in retinoic acid-induced embryonal carcinoma cells.

The mouse embryonal carcinoma cell line PCC7-Mz1 represents an advantageous model to study acquisition of polarity by neurons. During the first two days after differentiation is induced by the addition of retinoic acid, the neuronal derivatives develop extensions which for at least four more days do not differ from each other in growth characteristics, morphology, and marker expression. Beginning around differentiation day 6 and following the relocation of the nucleus from a central to a polar position in the cell soma, the morphology and marker expression changes dramatically: expression of MAP2 diminishes and eventually disappears in the thinner neurite (future axon), which originates at the nucleated pole, but remains strong in the branched, broad based neurite(s). The opposite changes in expression are observed for synaptophysin, together with a clustering of the vesicle protein in varicosity-like areas. Complete segregation of expression of the two markers is achieved around day 12, shortly followed by dendrite-specific location of MAP2 mRNA and the ability to generate and conduct action potentials. Our studies add several aspects to the process of neuronal polarity acquisition, as it was previously studied in primary cultures of embryonic neurons: (i) we monitored neuronal differentiation from the birth of neurons, rather than from later and less defined maturation stages, (ii) cell nucleus relocation may be associated with the induction of neuronal polarity, and (iii) functional competence of neurons is closely associated with previous acquisition of polarity. Acquisition of polarity by PCC7-Mz1 neuronal derivatives probably refers to de novo acquisition rather than to reestablishment of polarity.

Animals↗

PubMed1996

14-3-3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform-specific manner and reduce dephosphorylation of Ser-543 by endogenous protein phosphatases.

Three lines of evidence indicate that the 14-3-3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser-543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho-NR caused by 14-3-3 proteins. Second, sequence-specific and phosphorylation-dependent binding of the aforementioned synthetic peptide to the 14-3-3 proteins was demonstrated in vitro. Third, 14-3-3 proteins were required for the ATP-dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR-kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14-3-3 isoforms in the interaction with phospho-NR: omega> chi> upsilon>>> phi, psi.

14-3-3 Proteins↗

PubMed1996

The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein.

The inhibitor protein (IP) that inactivates spinach leaf NADH:nitrate reductase (NR) has been identified for the first time as a member of the eukaryotic 14-3-3 protein family based on three lines of evidence. First, the sequence of an eight amino acid tryptic peptide, obtained from immunopurified IP, matched that of a highly conserved region of the 14-3-3 proteins. Second, an authentic member of the 14-3-3 family, recombinant Arabidopsis GF14omega, caused inactivation of phospho-NR in a magnesium-dependent manner identical to IP. Third, an anti-GF14 monoclonal antibody cross-reacted with IP and anti-IP monoclonal antibodies cross-reacted with GF14omega.

14-3-3 Proteins↗

PubMed1996

Immunity to viruses in B cell-deficient mice: influence of antibodies on virus persistence and on T cell memory.

Mice rendered B cell deficient by targeted disruption of the immunoglobulin mu chain gene (IgM-/- mice) were used to analyze the role of antibodies and B cells in viral infections; homozygous IgM-/- mice were bred in a way to avoid transmission of maternal antibodies. After infection with vesicular stomatitis virus (VSV), IgM-/- mice developed paralytic disease and subsequently died, whereas C57BL/6 control mice or IgM-/- mice passively protected with VSV-neutralizing antibodies survived. Furthermore, IgM-/- mice showed increased natural killer (NK) activity upon exposure to either lymphocytic choriomeningitis virus (LCMV) or to poly(I).poly(C), while NK activity in untreated IgM-/- mice was within normal ranges. Cytotoxic T cell responses were comparable in IgM-/- and control mice infected either with VSV or with vaccinia virus or with low doses of LCMV (10(2) infectious focus-forming units [ifu]). After intracerebral infection with LCMV-Armstrong, CD8+ T cell-mediated lethal lymphocytic choriomeningitis developed independently of the presence of B cells and antibodies. After infection with high doses (2 x 10(6) - 5 x 10(6) ifu) of LCMV-WE or LCMV-Docile, IgM-/- mice exhibited a reduced capacity to control these primary infections and had elevated virus titers for prolonged times (> 60 days). Nevertheless, the cytotoxic T cell response against LCMV in the early phase of infection was comparable in IgM-/- and control mice, but disappeared in those IgM-/- mice which had a persistent viral infection. Cytotoxic T cell memory was apparently unimpaired in low-dose-primed IgM-/- mice, which were able to control the primary virus infection; both IgM-/- and control mice cleared a high intravenous dose of virus within 2 days after challenge infection. This indicates that an efficient T cell memory against LCMV was established in the absence of B cells.

Animals↗

PubMed1996

A frame shift mutation in a hot spot region of the nuclear autoantigen La (SS-B).

A hot spot region was identified in the exon 7 of the nuclear autoantigen La (SS-B). Two La cDNAs were identified which contained a frame shift mutation in the hot spot region. One La cDNA was isolated from a cDNA library made from peripheral blood lymphocytes of an autoimmune patient with primary Sjögren's Syndrome, the other La cDNA was isolated from a human liver cDNA library. The patient's La cDNA had a deletion and the liver La cDNA had an insert of an (A)-residue at the same position. Inserts of 4, 16 and 24 more or less homogeneous (A)-residues were found at the same site in the three La retropseudogenes. The hot spot region located in one of the major autoepitope regions of the La antigen. Both frame shift mutations resulted in premature stop codons. In case of the human liver La cDNA, the premature stop codon located a single amino acid downstream of the frame shift mutation, while it located eleven amino acids downstream of the frame shift mutation in the patient's La cDNA. In consequence, only the sequence of the La peptide encoded by the patient's La cDNA markedly differed from the corresponding La peptide sequence. Translation of the patient's mutant La mRNA in transfected mouse cells resulted in a C-terminally truncated La peptide. Due to the lack of the nuclear location signal it remained in the cytoplasm. The modified La peptide shared homology with (i) La protein itself and (ii) a series of DNA binding proteins including other autoantigens and viral proteins such as topoisomerase I, RNA dependent RNA polymerase of influenza virus and reverse transcriptase. The self-homology region includes the amino acids which the La protein shares with B1 Laminin. It represents a putative neo-epitope that could be involved in triggering of the autoimmune response.

3T3 Cells↗

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↗