Role of 67 kDa cell surface laminin binding protein of Leishmania donovani in pathogenesis

The role that interaction with laminin may play in Leishmania donovani infection was investigated. Binding of 125 I-radiolabeled laminin, in a liquid-phase assay, by the parasite was rapid, saturable, specific, reversible, and of high affinity. Using a Western blotting procedure, a 67 kDa laminin-binding protein (LBP) was identified from the membrane of both the promastigote and amastigote forms of L. donovani. Subsequently, the protein was purified by affinity chromatography. Immunofluorescence with a polyclonal anti-body against LBP as well as flow cytometric analysis demonstrated its presence at the parasite surface. After stimulation with phorbol-12-myristate-13-acetate (PMA), U937 cells exhibited the ability to adhere to laminin and LBP specifically inhibited this adhesion. The reduced parasite adhesion after tunicamycin treatment suggested the importance of sugar residues in cell adhesion. Although co-administration of either laminin or LBP or anti LBP antibody reduced parasite virulence, resulting in a lower level of infection in the BALB/c mouse model, an in vitro macrophage culture-enhanced level of infection was observed in the case of laminin-coated parasites. The results collectively suggest a role for LBP in the interaction of the parasite with extracellular matrix elements, which may constitute a basis for the homing of the parasite to its physiological address

Regulation of guanylyl cyclase by intracellular Ca<SUP>2+</SUP> in relation to the infectivity of the protozoan parasite, Leishmania donovani

A neuronal type Ca2+ stimulated nitric oxide synthase was earlier reported by us to be present in the protozoan parasite Leishmania donovani. As part of nitric oxide-cyclic GMP transduction signaling operative in higher eukaryotes and involved in the long-term potentiation, a soluble guanylyl cyclase has also been detected in this lower eukaryote. However, detailed biochemical characterization revealed the enzyme to be Ca2+ modulated and unstimulated by nitric oxide donors as opposed to higher eukaryotes. The possible role of intracellular Ca2+ level in the regulation of guanylyl cyclase activity as well as L. donovani infectivity was explored by measuring the intracellular survival of the parasites in mammalian macrophages after treatments, which decrease or elevate the intracellular Ca2+. Parasites loaded with intracellular Ca2+ chelators displayed significantly decreased infectivity and cyclic GMP level. In contrast, pretreatment with Ca2+ ionophores, which elevated Ca2+ levels in L. donovani, significantly enhanced the cyclic GMP level as well as the infectivity of the parasites. Moreover, treatment with selective inhibitors of soluble guanylyl cyclase also reduced infectivity, even in cases of calcium ionophore-treated parasites. The gene encoding the soluble guanylyl cyclase was cloned, sequenced and over expressed in bacterial system. The recombinant protein showed enzyme characteristics similar to that obtained in L. donovani promastigote cytosol. Together these results suggest a possible link between guanylyl cyclase, intracellular Ca2+ content and parasite infectivity

Targeting of Parasite-Specific Immunoliposome-Encapsulated Doxorubicin in the Treatmentof Experimental Visceral Leishmaniasis

A parasite-specific 51-kDa protein has been isolated from the membrane of macrophages infected with Leishmania donovani, the causative agent of visceral leishmaniasis. Active targeting of doxorubicin to infected macrophages was studied by incorporating it in immunoliposomes prepared by grafting F(ab)�2 of anti–51-kDa antibody onto the liposomal surface. In a 45-day mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by doxorubicin incorporated in immunoliposome (immunodoxosome) at a dose of 250 mg/kg/day that was given for 4 consecutive days. A similar dose of free and liposomal drug (doxosome) had 45% and 84% parasite suppressive effects, respectively. Immunodoxosome and doxosome were generally less toxic than the free drug, as determined by several clinical parameters of cardiotoxicity and liver toxicity. These results not only indicate the potential of doxorubicin as an effective chemotherapeutic agent but also establish the use of immunoliposomes as drug carrier in the therapy of leishmaniasis

