Biomedical subjects

M Schmid

Publications and source records attributed to M Schmid.

At least 127 records · Page 7Linked to original sources

PubMed2000

Collagens serve as an extracellular store of bioactive interleukin 2.

The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that interleukin 2 (IL-2), an important stimulator of T cell growth, preferentially binds to collagen types I, III, and VI and to a lesser degree to collagen types II, IV, and V, immobilized on polystyrene or nitrocellulose. These interactions are inhibited by denatured, single collagen chains or a subset of their cyanogen bromide peptides in a dose-dependent manner. Cross-inhibition experiments and ligand blotting of collagen-derived peptides point to a limited set of collagenous consensus sequences mediating the binding of IL-2. This interaction is saturable, with dissociation constants of approximately 10(-)(8) m, and estimated molar ratios of 4-6 molecules of IL-2 bound to one molecule of triple helical collagen. Furthermore, collagen-bound IL-2 stimulates proliferation of mouse lymphocytes. We conclude that its specific binding to the abundant interstitial collagens leads to a spatial pattern of bioavailable IL-2 which is dictated by the local organization of the collagenous extracellular matrix. This interaction may contribute to the particular phenotype of stromal lymphocytes and could be exploited for devising collagenous peptide analogues that modulate IL-2 bioactivity.

Animals↗

PubMed2000

A methylthioadenosine phosphorylase (MTAP) fusion transcript identifies a new gene on chromosome 9p21 that is frequently deleted in cancer.

Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines, and are also common in primary gliomas, lung cancers, and leukemias. Moving from the centromere to the telomere, this complex region encodes the tumor suppressor genes p15INK4B (CDKN2B), p14ARF, p16INK4A (CDKN2A), and the housekeeping gene methylthioadenosine phosphorylase (MTAP). However, not all chromosome 9p21 deletions in tumors include these tumor suppressor genes. Here we describe the partial sequence and the exact localization of a new gene on chromosome 9p21 centromeric of p15INK4B, that formed an in frame fusion transcript with MTAP in a glioma xenograft, and that is homozygously deleted in various malignant cell lines. Northern blot revealed corresponding 1.5 kb transcript in non-deleted cell lines as well as in normal lymphocytes. Using a RNA master blot membrane including 50 different tissues, we could show that this new transcript is expressed in all tissues of the adult but not or only at very low levels in most of the fetal tissues tested. The expression pattern is similar to that of p16INK4A. The localization as well as the deletion pattern makes this transcript a candidate for a new tumor suppressor gene.

Adult↗

PubMed2000

Identification of the active site of phosphoribosyl-dephospho-coenzyme A transferase and relationship of the enzyme to an ancient class of nucleotidyltransferases.

Malonate decarboxylase from Klebsiella pneumoniae contains an acyl carrier protein (MdcC) to which a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group is attached via phosphodiester linkage to serine 25. We have shown in the preceding paper in this issue that the formation of this phosphodiester bond is catalyzed by a phosphoribosyl-dephospho-coenzyme A transferase MdcG with the substrate 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA that is synthesized from ATP and dephospho-coenzyme A by the triphosphoribosyl transferase MdcB. The reaction catalyzed by MdcG is related to nucleotidyltransfer reactions, and the enzyme indeed catalyzes unphysiological nucleotidyltransfer, e.g., adenylyltransfer from ATP to apo acyl carrier protein (ACP). These unspecific side reactions are favored at high Mg(2+) concentrations. A sequence motif including D134 and D136 of MdcG is a signature of all nucleotidyltransferases. It is known from the well-characterized mammalian DNA polymerase beta that this motif is at the active site of the enzyme. Site-directed mutagenesis of D134 and/or D136 of MdcG to alanine abolished the transfer of the prosthetic group to apo ACP, but the binding of triphosphoribosyl-dephospho-CoA to MdcG was not affected. Evidence is presented that similar to MdcG, MadK encoded by the malonate decarboxylase operon of Malonomonas rubra and CitX from the operon encoding citrate lyase in Escherichia coli are phosphoribosyl-dephospho-CoA transferases catalyzing the attachment of the phosphoribosyl-dephospho-CoA prosthetic group to their specific apo ACPs.

Acyl Carrier Protein↗

PubMed2000

Regulation of chromatin structure by site-specific histone H3 methyltransferases.

