MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3′ UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed.     

Habitat complexity reduces prey vulnerability: An experimental analysis using aquatic insect predators and immature dipteran prey

The effects of alternative prey and structural complexity of habitat on the selection of mosquito larvae by aquatic insect predators were evaluated in the laboratory. The water bugs Anisops bouvieri, Diplonychus (= Sphaerodema) rusticus, and D. annulatus, and the odonate nymphs, Ceriagrion coromandelianum and Brachydiplax chalybea chalybea, selected mosquito larvae based on their abundance relative to chironomid larvae and on the levels of habitat complexity. The effect of one prey species on the other was asymmetrical, as indicated through prey selectivity values. Compared to open habitat, the presence of macrophytes reduced the vulnerability of mosquito larvae while the effect was reverse in the presence of sediments. When both sediment and macrophytes were present in habitats, all the predators except D. annulatus consumed more mosquito larvae than chironomid larvae. The clearance rate, an indicator of predatory efficiency, varied among the predator species and habitat types. The results suggest that the outcome of the interactions between insect predators and mosquito immatures was context-dependent and that it was mediated by the presence of alternative controphic species and the habitat complexity.     

Peptide-based Antibodies against Glutathione-binding Domains Suppress Superoxide Production Mediated by Mitochondrial Complex I

Complex I (NQR) is a critical site of superoxide () production and the major host of redox protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51- and 75-kDa subunits are known to be reversibly S-glutathionylated. Although several glutathionylated domains from NQR 51 and 75 kDa have been identified, their roles in the regulatory functions remain to be explored. To gain further insights into protein S-glutathionylation of complex I, we used two peptides of S-glutathionylated domain (²⁰⁰GAGAYICGEETALIESIEGK²¹⁹ of 51-kDa protein and ³⁶¹VDSDTLCTEEVFPTAGAGTDLR³⁸² of 75-kDa protein) as chimeric epitopes incorporating a “promiscuous” T-cell epitope to generate two polyclonal antibodies, AbGSCA206 and AbGSCB367. Binding of AbGSCA206 and AbGSCB367 inhibited NQR-mediated generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain (²¹SGDTTAPKKTSFGSLKDFDR⁴⁰ of 51-kDa peptide and ¹⁰⁰WNILTNSEKTKKAREGVMEFL¹²⁰ of 75-kDa peptide) were synthesized as chimeric epitopes to generate two polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated generation to a significant level. However, binding of Ab75 inhibited NQR-mediated generation by 35%. None of AbGSCA206, AbGSCB367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the glutathione-binding domain decreased electron leakage from the hydrophilic domain of NQR. When heart tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathionylation was enhanced in post-ischemic myocardium at the NQR 51-kDa subunit, but not at the 75-kDa subunit, indicating that the 51-kDa subunit of flavin subcomplex is more sensitive to oxidative stress resulting from myocardial infarction.     

Understanding mutations and protein stability through tripeptides

A novel methodology to predict the local conformational changes in a protein as a consequence of missense mutations is proposed. A pentapeptide at the locus of mutation plays the dominant role and it is analyzed in terms of tripeptides. A measure for spatial and temporal fluctuations in a pentapeptide is devised and validated. The method does not involve any prior knowledge of structural templates from sequence homology studies. Structural deformations can be predicted with about 70-80% reliability in any protein. Disease causing mutations and benign mutations have been addressed. In particular, p53, retinoblastoma protein and lipoprotein lipase are studied in detail.     

Chinch bug (Hemiptera: Blissidae) mouthpart morphology, probing frequencies, and locations on resistant and susceptible germplasm

Chinch bugs are common pests of many agronomic and horticulturally important crops and turfgrasses. Previous research has indicated that some grasses exhibit resistance to multiple chinch bug species, whereas others are resistant to only one species. The objectives of this research were to document differences in the probing frequencies and locations among Blissus species as well as differences in mouthpart morphology as a first step in understanding the differential responses of grasses to chinch bug feeding. Scanning electron microscopy detected differences in the total lengths of proboscises as well as individual mouthpart segments among the four species studied. Blissus occiduus Barber probed significantly more often on buffalograss, Buchlo? dactyloides (Nuttall) Engelmann, than any other plant material. Probing locations of B. occiduus and Blissus leucopterus leucopterus (Say) were similar on both B. occiduus-resistant and susceptible buffalograsses and KS94 sorghum, Sorghum bicolor (L.) Moench (B. occiduus-resistant, B. l. leucopterus-resistant). However, on 'Wheatland' sorghum (B. occiduus-resistant, B. l. leucopterus-susceptible), stylet tracts of B. l. leucopterus most often terminated in the bundle sheath cells, whereas those of B. occiduus generally terminated in the vascular tissues.     

