11 research outputs found
Isolation of a nitric oxide synthase from the protozoan parasite, Leishmania donovani
A soluble nitric oxide synthase (NOS) activity was purified 2800-fold from Leishmania donovani, the causative parasite of visceral leishmaniasis, by two-step affinity and anion-exchange chromatography. The purified enzyme ran as a prominent band of 110 kDa on SDS-PAGE whereas gel filtration experiments estimated the native molecular mass to be 230Ā±20 kDa indicating that the native enzyme exists as a dimer. The enzyme activity required NADPH and was blocked by EGTA. The enzyme kinetics, cofactor requirements, inhibition studies and Western blot analysis with brain anti-NOS antibody suggest its similarity with mammalian NOS isoform I
HUMAN UDP-GLUCURONOSYLTRANSFERASES SHOW ATYPICAL METABOLISM OF MYCOPHENOLIC ACID AND INHIBITION BY CURCUMIN
ABSTRACT: Although the promising immunosuppressant, mycophenolic acid (MPA), has many desirable properties and is widely prescribed for organ transplant recipients, its high oral dosage requirement is not understood. Whereas previous Northern blot analysis by Basu and colleagues (2004) located the mRNAs encoding MPA primary metabolizers, UDP-glucuronosyltransferases (UGTs) 1A7, 1A8, 1A9, and 1A10, in human gastrointestinal (GI) tissues, in situ hybridization analysis of mRNAs found that the isozymes were restricted to the mucosal layer of various GI organs. Concomitantly, MPA was glucuronidated by microsomes isolated from normal adjoining specimens. Microsomal studies showed the highest relative rates of metabolism in esophagus, ileum, duodenum, colon, and stomach at pH 6.4; only esophagus showed high pH 7.6 activity. Properties of the recombinant UGTs indicate that MPA is metabolized with pH 6.4 or 7.6 optimum. Activity for 1A7 and 1A9 increased with increasing concentrations up to 2.4 mM, with parallel production of both ether-and acylglucuronides; similarly, 1A8 and 1A10 reached plateaus at 1.6 mM, producing both glucuronides. K m values were 250 to 550 M. Between 400 and 1600 M MPA, isozymes generated between 15 and 42% of the acylglucuronides. In effect, high K m values (MPA) are associated with high concentrations to achieve saturation kinetics. Finally, transient inhibition of UGTs in human LS180 colon cells and mouse duodenum by the dietary agent, curcumin, has implications for in vivo pretreatment to reduce MPA glucuronidation to increase the therapeutic index
Synergistic Effect of Interferon-g and Mannosylated Liposome-Incorporated Doxorubicin in the Therapy of Experimental Visceral Leishmaniasis
Active targeting of doxorubicin to macrophages was studied by incorporating it in mannosecoated
liposomes by use of visceral leishmaniasis in BALB/c mice as the model macrophage
disease. Mannosylated liposomal doxorubicin was more effective than liposomal doxorubicin
or free doxorubicin. Because leishmaniasis is accompanied by immunosuppression, immunostimulation
by interferon (IFN)āg was evaluated to act synergistically with mannosylated
liposomal doxorubicin therapy. Combination chemotherapy with a suboptimal dose of IFNg
resulted in possibly complete elimination of spleen parasite burden. Analysis of mRNA
levels of infected spleen cells suggested that targeted drug treatment together with IFN-g, in
addition to greatly reducing parasite numbers, resulted in reduced levels of interleukin (IL)ā4
but increased levels of IL-12 and inducible nitric oxide synthase. Such combination chemotherapy
may provide a promising alternative for the cure of leishmaniasis, with a plausible
conversion of antiparasitic T cell response from a Th2 to Th1 pattern indicative of long-term
resistance
Purification and characterization of an organ specific haemorrhagic toxin from <i>Vipera russelli russelli </i>(Russell's viper) venom
114-120A haemorrhagic toxin (VRR-12) from Vipera
russelli russelli (Russell's viper) venom has been purified by ionexchange chromatography
on CM-Sephadex C-50 followed by size-exclusion HPLC to electrophoretically homogeneous
state. It is a 12 kDa single polypeptide having 1 mole of Ā Zn+2 ion. This toxin induces intense
intestinal haemorrhage and to a lesser extent skeletal muscle haemorrhage in mice.
It does not show detectable proteolytic and esterolytic activity with selected
substrates under specified conditions, haemolytic and phospholipase activity. When
VRR-12, preincubated with bivalent anti serum against Saw-scaled and Russell's
viper venom or EDTA was injected, haemorrhagic activity was not reduced, on the
otherhand preincubation with phenylmethyl sulphonyl fluoride reduced the activity
markedly. Biodistribution studies with 1251 VRR- 12 show that haemorrhagic manifestation
by this toxin is not a direct function of the fraction of the totally administered
toxin distributed to that tissue
Development of highly sensitive cell-based AKT kinase ELISA for monitoring PI3K beta activity and compound efficacy
Isolation of a laminin-binding protein from the protozoan parasite Leishmania donovani that may mediate cell adhesion
A Crystalline Compound (BM-ANF1) from the Indian Toad (Bufo melanostictus, Schneider) Skin Extract, Induced Antiproliferation and Apoptosis in Leukemic and Hepatoma Cell Line Involving Cell Cycle Proteins.
In our earlier communication, it was reported that Indian toad (Bufo melanostictus) skin extract (TSE) possesses
antiproliferative and apoptogenic activity in U937 and K562 cells [Giri et al., 2006. Antiproliferative, cytotoxic and
apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract in U937 and K562 cells. Toxicon 48 (4),
388ā400]. In the present study, a compound (BM-ANF1) has been isolated from the TSE by alumina gel column
chromatography, crystallized and evaluated for its antiproliferative and apoptogenic activity in U937, K562 and HepG2
cells. BM-ANF1 produced dose-dependent inhibition of U937, K562 and HepG2 cell growth. The antiproliferative activity
was reflected by the MTT assay and demonstrated by the reduced expression of proliferative cell nuclear antigen (PCNA).
Flow-cytometric analysis showed that BM-ANF1 arrested the cell cycle at G1 phase and enhanced annexin-V binding in
U937 and K562 cells. Scanning electron microscopic and fluorescent microscopic analysis of U937 and K562 cells revealed
the apoptogenic nature of the compound. Alkaline comet assay showed that BM-ANF1 produced DNA fragmentation.
The dose-dependent expression of caspase 3 indicated that the apoptogenic properties of BM-ANF1 were mediated
through the activation of downstream effector nucleases in the cancer cells. The increased expression of p53 and moderate
expression of p21Cip1/p27Kip1 due to BM-ANF1 treatment in HepG2 cells supported that the apoptogenic activity of
BM-ANF1 was mediated through p53 tumor-suppressor gene expression followed by the expression of p21Cip1 and p2
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)ļæ½2 of antiā51-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
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)ļæ½2 of antiā51-
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