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C Barth

Publications and source records attributed to C Barth.

At least 37 records · Page 2Linked to original sources
PubMed1996

Replicon rescue: a novel strategy to clone the genomic DNA flanking insertions of integrating shuttle vector DNA.

A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions. After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously. Restriction enzymes for cloning are so chosen that the only legitimate two fragment ligation yielding TetR clones involves a fragment spanning the boundary of the insertion. Replicon rescue was used successfully firstly in a test system to clone the chromosomal orl from a Klebsiella aerogenes strain, and secondly to recover a disrupted gene from a phototaxis-deficient mutant of Dictyostelium.

Animals↗
PubMed1996

Restricted TCR V beta repertoire in the human mixed lymphocyte reaction is determined by the TCR on the responder cell and not by the stimulating antigen on the stimulator cell.

The use of a restricted TCR repertoire has been reported in antigen-stimulated T cells. We have examined by flow cytometry and polymerase chain reaction the changes in the TCR variable beta (V beta) repertoire as they occur in the mixed leukocyte reaction. The HLA class I-negative lymphoblastoid B cell line, Daudi, an HLA class I-positive variant of the Daudi cell line, and the homozygous typing cell line HTC 9062 were used to stimulate HLA-mismatched responder cells from 8 individuals. The magnitude of HLA incompatibility in various combinations ranged from single DR10 and DR13 differences to full 6-antigen differences at the HLA-A, -B, and -DR loci. Using a panel of 19 V beta-specific oligonucleotide primers, changes in the level of TCR V beta mRNA were assessed. The observed V beta repertoire was not specific for the stimulator cell antigens used. We did not find a correlation between the number of mismatched HLA antigens and the number of V beta elements involved. It rather appeared that individual responder cells displayed a uniform V beta pattern following exposure to single and multiple HLA antigens. We conclude that the V beta repertoire was largely determined by factors associated with the TCR on the responder cells, and not as obviously influenced by the HLA antigen(s) on the stimulator cells.

Antigen Presentation↗
PubMed1996

Efficient circularization in Escherichia coli of linear plasmid multimers from Dictyostelium discoideum genomic DNA.

Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid multimers cyclize in E. coli by homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of the recA gene product but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. discoideum genome ensured that none of the E. coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of D. discoideum.

Animals↗
PubMed1996

Restricted T cell V beta repertoire in renal allografts during acute and chronic rejection.

Allo-MHC specific antigen recognition might not only be involved in acute, but also in chronic rejection. The clonotypic specificity of the T-cell receptor to recognize all-MHC is located in the variable (V) alpha and beta chain. A restricted T-cell receptor repertoire could support an immunological basis for chronic rejection. The novel feature of this study is that V beta repertoire was assessed in ongoing chronic rejection before end-stage renal failure and in acute rejection. V beta s 1 to 20 were quantitated by PCR in PBMC and biopsies of rejecting renal allografts. The V beta pattern in PBMC demonstrated a polyclonal distribution. However, the intragraft V beta repertoire was restricted to 1 to 3 dominant V betas and highly individual in 9 of 12 patients. Number and type of the HLA mismatch and the time interval between transplantation and biopsy did not correlate to the V beta distribution. The individual response is attributed to genetic predisposition factors of the recipient. Therefore, the restriction of the V beta repertoire indicates allo-MHC dependent immune processes not only in acute, but also in ongoing chronic rejection. Tailor-made antibodies against dominant V betas might offer specific individual immunosuppression in treating both acute and ongoing chronic rejection.

Graft Rejection↗
PubMed1996

Comparison of in-vitro falloposcopy with tubal histology in the diagnosis of fallopian tube pathology.

In order to assess the diagnostic quality of falloposcopy in relation to pathomorphology, a consecutive series of 30 Fallopian tubes obtained from surgical salpingectomy cases were prospectively examined by in-vitro falloposcopy and histology. Falloposcopy was performed using an over-the-wire catheterization system and a 0.5 mm falloposcope with 3000 pixels. Assessment of the specimens included the description of lumen geometry, intraluminal changes and status of the mucosal surface. Falloposcopy classified 14 tubes as normal and 16 pathological. Histology resulted in 17 normal versus 13 pathological tubes. Pathologies included lumen obstructions and dilatations, intraluminal synechiae and mucosal damage. Sensitivity and specificity of falloposcopy were calculated to be 0.85 and 0.71; positive and negative predictive values were 0.69 and 0.86. It was concluded that falloposcopic findings indeed reflect and successfully differentiate normal and pathological conditions allowing adequate and reproducible image interpretation. However, variations of the diagnostic accuracy with the type of pathology and the tubal segment have to be taken into account before clinical consequences are drawn from a falloposcopic investigation.

