Cardiomyocyte Antihypertrophic Effect of Adipose Tissue Conditioned Medium from Rats and Its Abrogation by Obesity is Mediated by the Leptin to Adiponectin Ratio

White adipocytes are known to function as endocrine organs by secreting a plethora of bioactive adipokines which can regulate cardiac function including the development of hypertrophy. We determined whether adipose tissue conditioned medium (ATCM) generated from the epididymal regions of normal rats can affect the hypertrophic response of cultured rat ventricular myocytes to endothelin-1 (ET-1) administration. Myocytes were treated with ET-1 (10 nM) for 24 hours in the absence or presence of increasing ATCM concentrations. ATCM supressed the hypertrophic response to ET-1 in a concentration-dependent manner, an effect enhanced by the leptin receptor antagonist and attenuated by an antibody against the adiponectin AdipoR1 receptor. Antihypertrophic effects were also observed with ATCM generated from perirenal-derived adipose tissue. However, this effect was absent in ATCM from adipose tissue harvested from corpulent JCR:LA-cp rats. Detailed analyses of adipokine content in ATCM from normal and corpulent rats revealed no differences in the majority of products assayed, although a significant increase in leptin concentrations concomitant with decreased adiponectin levels was observed, resulting in a 11 fold increase in the leptin to adiponectin ratio in ATCM from JCR:LA-cp. The antihypertrophic effect of ATCM was associated with increased phosphorylation of AMP-activated protein kinase (AMPK), an effect abrogated by the AdipoR1 antibody. Moreover, the antihypertrophic effect of ATCM was mimicked by an AMPK activator. There was no effect of ET-1 on mitogen-activated protein kinase (MAPK) activities 24 hour after its addition either in the presence or absence of ATCM. Our study suggests that adipose tissue from healthy subjects exerts antihypertrophic effects via an adiponectin–dependent pathway which is impaired in obesity, most likely due to adipocyte remodelling resulting in enhanced leptin and reduced adiponectin levels.     

A P4‐ATPase subunit of the Cdc50 family plays a role in iron acquisition and virulence in Cryptococcus neoformans

The pathogenic fungus Cryptococcus neoformans delivers virulence factors such as capsule polysaccharide to the cell surface to cause disease in vertebrate hosts. In this study, we screened for mutants sensitive to the secretion inhibitor brefeldin A to identify secretory pathway components that contribute to virulence. We identified an ortholog of the cell division control protein 50 (Cdc50) family of the noncatalytic subunit of type IV P‐type ATPases (flippases) that establish phospholipid asymmetry in membranes and function in vesicle‐mediated trafficking. We found that a cdc50 mutant in C. neoformans was defective for survival in macrophages, attenuated for virulence in mice and impaired in iron acquisition. The mutant also showed increased sensitivity to drugs associated with phospholipid metabolism (cinnamycin and miltefosine), the antifungal drug fluconazole and curcumin, an iron chelator that accumulates in the endoplasmic reticulum. Cdc50 is expected to function with catalytic subunits of flippases, and we previously documented the involvement of the flippase aminophospholipid translocases (Apt1) in virulence factor delivery. A comparison of phenotypes with mutants defective in genes encoding candidate flippases (designated APT1, APT2, APT3, and APT4) revealed similarities primarily between cdc50 and apt1 suggesting a potential functional interaction. Overall, these results highlight the importance of membrane composition and homeostasis for the ability of C. neoformans to cause disease.     

A novel promoter polymorphism (-71C>T) in KRTHB6 gene in Indian population

We have screened the basal promoter region, of KRTHB6 gene involving CAAT and TATA boxes in randomly selected 125 individuals of Indian origin by PCR-SSCP and DNA sequencing. We observed a novel promoter polymorphism (-71C>T) which could be differentiated by using LweI restriction enzyme. The frequency of -71 C allele, allele A (Accession no AY203963), was observed to be higher ( 0.712) in comparison to -71 T allele, allele B (0.288) (Accession no. AY037552).     

A study of phenotypic correlation with the genotypic status of HTM regions of KRTHB6 and KRTHB1 genes in monilethrix families of Indian origin