The organization of chromatin into higher-order structures influences chromosome function and epigenetic gene regulation. Higher-order chromatin has been proposed to be nucleated by the covalent modification of histone tails and the subsequent establishment of chromosomal subdomains by non-histone modifier factors. Here we show that human SUV39H1 and murine Suv39h1--mammalian homologues of Drosophila Su(var)3-9 and of Schizosaccharomyces pombe clr4--encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro. We mapped the catalytic motif to the evolutionarily conserved SET domain, which requires adjacent cysteine-rich regions to confer histone methyltransferase activity. Methylation of lysine 9 interferes with phosphorylation of serine 10, but is also influenced by pre-existing modifications in the amino terminus of H3. In vivo, deregulated SUV39H1 or disrupted Suv39h activity modulate H3 serine 10 phosphorylation in native chromatin and induce aberrant mitotic divisions. Our data reveal a functional interdependence of site-specific H3 tail modifications and suggest a dynamic mechanism for the regulation of higher-order chromatin.

Amino Acid Sequence↗

PubMed2000

Activation of nuclear factor-kB in the spinal cord of experimental allergic encephalomyelitis.

The DNA-binding activity of nuclear factor (NF-kB) was found to be induced in the spinal cord of rats with experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), from the onset of the disease. This activation of NF-kB persisted throughout the disease period and decreased thereafter in the recovery phase. Supershift analysis of NF-kB DNA-binding activity in nuclear extracts of spinal cords showed that RelA/p65 and p50 subunits but not c-Rel/p75, RelB/p68 and p52 subunits were involved in DNA binding. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB activation, markedly inhibited the in vivo activation of NF-kB in the spinal cord of EAE rats and attenuated the clinical symptoms of EAE. These studies suggest that activation of NF-kB plays an important role in the pathogenesis of EAE and inhibitors of NF-kB activation may have therapeutic importance in MS.

Animals↗

PubMed2000

Linkage and family-based association study of schizophrenia and the synapsin III locus that maps to chromosome 22q13.

The human synapsin III gene (synapsin III) is a member of a neuron-specific phosphoprotein gene family involved in short-term neurotransmitter release. We mapped synapsin III to chromosomal region 22q13 (13.1-13.31) by fluorescence in situ hybridization, a region that has been identified as a potential schizophrenia susceptibility locus. The dinucleotide repeat marker D22S280 located in intron 5 of synapsin III was genotyped in a linkage and family-based association study to assess the role of the synapsin III locus in the etiology of schizophrenia. In 12 pedigrees with periodic catatonia comprising 135 individuals, we found exclusion of linkage of marker D22S280 using lod score analysis with autosomal dominant/recessive models as well as affected only LOD score methods with dominant/recessive models. In a family-based association study of 61 unrelated parent-offspring trios with schizophrenia (according to the the Diagnostic and Statistical Manual of Mental Disorders, fourth edition [DSM-IV, American Psychiatric Association, 1994]), we found no association of individual D22S280 alleles to disease. Results of a multiallelic transmission/disequilibrium test (TDT(max) = 3.00; P = 0.55) challenged the possibility that D22S280 alleles appear with DSM-IV schizophrenia more frequently than expected. In addition, no evidence for gender differences or parent-of-origin effects were found. Thus, the synapsin III locus at chromosome 22q13 is not likely to contain a schizophrenia susceptibility gene.

Adult↗

PubMed2000

Characterization of the human and mouse HEY1, HEY2, and HEYL genes: cloning, mapping, and mutation screening of a new bHLH gene family.

Many basic helix-loop-helix (bHLH) transcription factors are known as key regulators of embryonic development or differentiation in various species. We have isolated and characterized three new hairy-related bHLH transcription factor genes from mouse and human (hairy and Enhancer-of-split related with YRPW motif; HEY1, HEY2, and HEYL). All three HEY genes have a similar genomic structure with five exons. Together with a highly related Drosophila homologue, they form a new bHLH gene subfamily that is different from both hairy and the known vertebrate Hes and Her genes. While the overall structure with the bHLH domain, Orange domain, and WRPW motif is similar, the last motif is changed to KPYRPWG in Hey1/2 and absent in HeyL. This and other sequence features suggest Hey proteins to have unique functional properties. The genes were mapped by fluorescence in situ hybridization and RH mapping to the following human chromosomes: (HEY1) 8q21, (HEY2) 6q21, and (HEYL) 1p34.3. Based on expression patterns and map location, HEY genes are candidates for several human or mouse disease loci. However, initial screening of DNA from affected individuals for two human disorders and four mouse mutants did not reveal any diagnostic alterations in the coding regions.

