Antineoplastic and Apoptotic Potential of Traditional Medicines Thymoquinone and Diosgenin in Squamous Cell Carcinoma

Thymoquinone (TQ) and diosgenin (DG), the active ingredients obtained from black cumin (Nigella sativa) and fenugreek (Trigonella foenum graecum), respectively, exert potent bioactivity, including anticancer effects. This study investigated the antineoplastic activity of these agents against squamous cell carcinoma in vitro and sarcoma 180–induced tumors in vivo. TQ and DG inhibited cell proliferation and induced cytotoxicity in A431 and Hep2 cells. These agents induced apoptosis by increasing the sub-G₁ population, LIVE/DEAD cytotoxicity, chromatin condensation, DNA laddering and TUNEL-positive cells significantly (P<0.05). Increased Bax/Bcl-2 ratio, activation of caspases and cleavage of poly ADP ribose polymerase were observed in treated cells. These drugs inhibited Akt and JNK phosphorylations, thus inhibiting cell proliferation while inducing apoptosis. In combination, TQ and DG had synergistic effects, resulting in cell viability as low as 10%. In a mouse xenograft model, a combination of TQ and DG significantly (P<0.05) reduced tumor volume, mass and increased apoptosis. TQ and DG, alone and in combination, inhibit cell proliferation and induce apoptosis in squamous cell carcinoma. The combination of TQ and DG is a potential antineoplastic therapy in this common skin cancer.     

CXCL10 Is Critical for the Generation of Protective CD8 T Cell Response Induced by Antigen Pulsed CpG-ODN Activated Dendritic Cells

The visceral form of leishmaniasis is the most severe form of the disease and of particular concern due to the emerging problem of HIV/visceral leishmaniasis (VL) co-infection in the tropics. Till date miltefosine, amphotericin B and pentavalent antimony compounds remain the main treatment regimens for leishmaniasis. However, because of severe side effects, there is an urgent need for alternative improved therapies to combat this dreaded disease. In the present study, we have used the murine model of leishmaniasis to evaluate the potential role played by soluble leishmanial antigen (SLA) pulsed-CpG-ODN stimulated dendritic cells (SLA-CpG-DCs) in restricting the intracellular leishmanial growth. We found that mice vaccinated with a single dose of SLA-pulsed DC stimulated by CpG-ODN were protected against a subsequent leishmanial challenge and had a dramatic reduction in parasite burden along with the generation of parasite specific cytotoxic T lymphocytes. Moreover, we demonstrate that the induction of protective immunity conferred by SLA-CpG-DCs depends entirely on the CXC chemokine IFN-γ-inducible protein 10 (CXCL10; IP-10). CXCL10 is directly involved in the generation of a parasite specific CD8⁺ T cell-mediated immune response. We observed significant reduction of CD8⁺ T cells in mice depleted of CXCL10 suggesting a direct role of CXCL10 in the generation of CD8⁺ T cells in SLA-CpG-DCs vaccinated mice. CXCL10 also contributed towards the generation of perforin and granzyme B, two important cytolytic mediators of CD8⁺ T cells, following SLA-CpG-DCs vaccination. Together, these findings strongly demonstrate that CXCL10 is critical for rendering a protective cellular immunity during SLA-CpG-DC vaccination that confers protection against Leishmania donovani infection.     

Two-dimensional gel-based alkaline proteome of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM (NCFM) is a well-documented probiotic bacterium isolated from human gut. Detailed 2D gel-based NCFM proteomics addressed the so-called alkaline range, i.e., pH 6-11. Proteins were identified in 150 of the 202 spots picked from the Coomassie Brilliant Blue stained 2D gel using MALDI-TOF-MS. The 102 unique gene products among the 150 protein identifications were assigned to different functional categories, and evaluated by considering a calculated distribution of abundance as well as grand average of hydrophobicity values. None of the very few available lactic acid bacteria proteome reference maps included the range of pI >7.0. The present report of such data on the proteome of NCFM fundamentally complements current knowledge on protein profiles limited to the acid and neutral pH range.     

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes.     

Characterization of a low-molecular-mass stimulator protein of Mg2+-independent Ca2+-ATPase: effect on phosphorylation/dephosphorylation, calcium transport and sperm-cell motility

A 14 kDa cytosolic protein purified from bovine brain homogenate has been recently reported as a stimulator of goat spermatozoa Mg2+-independent Ca2+-ATPase. In the present study, we demonstrate the formation of the [gamma-32P]ATP-labelled phosphoenzyme as the 110 kDa phosphoprotein and its rapid decomposition in presence of the stimulator protein. Together with the cross-reactivity of this 110 kDa protein with an anti-SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 2a antibody, the ATPase can now be conclusively said to belong to the SERCA family, which is activated by the stimulator. The ability of the stimulator to enhance the Ca2+ transport has been elucidated from 45Ca2+ uptake studies and was found to be sensitive to Ca2+ channel blockers. CD revealed an alpha-helical structure of the stimulator. The amino acid analysis suggests that it is composed primarily of hydrophobic and some acidic amino acid residues. The pI of 5.1 has been re-confirmed from two-dimensional electrophoresis. Immuno-cross-reactivity studies indicate that the stimulator or similar proteins are present in cytosolic fractions of liver, kidney or testes in different species, but brain is the richest source. Proteomic analyses of its trypsinized fragments suggest its similarity with bovine THRP (thyroid hormone-responsive protein). The physiological significance of the stimulator has been suggested from its ability to activate sperm-cell motility.     

