A sensitive and selective liquid chromatography/tandem mass spectrometry method for determination of MLN8237 in human plasma

We describe a selective and a highly sensitive high-performance liquid chromatography–electron spray ionization-collision induced dissociation-tandem mass spectrometry (HPLC–ESI-CID-MS/MS) assay for the Aurora A kinase inhibitor MLN8237 in human plasma. The intra-day precision based on the standard deviation of replicates of quality control samples ranged from 0.2 to 4% and with accuracy ranging from 96 to 102%. The inter-day precision ranged from 0.5 to 7% and the accuracy ranged from 93 to 105%. Stability studies showed that MLN8237 was stable both during the expected conditions for sample preparation and storage. The lower limit of quantification for MLN8237 was 5 ng/mL. The analytical method showed excellent sensitivity, precision, and accuracy. This method is robust and is being successfully employed in a Children's Oncology Group Phase 1 Consortium study of MLN8237 in children with cancer.     

Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.The ability to discriminate between bovine and other sources of fecal contamination is necessary for the accurate evaluation of human health risks associated with agricultural runoff and focused water quality management to make waters safe for human use. Many methods have been proposed to identify bovine fecal pollution using a variety of different microbiology and molecular techniques. One of the most widely used approaches utilizes a PCR to amplify a gene target that is specifically found in a host population. Currently, there are numerous PCR-based assays for the detection and/or quantitative assessment of bovine fecal pollution available for microbial source-tracking (MST) applications (1, 5-7, 11, 14, 17, 18, 21, 23). These assays target genes ranging from mitochondrial DNA to ribosomal rRNA to other functional genes involved in microorganism-host interactions.The majority of the reported bovine-associated PCR assays target 16S rRNA genes from the order Bacteroidales. This bacterial group constitutes a large proportion of the normal gut microbiota of most animals, including bovines (28), and contains subpopulations closely associated with other animal hosts such as swine, horse, and human (1, 3, 6, 18, 24). Host-associated PCR-based assays targeting Bacteroidales genetic markers have been used to investigate the sources and levels of fecal pollution at a number of beaches and inland watersheds, with variable levels of success (10, 13, 22, 27). Researchers have postulated that differences in host animal age, health, diet, and geographic location may influence bacterial community structures in the bovine gastrointestinal tract (2, 9, 26). Without a priori knowledge of the potential representational bias introduced by such factors, it may be difficult to use these assays with confidence as indicators of bovine fecal pollution.Assay specificity and sensitivity and the prevalence and abundance of genetic marker determinations are typically estimated from the systematic testing of a collection of reference fecal sources collected from known animal sources. However, the characterization of assay performance has been limited, in most cases, to animal sources originating from a particular geographic region or industry, such as dairy or beef. The determination of assay performance across a range of different host populations is essential as the field moves toward the implementation of PCR-based host-associated fecal pollution assessment approaches.We report a performance study of seven PCR and quantitative PCR (qPCR) assays targeting Bacteroidales genes reported to be associated with either ruminant (e.g., bovine, goat, sheep, deer, and others) or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations. Assay specificity was determined by testing 175 fecal DNA extracts from 24 different animal species. For qPCR assays, the abundance of each genetic marker was measured within each bovine population and compared to quantities of Bacteroidales 16S rRNA genetic markers. These analyses indicated large discrepancies in assay performance across different bovine populations.     

Effect of Organic Selenium Supplementation on Growth, Se Uptake, and Nutrient Utilization in Guinea Pigs

Forty weaned male guinea pigs (Cavia porcellus) of 152.6?±?7.96 g mean body weight were divided into four equal groups and fed a common basal diet comprised of 25% ground cowpea (Vigna unguiculata) hay, 30% ground maize (Zea mays) grain, 22% ground gram (Cicer arietinum) grain, 9.5% deoiled rice (Oryza sativa) bran, 6% soybean (Glycine max) meal, 6% fish meal, 1.5% mineral mixture (without Se), and ascorbic acid at 200 mg/kg to meet their nutrient requirements along with 0, 0.1, 0.2, and 0.3 ppm of organic selenium (Se) in groups I, II, III, and IV, respectively. Experimental feeding lasted for a period of 10 weeks, during which, daily feed intake and weekly body weights were recorded. Intake and digestibility of dry matter, organic matter, ether extract, crude fiber, and nitrogen-free extract as well as uptake of calcium and phosphorus were similar (P?>?0.05) among the four groups. Feed:gain ratio was also similar (P?>?0.05) in the four groups. However, digestibility of crude protein was significantly (P?<?0.001) higher in group II supplemented with 0.1 ppm organic Se as compared to other three group. Intake and absorption of Se was significantly (P?<?0.001) higher in all the Se supplemented groups as compared to control group. Average daily gain (ADG) was significantly (P?<?0.05) higher in group II (3.16 g/day) and III (3.38 g/day) as compared to group I (2.88 g/day). However, ADG in group IV (supplemented 0.3 ppm organic Se) was significantly (P?<?0.05) lower (2.83 g/day) than group II and III, but comparable (P?>?0.05) to group I. Findings of the present experiment suggests that Se requirements of guinea pigs are ≥0.2 ppm, as supplementation of 0.1 ppm organic Se in the diet (having 0.1 ppm Se) not only enhanced their growth rate but also improved the protein utilization.     