Amino Acid Sequence↗

PubMed2000

Radiation treatment for cervical esophagus: patterns of practice study in Canada, 1996.

PURPOSE: To assess the patterns of practice among Canadian radiation oncologists who treat esophageal cancers, using a trans-Canada survey, completed at the end of 1996. METHODS AND MATERIALS: One of 3 case presentations of different stages of cervical esophageal cancer was randomly assigned and sent to participating radiation oncologists by mail. Respondents were asked to fill in questionnaires regarding treatment techniques and to outline target volumes for the boost phase of radiotherapy. Radiation oncologists from 26 of 27 (96%) of all Canadian centers participated. RESULTS: High-energy X-rays (>/= 10 MV) were employed by 68% of the respondents in part of the treatment course. The majority (83%) of the radiation oncologists used at least two phases of treatment. Very few, 10 of 59 (17%), responses started with multifield treatment. The most frequently used prescription dose was 60 Gy/30 fractions/6 weeks, given with concurrent chemotherapy. Dose prescriptions were to the isocenter in 39 of 48 (81%) or to a particular isodose line in 9 of 48 (19%) of respondents. CONCLUSION: There was a variety of radiation treatment techniques in this trans-Canada survey. The majority of the patients had combined cisplatin-based chemoradiation. The isocenter was not used consistently as a dose prescription point.

Aged↗

PubMed2000

Gross chromosomal rearrangements and genetic exchange between nonhomologous chromosomes following BRCA2 inactivation.

Cancer-causing mutations often arise from gross chromosomal rearrangements (GCRs) such as translocations, which involve genetic exchange between nonhomologous chromosomes. Here we show that murine Brca2 has an essential function in suppressing GCR formation after chromosome breakage. Cells that harbor truncated Brca2 spontaneously incur GCRs and genomic DNA breaks during division. They exhibit hypersensitivity to DNA damage by interstrand cross-linkers, which even at low doses trigger aberrant genetic exchange between nonhomologous chromosomes. Therefore, genetic instability in Brca2-deficient cells results from the mutagenic processing of spontaneous or induced DNA damage into gross chromosomal rearrangements, providing a mechanistic basis for cancer predisposition.

Animals↗

PubMed2000

Essential role of tyrosine 229 of the oxaloacetate decarboxylase beta-subunit in the energy coupling mechanism of the Na(+) pump.

The membrane-bound beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coupled to Na(+) translocation and consumes a periplasmically derived proton. Upon site-directed mutagenesis of 20 polar and/or conserved residues within putative membrane-integral regions, the specific oxaloacetate decarboxylase activities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the network by which the proton of S382 is delivered to carboxybiotin, where it is consumed upon catalyzing the immediate decarboxylation of this acid-labile compound. Unlike S382 or D203, Y229 appears to be not involved in Na(+) binding, because in the Y229F orY229A mutants, the beta-subunit was protected from tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloacetate decarboxylase with a betaC291E mutation was unstable in the absence of Na(+) and dissociated into an alpha-gamma subcomplex and the beta-subunit. The enzyme could only be isolated in the presence of 0. 5 M NaCl. These results are consistent with the notion that the beta-subunit changes its conformation upon Na(+) binding.

Amino Acid Substitution↗

PubMed2000

A molecular coupling mechanism for the oxaloacetate decarboxylase Na+ pump as inferred from mutational analysis.

The oxaloacetate decarboxylase Na+ pump consists of subunits alpha, beta, and gamma, and contains biotin as the prosthetic group. Membrane-bound subunit beta catalyzes the decarboxylation of carboxybiotin coupled to Na+ translocation, and consumes a periplasmically derived proton. Site-directed mutagenesis of conserved amino acids of transmembrane helix VIII indicated that residues N373, G377, S382, and R389 are functionally important. The polar side groups of these amino acids may constitute together with D203 a network of ionizable groups which promotes the translocation of Na+ and the oppositely oriented H+ across the membrane. Evidence is presented that two Na+ ions are bound simultaneously to subunit beta during transport with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin, and a conformational switch exposes the bound Na+ ions toward the periplasm. After dissociation of Na+ and binding of H+, the cytoplasmically exposed conformation is regained.

