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Biomedical subjects

R Kuhn

Publications and source records attributed to R Kuhn.

At least 19 recordsLinked to original sources
PubMed1992

Down-regulation of the POU transcription factor SCIP is an early event in oligodendrocyte differentiation in vitro.

The POU-domain transcription factor SCIP (also known as Tst-1) has been implicated in the development of Schwann cells, the myelinating cells of the peripheral nervous system (PNS). We have investigated the possibility that SCIP also might play a role in the development of oligodendrocytes, the myelinating cells of the central nervous system (CNS). We purified oligodendrocyte precursors (O-2A progenitors) by immunoselection and cultured them in the presence of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), which together keep O-2A progenitors proliferating and prevent oligodendrocyte differentiation. Under these culture conditions, O-2A progenitors expressed high levels of SCIP mRNA and protein, and did not express myelin-specific genes. When oligodendrocyte differentiation was initiated by withdrawing the growth factors, SCIP mRNA was rapidly down-regulated, followed by a decline in SCIP protein and the sequential activation of myelin-specific genes. Rapid down-regulation of SCIP mRNA required continued protein synthesis. In O-2A progenitors that were cultured in the presence of PDGF alone, SCIP expression declined to an intermediate level, and low levels of the myelin gene products were induced. Thus, the level of SCIP expression in O-2A progenitors is inversely related to the level of myelin gene expression, suggesting that SCIP may be involved in the developmental switch from proliferation to differentiation in the oligodendrocyte lineage. When O-2A progenitors are cultured in the presence of 10% fetal calf serum, they differentiate into type-2 astrocytes rather than oligodendrocytes. SCIP mRNA was also down-regulated in type-2 astrocytes, which do not express myelin genes, so down-regulation of SCIP seems to be more closely linked to the cessation of cell proliferation per se than the expression of a particular differentiated phenotype.

Animals↗
PubMed1992

Trypanosoma cruzi in wild raccoons and opossums in North Carolina.

Trypanosoma cruzi was isolated from 1 of 12 (8.3%) opossums and 3 of 20 (15%) raccoons from the piedmont area of North Carolina. Although T. cruzi has been isolated previously from wild mammals in the southern United States, the present study is the first published report of naturally occurring T. cruzi infection of wild mammals in North Carolina. All 4 isolates were maintained successfully in axenic culture and in murine fibroblasts. In addition, intraperitoneal injection of 1 x 10(6) culture forms of 1 of the opossum isolates into C3H mice resulted in low but detectable parasitemias as early as day 6 of infection. These mice resolved parasitemia and survived infection. Intraperitoneal injection of 1 x 10(6) culture forms of a raccoon isolate resulted in the death of 3 out of 4 mice. Surprisingly, parasitemias were never detected in the peripheral blood of these mice. Infection of murine fibroblasts in vitro resulted in the presence of intracellular amastigote stages characteristic of T. cruzi.

Animals↗
PubMed1991

Axon-regulated expression of a Schwann cell transcript that is homologous to a 'growth arrest-specific' gene.

We have isolated a 1.8 kb cDNA (pCD25) clone that encodes a transcript that is differentially expressed during nerve regeneration. Nucleotide sequence comparison indicates 89.6% homology with the recently identified murine 'growth arrest-specific' gene gas3. The open reading frame of the CD25 transcript predicts a 17 kDa protein with four putative transmembrane regions. Steady-state levels of the CD25 mRNA are very much higher in sciatic nerve than in other tissues, and expression in sciatic nerve is confined to Schwann cells. Following nerve injury, the transcript levels rapidly declined in nerve segments distal to the site of lesion, but recovered upon nerve regeneration. In contrast, in distal stumps of permanently transected nerves, the mRNA level remained very low. Substantial amounts of the mRNA could be reinduced only upon anastomosis of these interrupted nerve stumps. Re-induction of the mRNA followed the elongation of regenerating axons through the distal nerve segment. Our data indicate that axons regulate expression of the CD25 mRNA in Schwann cells, and suggest that the CD25 protein functions during Schwann cell growth and differentiation.

Amino Acid Sequence↗
PubMed1991

A cluster of four genes selectively expressed in the male germ line of Drosophila melanogaster.

The gene Mst87F is exclusively expressed in the male germ line and is subject to translational regulation. The Mst87F mRNA is transcribed in the primary spermatocytes, stored for three days and then subsequently translated in the post-elongation period of spermiogenesis. Here we report on the isolation of a cluster of four small genes closely related in structure and function to Mst87F. These genes are located at polytene band 84D on the right arm of chromosome three and are named Mst84Da, Mst84Db, Mst84Dc and Mst84Dd. All four genes encode putative proteins composed primarily of a repetitive motif of cysteine-glycine-proline. The genes are exclusively expressed in the male germ line. The poly(A) tail of the Mst84D mRNAs increases in length at day three of pupal development, the time at which a similar change in Mst87F mRNA and translation has been shown to begin. In addition we have identified a conserved 12 base pair element within the 5' untranslated region (UTR) of each gene which is also found at an identical position in Mst87F and which has been demonstrated to be the structural element for translational control of Mst87F expression (Schäfer et al., 1990 EMBO J. 9, 4519-4525). We have mapped the gene cluster to a small deletion associated with a rotund mutation at 84D. Although flies with a homozygous deletion of the cluster still produce motile sperm, electron microscopic examination revealed numerous malformations in the ultrastructure of the axoneme resulting in a drastic reduction of motile sperm.

