• Volume 16, Issue 4

      December 1991,   pages  175-250

    • Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts

      S Srinivas T Nagashunmugam G Shanmugam

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      A DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [35S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S-phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0.25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.

    • Restriction enzyme digestion of heterochromatin inDrosophila nasuta

      P K Tiwari S C Lakhotia

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      In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.

    • Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

      K Cheirmaraj M V R Reddy B C Harinath

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      The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA2-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA2-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.

    • Immunoprophylaxis against filarial parasite,Brugia malayi: potential of excretory-secretory antigens in inducing immunity

      K Cheirmaraj V Chenthamarakshan M V R Reddy B C Harinath

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      The role of excretory-secretory antigens in inducing immunity in the host againstBrugia malayi microfilariae and infective larvae was studied byin vitro antibody dependent cell-mediated reaction as well asin vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised againstBrugia malayi microfilarial and infective larval excretory-secretory antigens(Bm Mf ESA andBm L3 ESA) promoted the adherence of peritoneal exudate cells toBrugia malayi microfilariae and infective larvaein vitro and induced cytotoxicity to the parasites within 48 h. The antiBm Mf ESA serum was more effective than antiBm L3 ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48–72 h. Further,Mastomys natalensis immunized againstBm Mf ESA and L3 ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretory-secretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.

    • Tissue distribution and antileishmanial activity of liposomised Amphotericin-B in Balb/c mice§

      Imran Ahmad Anshu Agarwal Ajay Pal P Y Guru B K Bachhawat C M Gupta

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      Antileishmanial activity and organ distribution of the antifungal drug Amphotericin-B in free and liposomised form have been studied in Balb/c mice infected withLeishmania donovani. Results indicate that Amphotericin-B in the liposomised form is significantly more active than the free form. This increase in the activity is perhaps related to the reduced drug toxicity rather than the altered drug distribution at the site of infection.

    • Plasminogen activator: Isolation and purification from Lymphosarcoma of ascites bearing mice

      M W Nulkar Rukmini Darad M Subramanian A R Pawse

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      Plasminogen activator secreted by lymphosarcoma (ascites) of mice was purified up to 163-fold by ammonium sulphate fractionation at 35% saturation and chromatography on p-aminobenzamidine-Sepharose 4B. The purified activator contained specific activity of 9980 IU/mg. The plasminogen activator displayed homogeneity by polyacrylamide slab gel electrophoresis and high performance liquid chromatography. The activator consisted of a single polypeptide chain with an apparent molecular weight of 66,000 daltons as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions as well as gel filtration on Sephadex G-100. Distinct differences between this activator and urokinase were discernible in respect of specific activities, fibrin affinity and immunochemical properties. The lymphosarcoma activator appears to be of tissue-type origin since it showed gross similarity to standard tissue plasminogen activator in terms of modes of binding to fibrin and immunological attributes.

    • Relationship between fatty acid binding proteins, acetyl-CoA formation and fatty acid synthesis in developing human placenta

      Tanya Das Gaurisankar Sa Anup K Bandyopadhyay Manju Mukherjea

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      The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-14 C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.

    • Effect of griseofulvin on lipid composition and membrane integrity inMicrosporum gypseum

      Indu Bala Chugh M P Gupta G K Khuller

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      The effect of griseofulvin on lipid constituents and membrane permeability ofMicrosporum gypseum has been investigated. Mycelia grown in medium containing griseofulvin (IC50 concentration) possessed a lower content of total lipids, phospholipids and sterols. This inhibitory effect was further supported by decreased incorporation of [14C] acetate in total lipids, total phospholipids and sterols. Decrease in total phospholipids was also reflected to a varying extent in all individual phospholipids. An increase in the unsaturated to saturated fatty acid ratio was observed in mycelia grown in medium containing griseofulvin. Membrane permeability was affected by griseofulvin as shown by increased K+-efflux and greater leakage of intracellular [32P] labelled components from prelabelled cells. Our results suggest that the antifungal activity of griseofulvin is partially due to its secondary effect on lipid constituents ofMicrosporum gypseum.

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