Amino Acid Sequence↗

PubMed2000

Atomic-scale structure and catalytic reactivity of the RuO(2)(110) surface

The structure of RuO(2)(110) and the mechanism for catalytic carbon monoxide oxidation on this surface were studied by low-energy electron diffraction, scanning tunneling microscopy, and density-functional calculations. The RuO(2)(110) surface exposes bridging oxygen atoms and ruthenium atoms not capped by oxygen. The latter act as coordinatively unsaturated sites-a hypothesis introduced long ago to account for the catalytic activity of oxide surfaces-onto which carbon monoxide can chemisorb and from where it can react with neighboring lattice-oxygen to carbon dioxide. Under steady-state conditions, the consumed lattice-oxygen is continuously restored by oxygen uptake from the gas phase. The results provide atomic-scale verification of a general mechanism originally proposed by Mars and van Krevelen in 1954 and are likely to be of general relevance for the mechanism of catalytic reactions at oxide surfaces.

Journal Article↗

PubMed2000

Intussusceptive angiogenesis: its role in embryonic vascular network formation.

Intussusceptive angiogenesis is a novel mode of blood vessel formation and remodeling, which occurs by internal division of the preexisting capillary plexus without sprouting. In this study, the process is demonstrated in developing chicken eye vasculature and in the chorioallantoic membrane by methylmethacrylate (Mercox) casting, transmission electron microscopy, and in vivo observation. In a first step of intussusceptive angiogenesis, the capillary plexus expands by insertion of numerous transcapillary tissue pillars, ie, by intussusceptive microvascular growth. In a subsequent step, a vascular tree arises from the primitive capillary plexus as a result of intussusceptive pillar formation and pillar fusions, a process we termed "intussusceptive arborization." On the basis of the morphological observations, a 4-step model for intussusceptive arborization is proposed, as follows: phase I, numerous circular pillars are formed in rows, thus demarcating future vessels; phase II, formation of narrow tissue septa by pillar reshaping and pillar fusions; phase III, delineation, segregation, growth, and extraction of the new vascular entity by merging of septa; and phase IV, formation of new branching generations by successively repeating the process, complemented by growth and maturation of all components. In contrast to sprouting, intussusceptive angiogenesis does not require intense local endothelial cell proliferation; it is implemented primarily by rearrangement and attenuation of the endothelial cell plates. In summary, transcapillary pillar formation, ie, intussusception, is a central and probably widespread process, which plays a role not only in capillary network growth and expansion (intussusceptive microvascular growth), but also in vascular plexus remodeling and tree formation (intussusceptive arborization).

Allantois↗

PubMed2000

Critical factors in the radioimmunoassay of endothelin-1, endothelin-3, and big endothelin-1 in human plasma.

The possible diagnostic or prognostic significance of changes in circulating level of endothelins in a variety of pathological conditions is currently of interest. Unfortunately, no consensus regarding optimization of sensitivity and extraction procedures for the reliable radioimmunoassay of endothelin-1 (ET-1), big endothelin-1 (BigET-1), and endothelin-3 (ET-3) currently exists. The object of the present study was to evaluate aspects of currently used extraction and assay procedures that limit accurate determination of ET in human plasma and define criteria to reduce variability. Critical parameters include the selectivity of commercial antibodies and the ability to remove interfering material after Sep-Pak absorption by selective washing with 24% ethanol in 4% acetic acid or methylene chloride in 0.1% trifluoroacetic acid. Assay sensitivity and specificity in the physiological range is improved by optimizing total binding parameters for the antibodies to give approximately 15-20% binding of radiolabeled peptide. With these modifications normal plasma values for ET-1, BigET-1, and ET-3 averaged 1.7 +/- 0.06, 2.5 +/- 0.3, and 5.8 +/- 0.2 pg/ml, respectively. These data suggest that such modifications may help to resolve many of the earlier difficulties concerning the role of ET under normal and pathological conditions.

Endothelin-1↗

PubMed2000

Noninvasive determination of genome size and ploidy level in fishes by flow cytometry: detection of triploid Poecilia formosa.

