Myelin membrane biogenesis by oligodendrocytes. Developmental regulation of low molecular weight GTP-binding proteins.

Oligodendrocytes synthesize dramatic amounts of myelin membrane. We hypothesized that this requires unique aspects of vesicular trafficking. Specific stages of the oligodendrocyte lineage were assayed for low molecular weight GTP-binding proteins implicated in the regulation of vesicular transport pathway (two dimensional gel electrophoresis, [alpha-32P]GTP overlay). Consistent with the hypothesis, as oligodendrocytes differentiate from early progenitors to mature myelin-producing cells, > or = 12 small GTP-binding proteins become up-regulated. Myelin membrane also has a complex pattern of GTP-binding proteins. Several of these proteins may be specific to oligodendrocytes, suggesting that oligodendrocytes may utilize cell-type specific GTP-binding proteins for biogenesis and maintenance of the myelin membrane.

Cloning of the 3' end of rat bax-alpha and corresponding developmental down-regulation in differentiating primary, cultured oligodendrocytes.

Bax-alpha is thought to form heterodimers with Bcl-2 and prevent apoptotic cell death. A sequence was isolated from oligodendrocyte cDNA corresponding to the uncloned 3' end of the rat bax-alpha coding region and part of the 3' UTR via a degenerate polymerase chain reaction (PCR)-based cloning method. The rat bax-alpha clone is 96 and 91% homologous to mouse and human clones, respectively, and the 3' UTR demonstrates high homology with the cloned human 3' UTR. Northern analysis demonstrated that the 1.0 kb bax-alpha mRNA species was predominant. bax-alpha mRNA is expressed in mitotic, oligodendrocyte progenitors, and is subsequently down-regulated 2-fold in differentiating oligodendrocytes.

Medical school admission and generalist physicians: a study of the class of 1985.

PURPOSE: To examine the medical school applications of physicians who are now established in their careers to see whether any of the applicants' data might have had predictive value for the admission process of a medical school wishing to increase its production of generalist physicians. METHOD: Three members of the Committee on Admissions of the University of North Carolina at Chapel Hill School of Medicine followed the same procedure they use when reviewing current medical school applicants in reading the applications of 148 graduates of the class of 1985. The readers recorded data from all parts of the American Medical College Application Service form--face sheet, personal statement, and the record of course work--and used alumni records and published physician locators to determine these class members' whereabouts and the specialties (and subspecialties) they were practicing 13 or 14 years after applying to medical school. RESULTS: Thirty-four percent of the class had elected generalist medical careers (the practice of family medicine, general internal medicine, or general pediatrics). A high service index (reflective of a demonstrated orientation toward community service prior to medical school matriculation) predicted strongly the choice of a generalist medical career. Conversely, the absence of any clear evidence of a service orientation predicted still more strongly a non-generalist career. Less strong predictors of a generalist practice included the selection of a generous number of non-science-content courses as an undergraduate, lower socioeconomic family origin, and a record of leadership in one or more extracurricular activities during college. CONCLUSION: If an admission committee informs itself of "what finally happens" to those it admits, its decisions can contribute to achieving whatever policy its medical school adopts with respect to the mix of physicians it wishes to produce.

C-terminal binding protein and poly(ADP)ribose polymerase 1 contribute to repression of the p21(waf1/cip1) promoter.

Transcriptional repression by the C-terminal binding protein (CtBP) is proposed to require nicotinamide adenine dinucleotide dehydrogenase (NAD(H). Previous studies have implicated CtBP in transcriptional repression of the p21(waf1/cip1) gene. Similarly, the NAD-dependent poly(adenosine diphosphate)ribose polymerase 1 (PARP1) may affect p21 expression via its NAD-dependent enzymatic activity; we therefore asked if PARP1 and CtBP were functionally linked in regulating p21 transcription. We found that restraint of basal p21 transcription requires both CtBP and PARP1. PARP inhibition attenuated activation of p21 transcription by both p53-independent and p53-dependent processes, in a CtBP-dependent manner. CtBP1+2 or PARP1+2 knockdown partially activated p21 gene expression, suggesting relief of a corepressor function dependent on both proteins. We localized CtBP-responsive repression elements to the proximal promoter region, and found ZBRK1 overexpression could also overcome DNA damage-dependent, but not p53-dependent activation through this region. By chromatin immunoprecipitation we find dismissal of CtBP from the proximal promoter following DNA-damage, and that PARP1 associates with a CtBP corepressor complex in nuclear extracts. We propose a model in which both CtBP and PARP functionally interact in a corepressor complex as components of a molecular switch necessary for p21 repression, and following DNA damage signals activation of p21 transcription by corepressor dismissal and co-activator recruitment.

