Nitric oxide as a potential pathological mechanism in demyelination: its differential effects on primary glial cells in vitro.

Because we believe that macrophage-derived nitric oxide contributes to pathology of demyelinating diseases, we have determined the differential effects of nitric oxide on primary rat glial cells in vitro. Enriched cultures of microglia, astrocytes and oligodendrocytes were treated with S-nitroso,N-acetyl-DL-penicillamine, a nitric oxide-releasing chemical. There was a significantly decreased function of one of the ferrosulfur-containing mitochondrial enzymes after S-nitroso,N-acetyl-DL-penicillamine/nitric oxide treatment in oligodendrocytes and astrocytes compared to microglia, which were much less sensitive to S-nitroso,N-acetyl-DL-penicillamine/nitric oxide at all concentrations.

Specific recognition of the human neuroendocrine receptor for vasoactive intestinal peptide by anti-peptide antibodies.

Rabbit polyclonal IgG antibodies were generated to three distinct synthetic peptide substituents of the human neuroendocrine-type 7 transmembrane-domain receptor for vasoactive intestinal peptide (VIP), including a portion of the amino-terminus, first extracellular loop, and carboxyl-terminus. Immunofluorescent staining of both human K293 cell transfectants, expressing recombinant VIP receptors, and HT-29 human intestinal epithelial cells, bearing native VIP receptors, was observed with each of the antibodies and was eliminated specifically after absorption of antibodies with the respective peptide immunogen. Each of the antibodies recognized the same approximately 70-kDa membrane proteins, extracted from both K293 cell transfectants and HT-29 cells, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis blots.

Differential sensitivity to nitric oxide in immortalized, cloned murine oligodendrocyte cell lines.

Using five different immortalized, cloned murine oligodendrocyte cells lines, we have assessed their sensitivity to the nitric oxide donor chemical S-nitroso, N-acetyl-DL-penicillamine (SNAP). The five lines were quite different in their sensitivity to SNAP as determined by assessment of viability, mitochondrial function, single-stranded DNA breaks, DNA/RNA/protein synthesis, morphology, and myelin gene expression. Single-stranded DNA breaks as well as mitochondrial and DNA damage occurred in viable cells.

Protein product of a human intronless calmodulin-like gene shows tissue-specific expression and reduced abundance in transformed cells.

The recently identified NB-1 mRNA is transcribed from a single intronless gene, previously thought to be an unexpressed calmodulin pseudogene. Although expression levels of the three known human calmodulin genes fluctuate only slightly in all cell types and tissues examined, NB-1 expression is limited to certain cells of pseudostratified and stratified epithelial tissues. Like calmodulin, the protein encoded by NB-1 is heat stable and binds to phenyl-Sepharose in a calcium-dependent manner.

Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells.

A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4-kilobase mRNA, designated NB-1, whose expression was decreased greater than 50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo[a]pyrene and Kirsten sarcoma virus.