Search PubMed⌕ Search

Biomedical subjects

F Koch

Publications and source records attributed to F Koch.

At least 109 records · Page 6Linked to original sources
PubMed1989

A single-step purification procedure and partial amino acid sequence analysis of picomole amounts of the rat T cell alloantigen RT6.2.

A single-step immunoaffinity purification procedure for the rat T cell marker RT6.2 is described which permits the isolation of microgram quantities of protein from the RT6.2+ T-T hybridoma EpD3. The amino terminus was sequenced directly from a polyvinylidene (PVDF) membrane blot prepared after SDS-PAGE. Further internal sequence data were obtained from peptides generated from purified RT6.2 digested with different endoproteases and separated by reverse-phase micro-HPLC. A computer search in data banks did not reveal any significant homology to other proteins.

ADP Ribose Transferases↗
PubMed1989

Murine epidermal Langerhans cells express significant amounts of class I major histocompatibility complex antigens.

Epidermal Langerhans cells (LC) are leukocytes that express major histocompatibility complex (MHC) class II antigens and function as antigen-presenting and accessory cells. Caughman et al. [Caughman, S. W., Sharrow, S. O., Shimada, S., Stephany, D., Mizuochi, T., Rosenberg, A. S., Katz, S. I. & Singer, A. (1986) Proc. Natl. Acad. Sci. USA 83, 7438-7442] reported that LC are deficient in surface expression of MHC class I antigens, implying a specialization of these cells to class II-restricted antigen presentation. To readdress this obviously important issue, we have studied murine epidermal sheets prepared from B6 X BALB/c----B6 bone marrow chimeras 5 months after irradiation and bone marrow reconstitution. This enabled us to distinguish class I of LC from that of surrounding keratinocytes. When sheets were analyzed by immunofluorescence microscopy with monoclonal antibodies specific for donor class I antigens, donor-derived LC but not LC of recipient origin were stained. Appropriate controls for antibody isotype and MHC haplotype were negative. LC in epidermal cell suspensions, prepared from normal BALB/c and BALB/cBy mice (MHC haplotype d), were analyzed by flow cytometry as well as immunofluorescence microscopy. LC were stained by monoclonal antibodies to class I antigens of haplotype d, but not by isotype-matched control antibodies to class I antigens of haplotype k. We also found that LC were virtually depleted from epidermal cell suspensions by treatment with monoclonal antibodies to class I antigens of haplotype d and complement but not by treatment with control monoclonal antibodies and complement. Our data, therefore, show that LC express MHC class I molecules on their surface.

Animals↗
PubMed1989

Evidence for normal thymic export of lymphocytes and an intact RT6a gene in RT6 deficient diabetes prone BB-rats.

Diabetes prone BB-rats develop an insulin-dependent diabetes mellitus between postnatal day 60 and 120 in a high incidence. Moreover, even before onset of the diabetes, these animals are deficient in the RT6+ subset of T lineage cells. We here put forward evidence that the export of cells from the thymus is normal in these rats, suggesting a maturational block in the periphery. We show furthermore by Southern blot analysis that diabetes prone BB-rats possess the RT6a gene which is not grossly altered in comparison to that of diabetes resistant strains.

Animals↗
PubMed1989

Construction of a rat T-cell hybridoma cDNA expression library and isolation of a cDNA clone for the rat T-cell differentiation marker RT6.2.

The phosphatidylinositol-linked T-cell surface protein RT6 occurs in two alloantigenic forms, RT6.1 and RT6.2. A lambda gt11 cDNA expression library was constructed from the RT6.2+ T-T hybridoma cell line EpD3 and screened with RT6-specific antisera. One of nine independent positive clones obtained in an initial screening of 500,000 recombinant phages was able to select antibodies capable of immunoprecipitating RT6.2 as well as RT6.1. Northern and Southern blot analyses with the cDNA insert of this clone provide additional strong evidence for its RT6.2 specificity. The 330 bp cDNA insert of this clone hybridizes with a 1.6 kb mRNA species present in RT6.2+ and RT6.1+ but absent from RT6- T-cell lines. In Southern blot analyses this insert detects a DNA restriction fragment length polymorphism between genomic DNAs from RT6.1 and RT6.2 rat strains. In addition, these analyses indicate that RT6.1 and RT6.2 are each encoded by a single gene and provide no evidence for the existence of additional RT6 alleles or a closely related gene family. Preliminary sequence analyses suggest that the RT6.2-specific partial cDNA found in these studies represents an internal EcoRI fragment of the corresponding cDNA and encodes part of the presumptive leader and the N-terminal domain of RT6.2. The immunological reactivity of the corresponding fusion protein indicates that the N-terminal domain of RT6.2 contains an allotype-specific epitope as well as at least one additional epitope shared with the alloantigenic counterpart RT6.1.

