Differential Postnatal Development of Monoamine Oxidases A and B in the Blood-Brain Barrier of the Rat

Abstract: We studied the monoamine metabolizing mitochondrial enzyme, monoamine oxidase (MAO), in cerebral microvessels obtained from postnatally developing rats by measuring the specific binding of [³H]pargyline, an irreversible inhibitor of MAO, and the rate of oxidation of three known MAO substrates: benzylamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and tryptamine. MAO activity increased postnatally, with the greatest increase occurring in the second week and reaching a peak at 3 weeks of age. A concomitant increase in MAO of the cerebral cortex also occurred, but was several-fold less than that of cerebral microvessels. Using clorgyline and deprenyl, relatively specific inhibitors of MAO-A and MAO-B, we showed that cerebral microvessels contain both forms of MAO at all ages, but there was a major preponderance in the postnatal development of MAO-B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat microvessels after [³H]pargyline binding also showed two distinct bands of radioactivity at all ages. These two bands corresponded to molecular weights of ∼6.5,000 for MAO-A and -60,000 for MAO-B. SDS-PAGE resuits of brain microvessels obtained from 1-, 14-, and 42-day-old rats confirm the differential postnatal development of MAO-B in rat brain microvessels.     

Amphomycin: A tool to study protein N-glycosylation

Radio-labelled amphomycin (³H-amphomycin) forms a complex with dolichylmonophosphate in presence of Ca²⁺. Complex formation has also been documented with retinylmonophosphate and perhydromonoeneretinylmonophosphate. Analysis of the space-filling model suggested both fatty acylated aspartic acid residue at the N-terminus of the lipopeptide and phosphate head group of dolichylmonophosphate are necessary for the complex formation. The binding ability of amphomycin is then utilized to localize dolichylmonophosphate in the microsomal membrane. Studies with microsomal membranes from hen oviduct suggested that dolichylmonophosphate is located in the cytoplasmic side of the membrane.     

Reactivity of glycoconjugate in membrane system II: Can a neutral glycolipid function as lectin receptor in the presence of gangliosides in plasma membrane?

To examine how surface Potential controls the reactivity of glycoconjugates at cell surface, the interaction of galactose-sPecific lectinse.g. peanut agglutinin,Ricinus cummunis agglutinin with liPosomes bearing asialo GM₁ were studied in the Presence of varying amount of ganglioside mixture, GM_n. The Presence of 5% GM_n causes comPlete slowing down of PreciPitin reaction and thereby make carbohydrate moiety of asialo GM₁ comPletely inaccessiblei.e. ‘cryPtic’. In contrast the Presence of 1–2% GM_n enhances the aPParent rate and amPlitude of the PreciPitin reaction as surface Potential becomes more negative. The relevance of the findings has been discussed in relation to the exPression and involvement of the cell-surface sialic acid residues during develoPment and differentiation.     

Interrelationships among three derivative forms of the immigrans-Hirtodrosophila radiation: evidence from heterochromatin and polytene chromosomes

The phylogenetic relationships among three derivative forms of the immigrans-Hirtodrosophila radiation viz. Chaetodrosophilella, Zaprionus and the immigrans species group are examined, by comparing the banding patterns of their polytene chromosomes and by analysing the nature of their heterochromatin. Based on the results of these studies it is concluded that D. quadrilineata has a very strong affinity with the members of the immigrans species group, while the genus Zaprionus represents a very distinct evolutionary lineage. This study further indicates that D. quadrilineata is a karyotypically primitive species having five pairs of rods and one pair of dots, while the karyotype of other members of the immigrans species group appears to have undergone modifications through fusions, fissions, inversions and the addition or deletion of heterochromatin to dots and other chromosomes.     

Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease

C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid). Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts. Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice. Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level. These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.     

Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.