Glycine receptors in the retinas of normal and spastic mutant mice

PURPOSE: Spastic mutant mice have abnormal gait and righting behavior, and the responses of their retinal ganglion cells have recently been shown to be abnormal. The former defects have been linked to a reduction of glycine-receptor density in the spinal cord of spastic mutants, but the cause of the retinal defects has not yet been determined. The authors thus tested for reduced glycine-receptor density in the mutant retina by comparing the levels of glycine receptors in the retinas of spastic mutant mice with those found in normal mice. METHODS: Indirect immunofluorescence histochemistry was employed, using monoclonal antibodies directed against the alpha- and beta-subunits of the receptor and against the 93-kd cytoplasmic receptor-associated protein, gephyrin. RESULTS: In normal mice, all glycine-receptor antibodies labeled two laminae of the inner plexiform layer (IPL): a broad band in the distal third of the IPL and a narrow band in the middle of the IPL. Lighter labeling was also seen in the outer plexiform layer with these antibodies. In spastic mutant mice, the glycine-receptor labeling of the IPL was reduced markedly. However, the overall structure of the spastic mutant retina was not disrupted because the distribution and intensity of both a presynaptic marker (synaptophysin) and a marker for the rod bipolar cell (protein kinase C) in the mutant retina were indistinguishable from those in normal retinas. CONCLUSIONS: The glycine-receptor distribution in normal mice was consistent with that previously reported for the rat and with the distribution of glycine responsiveness of dissociated rodent bipolar cells. The reduced levels of glycine receptors in spastic mice help explain the abnormal ganglion cell responses in the spastic mutant.     

CD164, a novel sialomucin on CD34(+) and erythroid subsets, is located on human chromosome 6q21

CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human CD34(+) hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in hematopoiesis by facilitating the adhesion of CD34(+) cells to bone marrow stroma and by negatively regulating CD34(+) hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes, defined by the monoclonal antibodies (MoAbs), 103B2/9E10 and 105A5. In this report, we show that these MoAbs, together with two other CD164 MoAbs, N6B6 and 67D2, show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical cord blood, and peripheral blood. Flow cytometric analyses revealed that, on average, 63% to 82% of human bone marrow and 55% to 93% of cord blood CD34(+) cells are CD164(+), with expression of the 105A5 epitope being more variable than that of the other identified epitopes. Extensive multiparameter flow cytometric analyses were performed on cells expressing the 103B2/9E10 functional epitope. These analyses showed that the majority (>90%) of CD34(+) human bone marrow and cord blood cells that were CD38(lo/-) or that coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)+. This CD164 epitope was generally detected on a significant proportion of CD34(+)CD71(lo/-) or CD34(+)CD33(lo/-) cells. In accord with our previous in vitro progenitor assay data, these phenotypes suggest that the CD164(103B2/9E10) epitope is expressed by a very primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD34(+)CD164(103B2/9E10)lo/- cells in bone marrow are mainly CD19(+) B-cell precursors, with the CD164(103B2/9E10) epitope subsequently appearing on CD34(lo/-)CD19(+) and CD34(lo/-)CD20(+) B cells in bone marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34(lo/-)CD164(103B2/9E10)+ subsets indicated that one of the most prominent populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/9E10) epitope is also found on developing myelomonocytic cells in bone marrow, being downregulated on mature neutrophils but maintained on monocytes in the peripheral blood. We have extended these studies further by identifying Pl artificial chromosome (PAC) clones containing the CD164 gene and have used these to localize the CD164 gene specifically to human chromosome 6q21.     

Vaults are up-regulated in multidrug-resistant cancer cell lines

Vaults are 13-MDa ribonucleoprotein particles composed largely of a 104-kDa protein, termed major vault protein or MVP, and a small vault RNA, vRNA. While MVP levels have been found to increase up to 15-fold in non-P-glycoprotein multidrug-resistant cell lines, the levels of vault particles have not been investigated. As both the function of vault particles and the mechanism of drug resistance in non-P-glycoprotein cells are unknown, we decided to determine whether vault synthesis was coupled to MDR. By cloning the human gene for vRNA and careful quantitation of the MVP and vRNA levels in MDR cells, we find that vRNA is in considerable excess to MVP. Sedimentation measurements of vault particles in multidrug resistance cells have indeed revealed up to a 15-fold increase in vault synthesis, coupled with a comparable shift of associated vRNA, demonstrating that vault formation is limited by expression of MVP or the minor vault proteins. The observation that vault synthesis is linked directly to multidrug resistance supports a direct role for vaults in drug resistance.     

Carbon dioxide field flooding minimizes residual intracardiac air after open heart operations

Transesophageal echocardiographic studies were used to monitor the presence of air bubbles in the heart after open heart operations. After cardiac valvular procedures all 22 patients managed with careful deairing procedures had persistence of air bubbles for at least 30 minutes and usually for 45 minutes. In 56 patients with CO2 field flooding, all foam disappeared in less than 1 minute in 48 patients and the remaining 8 had complete disappearance in 1 to 24 minutes. These observations demonstrate the ineffectiveness of the usual deairing maneuvers and the effectiveness of CO2 field flooding in displacing air.     