Amino Acid Sequence↗
PubMed1991

The gene encoding the transcription factor SCIP has features of an expressed retroposon.

SCIP is a POU domain transcription factor expressed by glial progenitor cells in the peripheral and central nervous systems (dividing Schwann cells and O-2A cells, respectively), where it appears to act as a repressor of myelin-specific genes. We have isolated genomic clones encoding the rat SCIP gene. Comparison of the structure of these clones with genomic Southern blots and SCIP cDNAs demonstrates that SCIP is encoded in a single-copy, intronless gene that has the general features of an expressed retroposon. This gene contributes to an extended CpG island. It is transcribed to produce a 3.1-kb mRNA that encodes a 451-amino-acid protein with a predicted molecular mass of 45 kDa. Immunopurified SCIP antibodies specifically recognize a nuclear protein of this size in cultured proliferating Schwann cells, and gel shift analyses demonstrate that this protein is the predominant octamer-binding protein in these cells.

Animals↗
PubMed1991

Growth factors and transcription factors in oligodendrocyte development.

O-2A progenitor cells, the precursors of oligodendrocytes in the central nervous system (CNS), probably originate in the subventricular germinal zones of the developing CNS, and subsequently migrate away from there to populate the rest of the CNS with oligodendrocytes. We are trying to understand how the O-2A progenitor cells interact with their changing environment as they migrate, and how this influences each stage of their development into mature, myelinating oligodendrocytes. In this article we summarize evidence that platelet-derived growth factor (PDGF) is important for stimulating O-2A progenitor cell proliferation in vivo, and describe our efforts to map the distribution of PDGF and its receptors in the developing rat CNS by in situ hybridization and immunohistochemistry. These studies suggest that, in the CNS, PDGF alpha-receptor subunits may be restricted to O-2A lineage cells that have started to migrate away from the subventricular zones towards their final destinations. Many neurons express the A and/or B chains of PDGF, and astrocytes express the A chain, but it is not yet clear which of these cell types might be the major source of PDGF for O-2A lineage cells in vivo. O-2A progenitor cells can be purified and maintained in a proliferating state in vitro by culturing in the presence of PDGF and bFGF. Under these conditions, the POU transcription factor SCIP/Tst-1 is expressed at a high level; when oligodendrocyte differentiation is initiated by withdrawing the growth factors, SCIP/Tst-1 mRNA is rapidly down-regulated, followed by a decline in SCIP/Tst-1 protein and sequential activation of myelin-specific genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Dendritic accessory cells derived from rat bone marrow precursors under chemically defined conditions in vitro belong to the myeloid lineage.

Serum-free conditions have been developed to differentiate dendritic cells from a non-adherent fraction of rat bone marrow precursors by action of the multipotential and macrophage colony-stimulating factors further supplemented with linoleic acid, vitamin E, and vitamin D3. Accessory activity was demonstrated by the high potency of the dendritic cells to stimulate autologous T cell proliferation, whereas such cells were negative for Fc receptor-dependent phagocytosis, a characteristic macrophage feature. While the dendritic cells were weakly positive for alpha-naphtylbutyrate esterase, they strongly expressed RT.1 class II antigens. Apparently, these cells represent a more differentiated phenotype since they expressed the nuclear A/C lamins. By addition of serum to the cultures, the dendritic cells developed into macrophages, which were also lamin A/C-positive as well as strongly positive for alpha-naphtylbutyrate esterase. Thus, these dendritic cells belong to the myeloid lineage, and it appears as if serum factor(s) control differentiation at a mature level. Suitable conditions could also be established for large-scale cultures of dendritic cells, which would be useful for applications requiring higher numbers of cells.

Animals↗
PubMed1990

Expression and activity of the POU transcription factor SCIP.

POU proteins have been shown to transcriptionally active cell-specific genes and to participate in the determination of cell fate. It is therefore thought that these proteins function in development through the stable activation of genes that define specific developmental pathways. Evidence is provided here for an alternative mode of action. The primary structure of SCIP, a POU protein expressed by developing Schwann cells of the peripheral nervous system, was deduced and SCIP activity was studied. Both in normal development and in response to nerve transection, SCIP expression was transiently activated only during the period of rapid cell division that separates the premyelinating and myelinating phases of Schwann cell differentiation. In cotransfection assays, SCIP acted as a transcriptional repressor of myelin-specific genes.

