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Biomedical subjects

A Schmarda

Publications and source records attributed to A Schmarda.

10 recordsLinked to original sources
PubMed1998

Characterization of the human gene coding for the swelling-dependent chloride channel ICln at position 11q13.5-14.1 (CLNS1A) and further characterization of the chromosome 6 (CLNS1B) localization.

Expression cloning revealed a chloride channel (ICln) that we found to be fundamental for the regulatory volume decrease in a variety of cells. The chromosomal localization of the human ICln-gene showed two loci, one at chromosome 11 in position q13.5-q14.1, termed CLNS1A, and a second one at chromosome 6 at position p12.1-q13, termed CLNS1B. In this study, we offer a detailed characterization of the CLNS1A gene and provide the exact position (6p12) and sequence data of CLNS1B, an intronless gene 91.3% homologous to the coding region of CLNS1A.

Amino Acid Sequence↗
PubMed1997

RHD/CE typing by polymerase chain reaction using sequence-specific primers.

BACKGROUND: Current DNA-based Rh system typing strategies may detect the two RH genes and their prevalent alleles, but they are known to fail sometimes, when rare RH alleles (e.g., D category phenotypes) are encountered. It is almost impossible to find a single DNA-based method that can accommodate the great heterogeneity within the human Rh system. STUDY DESIGN AND METHODS: An easy-to-perform DNA-based method for the detection of the two RH genes and their alleles, including variant RHD alleles, was developed. By the use of one RHD/C-, seven RHD-, and four RHCE-specific polymerase chain reactions, all triggered to work at identical thermocycling conditions, the DNA of 77 blood donors carrying weak D and that of 200 random donors with common D phenotype was investigated. In addition, 77 selected samples of ccDee and rare Rh system phenotypes were examined. RESULTS: Among 77 samples of weak D, one Rh33 and six DVI categories were detected, one of which showed new RHD-specific nucleotide patterns. In DFR and CCee samples, novel variant RHD alleles were found. RHD DNA types of 200 random donors were found to be concordant with their D phenotype. For RHE and RHe genotyping, a full correlation with serologic phenotypes was found. Our method for genotyping RHC and RHc failed in some cases, because of an already published RHc allelic variation, which we have called RHc(cyt48). An estimate of the frequency of this RHc(cyt48) allele in a white population was made. CONCLUSION: The presented exon-scanning RHD/CE polymerase chain reaction using sequence-specific primers complements current DNA-based Rh system typing strategies and is superior in the detection of variant RHD alleles.

Blood Donors↗
PubMed1996

Chromosomal localization of the genes (CLNS1A and CLNS1B) coding for the swelling-dependent chloride channel ICln.

ICln is a cloned chloride channel paramount for regulatory volume decrease. Two different loci that carry the coding region for ICln were identified in the human genome. By PCR strategies an intronless copy of the gene was located on chromosome 6 at position 6p12.1-6q13 (CLNS1B). By fluorescence in situ hybridization a copy carrying introns with a putative length of 19 kb was located at chromosome 11 on position 11q13.5-q14.1 (CLNS1A). The characterization and chromosomal localization of the ICln gene offer the opportunity to study the regulatory sites of this gene in greater detail and could be helpful in establishing linkages between ICln and potential human diseases.

Chloride Channels↗
PubMed1996

ABO glycosyltransferase genotyping by polymerase chain reaction using sequence-specific primers.

Serological typing for the classical ABO blood groups is routinely performed using anti-A and anti-B antisera of polyclonal or monoclonal origin, which are able to distinguish four phenotypes (A, B, AB, and O). Modern molecular biology methods offer the possibility of direct ABO genotyping without the need for family investigations. Typing can be done with small amounts of DNA and without detection of blood group molecules on the surface of red blood cells. We developed a system of eight polymerase chain reactions (PCR) to detect specific nucleotide sequence differences between the ABO alleles O1, O2, A1, A2, and B. PCR amplification using sequence-specific primers and detection of amplification products by agarose gel electrophoresis is one of the fastest genotyping methods and is easy to handle. With our method we tested the A1,2BO1,2 genotypes of 300 randomly chosen persons out of a pool of platelet donors and found the results to be consistent with ABO glycosyltransferase phenotypes. We also identified a presumably new ABO allele, which may be the result of a crossing-over event between alleles O1 and A2.

ABO Blood-Group System↗
PubMed1996

Blockade of swelling-induced chloride channels by phenol derivatives.

1. In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine Kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2. RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3. RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4. The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5. The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the ICln channel protein.

3T3 Cells↗
PubMed1996

ICln: a chloride channel paramount for cell volume regulation.

Cell volume regulation is a ubiquitous cell regulatory mechanism based on meticulously controlled ion transport mechanisms. Keeping the absolute volume constant seems to be of the highest priority for most cells and is achieved at the expense of altered intracellular ion concentrations. We have been able to demonstrate that ICln, a chloride channel cloned from epithelial cells, is paramount for the ability of swollen cells to regulate their volume back to that under resting conditions. A unique feature of ICln is the distinct sensitivity of these channels for nucleotides and nucleoside analogues added to the extracellular fluid. In addition, cromolyn sodium and nedocromil sodium, drugs used by patients with asthma, are able to impede the function of these channels.

Animals↗
PubMed1995

Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

Animals↗
PubMed1995

Antiviral drugs from the nucleoside analog family block volume-activated chloride channels.

BACKGROUND: The antiviral drugs AZT and acyclovir are generally used in the treatment of infections with human immunodeficiency virus (HIV) and herpes simplex virus (HSV). These substances are known to impede virus replication by premature nucleic acid chain termination. It is not yet clear, however, if this is the sole mechanism responsible for the antiviral and/or the numerous side effects observed in patients treated with these agents. We investigated the swelling-induced chloride current in fibroblasts, which we demonstrated is closely related or identical to a cloned epithelial chloride channel, ICln: This chloride channel can be blocked by nucleotides. MATERIALS AND METHODS: Electrophysiological, fluorescence optical, and volume measurements were made to determine the effect of nucleoside analogs on the swelling-dependent chloride current (ICl) in NIH 3T3 fibroblasts and in human T cell lymphoma (H9) cells and the cAMP-dependent chloride current in CaCo cells. RESULTS: AZT and acyclovir block the swelling-dependent chloride current and the chloride flux in fibroblasts, and the regulatory volume decrease (RVD) and ICl in H9 cells. This immediate effect can be substantially reduced by the simultaneous incubation of the cells with thymidine-5'-diphosphate (TDP) or uridine, both of which are by themselves unable to affect ICl. CONCLUSIONS: We show here a novel molecular mechanism by which antiviral drugs of the nucleoside analog family could lead to impairments of the kidney, bone marrow, gastrointestinal, and neuronal functions, and how these side effects could possibly be restricted by the presence of TDP or uridine.

3T3 Cells↗