The relation between serum markers in the second trimester and placental pathology. A study on extremely small for gestational age fetuses

To disclose the cytoprotective mechanism of 1,6-dihydro-2[2-(2-methyoxypropoxy)anilino]-6-oxo-5-pyrimidinecarb oxylic acid (CAS 98772-05-5, MAR-99), the effect of this compound on the microvascular injury in gastric mucosa induced by 99.5% ethanol in rats was studied. In this experiment, it was found that the elevation of vascular permeability observed at the early state of ethanol-induced gastric mucosal injury was closely correlated with the combined action of histamine and slow reacting substance (leukotriene C4, LTC4). MAR-99 (0.3-10 mg/kg p.o.) prevented dose-dependently the increase in vascular permeability. Furthermore, MAR-99 (10 mg/kg p.o.) improved the decrease in the number of histamine containing cells and histamine content, and prevented the production of LTC4. These results suggest that MAR-99 exerts its anti-microvascular injury effect by regulating the release of histamine and the production of LTC4 in glandular stomach against ethanol, and this effect may contribute to the anti-lesion effect of this compound.     

Perturbed dentate gyrus function in serotonin 5-HT2C receptor mutant mice

Serotonin systems have been implicated in the regulation of hippocampal function. Serotonin 5-HT2C receptors are widely expressed throughout the hippocampal formation, and these receptors have been proposed to modulate synaptic plasticity in the visual cortex. To assess the contribution of 5-HT2C receptors to the serotonergic regulation of hippocampal function, mice with a targeted 5-HT2C-receptor gene mutation were examined. An examination of long-term potentiation at each of four principal regions of the hippocampal formation revealed a selective impairment restricted to medial perforant path-dentate gyrus synapses of mutant mice. This deficit was accompanied by abnormal performance in behavioral assays associated with dentate gyrus function. 5-HT2C receptor mutants exhibited abnormal performance in the Morris water maze assay of spatial learning and reduced aversion to a novel environment. These deficits were selective and were not associated with a generalized learning deficit or with an impairment in the discrimination of spatial context. These results indicate that a genetic perturbation of serotonin receptor function can modulate dentate gyrus plasticity and that plasticity in this structure may contribute to neural mechanisms underlying hippocampus-dependent behaviors.     

Structure-activity relationships of astemizole derivatives for inhibition of store operated Ca2+ channels and exocytosis

The effects of a series of analogues of the antiallergic drug astemizole on the exocytosis of the enzyme beta-hexosaminidase were studied in a mast cell model, the rat basophilic leukemia (RBL-2H3) cell. Besides differences in the effects on Fc epsilonRI receptor-stimulated exocytosis, changes were also observed in Ca2+ influx and in the perturbation of the cell membrane. A strong correlation was found between the effects on antigen- and thapsigargin-stimulated 45Ca2+ influx. Furthermore, the inhibition of 45Ca2+ influx was correlated with the inhibition of beta-hexosaminidase release and membrane stabilization. It is concluded that the astemizole analogues are capable of inhibiting mast cell beta-hexosaminidase release through inhibition of Ca2+-store-operated Ca2+ channels (SOC). Compounds with high lipophilicity also released Ca2+ from intracellular stores. Lowering of the hydrophobicity by introduction of nitrogens or truncation at different sites in the astemizole structure decreased inhibitory activity on SOC channels. The inhibition of SOC channels cannot completely be ascribed to non-specific membrane effects. The piperidinyl-benzimidazole moiety was found to be important for inhibition of SOC channels. The observed differences in activity possibly depend on the way the compounds penetrate the membrane bilayer. Astemizole is an interesting new tool to study SOC channels and can be a lead for the design of mast cell-stabilizing antiallergic drugs.     

Global increases in seizure susceptibility in mice lacking 5-HT2C receptors: a behavioral analysis

OBJECTIVE: Vasodilation by beta-adrenergic receptors of smooth muscle cells appears to be impaired early after the onset of hypercholesteremia. The aim of this study was to analyze the modulation of beta-adrenergic receptor density and adenylyl cyclase activity in the presence of moderately elevated concentrations of LDL. The effects of beta 1- and beta 2-adrenergic receptor antagonists on LDL-induced receptor changes were studied. METHODS AND RESULTS: Media explants of porcine coronary arteries were incubated with moderately elevated LDL concentrations (0.7-3.9 mmol/l). The density of beta-adrenergic receptors was determined in plasma membranes using the radioligand [125I]iodocyanopinodolol. LDL (3.9 mmol/l) resulted in a decrease of beta-adrenergic receptor density (control 137 +/- 5 vs. 89 +/- 7 fmol/mg protein, P < 0.01). After removal of LDL and cultivation for an additional 3 days beta-adrenergic receptors increased to 129 +/- 5 fmol/mg. In the presence of the beta 1- or beta 2-adrenergic receptor antagonists the LDL-mediated decrease was inhibited. Addition of metoprolol after 3 days of LDL incubation caused a restoration of receptor density. The basal, isoproterenol- and forskolin-stimulated adenylyl cyclase activities were increased after LDL incubation by 180, 110 or 80%, respectively. CONCLUSION: Moderately elevated LDL levels decreased beta-adrenergic receptor density while adenylyl cyclase activity was simultaneously increased. beta 1- or beta 2-adrenergic receptor antagonists prevented this receptor decrease and might preserve the beta-adrenergic receptor density in the presence of moderately elevated LDL levels.