Structural and functional characterization and physiological significance of a stimulator protein of Mg2+-independent Ca2+-ATPase isolated from goat spermatozoa

Recently a low-molecular-mass protein purified from goat testes cytosol has been reported from our laboratory which is found to stimulate Mg²⁺-independent Ca²⁺-ATPase without any significant effect on Mg²⁺-dependent Ca²⁺-ATPase. In the present study, detailed structural and functional characterization, as well as the physiological significance of the protein has been described. The stimulatory effect is found to be inhibited by known inhibitors of P-type ATPases, vanadate and lanthanum chloride. Monitoring of the phosphoenzyme intermediate by autoradiography has shown that the stimulation of the ATPase is due to the enhancement in the rate of dephosphorylation of the overall reaction step. Along with the stimulation of the enzyme activity, the protein is found to enhance the calcium uptake. Amino acid analysis data show that the stimulator contains about 26% non-polar amino acid facilitating easy penetration to the hydrophobic core of the membrane bound ATPase. Circular dichroism analysis of the protein suggested the presence of all secondary structural elements. The Western-blotting experiment shows its expression level is the highest in goat testes. Peptide fragments obtained in MALDI-MS analysis when subjected to MSDB database search by MASCOT search engine reveals that the proteins of close similarity with the protein under study are actin related protein 2/3 complex subunit, peptidyl-prolyl cis-trans isomerase and gastrin releasing peptide precursor. Besides, the protein under study is also shown to decrease the forward motility of goat sperm without having any significant effect on the total motility indicating its possible role in fertility regulation.     

A novel association of a polymorphism in the first intron of adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia in Asian Indians

Adiponectin is an adipose tissue specific protein that is decreased in subjects with obesity and type 2 diabetes. The objective of the present study was to examine whether variants in the regulatory regions of the adiponectin gene contribute to type 2 diabetes in Asian Indians. The study comprised of 2,000 normal glucose tolerant (NGT) and 2,000 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. We identified two proximal promoter SNPs (−11377C→G and −11282T→C), one intronic SNP (+10211T→G) and one exonic SNP (+45T→G) by SSCP and direct sequencing in a pilot study (n = 500). The +10211T→G SNP alone was genotyped using PCR-RFLP in 4,000 study subjects. Logistic regression analysis revealed that subjects with TG genotype of +10211T→G had significantly higher risk for diabetes compared to TT genotype [Odds ratio 1.28; 95% Confidence Interval (CI) 1.07–1.54; P = 0.008]. However, no association with diabetes was observed with GG genotype (P = 0.22). Stratification of the study subjects based on BMI showed that the odds ratio for obesity for the TG genotype was 1.53 (95%CI 1.3–1.8; P < 10⁻⁷) and that for GG genotype, 2.10 (95% CI 1.3–3.3; P = 0.002). Among NGT subjects, the mean serum adiponectin levels were significantly lower among the GG (P = 0.007) and TG (P = 0.001) genotypes compared to TT genotype. Among Asian Indians there is an association of +10211T→G polymorphism in the first intron of the adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia.     

Molecular Cloning, Bacterial Overexpression and Characterization of L-myo- inositol 1- Phosphate Synthase from a Monocotyledonous Resurrection Plant, Xerophyta viscosa Baker

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS), an evolutionarily conserved enzyme-protein, catalyses the first and rate limiting step of inositol biosynthesis. Inositol and its derivatives play important roles in biological kingdom like growth regulation, membrane biogenesis, signal transduction and also acts as an osmolyte or osmoprotectant in abiotic stress tolerance. Here we report the cloning, sequencing and the characterization of the INO1 gene from Xerophyta viscosa (XINO1), a monocotyledonous resurrection plant. Nucleotide sequences of XINO1 show striking homology (70–99%) with a number of INO1 genes from plant sources particularly with the monocots. The gene is functionally identified through genetic complementation using a yeast inositol auxotrophic strain FY250. The gene is expressed in E. coli BL21, recombinant protein purified to homogeneity, biochemically characterized and compared with Oryza INO1 (RINO1) gene product. The XINO1 gene product is catalytically active in a broader range of lower temperature (between 10–40 °C) than the RINO1 gene- product. This is the first report of MIPS gene from any resurrection plant.     

Putative Virulence Traits and Pathogenicity of Vibrio cholerae Non-O1, Non-O139 Isolates from Surface Waters in Kolkata, India

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.