Expression of galactose-1-P-uridyltransferase in Chinese hamster × human galactosemia somatic cell hybrids

Lymphocytes from a patient with classic galactosemia (GALT deficiency) were hybridized with a Chinese hamster cell line. Electrophoretic evaluation of GALT in 31 independently derived interspecific hybrid clones failed to demonstrate expression of the human GALT gene even when human chromosome 9 was present. Possible mechanisms for this lack of expression are presented.This work was supported in part by Grants HD-04612 and HD-05615 from the National Institute of Child Health and Human Development.     

Release of intracellular Zn<Superscript>2+</Superscript> in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide

Several studies have shown intracellular Zn²⁺ release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn²⁺ release and subsequent cell death. Cortical neurons were loaded with the Zn²⁺ selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn²⁺ levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn²⁺. Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn²⁺ induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn²⁺ release appeared to peak at 1 μM spermine NONOate (123.8 ± 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn²⁺ levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn²⁺ in cortical neurons. The possibility that release of intracellular Zn²⁺ in response to NO could play a role in intracellular signaling is discussed.     

Mapping of multiple mouse loci related to the farnesyl pyrophosphate synthetase gene

The prenyltransferases are a class of enzymes involved in the synthesis of sterol and nonsterol isoprene compounds. We report here the chromosomal mapping of nine loci in the mouse that hybridize to the cDNA for the enzyme farnesyl pyrophosphate synthetase (FPS), a prenyltransferase that catalyzes the synthesis of an intermediate common to both the sterol and nonsterol branches of the isoprene biosynthetic pathway. Mapping was performed with genomic DNA from a mouse-hamster somatic cell hybrid panel, and by linkage analysis with recombinant inbred strains and the progeny of an interspecific backcross. The mapped loci have been designated farnesyl pyrophosphate synthetase-like-1 (Fpsl-1) on mouse Chromosome (Chr) 3; Fpsl-2 on Chr 4; Fpsl-3, Fpsl-4, and Fpsl-5, dispersed on Chr 10; Fpsl-6 on Chr 12; Fpsl-7 on Chr 13; Fpsl-8 on Chr 17; and Fpsl-9 on Chr X. It is presently unclear which of these loci encode active prenyltransferases and which may correspond to pseudogenes. The strongly hybridizing loci provide convenient genetic markers for seven mouse chromosomes.     

Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes

Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1^–/–; Shi TS et al. J Clin Invest 103: 331, 1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1^–/– mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1^–/– erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 ± 3.2 in 4.1^–/– vs. 9.8 ± 1.3 mmol/10¹³ cell x h in control mice), with an abnormal dependence on osmolality (EC₅₀ = 417 ± 42 in 4.1^–/– vs. 460 ± 35 mosmol/kgH₂O in control mice), suggestive of an upregulated functional state. While the affinity for internal protons was not altered (K_0.5 = 489.7 ± 0.7 vs. 537.0 ± 0.56 nM in control mice), the V_max of the H-induced Na/H exchange activity was markedly elevated in 4.1^–/– erythrocytes (V_max 91.47 ± 7.2 compared with 46.52 ± 5.4 mmol/10¹³ cell x h in control mice). Na/H exchange activation by okadaic acid was absent in 4.1^–/– erythrocytes. Altogether, these results suggest that erythroid protein 4.1 plays a major role in volume regulation and physiologically downregulates Na/H exchange in mouse erythrocytes. Upregulation of the Na/H exchange is an important contributor to the elevated cell Na content of 4.1^–/– erythrocytes. spherocytosis; cell Na; Na/H exchange     

Molecular Analysis of Divergence in Tachinid Uzi (<Emphasis Type="Italic">Exorista Sorbillans</Emphasis>) Populations in India

Exorista sorbillans is a tachinid endoparasitoid of silkworm, Bombyx mori, and is globally known as uzi. It causes economic injury to the cocoon crop in silkworm cultivating areas of India, except those above 400 m above mean sea level (AMSL) in the foothills of the Himalayas (Darjeeling). It is reported that the sericulture tract of south India became infected with this pest only since 1980 through an accidental transportation of cocoons from West Bengal. To ascertain whether the genome of this parasitoid is differentiating into discrete gene pools in contrasting geo-climatic conditions, molecular profiling of four populations (Es _Annatapur, Es_Ramanagaram, Es _Channapatna and Es_Kodathi from south India and Es_Murshidabad from Murshidabad, West Bengal was undertaken with 13 ISSR, 3 RAPD and six non-random primers designed from various repeat sequences of B. mori . MANOVA indicated significance for the Roy’s largest root estimate (55.4; F =18.47; p = 0.002) for the variability contributed by the replication. Further, hierarchical clustering done on the basis of Euclidean distance matrix and Nei’s unbiased Phylip clustering put Es_Murshidabad at the maximum distance from those of south India and 29 markers could also be identified which significantly differentiateEs_Murshidabad from others. However, Nei’s statistics for gene diversity in sub-populations reveal considerably high gene-flow (3.44 and 2.51) among the populations around Bangalore. The gene-flow between Es_Murshidabad and other population is lowest but cannot be ignored. The comparison of endosymbiont specific 16SrRNA and fts Z gene (partial) sequences through clustalW (gcgMSF) revealed a closer relationship of Es_Murshidabad withEs_Annatapurand Es _Ramanagaram and is not congruent with the relationships discussed above. The significance of this maiden study with a tachinid fly-pest is discussed in the context of understanding the diversification of Uzi fly-pest and also establishing this pest as a relevant biological material for studying microevolution in future.     

Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet. That there may be multiple sites of interaction with PS in the spectrin sequence has been inferred, but they have not hitherto been identified. Here we have explored the interaction of PS-containing liposomes with native alpha- and beta-spectrin chains and with recombinant spectrin fragments encompassing the entire sequences of both chains. We show that both alpha-spectrin and beta-spectrin bind PS and that sites of high affinity are located within 8 of the 38 triple-helical structural repeats which make up the bulk of both chains; these are alpha8, alpha9-10, beta2, beta3, beta4, beta12, beta13, and beta14, and PS affinity was also found in the nonhomologous N-terminal domain of the beta-chain. No other fragments of either chain showed appreciable binding. Binding of spectrin and its constituent chains to mixed liposomes of PS and phosphatidylcholine (PC) depended on the proportion of PS. Binding of spectrin dimers to PS liposomes was inhibited by single repeats containing PS binding sites. It is noteworthy that the PS binding sites in beta-spectrin are grouped in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane may modulate its interactions with the proteins and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.     

Cooperative action between band 3 and glycophorin A in human erythrocytes: immobilization of band 3 induced by antibodies to glycophorin A.

The ability of transmembrane receptor proteins to change their association with the cytoskeleton in response to ligand binding seems to be a key mechanism of signal transduction across membranes. To investigate the molecular features of this mechanism we have used the red cell membrane as a model system to study signal transduction through the integral protein, glycophorin A. In these studies the lateral mobility of integral proteins was measured in situ by fluorescence recovery after photobleaching, and membrane rigidity was characterized by micropipette aspiration technique. We found that binding either a monoclonal antibody or its monovalent Fab to the exoplasmic domain of glycophorin A in normal red cells immobilized the receptor and rigidified the membrane. Further, immobilization and rigidification did not occur when antibodies were bound to Miltenberger V cells containing a mutant form of glycophorin A lacking the cytoplasmic domain. These results imply that the site of the immobilization/rigidification lies within the membrane skeletal structure, not in exofacial receptor crosslinking, and requires the extended cytoplasmic domain of normal glycophorin A. In addition, we found that glycophorin A immobilization and membrane skeletal rigidification were accompanied by immobilization of band 3 receptors. This unexpected result indicates a cooperative coupling between liganded glycophorin A, band 3, and the membrane skeleton. We speculate that cooperation of this type may represent a general mechanism for cytoskeletal linkage and transformation initiated by receptors with short cytoplasmic sequences, such as integrins.     

Erythrocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations

Skeletal proteins play an important role in determining erythrocyte membrane biophysical properties. To study whether membrane deformability and stability are regulated by the same or different skeletal protein interactions, we measured these two properties, by means of ektacytometry, in biochemically perturbed normal membranes and in membranes from individuals with known erythrocyte abnormalities. Treatment with 2,3-diphosphoglycerate resulted in membranes with decreased deformability and decreased stability, whereas treatment with diamide produced decreased deformability but increased stability. N-ethylmaleimide induced time-dependent changes in membrane stability. Over the first minute, the stability increased; but with continued incubation, the membranes became less stable than control. Meanwhile, the deformability of these membranes decreased with no time dependence. Biophysical measurements were also carried out on pathologic erythrocytes. Membranes from an individual with hereditary spherocytosis and a defined abnormality in spectrin-protein 4.1 association showed decreased stability but normal deformability. In a family with hereditary elliptocytosis and an abnormality in spectrin self-association, the membranes had decreased deformability and stability. Finally, membranes from several individuals with Malaysian ovalocytosis had decreased deformability but increased stability. Our data from both pathologic membranes and biochemically perturbed membranes show that deformability and stability change with no fixed relationship to one another. These findings imply that different skeletal protein interactions regulate membrane deformability and stability. In light of these data, we propose a model of the role of skeletal protein interactions in deformability and stability.     

The gene for human liver arginase (ARG1) is assigned to chromosome band 6q23.

The human liver arginase gene, whose deficiency is responsible for argininemia (McKusick no. 20780), has been assigned to 6q23 through a combination of somatic cell hybrid analysis and in situ hybridization using a 1,550-base pair (bp) human DNA probe for this gene.     

A detailed genetic map of the long arm of chromosome 11

We describe 14 new restriction fragment length polymorphisms, corresponding to 13 loci on the long arm of chromosome 11. A detailed genetic map of chromosome 11q has been constructed from these and other loci (a total of 31 loci) typed in 59 reference families. The 23 most informative markers were selected to establish a map with a strongly supported order; regional localizations are provided for eight other markers. The loci span 88 cM in males and 148 cM in females and form a dense continuum on 11q. These ordered polymorphic markers will be of help in studying the genes responsible for several diseases that have been localized to this region, including genes responsible for multiple endocrine neoplasia type I (MEN1), ataxia telangiectasia (AT), tuberous sclerosis (TSC), and some forms of asthma and rhinitis.