We investigated 21 affected individuals in two unrelated monilethrix families of Indian origin and identified point mutation (g.4624G>A) in the HTM motif (exon-7) of the KRTHB6 gene in all the affected members leading to E413K change in this basic keratin. The HTM motif of KRTHB1, however, showed previously unreported two allelic variants, one with three novel variations (SNPs) in cis: g.4421insT (intronic); g.4461T>C (exonic); g.4485A>G (exonic) and second with only intronic variation (SNP) (g.4421insT). Interestingly, the two distinct phenotypes of: localized severe hair defect with beaded appearance confined to the scalp of all the affected members of Family 1 and of generalized unbeaded hair defect of moderate severity in Family 2, segregated in the two families, respectively, correlating with the two separate genotypes for the functionally critical HTM region of KRTHB1 gene in the background of E413K mutation in the KRTHB6 gene. Presence of E413K mutation in the HTM of KRTHB6 gene was not observed in the background of the allelic variant with three SNPs in KRTHB1 gene in homozygous condition in all the affected members of Family 1, affected with a localized but severe form of the disease. However, the same (E413K) mutation existed in the KRTHB6 gene in the background of the allelic variant with three SNPs in the KRTHB1 gene in homozygous condition, consistently in all the affected members of Family 2, where all its affected members showed the segregation of a milder form of the disease. Presence of both E413K mutation in the KRTHB6 and the variations in the KRTHB1 genes were not observed together in randomly selected 150 unaffected controls outside the two affected families. This is also the first report of HTM mutation of KRTHB6 gene in monilethrix cases of Indian origin and the first report of SNPs in the KRTHB1 gene in literature to our knowledge.     

Functional IFNG polymorphism in intron 1 in association with an increased risk to promote sporadic breast cancer

Interferon (IFN)- is an important Th1 cytokine, which plays a role in immune surveillance and anti-tumor activity. A case-control study involving 54 sporadic breast cancer patients and 144 healthy controls was carried out to explore if the genotype variation of a proposed non-specific enhancer element with a dinucleotide (CA)_n repeat in intron 1 has a role in the susceptibility to promote sporadic breast cancer. Genotype analysis carried out by single-strand length polymorphism and confirmed by sequencing showed an increased frequency of (CA)₁₂ allele (P<0.001) and decreased frequencies of (CA)₁₅ (P<0.01) and (CA)_>15 (p<0.001) alleles in sporadic breast cancer patients as compared to controls. Further, in vitro reporter assays for (CA)₁₂ and (CA)₁₅ alleles suggested these to be associated with decreased and increased expressions, respectively, suggesting the (CA)₁₂/(CA)₁₂ background to act as one of the factors that could lead to low production of IFN-. The study concludes that such genetic background for a proposed non-specific enhancer element with (CA)_n repeat within intron 1 of the IFNG gene might put the individuals with this genotype at higher risk to promote the development of sporadic breast cancer due to a resultant compromised immune surveillance.Anjana Saha and Ashish Dhir contributed equally.     

Management of the late blight (Phytophthora infestans) disease of potato in the southern hills of India

Late blight of potato is considered to be the most devastating problem causing severe yield losses in potato worldwide. Among the different management strategies, the use of resistant cultivars is the most viable option, but the non‐availability of enough quantity of quality seed materials of resistant cultivars forces the farmers to grow susceptible cultivars with proper fungicide scheduling. Therefore, in the present study, chemical control using fungicide has been attempted with newer molecules in the susceptible cultivar along with a resistant cultivar as a positive control. All the tested fungicides were found safe, and no phytotoxicity was observed with any chemical at the applied rate. In resistant cultivar, no late blight was appeared in both the years, whereas maximum AUDPC was observed in the untreated control (276.3) and minimum (41.7) in mancozeb‐cymoxanil + mancozeb based scheduling which was found on par with chlorothalonil‐famoxadone + cymoxanil (51.3) and chlorothalonil‐ametoctradin + dimethomorph (53.5) based scheduling. Among the treatments, resistant cultivar, Kufri Girdhari followed by chlorothalonil‐ametoctradin + dimethomorph and mancozeb‐cymoxanil + mancozeb based fungicidal scheduling were proven as the best treatments for both the crop seasons resulting in the highest yield parameters. The disease severity showed a strong negative correlation with the tuber yield of potatoes in both the years. Based on overall observations including BC ratio, it can be concluded that, wherever seed material of resistant cultivar is available farmers should grow the same or else with susceptible cultivars the fungicidal scheduling based on mancozeb‐cymoxanil + mancozeb or chlorothalonil‐ametoctradin + dimethomorph can be followed to obtain the maximum returns with effective management of late blight at the southern hills of India.     

Role of clathrin‐mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life‐threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non‐iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin‐mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin‐containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.     

Comparison of Established Cell Lines at Different Passages by Karyotype and Comparative Genomic Hybridization

Two established cancer cell lines, MCF-7 and Ishikawa, were both obtained directly from a cell repository and through another laboratory. The karyotypes from the two MCF-7 cell lines had up to 83 chromosomes and similarities for chromosomal gain and structural abnormalities. The two Ishikawa cell lines had up to 60 chromosomes with only a missing X as the common chromosome abnormality. CGH studies were performed by co-hybridizing the two Ishikawa or MCF-7 cell lines to normal metaphases. The differences seen between the two MCF-7 cell cultures reflect changes due to passage number and culture conditions. For Ishikawa, DNA polymorphic data and mutation studies suggest that the two cell lines are not derived from the same established tumor cell line. Our study shows the utilization of CGH in comparing cell lines originating from the same specimen. Our study also demonstrates the necessity for periodically evaluating cell lines to confirm their origin.