Evidence for aromatic ring reduction in the biodegradation pathwayof carboxylated naphthalene by a sulfate reducing consortium

Naphthalene was used as a model compound in order to study the anaerobic pathway of polycyclic aromatic hydrocarbon degradation. Previously we had determined that carboxylation is an initial step for anaerobic metabolism of naphthalene, but no other intermediate metabolites were identified (Zhang & Young 1997). In the present study we further elucidate the pathway with the identification of six novel naphthalene metabolites detected when cultures were fed naphthalene in the presence of its analog 1-fluoronaphthalene. Results from cultures supplemented with either deuterated naphthalene or non-deuterated naphthalene plus [¹³C]bicarbonate confirm that the metabolites originated from naphthalene. Three of these metabolites were identified by comparison with the following standards: 2-naphthoic acid (2-NA), 5,6,7,8-tetrahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. The presence of 5,6,7,8-tetrahydro-2-NA as a metabolite of naphthalene degradation indicates that the first reduction reaction occurs at the unsubstituted ring, rather than the carboxylated ring. The overall results suggest that after the initial carboxylation of naphthalene, 2-NA is sequentially reduced to decahydro-2-naphthoic acid through 5 hydrogenation reactions, each of which eliminated one double bond. Incorporation of deuterium atoms from D₂O into 5,6,7,8-tetrahydro-2-naphthoic acid suggests that water is the proton source for hydrogenation.     

Luminescent α-diimine complexes of ruthenium(II) containing scorpionate ligands

We describe the synthesis, characterization, and reactivity of several Ru(II) complexes of the type cis-L₂Ru(Z)ⁿ⁺, where L is an α-diimine [e.g. 2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen)] ligand and Z is a bis-coordinated scorpionate ligand such as tris-(1-pyrazolyl)methane (HC(pz)₃, PZ=1-pyrazolyl; n=2) or tetrakis-(1-pyrazolyl)borate anion (B(pz)₄ ⁻; n=1). The complexes each exhibit strong visible absorption assigned as a π*(L)←dπ(Ru) metal-to-ligand charge-transfer (MLCT) transition characteristic of the cis-L₂Ru²⁺ kernel. A corresponding MLCT excited state emission is observed in room temperature CH₃CN solution, although emission energies, lifetimes, and quantum yields are reduced relative to Ru(bpy)₃ ²⁺. Electronic spectra and cyclic voltammetry measurements indicate that the relative π-acceptor abilities of the coordinated Z are: Z=(1H-pyrazolyl)₂(pz)₂B(pz)₂<(pyridine)₂<(pz)₂CH(pz). Uncoordinated pz groups of cis-(bpy)₂Ru(pz)₂B(pz)₂ ⁺ can be reacted to form a sterically hindered, localized-valence (K_com33 l mol⁻¹) cis,cis-(bpy)₂Ru^II(pz)₂B(pz)₂Ru^II(bpy)₂ ³⁺ dimer. The dimer properties are interpreted by comparison to the known cis-(bpy)₂Ru^II(pz)₂Ru^II(bpy)₂ ²⁺ analog. The dimer is photoreactive and undergoes an asymmetrical photocleavage in CH₃CN (yielding cis-(bpy)₂Ru^III(pz)₂B(pz)₂ ²⁺ and cis-(bpy)₂Ru^II(CH₃CN)₂ ²⁺), similar to the corresponding thermal reaction observed for the mixed-valence cis-(bpy)₂Ru^II(pz)₂Ru^III(bpy)₂ ³⁺ system.     

Determinants of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Activation by Soluble CD4 and Monoclonal Antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1β, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Genome-Wide Association Studies in an Isolated Founder Population from the Pacific Island of Kosrae

It has been argued that the limited genetic diversity and reduced allelic heterogeneity observed in isolated founder populations facilitates discovery of loci contributing to both Mendelian and complex disease. A strong founder effect, severe isolation, and substantial inbreeding have dramatically reduced genetic diversity in natives from the island of Kosrae, Federated States of Micronesia, who exhibit a high prevalence of obesity and other metabolic disorders. We hypothesized that genetic drift and possibly natural selection on Kosrae might have increased the frequency of previously rare genetic variants with relatively large effects, making these alleles readily detectable in genome-wide association analysis. However, mapping in large, inbred cohorts introduces analytic challenges, as extensive relatedness between subjects violates the assumptions of independence upon which traditional association test statistics are based. We performed genome-wide association analysis for 15 quantitative traits in 2,906 members of the Kosrae population, using novel approaches to manage the extreme relatedness in the sample. As positive controls, we observe association to known loci for plasma cholesterol, triglycerides, and C-reactive protein and to a compelling candidate loci for thyroid stimulating hormone and fasting plasma glucose. We show that our study is well powered to detect common alleles explaining ≥5% phenotypic variance. However, no such large effects were observed with genome-wide significance, arguing that even in such a severely inbred population, common alleles typically have modest effects. Finally, we show that a majority of common variants discovered in Caucasians have indistinguishable effect sizes on Kosrae, despite the major differences in population genetics and environment.     

