Polaris, a Protein Involved in Left-Right Axis Patterning, Localizes to Basal Bodies and Cilia

Mutations in Tg737 cause a wide spectrum of phenotypes, including random left-right axis specification, polycystic kidney disease, liver and pancreatic defects, hydrocephalus, and skeletal patterning abnormalities. To further assess the biological function of Tg737 and its role in the mutant pathology, we identified the cell population expressing Tg737 and determined the subcellular localization of its protein product called Polaris. Tg737 expression is associated with cells possessing either motile or immotile cilia and sperm. Similarly, Polaris concentrated just below the apical membrane in the region of the basal bodies and within the cilia or flagellar axoneme. The data suggest that Polaris functions in a ciliogenic pathway or in cilia maintenance, a role supported by the loss of cilia on the ependymal cell layer in ventricles of Tg737orpk brains and by the lack of node cilia in Tg737Δ2-3βGal mutants.

POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.

Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.

The dentate gyrus of the hippocampus is one of the few areas of the adult brain that undergoes neurogenesis. In the present study, cells capable of proliferation and neurogenesis were isolated and cultured from the adult rat hippocampus. In defined medium containing basic fibroblast growth factor (FGF-2), cells can survive, proliferate, and express neuronal and glial markers. Cells have been maintained in culture for 1 year through multiple passages. These cultured adult cells were labeled in vitro with bromodeoxyuridine and adenovirus expressing beta-galactosidase and were transplanted to the adult rat hippocampus. Surviving cells were evident through 3 months postimplantation with no evidence of tumor formation. Within 2 months postgrafting, labeled cells were found in the dentate gyrus, where they differentiated into neurons only in the intact region of the granule cell layer. Our results indicate that FGF-2 responsive progenitors can be isolated from the adult hippocampus and that these cells retain the capacity to generate mature neurons when grafted into the adult rat brain.

Platelet-derived growth factor stimulates the secretion of hyaluronic acid by proliferating human vascular smooth muscle cells.

Total glycans from the cell layer and the culture medium of human vascular smooth muscle cells (VSMC) that had been cultivated in the presence of platelet-derived growth factor (PDGF) were isolated and purified by gel filtration after Pronase and DNase digestion and alkaliborohydride treatment. Measurements of the content of neutral hexoses and uronic acids revealed that PDGF stimulates total glycan synthesis by proliferating VSMC in a linear fashion from 24 h to 72 h of incubation. In contrast, total glycan synthesis by human fibroblasts, epithelial cells, or endothelial cells was not affected by PDGF, indicating cell-type specificity. Chemical, biochemical, and enzymological characterization of the total glycans synthesized by VSMC showed that PDGF stimulates the secretion of a 340-kDa glycan molecule in a time-dependent manner from 24 h to 72 h. This molecule is highly acidic, shares a common structure with hyaluronic acid, and exhibits a potent antiproliferative activity on VSMC. These results suggest that VSMC in response to PDGF are capable of controlling their own growth and migration by the synthesis of a specific form of hyaluronic acid with antiproliferative potency, which may be involved in the regulation of the local inflammatory responses associated with atherosclerosis.

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.