Agni's fungi: heat-resistant spores from the Western Ghats, southern India

This study concerns the thermotolerance of spores of mesophilic fungi isolated from a tropical semi-arid habitat subject to dry season fire in the Western Ghats, southern India. Among 25 species of Ascomycota isolated from leaf litter, nine were able to grow after incubation in a drying oven for 2h at 100°C; the spores of two of these species survived 2h incubation at 110°C, and one survived exposure to 115°C for 2h. The range of thermotolerance among mesophilic fungi isolated from the leaf litter was surprising: filamentous fungi from other habitats, including species that colonize scorched vegetation after fires and thermophilic forms occurring in self-heating plant composts, cannot survive even brief exposure to such high temperatures. It is possible that the exceptional heat resistance of the Indian fungi is related to adaptations to surviving fires. Genetic analysis of the physiological mechanisms of heat resistance in these fungi offers prospects for future biotechnological innovations. The discovery of extreme thermotolerance among common saprotrophs shows that this physiological trait may be more widespread than recognized previously, adding to concern about the evolution of opportunistic pathogens on a warmer planet. The fungi in this study are among the most heat-resistant eukaryotes on record and are referred to here as 'Agni's Fungi', after the Hindu God of Fire.     

Substituted aminobenzimidazole pyrimidines as cyclin-dependent kinase inhibitors

A series of aminobenzimidazole-substituted pyrimidines were synthesized and evaluated for biochemical activity against CDK1. A high-speed parallel synthesis approach enabled the identification of a potent lead series having improved potency in the CDK1 assay (IC(50)<10nM). Cell cycle analysis showed that the compounds induced a G2/M block. Docking studies were carried out with a CDK1 homology model, and provide a rationale for the observed activities.     

Time-course of mitochondrial gene expressions in mice brains: implications for mitochondrial dysfunction, oxidative damage, and cytochrome c in aging

The study of aging is critical for a better understanding of many age-related diseases. The free radical theory of aging, one of the prominent aging hypotheses, holds that during aging, increasing reactive oxygen species in mitochondria causes mutations in the mitochondrial DNA and damages mitochondrial components, resulting in senescence. Understanding a mitochondrial gene expression profile and its relationship to mitochondrial function becomes an important step in understanding aging. The objective of the present study was to determine mRNA expression of mitochondrial-encoded genes in brain slices from C57BL6 mice at four ages (2, 12, 18, and 24 months) and to determine how these altered mitochondrial genes influence age-related changes, including oxidative damage and cytochrome c in apoptosis. Using northern blot analysis, in situ hybridization, and immunofluorescence analyses, we analyzed changes in the expression of mitochondrial RNA encoding the mitochondrial genes, oxidative damage marker, 8-hydroxyguanosine (8-OHG), and cytochrome c in brain slices from the cortex of C57BL6 mice at each of the four ages. Our northern blot analysis revealed an increased expression of mitochondrial-encoded genes in complexes I, III, IV, and V of the respiratory chain in 12- and 18-month-old C57BL6 mice compared to 2-month-old mice, suggesting a compensatory mechanism that allows the production of proteins involved in the electron transport chain. In contrast to the up-regulation of mitochondrial genes in 12- and 18-month-old C57BL6 mice, mRNA expression in 24-month-old C57BL6 mice was decreased, suggesting that compensation maintained by the up-regulated genes cannot be sustained and that the down-regulation of expression results in the later stage of aging. Our in situ hybridization analyses of mitochondrial genes from the hippocampus and the cortex revealed that mitochondrial genes were over-expressed, suggesting that these brain areas are critical for mitochondrial functions. Our immunofluorescence analysis of 8-OHG and cytochrome c revealed increased 8-OHG and cytochrome c in 12-month-old C57BL6 mice, suggesting that age-related mitochondrial oxidative damage and apoptosis are associated with mitochondrial dysfunction. Our double-labeling analysis of in situ hybridization of ATPase 6 and our immunofluorescence analysis of 8-OHG suggest that specific neuronal populations undergo oxidative damage. Further, double-labeling analysis of in situ hybridization of ATPase 6 and immunofluorescence analysis of cytochrome c suggest cytochrome c release is related to mitochondrial dysfunction in the aging C57BL6 mouse brain. This study also suggests that these mitochondrial gene expression changes may relate to the role of mitochondrial dysfunction, oxidative damage, and cytochrome c in aging and in age-related diseases such as Alzheimer's disease and Parkinson's disease.     

Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H⁺ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca²⁺ chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca²⁺ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca²⁺ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca²⁺ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.     

Anthocyanins in Cornus alternifolia, Cornus controversa, Cornus kousa and Cornus florida fruits with health benefits

The anthocyanins in native Cornus alternifolia, Cornus controversa, Cornus kousa and Cornus florida were quantified by HPLC and characterized by spectroscopic methods. The analyses of C. alternifolia and C. controversa revealed that both contained , and , respectively. Similarly, C. florida and C. kousa showed identical anthocyanin profiles with major anthocyanins as and cyanidin 3-O-glucoside (6), respectively. The amount of anthocyanins , and in C. alternifolia and C. controversa were 8.21, 8.44 and 0.02 mg; and 7.74, 5.92, and 0.02 mg/g of fresh fruits, respectively. The anthocyanins and in C. kousa and C. florida were 0.02 and 0.16 mg; and 0.62 and 0.03 mg/g fresh fruits, respectively. Anthocyanins and were not studied earlier for their inhibition of lipid peroxidation, cyclooxygenase enzymes (COX-1 and COX-2), and tumor cell proliferation. At 50 microg/mL, anthocyanins and inhibited lipid peroxidation by 71% and 68%, respectively. Similarly, they inhibited COX-1 enzymes by 39% and 49% and COX-2 enzyme by 54% and 48%, respectively, at 100 microg/mL. Anthocyanin displayed 50% growth inhibition (IC(50)) at 21, 25, 50, 60, and 75 microg/mL, against HCT-116 (colon), MCF-7 (breast), NCI-H460 (lung), SF-268 (central nervous system CNS), and AGS (stomach) human tumor cell lines, respectively. Similarly, IC(50) values for anthocyanin were 38, 30, 76, 100, and 100 microg/mL against HCT-116, MCF-7, NCI H460, SF-268, and AGS, respectively. This is the first report of the quantification and biological activities of anthocyanins in C. alternifolia, C. kousa and C. florida in addition to the anthocyanins not previously quantified in C. controversa.     

In silico mining of microsatellites and analysis of genetic diversity among inter‐ and intra‐generic aphids of the subfamily Aphidinae

Nearly 5 000 aphid species damage crops, either by sucking plant sap or as disease‐transmitting vectors. Microsatellites are used for understanding molecular diversity and eco‐geographical relationships among aphid species. Expressed sequence tag (EST)‐microsatellite motifs were identified through an in silico approach using inbuilt simple sequence repeat mining tools in aphid EST dataset. Microsatellite mining revealed one in every five aphid genes as containing a repeat motif, and out of 9 290 EST microsatellites mined from Aphis gossypii Glover and Acyrthosiphon pisum (Harris) (both Hemiptera: Aphididae), 80% were of A and/or T (AT, ATA, AAT, AATA, and ATTT) motifs, and the rest contained G and/or C motifs. All microsatellite sequences were annotated using BLAST. Primers for EST microsatellites were designed using the Primer 3.0 tool. 106 primer pairs of both dinucleotide repeats (DNRs) and trinucleotide repeats (TNRs), representing open reading frames (ORFs) and untranslated regions (UTRs), were synthesized to amplify 15 aphid species belonging to the subfamily Aphidinae, collected from diverse hosts. Four hundred forty‐five polymorphic alleles were amplified. Fifty TNR and 23 DNR microsatellites amplified across the species studied. Polymorphism information content values of microsatellites ranged from 0.23 to 0.91, amplifying 2–16 alleles. Genetic similarity indices were estimated using the ‘NTSYS‐pc’ software package. Unweighted pair group with arithmetic mean and principal component analysis resolved taxonomic relationships of the aphid species studied. The new aphid microsatellites developed will provide valuable information to researchers to study Indian aphid species diversity and genetic relationships.