Molecular evidence for the expression of nicotinic acetylcholine receptor alpha-chain in mouse thymus.

The presence and structure of nicotinic acetylcholine receptor (nAChR) in the thymus has been a subject of interest for many years because of its possible role in the pathogenesis of the autoimmune disease myasthenia gravis. Using the polymerase chain reaction with primers specific for the alpha-chain of nAChR (nAChR-alpha), an 880-bp homologous band was found after amplification of cDNA prepared from mouse thymus, thymic medullary and cortical epithelial cell lines, but not from thymocytes or kidney. Sequencing of the polymerase chain reaction product from the thymus and thymic medullary and cortical epithelial lines showed identity with skeletal muscle nAChR-alpha over the region examined. This region includes the domains of the molecule on which B cell and T cell autoantigenic targets have been described. No evidence was found in mouse tissue for the exon 3A, which has been described in human muscle and the human rhabdomyosarcoma cell line TE671. Our results provide evidence at the RNA level for the expression of the nAChR-alpha on stromal cells but not on thymocytes in normal murine thymus and are consistent with a role for intrathymic autoantigen expression in the pathogenesis of myasthenia gravis.     

Biochemical effects of mild iron deficiency and cold acclimatization on rat skeletal muscle

Most of the previous studies on the effects of iron deficiency on skeletal muscle respiratory capacity and work performance have been investigated in severe or moderate iron-deficiency anemia. We report here that even in mild iron deficiency where the hemoglobin concentration was 10 g/dl and the iron stores in livers and spleen were not completely depleted, a marked reduction in succinate dehydrogenase was observed in skeletal muscles but not in heart. Similarly, cytochrome oxidase activities were reduced. Although no significant change in glycerophosphate dehydrogenase was detected in the iron-deficient rats, exposure to cold in this group greatly reduced this enzyme activity. As cold acclimatization accelerates marrow erythropoiesis (20) which in turn, demands more iron, it seems that in the iron-insufficient state, this iron demand for marrow activity may persist at the expense of the tissue iron pool, resulting in a marked reduction in glycerophosphate dehydrogenase activities. Since succinate dehydrogenase plays a significant role in the impairment of mitochondrial function and early fatigue of iron-deficient muscle (11), the present study shows that even in mild iron deficiency, some loss of muscle functions could result as succinate dehydrogenase activities were greatly reduced.     

Production of pro-opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by POMC-converting enzyme

Pro-opiomelanocortin (POMC) was expressed in CV-1 (green monkey kidney) cells using a vaccinia virus transient expression system [(1986) Proc. Natl. Acad. Sci. USA 83, 8122]. The system involved infection of cells with a recombinant vaccinia virus carrying the T7 RNA polymerase gene and transfection with a plasmid containing the mouse POMC sequence flanked by the T7 RNA polymerase promoter at its 5'-end and the T7 RNA polymerase terminator at its 3'-end. Assay of the medium from transfected cells showed that 1-2 micrograms of immunoreactive ACTH was produced/10(6) cells. Analysis of the same medium by SDS-PAGE/Western blots revealed a band of 30-36 kDa, which was immunostained with both ACTH and beta-endorphin antisera. Labeling the transfected cells with [3H]Arg, followed by immunoprecipitation and SDS-PAGE showed the synthesis of a major peak of POMC, 33 kDa. Purified [3H]POMC expressed by CV-1 cells was cleaved in vitro by bovine intermediate lobe secretory vesicle pro-opiomelanocortin-converting enzyme to ACTH intermediates (19-25 kDa), beta-lipotropin and beta-endorphin. Thus, this work has demonstrated a technique for expressing microgram quantities of prohormones in mammalian cells, suitable for use as substrates for prohormone-converting enzymes in vitro.     

NEUROSECRETORY CELL PROTEIN METABOLISM IN THE LAND SNAIL,OTALA LACTEA

—Protein synthesis in an identified molluscan neurosecretory cell of the land snail, Otala lactea was examined using three different types of polyacrylamide gel electrophoresis. Cells taken from active snails synthesized specific low molecular weight proteins while those from aestivated snails did not. Most of the newly synthesized low molecular weight proteins in the active snails were lost from the cell body when the preparations was chased for 19 h in label-free enriched medium in the presence of anisomycin, an inhibitor of protein synthesis. If colchicine, a blocker of axonal transport, was included in the chase medium, the proteins present following a pulse were largely replaced by smaller molecular weight species. The results suggest that specific low molecular weight proteins are converted to smaller species and then transported from the cell body.     

Chlormethiazole in treatment of status epilepticus.

Chlormethiazole (Heminevrin) was successful in controlling fits in seven out of nine episodes of intractable status epilepticus. It was administered as a constant intravenous injection at rates of up to 0.7g/h. No serious side effects were encountered, and the drug deserves wider recognition as a useful therapeutic agent in the management of status epilepticus.     

Involvement of regulatory volume decrease in the migration of nasopharyngeal carcinoma cells

The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5‘-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of nonmigrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.     

Location of a second partitioning region (ParL) on the F plasmid

Abstract The leading region of the F plasmid has been found to extend the maintenance of the normally unstable plasmid vector pACYC184. This ability is due to effective partitioning of plasmid molecules at cell division. Cloning, deletion analysis and transposon mutagenesis have located the partitioning region (ParL) to be encoded within 63.65–64.11F. Complementation studies indicated that parL is a cis -acting locus.     

Host ethnicity and virus genotype shape the hepatitis B virus-specific T-cell repertoire

Repertoire composition, quantity, and qualitative functional ability are the parameters that define virus-specific T-cell responses and are linked with their potential to control infection. We took advantage of the segregation of different hepatitis B virus (HBV) genotypes in geographically and genetically distinct host populations to directly analyze the impact that host and virus variables exert on these virus-specific T-cell parameters. T-cell responses against the entire HBV proteome were analyzed in a total of 109 HBV-infected subjects of distinct ethnicities (47 of Chinese origin and 62 of Caucasian origin). We demonstrate that HBV-specific T-cell quantity is determined by the virological and clinical profiles of the patients, which outweigh any influence of race or viral diversity. In contrast, HBV-specific T-cell repertoires are divergent in the two ethnic groups, with T-cell epitopes frequently found in Caucasian patients seldom detected in Chinese patients. In conclusion, we provide a direct biological evaluation of the impact that host and virus variables exert on virus-specific T-cell responses. The discordance between HBV-specific CD8 T-cell repertoires present in Caucasian and Chinese subjects shows the ability of HLA micropolymorphisms to diversify T-cell responses and has implications for the rational development of therapeutic and prophylactic vaccines for worldwide use.