Amino Acid Sequence↗
PubMed1990

Free flow electrophoresis for the purification of proteins: I. Zone electrophoresis and isotachophoresis.

The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97% alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.

Alcohol Dehydrogenase↗
PubMed1990

Free-flow electrophoresis for the purification of proteins: II. Isoelectric focusing and field step electrophoresis.

Two modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of alpha-amylase from an E. coli protein extract. Furthermore the principle of field step electrophoresis is presented. The most important factors influencing the efficiency: (i) residence time, (ii) conductivity of the sample and (iii) sample zone width, are discussed. The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling-up field step electrophoresis. This approach permits a throughput of about 20 mL sample solution per minute.

Bacterial Proteins↗
PubMed1990

A conserved element in the leader mediates post-meiotic translation as well as cytoplasmic polyadenylation of a Drosophila spermatocyte mRNA.

We have previously shown that Mst87F (previously called mst(3)g1-9), a gene which is exclusively expressed in the male germ line of Drosophila melanogaster, is subject to negative translational control. While transcription of this gene takes place premeiotically, translation occurs only after the elongation of spermatids is complete. We report here the identification of a sequence element within the first 45 nucleotides of the leader which is crucial for this translational regulation. Sequence comparison with six other genes, which form a gene family with Mst87F, shows the conservation of a twelve nucleotide element within this leader segment. It is found in all genes at positions +28 to +39 of the leader. Deletion of this element or alteration of two nucleotides by in vitro mutagenesis both lead to the breakdown of the translational control mechanism. The poly(A) tail of the Mst87F mRNA becomes longer and heterogeneous in length when the mRNA is recruited for translation. We present evidence that the control for this additional polyadenylation also resides within the conserved element of the leader.

Animals↗
PubMed1990

Subclinical course of cholesterol ester storage disease (CESD) diagnosed in adulthood. Report on two cases with remarks on the nature of the liver storage process.

An extremely benign variant of cholesterol ester storage disease (CESD) was diagnosed in two female patients aged 43 and 56 years. In one of them the course was entirely subclinical until a stroke at the age of 47, most probably a complication of secondary hyperlipoproteinaemia. The diagnosis was made accidentally in vivo during extensive examination for concomitant monoclonal gammapathy. The other patient (aged 56), still displays a fairly stable course with minor dyspeptic symptoms. The clinical findings in both patients were confined to moderate well tolerated hepatomegaly, hyperlipoproteinaemia of IIb type and xanthelasmata. Acid lipase activity was markedly deficient in peripheral leukocytes and cultured fibroblasts. These cases represent a rare adult variant the existence of which should be borne in mind in the differential diagnosis of chronic liver disease in advanced age and of hyperlipoproteinaemic states. The diagnostic criteria for the routine clinicopathological steps are summarized with emphasis on a special lipopigment deposition pattern, encompassing inhibition and modification of lipofuscin generation in hepatocytes and an excess of ceroid production in both portal and intralobular histiocytes. The varied ultrastructural appearance of the lysosomal limiting membrane complex is described.

Acetylesterase↗
PubMed1990

A monoclonal antibody recognizing selectively rat granulocytes.

In the present study, a murine monoclonal antibody, RK-4 (IgG1) specific for rat granulocytes and their precursors in blood and bone marrow is described. Results obtained by immunocytological and flowcytometry analysis showed this mAb to recognize selectively a surface antigen on neutrophils, eosinophils and basophils. Analysis of cryosections of various lymphatic and non-lymphatic organs demonstrates that this mouse antibody does not cross-react with any other structures. By flow cytometric analysis we found peripheral blood leukocytes to consist of about 15-20% granulocytes. RK-4 is therefore a valuable tool for studying differentiation and function of granulocytes under various conditions.

Animals↗
PubMed1990

Inhibition of epoxide hydrolase by valproic acid in epileptic patients receiving carbamazepine.

The effect of valproic acid (VPA) on the disposition of carbamazepine-10,11-epoxide (epoxide) was studied in five epileptic patients on chronic carbamazepine (CBZ) therapy. The individual pharmacokinetic parameters influencing epoxide disposition were determined in the presence and absence of VPA. VPA significantly decreased the clearance of unbound epoxide (an in vivo index of epoxide hydrolase activity), but did not appear to affect epoxide formation. VPA also increased the free concentrations of both CBZ and epoxide.

Adolescent↗
PubMed1990

The gene encoding peripheral myelin protein zero is located on mouse chromosome 1.

We have used somatic cell hybrids to map the gene encoding protein zero (P0), the major structural protein of peripheral myelin. Analysis of Southern blots of DNA obtained from these hybrids allows us to unambiguously assign the P0 gene to mouse chromosome 1. This observation indicates that mutations in the P0 gene do not account for Trembler, a chromosome 11 mutation that specifically affects myelination in the peripheral nervous system.

Animals↗