Animals↗
PubMed1988

Granulocyte/macrophage colony-stimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells.

Freshly isolated, murine epidermal Langerhans cells (LC) are weak accessory cells for primary T cell-dependent immune responses, but increase their stimulatory capacity at least 20-fold progressively over a 3-d culture with keratinocytes. We have studied the mediators of LC maturation. LC enriched from 12-h epidermal cultures by negative selection do not survive when cultured for 60 h in standard medium. LC survive and show increased stimulatory capacity for oxidative mitogenesis and the primary MLR when 30% keratinocyte-conditioned medium is included. Of the three cytokines that are known to be produced by keratinocytes, only granulocyte/macrophage CSF (GM-CSF) maintains viability and increases stimulatory capacity. IL-1 alone does not keep LC alive, but further enhances the stimulatory activity when combined with GM-CSF. IL-3 has no effect. The increase in LC stimulatory capacity is not due to increased Ia antigen expression, which does not change between 12 and 60 h. Function is not simply due to improved viability, as GM-CSF does not enhance the function of 12-h LC when added to the short-term oxidative mitogenesis assay. By generating LC with strong stimulating activity for resting T cells, GM-CSF and IL-1 may be critical in the sensitization phase of T cell-mediated immunity.

Animals↗
PubMed1988

Production of a rat T cell hybridoma that stably expresses the T cell differentiation marker RT6.2.

The rat T cell alloantigen RT6.2 shows a slow rate of synthesis in isolated T cells which hampers studies on the metabolism of RT6.2 in these cells (1). In order to facilitate further molecular and functional characterization of this molecule we have established a T-T hybridoma cell line which stably expresses RT6.2. Only 1 of 102 T cell hybridomas obtained upon fusion of the rat thymoma C58NT with DA rat lymph node cells expressed this antigen. This clone--EpD3--initially showed a relatively slow rate of cell division and a highly unstable pattern of expression of RT6.2. Thus, the relative number of RT6.2 bearing cells consistently decreased during cultivation of EpD3 and of EpD3-derived subclones. Cell separation studies suggest that the switch in RT6.2 phenotype of EpD3 cultures was due to the appearance of variant cells of RT6.2- phenotype with a higher proliferative capacity and was not induced by factors in the culture medium. By repeated panning and subcloning procedures to select for RT6.2 expressing cells, a subline of EpD3--EpD3/87--was obtained which stably expresses high levels of RT6.2. Metabolic labeling studies of RT6.2 in EpD3 show that it is synthesized very efficiently in these cells and support our previous suggestion that RT6.2 does not bear any classic oligosaccharide side chains. Moreover, RT6.2 can be released almost completely from EpD3 cells by phosphatidylinositol-specific phospholipase C (PI-PLC).

Animals↗
PubMed1988

The rat T-cell differentiation marker RT6.1 is more polymorphic than its alloantigenic counterpart RT6.2.

Utilizing allotype-specific antibodies to immunoprecipitate RT6.2 from DA.6B rat lymphocyte lysates, we have shown this antigen recently to be composed of two related, non-glycosylated polypeptides with apparent molecular weights (MW) of 24,000 and 26,000 (reducing conditions), which evidently are anchored in the cell membrane by covalent linkage to phosphatidylinositol. The present report shows that RT6.1 allotype-specific antibodies precipitate a more complex pattern of bands from LEW.6A rat lymphocyte lysates. These consist of an endo-F-resistant RT6.2-like 25/27,000 MW doublet (reducing conditions) as well as at least five additional endo-F-sensitive, endo-H-resistant polypeptides of 30,000, 32,000, 33,000, 34,000, 35,000 MW. Endo-F treatment seems to convert the additional higher to the lower molecular weight forms. In contrast to the endo-F-resistant 25/27,000 MW doublet, the higher MW forms of RT6.1 partly bind to lentil lectin and concanavalin A (Con A). Two-dimensional electrophoretic analyses (NEPHGE/SDS-PAGE) reveal similar patterns of charge heterogeneity of the lower and higher MW forms of RT6.1. Neuraminidase treatment does not affect the pIs of the lower MW forms but shifts the pIs of the higher MW forms to those of the lower ones. All forms of RT6.1 evidently employ the covalent linkage to phosphatidylinositol for membrane anchorage. Identical patterns of molecular forms--the doublet for RT6.2, the polymorphic pattern for RT6.1--are observed upon immunoprecipitation of the alloantigens from the lysates of corresponding series of inbred strains of rats with both allotype-specific antibodies and a polyclonal rabbit serum recognizing a common determinant on both alloantigens.

