Effect of probiotic fermented milk and chlorophyllin on gene expressions and genotoxicity during AFB₁-induced hepatocellular carcinoma

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B? (AFB?) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B? at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB? control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (P<0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B? control group. The c-myc, bcl-2, cyclin D1 and rasp-21 level was found to be highest in AFB? control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB?-induced molecular alterations in hepatic cells during carcinogenesis.     

Development and validation of a robust QSAR model for prediction of carcinogenicity of drugs

Carcinogenicity is one of the toxicological endpoints causing the highest concern. Also, the standard bioassays in rodents used to assess the carcinogenic potential of chemicals and drugs are extremely long, costly and require the sacrifice of large numbers of animals. For these reasons, we have attempted development of a global quantitative structure-activity relationship (QSAR) model using a data set of 1464 compounds (the Galvez data set available from http://www.uv.es/-galvez/tablevi.pdf), including many marketed drugs for their carcinogenesis potential. Though experimental toxicity testing using animal models is unavoidable for new drug candidates at an advanced stage of drug development, yet the developed global QSAR model can in silico predict the carcinogenicity of new drug compounds to provide a tool for initial screening of new drug candidate molecules with reduced number of animal testing, money and time. Considering large number of data points with diverse structural features used for model development (n(training) = 732) and model validation (n(test) = 732), the model developed in this study has an encouraging statistical quality (leave-one-out Q2 = 0.731, R2pred = 0.716). Our developed model suggests that higher lipophilicity values and conjugated ring systems, thioketo and nitro groups contribute positively towards drug carcinogenicity. On the contrary, tertiary and secondary nitrogens, phenolic, enolic and carboxylic OH fragments and presence of three-membered rings reduce the carcinogenicity. Branching, size and shape are found to be crucial factors for drug-induced carcinogenicity. One may consider all these points to reduce carcinogenic potential of the molecules.     

Signalling to actin: role of C3G, a multitasking guanine-nucleotide-exchange factor

C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.     

Cytoskeletal remodeling by C3G to induce neurite-like extensions and inhibit motility in highly invasive breast carcinoma cells

Cytoskeletal remodeling is responsible for cell plasticity and facilitates differentiation, motility and adherence related functions. C3G (RAPGEF1), an exchange factor for Ras family of small GTPases, regulates cytoskeletal reorganization to induce filopodia in epithelial cells and neurite growth in neuroblastoma cells. Here we show that C3G overexpression induces neurite-like extensions (NLE) in MDA-MB-231 and BT549 breast carcinoma cells and not in a variety of other cancer cell lines examined. These processes were actin-rich with nodes, branches and microspikes. C3G associates with the cytoskeleton and its expression enabled stabilization of microtubules. NLE formation was dependent on Rap, Rac and Cdc42. C3G expression was associated with a decrease in cellular β-catenin levels specifically in MDA-MB-231 and BT549 cells. β-Catenin stabilization induced by GSK-3β inhibition, or coexpression of β-catenin, reduced C3G induced NLE formation. Time lapse analysis showed reduced motility of C3G expressing cells compared to GFP expressing cells. Our results suggest that C3G overexpression can induce phenotypic characteristics of neuronal cells in highly invasive breast cancer cells and inhibit their motility.     

A novel haplotype within C-reactive protein gene influences CRP levels and coronary heart disease risk in Northwest Indians

According to several epidemiological and clinical studies, the concentration of C-reactive protein (CRP) in blood is associated with the risk of coronary heart disease (CHD). However, these studies are limited in high incidence and prevalence area of North-West India. The present case control study investigated the contribution of three relevant CRP single nucleotide polymorphisms: ?717A>G located in the promoter region (rs2794521), +1059G>C on exon2 (rs1800947) and +1444C>T in the 3′ UTR (rs1130864) in 180 angiographically verified CHD cases and 175 control subjects. Minor allele frequencies (G, C and T) of rs2794521, rs1800947 and rs1130864 are observed to be 21.1, 11.7, 29.4 and 11.4, 10.0, 19.7 % in CHD cases and controls respectively. AA genotype of ?717A>G and TT genotype of +1444C>T were significantly associated (P = 0.02 & 0.03 respectively) with the risk of CHD whereas, +1059G and +1444T were found to be strongly related (P = 0.023 & P = 0.008 respectively) with multivariable adjusted CRP levels. AGT Haplotype was significantly associated with the adjusted CRP levels (P < 0.05). Disease association analysis revealed that haplotype AGT influences CHD risk (OR 2.4, 95 % CI 1.23–4.84, P = 0.006) which exacerbates after correcting the confounding effects of risk variables (OR 2.5, 95 % CI 1.27–4.99, P = 0.004). With the global index of Akaike information criterion, it has been observed that the carrying each single unit of this susceptibility haplotype increases CHD risk by a value of 2.41 ± 0.439 (β ± SE) in the recessive mode.     

The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K⁺ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca²⁺ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca²⁺ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.     

Encapsulation of Sorbitan Ester-Based Organogels in Alginate Microparticles

Leaching of the internal apolar phase from the biopolymeric microparticles during storage is a great concern as it undoes the beneficial effects of encapsulation. In this paper, a novel formulation was prepared by encapsulating the sunflower oil-based organogels in alginate microparticles. Salicylic acid and metronidazole were used as the model drugs. The microparticles were prepared by double emulsion methodology. Physico-chemical characterization of the microparticles was done by microscopy, FTIR, XRD, and DSC studies. Oil leaching studies, biocompatibility, mucoadhesivity, in vitro drug release, and the antimicrobial efficiency of the microparticles were also performed. The microparticles were found to be spherical in shape. Gelation of the sunflower oil prevented leaching of the internal phase from the microparticles. Release of drugs from the microparticles followed Fickian kinetics and non-Fickian kinetics in gastric and intestinal environments, respectively. Microparticles showed good antimicrobial activity against both Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. The results suggested that the developed formulations hold promise to carry oils without leakage of the internal phase. Encapsulation of organogels within the microparticles has improved the drug entrapment efficiency and improved characteristics for controlled delivery applications.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0147-2) contains supplementary material, which is available to authorized users.KEY WORDS: alginate, drug delivery, leaching, microparticles, organogels     

Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding,Organization of Active Site Residues and Stereopreferences

Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.