BACKGROUND: In order to understand the evolutionary significance of single triploids among the mostly diploid Poecilia formosa we have developed a simple, noninvasive technique for DNA content and ploidy determination. METHODS: From dorsal fin clips of 14 different fish species single cell suspensions were obtained by chopping the material in 2.1% citric acid/0.5% Tween20, passing it through a 0. 6-gauge needle and incubating it for 20 min at room temperature (RT) with gentle agitation. After overnight fixation in 70% ethanol, the cells were treated with 1ml 0.5% pepsin/0.1 M HCl for 15 min at RT before adding DAPI to a final volume of 2 ml. The cells were stained for 1-3 h and then analyzed by flow cytometry. RESULTS: We obtained good measurements with CVs ranging from 1.23% to 3.36%. The poeciliid species measured contain from 1.6 to 2.0 pg/nucleus, Oryzias latipes (Medaka) exhibits a nuclear DNA content of 2.2 pg, Danio rerio (zebrafish) 4.6 pg, Tetraodon fluviatilis (freshwater fugu) 0.70 pg. All values except zebrafish are in good agreement with the literature. CONCLUSIONS: The identification of living specimens of different ploidy for breeding experiments, behavioral studies and tissue transplantations is now made possible. With slight modifications the method can be extended to a field technique, providing therefore a useful tool for a variety of researchers.

Animals↗

PubMed2000

Effects of nicotine on bone and calciotropic hormones in growing female rats.

Limited research in young adults and immature animals suggests a detrimental effect of tobacco on bone during growth. This study investigated the effects of nicotine, the major alkaloid component of tobacco, on calciotropic hormone concentrations and bone status in growing female rats. One-month-old animals received either saline (n = 10), nicotine at 3.0 mg/kg/day (n = 10), or nicotine at 4.5 mg/kg/day (n = 10) administered subcutaneously via osmotic minipumps for either 2 or 3 months. Sera, femora, tibiae, and lumbar vertebrae (3-5) were collected at necropsy. The concentrations of serum calcium, phosphorus, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone, calcitonin, and insulin-like growth factor-I were determined. Bone variables evaluated included mineral content and density (vertebrae and femora), cancellous and cortical histomorphometry (tibiae), and bone strength (vertebrae and femora). Statistically significant differences in serum mineral and hormone concentrations were not associated with nicotine dose or exposure time. No significant nicotine treatment effects were detected for bone mineral content and density, bone histomorphometry, or bone strength. We conclude that nicotine treatment for 2 or 3 months at serum concentrations in the upper range of those found in smokers has no detrimental effect on bone mass, volume, or strength in the growing rat.

Animals↗

PubMed2000

hKCNN3 which maps to chromosome 1q21 is not the causative gene in periodic catatonia, a familial subtype of schizophrenia.

The human calcium-activated potassium channel gene (hKCNN3, hSKCa3) contains two tandemly arranged, multiallelic CAG repeats located in exon 1 which result in short to moderate polyglutamine stretches of unknown functional significance. Case-control and family-based association studies suggested an association of hKCNN3 repeats with susceptibility for schizophrenia. Twelve multiplex pedigrees with periodic catatonia, a schizophrenia subtype with major gene effect and patterns of anticipation, were genotyped using the multiallelic hKCNN3 repeat polymorphism. Using a dominant model of inheritance with sex- and age-dependent penetrance classes, cumulative results showed exclusion of linkage of hKCNN3 to periodic catatonia under the assumption of genetic homogeneity with lod score of -48.01 at zero recombination fraction. Our results provide evidence that hKCNN3 is not the causative gene in the familial schizophrenia subtype of periodic catatonia. By fluorescent in situ hybridization we confirmed the assignment of hKCNN3 to chromosome 1q21 near the heterochromatin region. Linkage mapping showed segregation with marker D1S498 (theta = 0.05) and placed hKCNN3 in the genetic linkage map in a cluster of genes near the centromeric region of chromosome 1.

Adult↗

PubMed2000

Amplification of a long terminal repeat-like element on the Y chromosome of the platyfish, Xiphophorus maculatus.

The platyfish (Xiphophorus maculatus), in which sex chromosomes are evident from stable and predictable inheritance of sex, is one of the best-studied lower vertebrates with respect to sex determination. In order to identify the structural equivalent for this in the karyotype, which does not contain heteromorphic pairs of chromosomes, two sex-linked molecular probes were used for fluorescent in situ hybridization analysis. One probe, derived from the melanoma oncogene locus ONC-Xmrk, stained both the X and the Y chromosome. This cytogenetic analysis mapped the sex-determining locus to the subtelomeric region of a medium-sized telocentric chromosome. Another probe, a repetitive element (XIR), specifically labeled the Y chromosome in metaphase spreads and in interphase nuclei. The sex chromosomes of X. maculatus can be considered to be at an early stage of evolution of gonosomes. Expansion of the XIR repeat is obviously one of the earliest of the molecular events that lead to divergence of the Y chromosome and recombinational isolation of the sex-determining locus.

Alleles↗