Validation of the LC-MS/MS method for the quantification of mevalonic acid in human plasma and determination of the matrix effect

A simple, specific, and sufficiently sensitive liquid chromatography-tandem mass spectrometry (negative-ion electrospray ionization) methodology to determine mevalonic acid (MVA) in human plasma is described, and its application to the analysis of rat plasma MVA levels after rosuvastatin administration is demonstrated. The method was validated over the linearity range of 0.5-50.0 ng/ml (r(2) > 0.99) using deuterated MVA as an internal standard. The lower limit of quantification was 0.5 ng/ml. The assay procedure involved the isolation of MVA from plasma samples using solid-phase extraction. Chromatographic separation was achieved on a HyPurity Advance column with a mobile phase consisting of ammonium formate buffer (10 mM, pH 8.0) and acetonitrile (70:30, v/v). Excellent precision and accuracy were observed. MVA and deuterated mevalonolactone were stable in water and plasma under different storage and processing conditions. The recovery observed was low, which was attributable to a significant matrix effect. A significant decrease (30-40%; P < 0.05) was observed in rat plasma MVA levels after rosuvastatin administration.     

Body mass index and chronic energy deficiency of adult males of Central Indian populations

Data on body weight, height, and sitting height from 11,496 adult males, age 18-62 years, belonging to 38 different populations of five major social groups (scheduled tribes, scheduled castes, "other backward castes," general castes, and Muslims) of Central India were taken for our analysis to assess the nutritional status of these groups. Cormic index and body mass index (BMI) were computed, and an analysis of variance (ANOVA) was carried out among different populations as well as among social groups separately on Cormic index and BMI. Shape, size, and generalized distances among the different social groups were computed and dendrograms were drawn. The level of malnutrition is the lowest among the general castes. The opposite is the case with the scheduled castes and scheduled tribes. Comparison of the coefficient of variation shows that there is variation in weight and BMI but that there is no marked variation in the other anthropometric variables. The ANOVA on Cormic index and BMI suggests that the people within a population are more homogeneous than the people between populations. There is a positive but statistically insignificant correlation between Cormic index and BMI. The five social groups differ more in size distance than in shape distance. According to the dendrogram of generalized distance values, the Muslims and the general castes can be grouped into one cluster and the scheduled castes, scheduled tribes, and other backward castes can be grouped into another cluster.     

Flow-activated chloride channels in vascular endothelium. Shear stress sensitivity, desensitization dynamics, and physiological implications

Although activation of outward rectifying Cl(-) channels is one of the fastest responses of endothelial cells (ECs) to shear stress, little is known about these channels. In this study, we used whole-cell patch clamp recordings to characterize the flow-activated Cl(-) current in bovine aortic ECs (BAECs). Application of shear stress induced rapid development of a Cl(-) current that was effectively blocked by the Cl(-) channel antagonist 5-nitro-2-(3-phenopropylamino)benzoic acid (100 microM). The current initiated at a shear stress as low as 0.3 dyne/cm(2), attained its peak within minutes of flow onset, and saturated above 3.5 dynes/cm(2) approximately 2.5-3.5-fold increase over pre-flow levels). The Cl(-) current desensitized slowly in response to sustained flow, and step increases in shear stress elicited increased current only if the shear stress levels were below the 3.5 dynes/cm(2) saturation level. Oscillatory flow with a physiological oscillation frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the Cl(-) current, whereas lower oscillation frequencies led to partial recovery of the current. Nonreversing pulsatile flow, generally considered protective of atherosclerosis, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the Cl(-) current is responsive to shear stress rather than shear rate. Blocking the flow-activated Cl(-) current abolished flow-induced Akt phosphorylation in BAECs, whereas blocking flow-sensitive K(+) currents had no effect, suggesting that flow-activated Cl(-) channels play an important role in regulating EC flow signaling.     

Zinc deficiency (Khaira) as influenced by planting schemes and plant densities in rice

Summary Visual zinc deficiency symptoms were recorded under two planting schemes and at three plant densities of four different duration rice varieties. Increase in plant density resulted in significant reduction in zinc deficiency (Khaira) symptoms. However planting schemes did not show very abrupt differences for the appearance of deficiency symptoms.