Endoscopy↗
PubMed1996

A proteoglycan that activates fibroblast growth factors during early neuronal development is a perlecan variant.

Cells in the early embryonic vertebrate nervous system are dependent on members of the fibroblast growth factor (FGF) family for their proliferation and subsequent differentiation. These growth factors will only bind to their specific high affinity cell surface receptors after formation of a ternary complex with the glycosaminoglycan heparan sulfate. Such specific heparan sulfates are secreted as proteoglycans from neural precursor cells and localise to their surfaces. One such proteoglycan, HSPG-PRM (Perlecan-related molecule), was isolated through its ability to potentiate neural cell responses to either FGF-1 or FGF-2. In this study, we have verified the relative molecular mass of the core protein of PRM as 45,000 and obtained partial amino acid sequence from it. The sequences bore significant homology to native perlecan. A probe generated by reverse transcriptase polymerase chain reaction using oligonucleotides designed from the protein sequence used on northern blots of RNA from a neuroepithelial cell line detected perlecan at 12.6 kilobases, as well as novel transcripts at 6.5 and 3.5 kilobases. The latter species appears by virtue of its size and abundance to be the novel PRM transcript. PRM appears to be encoded by the same gene as perlecan, as genomic Southern blotting only detected a single gene. Polyclonal antibodies raised against the PRM molecule detected a single proteoglycan species at 290x10(3) with a core protein of 45x10(3). Polyclonal anti-perlecan antibodies cross-reacted with PRM confirming their relatedness, although immunohistochemical studies revealed a differential staining pattern for PRM as compared to perlecan within the developing nervous system. The PRM molecule was shown to be localised to several different tissues of the developing embryo, indicating that it plays a broad role. We conclude that PRM is a variant of perlecan that is differentially glycosylated in a manner that confers highly specific functions at critical stages of neural development and tissue growth.

Alternative Splicing↗
PubMed1995

Application of a fast charge-pulse technique to study the effect of the dipolar substance 2,4-dichlorophenoxyacetic acid on the kinetics of valinomycin mediated K(+)-transport across monoolein membranes.

We report on a modified charge-pulse relaxation technique applied at planar lipid membranes. The method has an improved time resolution of 20-30 ns. It is based on the capacitive coupling of a voltage-jump to the membrane. The method was used to study the fast relaxation processes induced by valinomycin/K+ in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). The change of the rate constants of the ion carrier valinomycin was analysed as a consequence of the adsorption of the dipolar substance 2,4-D to the membrane/water interface of monoolein membranes. The effect of 2,4-D can be explained solely via the influence of the introduced dipole potential, VD. The latter was found to act (primarily) on the inner membrane barrier experienced by the positively charged carrier-ion complex and on the interfacial barriers responsible for complex formation and dissociation. No evidence for a change of the microviscosity of the membrane interior was obtained.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1995

Radiation-induced and free radical-mediated inactivation of ion channels formed by the polyene antibiotic amphotericin B in lipid membranes: effect of radical scavengers and single-channel analysis.

This paper is part of a study on the effect of ionizing radiation on ion channels in biological membranes. Ion channels formed by polyene antibiotics amphotericin B or nystatin represent clusters of conjugated double bonds. As a consequence of this structural peculiarity, the conductance of lipid membranes--in the presence of polyene channels--has been found to decrease by several orders of magnitude at comparatively small doses of ionizing radiation. The phenomenon shows an inverse dose-rate behaviour similar to that of radiation-induced lipid peroxidation. We report on experiments performed in the presence of various radical scavengers, at varying cholesterol concentrations, and with different lipids. They support the view that channel inactivation is due to free radical-induced peroxidation of the polyenes leading to a destabilization of the barrel-like structure of the ion channels. Radiation-induced channel closing is shown for the first time at the level of single-ion channels.

Amphotericin B↗
PubMed1994

Non-responsiveness to hepatitis B vaccination in haemodialysis patients: association with impaired TCR/CD3 antigen receptor expression regulating co-stimulatory processes in antigen presentation and recognition.