Polyomavirus large T mutants affected in retinoblastoma protein binding are defective in immortalization.

To clarify the relationship between the various activities of the polyomavirus large T antigen and the contribution of this oncogene to neoplastic transformation, we constructed a series of mutants with small deletions or single-amino-acid substitutions in two separate regions of the protein. These sequences were targeted because they showed considerable similarity to conserved regions 1 and 2 of adenovirus E1A which are thought to be binding sites for the retinoblastoma gene product (pRB). The pRB-binding properties of the large T mutants were assessed with an in vitro coimmunoprecipitation assay. pRB binding was readily detected with wild-type large T, but coprecipitation was completely abolished by as little as a single amino acid substitution (Asp-141----Glu or Glu-146----Asp) in region 2 of the polyomavirus large T antigen. Mutants defective in pRB binding were unable to immortalize primary rat embryo fibroblasts, suggesting that association with pRB is an important component of immortalization mediated by polyomavirus large T. The mutations in region 1 affected pRB binding only marginally, yet some of them severely impaired immortalization, indicating that pRB binding may be essential but not sufficient for immortalization.     

Optimization of a series of quinazolinone-derived antagonists of CXCR3

The evaluation of the CXCR3 antagonist AMG 487 in clinic trials was complicated due to the formation of an active metabolite. In this Letter, we will discuss the further optimization of the quinazolinone series that led to the discovery of compounds devoid of the formation of the active metabolite that was seen with AMG 487. In addition, these compounds also feature increased potency and good pharmacokinetic properties. We will also discuss the efficacy of the lead compound 34 in a mouse model of cellular recruitment induced by bleomycin.     

Iron supplementation promotes in vitro shoot induction and multiplication of <Emphasis Type="Italic">Baptisia australis</Emphasis>

C₄ plants can efficiently accumulate CO₂ in leaves and thus reduce wasteful oxygen fixation by the RuBisCO enzyme. Three C₄ enzymes, namely carbonic anhydrase (CA), phosphoenol pyruvate (PEPC) and pyruvate orthophosphate dikinase (PPDK), were over expressed in Oryza sativa L. ssp. indica var. Khitish under the control of green tissue specific promoters PD_54o, PEPC and PPDK, respectively. Integration of these genes was confirmed by Southern hybridization. The relative expression of PEPC, CA and PPDK were, respectively, 6.75, 6.57 and 3.6-fold higher in transgenic plants compared to wild type plants (control). Photosynthetic efficiency of the transgenic plants increased significantly along with a 12?% increase in grain yield compared to wild type plants. Compared to control plants, transgenic plants also showed phenotypic changes such as increased leaf blade size, root biomass, and plant height and anatomical changes such as greater leaf vein number, bundle sheath cells, and bulliform cells. Our findings indicate that the combined over expression of these three enzymes is an efficient strategy for incorporating beneficial physiological and anatomical features that will enable subsequent yield enhancement in C₃ rice plants.     

Haplotype distribution of the human phenylalanine hydroxylase locus in Scotland and Switzerland.

RFLP haplotypes at the phenylalanine hydroxylase (PAH) locus were determined in 45 nuclear Caucasian families from Switzerland and Scotland. The RFLPs at the PAH locus are highly informative, and prenatal diagnosis is possible in 85% of the families studied. The data were combined with the profiles previously observed in the Danish population, in order to study the variation in RFLP haplotype distribution among European populations. A total of 22 different haplotypes were observed in Denmark, Switzerland, and Scotland. Fifteen and 19 haplotypes are associated with the normal (non-PKU) and with the mutant chromosomes, respectively. The haplotype distribution and the allele frequency of normal chromosomes remain constant between Denmark, Switzerland, and Scotland. However, both the haplotype distribution and allele frequencies of mutant chromosomes show significant variation between the three countries. Our results suggest there may be additional mutations in the PAH gene that cause PKU.     

Large-scale recording of neurons by movable silicon probes in behaving rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design. Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (http://www.neuronexustech.com/; http://www.sbmicrosystems.com/; http://www.acreo.se/) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.