Notes on the history of group practice: the tradition of the dispensary.

According to author Donald Madison, M.D., medical group practice is, "an original American phenomenon...its appearance coincided with specialization in medicine and much of the early growth was fed by the experiences of American physicians who served...during World War I." Madison answers the questions of how, where, when and why group practice developed as it did.

Large medical group-practice organizations and employed physicians: a relationship in transition.

Out of the revolution in medical practice is being forged a new type of group-practice organization--larger and more complex, more tightly administered, and more strategically aware than its antecedents. A typology is offered to contribute to an understanding of the changing physician/group-practice relationship. Drawing upon historical and contemporary literature, initial and following-up field observations, and extensive interviews, large medical group-practice organizations are analyzed according to basic orientation toward the health care market, and to a belief in how medical practice should be organized. The revolution in practice will be stamped on future health care arrangements, and will be transmitted into the professional culture of medicine.

Location patterns of recent physician settlers in rural America.

The location patterns of young physicians who settled in the most rural communities of America between 1973 and 1976 are analyzed. The majority of these recent rural settlers were primary care practitioners. They tended to be the alumni of state university medical schools in states with large rural populations. Foreign medical graduates were heavily represented. The principal finding-a tendency toward further concentration of rural physicians within existing medical communities-suggests that those rural communities with the greatest needs amy remain underserved without the assistance of organized external programs.

Orf virus infection in pregnancy.

Orf virus infection is endemic among sheep and goats, and can occur in humans who handle these animals. Orf virus infection in humans causes a characteristic skin lesion, and systemic symptoms can occur. Very little is known about Orf virus infection in human pregnancy. A case of Orf virus infection, with onset at 33 weeks gestation, is presented. There were no pathological findings in the infant born at term, or in the placenta.

Transient transfection of oligodendrocyte progenitors by electroporation.

The transient transfection of transgenes into oligodendrocytes offers an important tool for studying the function of proteins during myelin formation. Currently established procedures, however, have generally resulted in low survival rates and low levels of uptake of the transgene into primary oligodendrocyte progenitors. We describe an electroporation method which yields transient transfection of oligodendrocyte progenitors of up to 10-15% of the surviving cells, and provides approximately 10(4) surviving, transfected cells per electroporation reaction. In recent applications transgene expression persisted as the transfected progenitors progressed through subsequent stages of the oligodendrocyte lineage. This technique is expected to facilitate the study of the function of key proteins and lipids during the development of primary cultured oligodendrocytes.

Cloning and characterization of MVP17: a developmentally regulated myelin protein in oligodendrocytes.

The remarkable quantities of myelin membrane produced by oligodendrocytes has led us to examine the mechanisms involved in the sorting and transport of proteins and lipids during myelinogenesis. Noting that it has been proposed that proteins destined for the apical surface of polarized epithelial cells co-cluster with glycolipid-rich microdomains during sorting and transport from the trans-Golgi network (Simons and van Meer: Biochemistry 27:6197-6202, 1988; Simons and Wandinger-Ness: Cell 62:207-210, 1990), we hypothesized that the glycolipid-rich oligodendrocytes may adopt this mechanism for myelinogenesis. Protein-lipid complexes from oligodendrocytes and myelin were isolated utilizing detergent insolubility and two-dimensional gel electrophoresis. A developmentally regulated protein, MVP17 (myelin vesicular protein of 17 kDa), was identified. Microsequencing of the N-terminal peptide revealed a high homology to human T-cell MAL protein (Alonso and Weissman: Proc Natl Acad Sci USA 84:1997-2001, 1987). The corresponding MVP17 cDNA was isolated from an oligodendrocyte cDNA library. The predicted protein sequence showed 88.9% identity with MAL, and the hydrophobicity profile suggested four transmembrane domains. In vitro translation demonstrated a signal at the deduced Mr of approximately 17 kDa. Northern analyses indicated that MVP17 mRNA expression is restricted to brain and kidney and that this expression is up-regulated in oligodendrocytes and brain during the period of active myelination. These data suggest that MVP17 is involved in myelin biogenesis and/or myelin function.

Differential expression of rab3 isoforms in oligodendrocytes and astrocytes.

