Hexavalent uranium supports growth of Anaeromyxobacter dehalogenans and Geobacter spp. with lower than predicted biomass yields

The stimulation of bacteria capable of reducing soluble U(VI) to sparingly soluble U(IV) is a promising approach for containing U(VI) plumes. Anaeromyxobacter dehalogenans is capable of mediating this activity; however, its ability to couple U(VI) reduction to growth has not been established. Monitoring the increase in 16S rRNA gene copy numbers using quantitative real-time PCR (qPCR) in cultures provided with U(VI) as an electron acceptor demonstrated growth, and 7.7-8.6 x 10(6) cells were produced per mumole of U(VI) reduced. This biomass yield was lower than predicted based on the theoretical free energy changes associated with U(VI)-to-U(IV) reduction. Lower than predicted growth yields with U(VI) as electron acceptor were also determined in cultures of Geobacter lovleyi and Geobacter sulfurreducens suggesting that U(VI) reduction is inefficient or imposes an additional cost to growing cells. These findings have implications for U(VI) bioremediation because Anaeromyxobacter spp. and Geobacter spp. contribute to radionuclide immobilization in contaminated subsurface environments.     

Identification of FAK substrate peptides via colorimetric screening of a one‐bead one‐peptide combinatorial library

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is associated with regulating cellular functions such as cell adhesion and migration and has emerged as an important target for cancer research. Short peptide substrates that are selectively and efficiently phosphorylated by FAK have not been previously identified and tested. Here we report the synthesis and screening of a one‐bead one‐peptide combinatorial library to identify novel substrates for FAK. Using a solid‐phase colorimetric antibody tagging detection platform, the peptide beads phosphorylated by FAK were sequenced via Edman degradation and then validated through radioisotope kinetic studies with [γ‐³²P] ATP to derive Michaelis–Menton constants. The combination of results gathered from both colorimetric and radioisotope kinase assays led to the rational design of a second generation of FAK peptide substrates. Out of all the potential peptide substrates evaluated, the most active was GDYVEFKKK with a K_M = 92 μM and a V_max = 1920 nmol/min/mg. Peptide substrates discovered within this study may be useful diagnostic tools for future kinase investigations and may lead to novel therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles

Aberrant DNA methylation is a common epigenetic alteration found in colorectal adenomas and cancers and plays a role in cancer initiation and progression. Aberrantly methylated DNA loci can also be found infrequently present in normal colon tissue, where they seem to have potential to be used as colorectal cancer (CRC) risk biomarkers. However, detection and precise quantification of the infrequent methylation events seen in normal colon is likely beyond the capability of commonly used PCR technologies. To determine the potential for methylated DNA loci as CRC risk biomarkers, we developed MethyLight droplet digital PCR (ddPCR) assays and compared their performance to the widely used conventional MethyLight PCR. Our analyses demonstrated the capacity of MethyLight ddPCR to detect a single methylated NTRK3 allele from among more than 3125 unmethylated alleles, 25-fold more sensitive than conventional MethyLight PCR. The MethyLight ddPCR assay detected as little as 19 and 38 haploid genome equivalents of methylated EVL and methylated NTRK3, respectively, which far exceeded conventional MethyLight PCR (379 haploid genome equivalents for both genes). When assessing methylated EVL levels in CRC tissue samples, MethyLight ddPCR reduced coefficients of variation (CV) to 6–65% of CVs seen with conventional MethyLight PCR. Importantly, we showed the ability of MethyLight ddPCR to detect infrequently methylated EVL alleles in normal colon mucosa samples that could not be detected by conventional MethyLight PCR. This study suggests that the sensitivity and precision of methylation detection by MethyLight ddPCR enhances the potential of methylated alleles for use as CRC risk biomarkers.     

Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes

The liver is a major insulin‐responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin‐induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin‐induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin‐induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML‐12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin‐induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin‐induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin‐induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin‐induced glucose uptake and glycogenesis in hepatocytes. J. Cell. Biochem. 113: 2064–2076, 2012. © 2012 Wiley Periodicals, Inc.     

Maintenance of redox state and pancreatic beta-cell function: role of leptin and adiponectin