Cooperative Activation of Cyclin D1 and Progesterone Receptor Gene Expression by the SRC-3 Coactivator and SMRT Corepressor

Although the ability of coactivators to enhance the expression of estrogen receptor-α (ERα) target genes is well established, the role of corepressors in regulating 17β-estradiol (E2)-induced gene expression is poorly understood. Previous studies revealed that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor is required for full ERα transcriptional activity in MCF-7 breast cancer cells, and we report herein the E2-dependent recruitment of SMRT to the regulatory regions of the progesterone receptor (PR) and cyclin D1 genes. Individual depletion of SMRT or steroid receptor coactivator (SRC)-3 modestly decreased E2-induced PR and cyclin D1 expression; however, simultaneous depletion revealed a cooperative effect of this coactivator and corepressor on the expression of these genes. SMRT and SRC-3 bind directly in an ERα-independent manner, and this interaction promotes E2-dependent SRC-3 binding to ERα measured by co-IP and SRC-3 recruitment to the cyclin D1 gene as measured by chromatin IP assays. Moreover, SMRT stimulates the intrinsic transcriptional activity of all of the SRC family (p160) coactivators. Our data link the SMRT corepressor directly with SRC family coactivators in positive regulation of ERα-dependent gene expression and, taken with the positive correlation found for SMRT and SRC-3 in human breast tumors, suggest that SMRT can promote ERα- and SRC-3-dependent gene expression in breast cancer

Targeting of Parasite-Specific Immunoliposome- Encapsulated Doxorubicin in the Treatment of Experimental Visceral Leishmaniasis

A parasite-specific 51-kDa protein has been isolated from the membrane of macrophages infected with Leishmania donovani, the causative agent of visceral leishmaniasis. Active targeting of doxorubicin to infected macrophages was studied by incorporating it in immunoliposomes prepared by grafting F(ab)�2 of anti–51- kDa antibody onto the liposomal surface. In a 45-day mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by doxorubicin incorporated in immunoliposome (immunodoxosome) at a dose of 250 mg/kg/day that was given for 4 consecutive days. A similar dose of free and liposomal drug (doxosome) had 45% and 84% parasite suppressive effects, respectively. Immunodoxosome and doxosome were generally less toxic than the free drug, as determined by several clinical parameters of cardiotoxicity and liver toxicity. These results not only indicate the potential of doxorubicin as an effective chemotherapeutic agent but also establish the use of immunoliposomes as drug carrier in the therapy of leishmaniasis

The Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Corepressor Is Required for Full Estrogen Receptor α Transcriptional Activity▿

Multiple factors influence estrogen receptor α (ERα) transcriptional activity. Current models suggest that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor functions within a histone deactylase-containing protein complex that binds to antiestrogen-bound ERα and contributes to negative regulation of gene expression. In this report, we demonstrate that SMRT is required for full agonist-dependent ERα activation. Chromatin immunoprecipitation assays demonstrate that SMRT, like ERα and the SRC-3 coactivator, is recruited to an estrogen-responsive promoter in estrogen-treated MCF-7 cells. Depletion of SMRT, but not histone deacetylases 1 or 3, negatively impacts estradiol-stimulated ERα transcriptional activity, while exogenous expression of SMRT's receptor interaction domains blocks ERα activity, indicating a functional interaction between this corepressor and agonist-bound ERα. Stimulation of estradiol-induced ERα activity by SMRT overexpression occurred in HeLa and MCF-7 cells, but not HepG2 cells, indicating that these positive effects are cell type specific. Similarly, the ability of SMRT depletion to promote the agonist activity of tamoxifen was observed for HeLa but not MCF-7 cells. Furthermore, impairment of agonist-stimulated activity by SMRT depletion is specific to ERα and not observed for receptors for vitamin D, androgen, or thyroid hormone. Nuclear receptor corepressor (N-CoR) depletion increased the transcriptional activity of all four tested receptors. SMRT is required for full expression of the ERα target genes cyclin D1, BCL-2, and progesterone receptor but not pS2, and its depletion significantly attenuated estrogen-dependent proliferation of MCF-7 cells. Taken together, these data indicate that SMRT, in conjunction with gene-specific and cell-dependent factors, is required for positively regulating agonist-dependent ERα transcriptional activity

