Inhibition of diverse opportunistic viruses by structurally optimized retrograde trafficking inhibitors

Opportunistic viruses are a major problem for immunosuppressed individuals, particularly following organ or stem cell transplantation. Current treatments are non-existent or suffer from problems such as high toxicity or development of resistant strains. We previously published that a trafficking inhibitor that targets a host protein greatly reduces the replication of human cytomegalovirus. This inhibitor was also shown to be moderately effective against polyomaviruses, another family of opportunistic viruses. We have developed a panel of analogues for this inhibitor and have shown that these analogues maintain their high efficacy against HCMV, while substantially lowering the concentration required to inhibit polyomavirus replication. By targeting a host protein these compounds are able to inhibit the replication of two very different viruses. These observations open up the possibility of pan-viral inhibitors for immunosuppressed individuals that are effective against multiple, diverse opportunistic viruses.     

Role of siderophore in iron uptake in cowpea Rhizobium GN1 (peanut isolate): Possible involvement of iron repressible outer membrane proteins

Abstract Siderophore produced by cowpea Rhizobium GN1 (Peanut isolate) was shown to be involved in iron uptake by this organism. Siderophore enhanced iron uptake in iron-starved cells. SDS-PAGE analysis of the outer membrane proteins showed two iron repressible outer membrane proteins with approximate molecular mass of 80 kDa and 76 kDa. A siderophore non-producing mutant, which was unable to grow on a medium containing synthetic iron chelators unless and until iron was added exogenously in the medium, could use siderophore of the wild-type for iron uptake indicating that the receptor for Fe-siderophore complex was intact in the mutant.     

Functional analysis of kinetochore assembly in Caenorhabditis elegans

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical "kinetochore null" phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A-containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.     

Signal transduction and biological function of placenta growth factor in primary human trophoblast

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family of angiogenic factors, is prominently expressed by trophoblast. In addition to its role as a paracrine angiogenic factor within the placenta and endometrium, presence of its receptor, Flt-1, on trophoblast suggests that PlGF also may have an autocrine role(s) in regulating trophoblast function. To elucidate its role in trophoblast, we examined the signal transduction and functional responses of primary human trophoblast to PlGF. Exogenous PlGF induced specific activation of the stress-activated protein kinase (SAPK) pathways, c-Jun-N terminal kinase (JNK) and p38 kinase, in primary term trophoblast with little to no induction of the extracellular signal regulated kinase (ERK-1 and -2) pathways. In contrast, PlGF induced significant ERK-1 and -2 activity in human umbilical vein endothelial cells but did not induce JNK or p38 activity. PlGF-induced activation of the SAPK signaling pathways protected trophoblast from growth factor withdrawal-induced apoptosis, but it did not protect trophoblast from apoptosis induced by the pro-inflammatory cytokines, interferon gamma and tumor necrosis factor alpha. These results provide the first direct evidence of a biochemical and functional role for PlGF/Flt-1 in normal trophoblast and suggest that aberrant PlGF expression during pregnancy may impact upon trophoblast function as well as vascularity within the placental bed.     

RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.