Whereas oxidative stress is linked to cellular damage, reactive oxygen species (ROS) are also believed to be involved in the propagation of signaling pathways. Studies on the role of ROS in pancreatic beta‐cell physiology, in contrast to pathophysiology, have not yet been reported. In this study we investigate the importance of maintaining cellular redox state on pancreatic beta‐cell function and viability, and the effects of leptin and adiponectin on this balance. Experiments were conducted on RINm and MIN6 pancreatic beta‐cells. Leptin (1–100 ng/ml) and adiponectin (1–100 nM) increased ROS accumulation, as was determined by DCFDA fluorescence. Using specific inhibitors, we found that the increase in ROS levels was mediated by NADPH oxidase (Nox), but not by AMP kinase (AMPK) or phosphatidyl inositol 3 kinase (PI3K). Leptin and adiponectin increased beta‐cell number as detected by the XTT method, but did not affect apoptosis, indicating that the increased cell number results from increased proliferation. The adipokines‐induced increase in viability is ROS dependent as this effect was abolished by N‐acetyl‐L‐cysteine (NAC) or PEG‐catalase. In addition, insulin secretion was found to be regulated by alterations in redox state, but not by adipokines. Finally, the effects of the various treatments on activity and mRNA expression of several antioxidant enzymes were determined. Both leptin and adiponectin reduced mRNA levels of superoxide dismutase (SOD)1. Adiponectin also decreased SOD activity and increased catalase and glutathione peroxidase (GPx) activities in the presence of H₂O₂. The results of this study show that leptin and adiponectin, by inducing a physiological increase in ROS levels, may be positive regulators of beta‐cell mass. J. Cell. Biochem. 113: 1966–1976, 2012. © 2012 Wiley Periodicals, Inc.     

Love the one you're with: proximity determines paternity success in the barnacle Tetraclita rubescens

A species' mating system sets limits on the strength of sexual selection. Sexual selection is widespread in dioecious species, but is less well documented in hermaphrodites, and may be less important. We used four highly polymorphic microsatellite markers to assign paternity to broods of the hermaphroditic eastern Pacific volcano barnacle Tetraclita rubescens. These data were used to describe the species' mating system and to examine factors affecting male reproductive success. Tetraclita can sire broods over distances of 11.2 cm, but proximity to the sperm recipient had a highly significant effect on the probability of siring success. There was no effect of body size or the mass of male reproductive tissues on siring success. Broods showed relatively low frequencies of multiple paternity; even at high densities, 75% of broods had only one father. High frequencies of single‐paternity broods imply either that this species does not compete via sperm displacement, or that sperm displacement is extremely effective, potentially explaining the lack of a positive relationship between male investment and paternity. In addition, there was low variance in siring success among individuals, suggesting a lack of strong sexual selection on male traits. Low variance among sires and the strong effect of proximity are probably driven by the unusual biology of a sessile copulating species.     

Amino acid changes in Drosophila alphaPS2betaPS integrins that affect ligand affinity

We developed a ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2betaPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavbeta3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2betaPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the betaPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the betaPS I domain (betaPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2betaPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin beta subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the betaPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because betaPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.     

Effects of hydrophilic and hydrophobic kaolin-based particle films on pea aphid (Homoptera: Aphididae) and its entomopathogen Pandora neoaphidis (Entomophthorales: Entomophthoraceae)

Hydrophobic and hydrophilic kaolin-based particle films are effective for control of insect pests in certain agricultural crops. How these products interact with potential biological control agents is not well documented. This study was conducted to evaluate the effects of the hydrophobic (M96-018) and hydrophilic (Surround WP) kaolin-based particle films (Engelhard Corporation, Iselin, NJ) on pea aphid, Acyrthosiphon pisum (Harris), on peas (Pisum spp.), and on the fungal aphid pathogen Pandora neoaphidis (Remaudière and Hennebert) Humber. Over two field seasons (2001 and 2002) in northern Idaho, applications of M96-018 significantly reduced the rate of pea aphid increase on pea, but Surround WP, tested only in 2001, did not reduce aphid population growth rate. Neither particle film treatment was as effective as a standard application of esfenvalerate (DuPont Asana). In the laboratory, particle films suppressed pea aphid populations by up to 30%. M96-018 seemed to have some repellent activity based on aphid distributions after treating plants. When applied along with P. neoaphidis conidia, M96-018 but not Surround WP caused higher percentage of infection mortality of pea aphids by P. neoaphidis than occurred on controls treated only with P. neoaphidis conidia. P. neoaphidis conidia deposited on glass slides coated with M96-018, produced more germ tubes and secondary conidia than those deposited on untreated glass slides or slides treated with Surround WP. This result suggests that greater infection of pea aphids on plants treated with M96-018 is in part a result of a direct enhancement of fungal germination by the particle film.     

UV resonance Raman investigation of a 3(10)-helical peptide reveals a rough energy landscape

We used UVRRS at 194 and 204 nm excitation to examine the backbone conformation of a 13-residue polypeptide (gp41(659-671)) that has been shown by NMR to predominantly fold into a 3(10)-helix. Examination of the conformation sensitive AmIII(3) region indicates the peptide has significant populations of beta-turn, PPII, 3(10)-helix, and pi-helix-like conformations but little alpha-helix. We estimate that at 1 degree C on average six of the 12 peptide bonds are in folded conformations (predominantly 3(10)- and pi-helix), while the other six are in unfolded (beta-turn/PPII) conformations. The folded and unfolded populations do not change significantly as the temperature is increased from 1 to 60 degrees C, suggesting a unique energy landscape where the folded and unfolded conformations are essentially degenerate in energy and exhibit identical temperature dependences.