The study was undertaken to evaluate the relationship between non-responsiveness to hepatitis B (HBV) vaccination in haemodialysed patients and HBs antigen (Ag) presentation and recognition depending on TCR/CD3 receptors expression. We have found that the cause of the blunted response to HBV vaccination is multifactorial and seems to be associated with the following: (1) A reduced number of TCR/CD3 antigen receptor complexes on freshly isolated uraemic CD4 T cells, especially in non-responders. (2) The blunted proliferative response of uraemic CD4 T cells isolated from non-responders and stimulated for 6 days by autologous monocytes presenting HBsAg was associated with the decreased density of the TCR/CD3 receptors. (3) Moreover, in uraemic non-responders the expression of adhesion and accessory molecules on monocytes (intercellular adhesion molecule-1/ICAM-1, HLA-DR/Ia/) was significantly decreased following the culture with autologous monocytes serving as HBsAg-presenting cells. CD4 molecules and lymphocyte function antigen-1 beta/LFA-1 beta/ on helper-inducer T cells were increased before and after the culture. (4) These findings were also associated with a diminished binding capacity of IL-1 beta and IL-6 to their receptors on helper-inducer T cells. (5) IL-2, IFN-gamma and IL-4 production was decreased in uraemic non-responders, especially after 72 h of the culture. (6) Inhibited proliferation of helper-inducer T cells in uraemic non-responders was only partially reversible in the presence of exogenous IL-1 beta, IL-6, IL-2 and IFN-gamma. (7) HLA typing of uraemic non-responders was associated with extended haplotype: HLA A1,B8,DR3,DR7,DQ2.

Adult↗
PubMed1993

Inactivation by ionizing radiation of ion channels formed by polyene antibiotics amphotericin B and nystatin in lipid membranes: an inverse dose-rate behavior.

The phenomena reported are part of a study about the effects of ionizing radiation on membrane transport. We found that the conductance of lipid membranes in the presence of the polyene-antibiotics nystatin or amphotericin B is reduced to virtually zero following irradiation. Ion channels formed by these substances seem to represent extremely sensitive structures being inactivated by radiation doses in the range of a few Centigray (1 cGy = 1 rad) at sufficiently small dose rates. Inactivation shows a so-called inverse dose-rate behavior, i.e., at constant radiation dose the effect increases with decreasing dose rate. Similar to radiation-induced lipid peroxidation the phenomenon may be understood on the basis of a radical chain mechanism initiated by free radicals of water radiolysis. The process--via peroxidation of the polyene part of the molecules--is suggested to modify the hydrophobic exterior and to destabilize the barrel-like structure of the ion channels.

Amphotericin B↗
PubMed1991

Radiation inactivation of ion channels formed by gramicidin A. Protection by lipid double bonds and by alpha-tocopherol.

The conductance induced by the channel-forming peptide gramicidin A in lipid membranes is reduced by many orders of magnitude on exposure of the membrane and its aqueous environment to ionizing radiation. This results from an interaction of free radicals of water radiolysis with the tryptophan residues of gramicidin A. The sensitivity of the ion channels towards irradiation is strongly reduced in the presence of either vitamin E or of highly unsaturated lipids. An increase of the D37 dose up to a factor of 50 was found. The phenomena are interpreted via a reduction of the effective concentration of free radicals (such as OH.) in the membrane by reaction with unsaturated fatty acid residues or with vitamin E.

Electric Conductivity↗
PubMed1991

Estimation of carrier density and turnover rate of the Na+/H+ exchanger in human platelets using 5-(N-methyl-N-[3H]isobutyl)- amiloride.

We used the radiolabelled inhibitor of Na+/H+ exchange 5-(N-methyl-N-[3H]isobutyl)amiloride ([3H]-MIA) for assessment of the amount of Na+/H+ exchanger in intact human blood platelets. The inhibition constant, KI, of unlabelled MIA toward the antiport was determined at 100 nM. Washed platelets were incubated for 5 s with different concentrations of [3H]-MIA in the presence or absence of an excess concentration of unlabelled amiloride (400 microM). The platelets were rapidly centrifuged and the radioactivity in the pellet was determined. Scatchard analysis revealed one single class of specific binding sites (KD = 63 nM) and a maximum binding capacity of 500 sites/cell. The turnover rate of the Na+/H(+)-exchanger in unstimulated platelets was estimated at 800/s at 25 degrees C.

Amiloride↗
PubMed1991

Attenuation of myocardial ischemia during coronary occlusion by ultrashort-acting beta adrenergic blockade.