Thearubigin, the major polyphenol of black tea, ameliorates mucosal injury in trinitrobenzene sulfonic acid-induced colitis

Inflammatory bowel disease is characterized by oxidative and nitrosative stress, leukocyte infiltration and upregulation of proinflammatory cytokines. The aim of the present study was to examine the protective effects of thearubigin, an anti-inflammatory and anti-oxidant beverage derivative, on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice, a model for inflammatory bowel disease. Intestinal lesions (judged by macroscopic and histological score) were associated with neutrophil infiltration (measured as increase in myeloperoxidase activity in the mucosa), increased serine protease activity (may be involved in the degradation of colonic tissue) and high levels of malondialdehyde (an indicator of lipid peroxidation). Both nitric oxide (NO) and O2- were increased with concomitant upregulation in the mRNA expression of proinflammatory cytokine response and inducible NO synthase (iNOS). Dose-response studies revealed that pretreatment of mice with thearubigin (40 mg kg-1 day-1, i.g. for 10 days) significantly ameliorated the appearance of diarrhoea and the disruption of colonic architecture. Higher dose (100 mg kg-1) had comparable effects. This was associated with a significant reduction in the degree of both neutrophil infiltration and lipid peroxidation in the inflamed colon as well as decreased serine protease activity. Thearubigin also reduced the levels of NO and O2- associated with the favourable expression of T-helper 1 cytokines and iNOS. Consistent with these observations, nuclear factor kappa B (NF-&#954;B) activation in colonic mucosa was suppressed in thearubigin-treated mice. The results of this study suggest that thearubigin, the most predominant polyphenol of black tea, exerts beneficial effects in experimental colitis and may, therefore, be useful in the treatment of inflammatory bowel disease

Unique Roles of p160 Coactivators for Regulation of Breast Cancer Cell Proliferation and Estrogen Receptor-α Transcriptional Activity

Each of the three members of the p160 steroid receptor coactivator (SRC) family of coactivators (SRC-1, SRC-2 and SRC-3) stimulates estrogen receptor (ER)-α function in trans-activation assays. Consequently, we sought to elucidate their contributions to the ER-regulated processes of cell proliferation, apoptosis, and the expression of ERα target genes in MCF-7 breast cancer cells. The small interfering RNA depletion of SRC-2 or SRC-3 but not SRC-1 inhibited growth of MCF-7 cells, and this was reflected in decreased cell cycle progression and increased apoptosis in SRC-2- or SRC-3-depleted cells as well as a reduction in ERα transcriptional activity measured on a synthetic reporter gene. However, only SRC-3 depletion blocked estradiol stimulated cell proliferation. Depletion of SRC-1 did not affect these events, and together this reveals functional differences between each of the three SRC family coactivators. Regulation of the endogenous ERα target gene, c-myc was not affected by depletion of any of the p160 coactivators although depletion of each of them decreased pS2 mRNA expression in estradiol-treated MCF-7 cells. Moreover, progesterone receptor and cyclin D1 gene expression were decreased in SRC-3 small interfering RNA-treated cells. Expression of mRNA and protein levels for the antiapoptotic gene, Bcl-2 was dependent on SRC-3 expression, whereas Bcl-2 protein but not mRNA expression also was sensitive to SRC-1 depletion. Together these data indicate that the closely related p160 coactivators are not functionally redundant in breast cancer cells because they play gene-specific roles in regulating mRNA and protein expression, and they therefore are likely to make unique contributions to breast tumorigenesis