Detection of reverse transcriptase activity by enzyme-linked immunosorbent assay in human immunodeficiency virus type 1.

An enzyme-linked immunosorbent assay (ELISA) using biotin-labelled oligo-dT primer and digoxigenin (Dig)-dUTP was designed to measure the reverse transcriptase (RT) activity of human immunodeficiency virus type 1 (HIV-1). The ELISA system involves the selective detection step of a newly synthesized cDNA by two specific bindings, biotin-streptavidin binding and alkaline phosphatase (AP)-conjugated anti-Dig-Dig binding, and the enzymatic amplification step to increase coloring generated by AP. This method was used to measure the activity of RT in the culture supernatants of peripheral leukocytes obtained from four anti-HIV-1-positive persons cocultivated with those from four anti-HIV-1-negative persons. RT activity was detected in all of four anti-HIV-1-positive culture supernatants but not in those cultivated with anti-HIV-1-negative supernatants alone. Thus, our improved ELISA for detection of HIV-1 appears to be sensitive enough and useful for routine laboratory work. This non-radioactive method will also be useful for detecting other retroviruses and for screening of RT inhibitors.</p

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

High performance global exploration of isomers and isomerization channels on quantum chemical potential energy surface of H5C2NO2

High performance global exploration of isomers and isomerization channels on the quantum chemical potential energy surface (PES) is performed for H5C2NO2 by using the scaled hypersphere search‐anharmonic downward distortion following (SHS‐ADDF) method. A multi‐node operation, NeoGRRM, has achieved high performance exploration calculations for the large system by submitting SHS‐ADDF sub‐jobs into many cores in parallel and unifying the results of sub‐jobs into the total lists of the main‐job. Global exploration of equilibrium (EQ) and transition‐state structures at the level of B3LYP/6‐31G(d) gave 3210 EQs and 23278 TSs. Nine compounds were found in the low energy regions of 0–100 kJ/mol; the lowest energy compound is N‐methylcarbamic acid, the second is methyl carbamate, and the third is glycine (the most fundamental amino acid). Interconversion pathways between the conformers of each of the low energy compounds were surveyed. Isomerization channels around glycine were explored in detail. The lowest energy barriers around some of the EQs turned to be negative after zero‐point energy corrections. This indicates that those structures cannot exist as independent structures because they spontaneously collapse into more stable structures. The global PES search showed various interesting dissociating channels which indicate synthon reaction pathways in the reverse directions

Simulation code for estimating external gamma-ray doses from a radioactive plume and contaminated ground using a local-scale atmospheric dispersion model.

In this study, we developed a simulation code powered by lattice dose-response functions (hereinafter SIBYL), which helps in the quick and accurate estimation of external gamma-ray doses emitted from a radioactive plume and contaminated ground. SIBYL couples with atmospheric dispersion models and calculates gamma-ray dose distributions inside a target area based on a map of activity concentrations using pre-evaluated dose-response functions. Moreover, SIBYL considers radiation shielding due to obstructions such as buildings. To examine the reliability of SIBYL, we investigated five typical cases for steady-state and unsteady-state plume dispersions by coupling the Gaussian plume model and the local-scale high-resolution atmospheric dispersion model using large eddy simulation. The results of this coupled model were compared with those of full Monte Carlo simulations using the particle and heavy-ion transport code system (PHITS). The dose-distribution maps calculated using SIBYL differed by up to 10% from those calculated using PHITS in most target locations. The exceptions were locations far from the radioactive contamination and those behind the intricate structures of building arrays. In addition, SIBYL's computation time using 96 parallel processing elements was several tens of minutes even for the most computationally expensive tasks of this study. The computation using SIBYL was approximately 100 times faster than the same calculation using PHITS under the same computation conditions. From the results of the case studies, we concluded that SIBYL can estimate a ground-level dose-distribution map within one hour with accuracy that is comparable to that of the full Monte Carlo simulation

Targeted inactivation of synaptic HRG4 (UNC119) causes dysfunction in the distal photoreceptor and slow retinal degeneration, revealing a new function