To assess the effect of the ultrashort-acting beta blocker esmolol on ischemia induced by acute coronary occlusion, we studied 16 patients undergoing coronary angioplasty. Doppler echocardiography and ECG monitoring were performed continuously before, during, and after balloon occlusion in the drug-free state and during esmolol infusion. Fourteen of the 16 patients had ST segment elevation during balloon inflation. However, maximal ST segment elevation (2.1 +/- 1.5 mm vs 1.7 +/- 1.3 mm, p less than 0.001) and duration of ST segment elevation (68 +/- 20 seconds vs 54 +/- 19 seconds, p less than 0.05) were both significantly reduced during esmolol infusion. Furthermore, the decrease in ejection fraction seen during drug-free balloon occlusions was significantly blunted during esmolol infusion. In the baseline state ejection fraction decreased from 55% to 38% (p less than 0.05) during coronary occlusion compared with a decrease from 52% to 49% (p = NS) during esmolol infusion. In addition, esmolol appeared to delay the onset of segmental wall motion abnormalities after coronary occlusion, occurring at a mean of 40 seconds after balloon inflation versus a mean of 31 seconds in the absence of beta blockade (p less than 0.05). Thus the use of ultrashort-acting beta blockade appears to diminish the extent and delay the onset of myocardial ischemia during acute coronary occlusion.

Adrenergic beta-Antagonists↗
PubMed1991

Ultra short-acting intravenous beta-adrenergic blockade as add-on therapy in acute unstable angina.

To assess the efficacy and safety of the ultra short-acting beta-blocking agent, esmolol, in acute unstable angina, we administered esmolol to 21 patients who had persistent angina despite conventional medical therapy. Following a baseline Doppler echocardiographic examination, esmolol was titrated to reduce the rate-pressure product by at least 20%. Once the patients had been receiving a maintenance dosage for 30 minutes, Doppler echocardiographic studies were repeated. Mean esmolol dose at target response was 17 +/- 16 mg/min, with the dosage range of 8 to 24 mg/min. Esmolol was effective in alleviating anginal chest pain in 18 of the 21 patients. Seven patients eventually underwent percutaneous transluminal coronary angioplasty (PTCA) and eight had coronary bypass surgery. The remainder were discharged receiving medical therapy including oral beta-blockade. During esmolol therapy, heart rate and blood pressure decreased significantly (86 +/- 14 to 68 +/- 12 beats/min and 125 +/- 16 to 103 +/- 20 mm Hg, both p less than 0.001). Cardiac output decreased from 5.4 +/- 1.3 to 4.5 +/- 1.1 L/min (p less than 0.001) secondary to a decrease in heart rate as stroke volume remained unchanged. Left ventricular ejection fraction increased from 47 +/- 12 to 49 +/- 13 with esmolol therapy, although this change was not statistically significant. Both the one third filling fraction as well as E/A ratio (ratio of early-to-late diastolic filling velocities) increased with esmolol therapy (35 +/- 8% to 38 +/- 8% and 0.73 +/- 0.2 to 0.85 +/- 0.23, both p less than 0.005), indicating improvement in left ventricular diastolic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1990

Transcriptional modulation of human IL-6 gene expression by verapamil.

Calcium channel-blocking agents interfere with the initial increase of cytosolic calcium that follows mitogenic stimulation of T lymphocytes. In cultures of mitogen-stimulated PBMC, verapamil also blocked T cell accumulation of cytoplasmic IL-2-encoding mRNA. In sharp contrast, the addition of verapamil to PHA and PMA-stimulated PBMC augmented the mitogen-stimulated increases in nuclear transcription of IL-6-encoding mRNA, steady state levels of IL-6 encoding mRNA, and release of IL-6 bioactivity. These experiments indicated that an increased IL-6 transcriptional rate rather than stabilization of transcripts accounted for the increased cytoplasmic IL-6 mRNA levels and subsequent expression of IL-6 bioactivity. These effects were not produced by nicardipine, another potent calcium channel blocker, or EGTA. We suggest that a non-calcium-dependent, IL-6 regulatory factor, absent or inactive in verapamil-treated cultures, inhibits IL-6 gene activation in mitogen-stimulated PBMC. Failure to express this inhibitory factor would result in IL-6 gene superinduction at a transcriptional level.

Blotting, Northern↗
PubMed1990

[Ultraviolet exposure in the environment of welding work sites].

It has been checked if the "limits of the acceptable doses of ultraviolet radiation of monochromatic radiation at the workplace" (NIOSH-standard of USA) are adhered to in the vicinity of welders' work stations in a factory hall. For recording of UV-doses polysulphon films have been used. According to these investigations non-welders are exposed to doses of UV-radiation which exceed the limits recommended by about eight times. Clinical examinations of these persons showed chronic damage of the external parts of the eyes in a higher percentage than in a control group. It is pointed to the necessity of taking protective measures to shield persons working in the vicinity of welding job sites from ultraviolet radiation.

Dose-Response Relationship, Radiation↗