A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.     

ATM and the catalytic subunit of DNA-dependent protein kinase activate NF-kappaB through a common MEK/extracellular signal-regulated kinase/p90(rsk) signaling pathway in response to distinct forms of DNA damage

We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor kappaB (NF-kappaB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90(rsk) signaling cascade in a p53-independent fashion. In turn, p90(rsk) interacts with the IkappaB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-kappaB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-kappaB pathway that opposes the apoptotic response following DNA damage.     

Structural basis of T-cell specificity and activation by the bacterial superantigen TSST-1

Superantigens (SAGs) bind simultaneously to major histocompatibility complex (MHC) and T-cell receptor (TCR) molecules, resulting in the massive release of inflammatory cytokines that can lead to toxic shock syndrome (TSS) and death. A major causative agent of TSS is toxic shock syndrome toxin-1 (TSST-1), which is unique relative to other bacterial SAGs owing to its structural divergence and its stringent TCR specificity. Here, we report the crystal structure of TSST-1 in complex with an affinity-matured variant of its wild-type TCR ligand, human T-cell receptor beta chain variable domain 2.1. From this structure and a model of the wild-type complex, we show that TSST-1 engages TCR ligands in a markedly different way than do other SAGs. We provide a structural basis for the high TCR specificity of TSST-1 and present a model of the TSST-1-dependent MHC-SAG-TCR T-cell signaling complex that is structurally and energetically unique relative to those formed by other SAGs. Our data also suggest that protein plasticity plays an exceptionally significant role in this affinity maturation process that results in more than a 3000-fold increase in affinity.     

Differential induction of three pathogenesis-related genes, PR10, PR1b and PR5 by the ethylene generator ethephon under light and dark in rice (Oryza sativa L.) seedlings

Ethylene has been shown to be involved in triggering pathogenesis-related (PR) gene expression mainly in dicotyledonous species; however, less attention has been devoted identifying and characterizing PR genes in rice (Oryza sativa L.), a monocot and a model of cereal crop genera. Here, we demonstrate that ethylene induces at least three important rice PR genes, the PR10, PR1 basic (PR1b), and PR5 genes in rice (cv. Nipponbare) seedling leaf, upon treatment with the ethylene generator, ethephon (ET), in a light-, time- and dose-dependent manner. Induction of these PR genes was partially blocked by cycloheximide (CHX), a eukaryotic cytoplasmic protein synthesis inhibitor, which indicates an involvement of cytoplasmic de novo protein synthesis in their induction. These results clearly indicate a dynamic role for ethylene in PR genes induction in rice.     

Extent and nature of contacts between protein molecules in crystal lattices and between subunits of protein oligomers

A survey was compiled of several characteristics of the intersubunit contacts in 58 oligomeric proteins, and of the intermolecular contacts in the lattice for 223 protein crystal structures. The total number of atoms in contact and the secondary structure elements involved are similar in the two types of interfaces. Crystal contact patches are frequently smaller than patches involved in oligomer interfaces. Crystal contacts result from more numerous interactions by polar residues, compared with a tendency toward nonpolar amino acids at oligomer interfaces. Arginine is the only amino acid prominent in both types of interfaces. Potentials of mean force for residue–residue contacts at both crystal and oligomer interfaces were derived from comparison of the number of observed residue–residue interactions with the number expected by mass action. They show that hydrophobic interactions at oligomer interfaces favor aromatic amino acids and methionine over aliphatic amino acids; and that crystal contacts form in such a way as to avoid inclusion of hydrophobic interactions. They also suggest that complex salt bridges with certain amino acid compositions might be important in oligomer formation. For a protein that is recalcitrant to crystallization, substitution of lysine residues with arginine or glutamine is a recommended strategy. Proteins 28:494–514, 1997. © 1997 Wiley-Liss, Inc.     

An essential role for Grk2 in Hedgehog signalling downstream of Smoothened

The G‐protein‐coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock‐down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP‐dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase‐dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C‐terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho‐null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.