HRG4 (UNC119) is a photoreceptor protein predominantly localized to the photoreceptor synapses and to the inner segments to a lesser degree. A heterozygous truncation mutation in HRG4 was found in a patient with late onset cone–rod dystrophy, and a transgenic (TG) mouse expressing the identical mutant protein developed late onset retinal degeneration, confirming the pathogenic potential of HRG4. Recently, the dominant negative pathogenic mechanism in the TG model was shown to involve increased affinity of the truncated mutant HRG4 for its target, ARL2, which leads to a delayed decrease in its downstream target, mitochondrial ANT1, mitochondrial stress, synaptic degeneration, trans-synaptic degeneration, and whole photoreceptor degeneration by apoptosis. In this study, the mouse HRG4 (MRG4) gene was cloned and targeted to construct a knock-out (KO) mouse model of HRG4 in order to study the effects of completely inactivating this protein. The KO model was examined by genomic Southern blotting, Western blotting, immunofluorescence, funduscopy, LM and EM histopathology, ERG, and TUNEL analyses. The KO model developed a slowly progressive retinal degeneration, characterized by mottling in the fundus, mild thinning of the photoreceptor layer, and increase in apoptosis as early as 6 months, dramatic acceleration at ∼17 months, and virtual obliteration of the photoreceptors by 20 months. When compared to retinal degeneration in the TG model, significant differences existed in the KO consisting of more severe and early photoreceptor death without evidence of early synaptic and trans-synaptic degeneration as seen in the TG, confirmed by LM and EM histopathology, ERG, and Western blotting of synaptic proteins. The results indicated a dysfunction in the KO outside the synapses in the distal end of photoreceptors where MRG4 is also localized. Differences in the phenotypes of retinal degeneration in the KO and TG models reflect a dysfunction in the two opposite ends of photoreceptors, i.e., the distal inner/outer segments and proximal synapses, respectively, indicating a second function of MRG4 in the distal photoreceptor and dual functionality of MRG4. Thus, inactivation of MRG4 by gene targeting resulted in a retinal degeneration phenotype quite different from that previously seen in the TG, attesting to the multiplicity of MRG4 function, in addition to the importance of this protein for normal retinal function. These models will be useful in elucidating the functions of HRG4/MRG4 and the mechanism of slow retinal degeneration

Identification of Key Uric Acid Synthesis Pathway in a Unique Mutant Silkworm <i>Bombyx mori</i> Model of Parkinson’s Disease

<div><p>Plasma uric acid (UA) levels decrease following clinical progression and stage development of Parkinson’s disease (PD). However, the molecular mechanisms underlying decreases in plasma UA levels remain unclear, and the potential to apply mutagenesis to a PD model has not previously been discovered. We identified a unique mutant of the silkworm <i>Bombyx mori</i> (<i>B.mori</i>) <i>op</i>. Initially, we investigated the causality of the phenotypic “<i>op”</i> by microarray analysis using our constructed KAIKO functional annotation pipeline. Consequently, we found a novel UA synthesis-modulating pathway, from DJ-1 to xanthine oxidase, and established methods for large-scale analysis of gene expression in <i>B. mori</i>. We found that the mRNA levels of genes in this pathway were significantly lower in <i>B. mori op</i> mutants, indicating that downstream events in the signal transduction cascade might be prevented. Additionally, levels of <i>B.mori</i> tyrosine hydroxylase (TH) and DJ-1 mRNA were significantly lower in the brain of <i>B. mori op</i> mutants. UA content was significantly lower in the <i>B. mori op</i> mutant tissues and hemolymph. The possibility that the <i>B. mori op</i> mutant might be due to loss of DJ-1 function was supported by the observed vulnerability to oxidative stress. These results suggest that UA synthesis, transport, elimination and accumulation are decreased by environmental oxidative stress in the <i>B. mori op</i> mutant. In the case of <i>B. mori op</i> mutants, the relatively low availability of UA appears to be due both to the oxidation of DJ-1 and to its expenditure to mitigate the effects of environmental oxidative stress. Our findings are expected to provide information needed to elucidate the molecular mechanism of decreased plasma UA levels in the clinical stage progression of PD.</p></div