The small GTP-binding protein Rab3a is involved in regulated secretory pathways and is enriched in synaptic and neuroendocrine secretory vesicles. We have reported previously the developmental regulation of Rab3a in oligodendrocytes in culture and purified central nervous system myelin (Huber et al.: FEBS Lett 347: 273-278, 1994). Since multiple rab3 isoforms exist in the brain and may be associated with different secretory pathways, we have investigated the differential expression of the rab3 isoforms in oligodendrocytes, astrocytes, and Schwann cell line RT4-D6P2T. The expression of specific rab3 isoforms (rab3a-c) was detected by polymerase chain reaction (PCR) amplification and confirmed by sequence analyses. These data show that in addition to the previously reported expression in neurons, the two macroglial populations, astrocytes and oligodendrocytes, also express rab3 isoforms. Rab3b was preferentially amplified from purified, cultured astrocytes, while rab3a and rab3c were preferentially amplified from highly enriched populations of both cultured oligodendrocytes and those isolated directly from the brain by immunopanning. No novel rab3 isoform was detected in glia. These results indicate that glial cells in the brain express specific isoforms of the vesicular trafficking Rab3 protein family.

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes.

Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane-directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP-2/synaptobrevin-2, syntaxin-2 and -4, nsec-1/munc-18-1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP-2, the small GTP-binding protein Rab3a, and the target SNARE syntaxin-4 were up-regulated. VAMP-2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network; in situ anti-VAMP-2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane-directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.

Endothelial seeding of polytetrafluoroethylene popliteal bypasses. A preliminary report.

At St. Vincent Hospital, Indianapolis, 17 patients have undergone femoropopliteal bypass operations with polytetrafluoroethylene (PTFE) grafts that were seeded with enzymatically harvested, autogenous endothelium. Three patients received seeded grafts because satisfactory veins were not available. Twenty-eight patients alternately received seeded or unseeded, externally supported e-PTFE grafts. Graft patency was evaluated by clinical criteria and changes in the Doppler ankle-brachial systolic pressure ratios at 2 and 30 days postoperatively and at 3-month intervals thereafter. Occlusions were defined arteriographically if the clinical situation or the Doppler findings deteriorated. Smoking histories were taken, and carboxyhemoglobin (COHgb) levels were sampled 1 month postoperatively. Cumulative patency after 3 months was 93.3% +/- 6.5% for seeded and 84.0% +/- 10.4% for unseeded grafts. After 1 year it was 81.6% +/- 12.3% for seeded grafts and 30.8% +/- 18.7% for unseeded grafts (p = 0.02). Thus far all but one of the occlusions have occurred in patients with a history of smoking or with a COHgb level greater than 1.5%, whereas all of the seeded grafts in nonsmokers with COHgb levels less than or equal to 1.5% are patent. We conclude that endothelial seeding of PTFE femoropopliteal grafts is feasible. During this preliminary study period, a small number of patients had favorable patency rates with seeded as compared with unseeded grafts, especially among smokers. A multiple-institution study will be needed to establish the role of endothelial cell seeding in the treatment of vascular occlusions of the femoral and popliteal arteries.

TPO1, a member of a novel protein family, is developmentally regulated in cultured oligodendrocytes.

Although the myelin membrane contains only a small set of major proteins, more sensitive assays indicate the presence of a plethora of uncharacterized proteins. We have used an antibody perturbation approach to reversibly block the differentiation of prooligodendroblasts into myelinating cells, and, in combination with a differential screening procedure, identified novel mRNAs that are activated during this period. One cDNA, TPO1, recognizes a 5.5-kb mRNA that is strongly up-regulated in oligodendrocytes after release of the differentiation block and that is expressed at high levels in brain tissue during active myelination. This cDNA represents at least two mRNAs differing from each other in their 5'-termini. The TPO1 cDNA contains an open reading frame of 1,380 bp, encoding a protein of 51.8 kDa with a predicted pI of 9.1 that contains two regions homologous to nonclassic zinc finger motifs. Subcellular localization studies suggest the enriched presence of TPO1 in spherical structures along the major cytoplasmic processes of oligodendrocytes. TPO1, along with homologues expressed in testis, placenta, and PC12 cells, form a novel family of proteins with multiple hydrophobic domains possibly serving as membrane spanning regions. We postulate that in oligodendrocytes, TPO1 encodes a protein factor involved in myelin biogenesis.