Animals↗
PubMed1987

The effect of long-term irradiation with fluorescent light on the rat fundus.

In the rat, continuous exposure to so-called "cold light" of 800 lux leads to a nearly complete loss of retinal photoreceptors, which occurs within 4 weeks. This process is followed by vascularization of the retinal pigment epithelium (RPE). The origin, extent, and expansion of such proliferations within 6 months after the onset of irradiation were evaluated by means of light microscopy, transmission electron microscopy, scanning electron microscopy, corrosion cast preparation, india ink injection, and trypsin digest preparation. The vessels within the RPE were found to be fenestrated and to form a network with the vessels of the neurosensory retina. Invasion of Bruch's membrane and formation of chorioretinal anastomoses were not observed. The occurrence of vessels within the RPE coincided with a massive regression of capillaries in the neurosensory retina.

Animals↗
PubMed1986

Release of the rat T cell alloantigen RT-6.2 from cell membranes by phosphatidylinositol-specific phospholipase C.

The mechanism by which the rat T cell alloantigen, RT-6.2, is attached to the membrane was investigated. Treatment of rat lymph node and T-hybridoma cells with phosphatidylinositol-specific phospholipase C (PI-PLC) caused a substantial reduction in the amount of RT-6.2 on the cell surface. No significant release of a rat T helper marker (visualized by the mAb W3/25) was observed in response to PI-PLC treatment. This is in sharp contrast to the effects of trypsin, which removes most of the T helper marker but had little effect on RT-6.2. SDS-PAGE analysis of the RT-6.2 released by PI-PLC indicated that the Mr was not significantly changed by this treatment. Phase separation of the released RT-6.2 in Triton X-114 showed that the PI-PLC had converted it from an amphiphilic membrane form to a water-soluble form, apparently by removing its hydrophobic membrane anchoring domain. These results strongly suggest that RT-6.2, in common with Thy-1 and several other cell surface proteins, is anchored in the membrane by the 1,2-diacylglycerol moiety of a covalently attached phosphatidylinositol molecule.

Animals↗
PubMed1986

Biochemical characterization of the T-cell alloantigen RT-6.2.

This study presents the partial molecular characterization of the alloantigenic rat T-cell marker RT-6. In contrast to most lymphocyte surface membrane proteins, RT-6 proved to be resistant to short-term (15 min) solubilization by TX-100 at 4 degrees, but could be efficiently solubilized by long-term (16 hr) exposure at 4 degrees or short-term (15 min) exposure at 37 degrees. SDS-PAGE analyses of RT-6.2 immunoprecipitates under non-reducing conditions revealed two bands with apparent molecular weights of 21,000 and 24,000 (25,000 and 28,000 under reducing conditions). Radiolabelled sugars failed to be incorporated metabolically into RT-6.2, neither endoglycosidase-F and 0-glycanase digestion nor NaOH treatment caused any detectable decrease in the molecular weight of RT-6.2, and RT-6.2 failed to bind to any of a series of agarose-coupled lectins with a wide of sugar-binding specificities. These observations suggest that RT-6.2 may lack carbohydrate moieties.

Animals↗
PubMed1985

Comparison of the septal areas in New Guinean and European brains.

In 1966, septal nuclei of unusual size in brains of kuru-diseased Fore people from the Eastern Highlands of New Guinea were reported (Beck and Gajdusek, 1966). Microscopic investigation which did not reveal any pathological changes in these nuclei led to the question of whether their large size could be a characteristic feature of the Fore (and perhaps also of other ethnic groups of New Guinea), rather than a consequence of the disease. We had at our disposal brains from patients of coastal areas of New Guinea who did not suffer from kuru. The septal areas of eight cases were investigated by serial sections and compared to those of nine European brains. No difference in size comparable to that reported in the earlier study could be detected. We conclude that a possible pecularity in the size of the septal nuclei of Fore people cannot be generalized to the entire population of New Guinea.

Adolescent↗