Withanolide Z, a new chlorinated withanolide from Withania somnifera

Reverse-phase preparative HPLC analysis of the n-butanol fraction of the methanolic extract of Withania somnifera Dunal (leaves) afforded a novel chlorinated withanolide, namely withanolide Z (1), along with four known withanolides, withanolide B (2), withanolide A (3), 27-hydroxywithanolide B (4) and withaferin A (5). Their structures were elucidated by IR, MS, CD and a combination of 1 D and 2 D NMR spectral analyses. The Leishmania donovani DNA topoisomerase I inhibitory activities of the isolated compounds were determined.     

Interaction of different hemoglobinopathies in Eastern India with a view to establish genotype–phenotype correlation

Analysis of the molecular basis of hemoglobinopathies provides an opportunity to define genotype–phenotype variations as well as establish the origin of mutation. The present study deals with a large cohort of 1,661 cases referred to the counseling unit and 889 individuals from random screening of the population of Tripura. Characterization of mutation in 291 cases (582 alleles) was performed by the PCR-ARMS method using genomic DNA. The haplotype of 56 β^E mutation-bearing chromosomes were identified by the RFLP-PCR method. Genotypes were constructed and correlated with hematological and clinical phenotypes. IVS-1nt 5 (G→C) mutation was observed as the most frequent mutation, followed by codon 30 (G→C). Production of HbE was significantly (P < 0.001) higher in nontransfusion-dependent Eβ-thalassemia patients. β^E mutation was observed only on four haplotypes linked to framework 2. Type 2 haplotype was observed mainly from chromosomes of Tripura origin, but none from South Bengal. Homozygous E individuals with 1//1 genotype were significantly (P < 0.01) more anemic compared to individuals with 2//2 genotype. This work creates a database of hemoglobinopathy mutations for the population of Eastern India which will facilitate prenatal diagnosis and counseling. Am. J. Hum. Biol. 12:454–459, 2000. © 2000 Wiley-Liss, Inc.     

Anticancer Activity of Solvent Extracts of Hexogonia glabra against Cervical Cancer Cell Lines

Objective: In this study, we aimed to harness some solvent extracts of one wild mushroom Hexagonia glabra and test their anti-cancer activity against cervical human cell lines, namelyHeLa, SiHa, and CaSki. Methods: It includes cell morphological study by microscope, nuclear morphology by DAPI staining under fluorescence microscopy, apoptosis assay by fluorescence technique, anti-proliferation by MTT assay and expression of apoptotic and anti-apoptotic genes by Western blotting and cell cycle analysis was done. Results: The selected cervical cancer cells were treated separately with 150 µg/mL of three extracts, namely of ethanolic (EE), ethyl acetate (EAE), and water extract (WE) and exhibited features like round, shrink and dead. All extracts caused apoptosis in cell lines and EE had the highest effect in this regard. The percentages of apoptotic cells in HeLa, SiHa and CaSki, at the same concentration of EE were 79.23, 75.42, and 76.36% respectively. Cytotoxicity assay showed that all three extracts (50 – 250 μg/mL) were potent for inhibition of cell growth of three cell lines and again EE had the highest effect. The percentages of cell growth inhibition in HeLa, SiHa, and CaSki cells treated with EE at 24 h at 50 µg/mL were 45.79±4.11, 41.66±4.03, and 36.72±2.67, while they were 74.23±7.45, 62.31±5.97, and 54.23±5.04 at 150 µg/mL concentration. At 250 µg/mL concentration, the percentages of cell growth inhibition were 94.25 ±8.11, 90.02 ±8.67, and 85.43±6.21, respectively. The expression of apoptotic gene (Caspase 3, 9) and tumor guard gene (p53), as their proteins in Western blotting increased . However, anti-apoptotic BcL2 gene of all cell lines was decreased following treatment with extracts. In addition, the cell cycle analysis (CaSki cell) showed that treatment (EE) arrested at G2/M check point cell cycle. Conclusion: All extracts of this mushroom were active in arresting growth of three cell lines and EE had the highest effect, indicating that this mushroom can be a valuable source of anticancer agents.     

Estrogen increases intracellular p26Bcl-2 to p21Bax ratios and inhibits taxol-induced apoptosis of human breast cancer MCF-7 cells

Recent studies have demonstrated that following estrogen ablation, estrogen responsive breast cancer cells undergo apoptosis. In addition, estrogen receptor (ER) expression has been strongly correlated with the expression of the bcl-2 gene product, p26Bcl-2 protein, which is known to inhibit apoptosis. In the present studies, we investigated whether estrogen affects the intracellular levels of p26Bcl-2 and thereby modulates taxol-induced apoptosis of estrogen responsive human breast cancer MCF-7 cells. Transfer of MCF-7 cells to a culture-medium without estrogens reduced their intracellular p26Bcl-2 levels by 50%. Inclusion of 0.1 M estradiol in the medium produced approximately a four-fold increase in p26Bcl-2, but not p29Bcl-xL or p21Bax levels; the expression of the c-myc and mdr-1 genes remained unchanged. Estradiol-induced four-fold increase in the ratio of the p26Bcl-2 to p21Bax levels caused a significant decline in the lethal, kilobase size DNA fragments of apoptosis, which had resulted when MCF-7 cells were cultured in a medium without estrogen. In addition, in MCF-7 cells, estradiol-induced increase in the intracellular p26Bcl-2 to p21Bax ratios was associated with a significant reduction in the large-sized DNA fragmentation induced by treatment with taxol. The increased ratios also protected MCF-7 cells against taxol-mediated cytotoxicity as assessed by the MTT assay. These results suggest that by modulating p26Bcl-2 levels, estrogens may affect the antitumor activity of taxol and potentially of other anti-breast cancer drugs against estrogen responsive human breast cancer cells.     

Calpeptin provides functional neuroprotection to rat retinal ganglion cells following Ca2+ influx

Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 microM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection.     

Cutaneous Crohn''s disease mimicking Melkersson–Rosenthal syndrome: treatment with methotrexate

A woman with a 5-year history of unilateral orofacial granulomatosis required repeated evaluations (including sequential colonoscopies) to establish the diagnosis of cutaneous Crohn's disease, a condition that proved responsive to low doses of oral methotrexate administered weekly. To our knowledge this is the first report describing the use of methotrexate for treatment of orofacial granulomatosis caused by underlying Crohn's disease.