Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector

Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients, created as a hematologic delivery test system, were transduced. As with the fibroblasts, transduced patient B cell lines demonstrated both endogenous enzyme correction and a small amount of secretion together with uptake by uncorrected cells. These studies demonstrate that endogenous metabolic correction in transduced cells, combined with secretion, may provide a continuous source of corrective material in trans to unmodified patient bystander cells (metabolic cooperativity).     

寻求油气勘探的一致性：一种控制远景圈闭体积不确定性的系统方法

单个成藏层带的勘探结果表明，虽然油气发现规模变化很大，但其分布的某些性质对评价未钻探的远景圈闭有用，例如P99的上限。但这样的信息很少成为未钻探远景圈闭评价的必要组成部分。如果能将其纳入工作流程，历史数据就可以帮助限定与储层特性有关的自然分布，还可帮助控制地质和商业风险。在评价新远景圈闭时，可以计算高值情形的确定性储量体积，但获得这种储量的概率其本身主要是猜测性的；而低体积情形的计算更难控制。因此，概率方法已成处理勘探不确定性的标准方法。但在确定圈闭规模或含油气岩石总体积（hobGRV）时存在一个问题，由盖层位置、储层和流体界面的综合累计概率所确定的分布更为如此，因为在钻探之前，我们没有这些界面的直接数据。遗憾的是，这个问题没有解决办法，所以我们开发了一种质量控制手段，它利用确定性输入参数来检验概率输出结果的真实性。这种手段可以称为实点资源迭代（RPRI），其主要目的是提高体积预测的一致性。RPRI使用客观指标来计算两种确定性情况，据此就可以生成一个完整的油气发现规模分布。然后用简单的统计数据和由历史数据得出的信息对有关结果进行迭代。这种方法的关键在于根据最后闭合等深线（LCC）相对于构造顶点的深度来确定标准hcbGRV。这种方法不但快速、透明和可以重复，而且根据预测真实低值储量体积的经验可以避免过分乐观。其输出结果与概率方法的相似，意味着很容易对比和调整这两种方法。RPRI还可用于为特定的概率输出结果生成图件和储层参数，从而为经济评价和方案规划提供真实的依据。     

Predicting employment status at 2 years' postdischarge from spinal cord injury rehabilitation.

Objectives: This exploratory study examined the extent to which postinjury employment was predictable when patients were followed up 2 years' postdischarge from a specialist Spinal Cord Injury (SCI) Unit. Participants and Design: Seventy-two individuals with SCI for whom there were discharge Functional Independence Measure (FIM) scores, 60 complete sets of data were available for Discriminant Function Analysis. Results: Using a combination of variables assessed prior to or shortly after discharge from rehabilitation, complemented by psychosocial variables assessed at the time of follow-up, moderate classification accuracy was achieved with respect to employment status at the 2-year follow-up period (72%), with the set of predictor variables being more accurate at predicting those subsequently in paid employment (83%) than those not (67%). Conclusions: The main implication of the study results is that nontraditional variables (i.e., variables other than injury and demographic variables), including, particularly, contextual environmental variables such as community integration, access to transport, and social support, are worthy of further research, especially because many of these are amenable to rehabilitation program interventions, and thus may facilitate the attainment of enhanced rates of postdischarge employment among those living with SCI. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

PALYNOLOGICAL AND GEOCHEMICAL ANALYSIS OF CARBONIFEROUS BOREHOLE AND OUTCROP SAMPLES FROM THE ISLE OF MAN

Palynological and geochemical analyses were performed on Namurian (mid-Carboniferous) samples from the Ballavaarkish and Shellag Point boreholes from the north of the Isle of Man, and on Dinantian/Namurian outcrop samples from Black Marble Quarry in the south of the island. The boreholes yielded a rich and diverse (over 60 species) Namurian B-C (Kinderscoutian-Yeadonian) palynomorph assemblage equivalent to the KV/FR Zones of Clayton et al. (1977). Reworked Brigantian-Namurian A palynomorphs of the NC/TK Zones were recorded in the Shellag Point borehole. Offshore seismic data indicates a period of major fault activity in the early Namurian which may support this concept of early Namurian reworking. Amorphous organic matter indicating anoxic/dysoxic depositional conditions together with prasinophyte algae and goniatites suggesting a marine environment were recorded in the Shellag Point borehole. Vitrinite reflectance and spore colouration measurements coupled with Tmax data from Rock-Eval pyrolysis indicate that the Namurian shales from the Shellag Point borehole are currently in the oil window. Rock-Eval data and kerogen analyses indicate that these shales represent moderate to good gas-prone source rocks which have yet to attain full maturity. The Namurian samples from Ballavaarkish are mainly in the oil window or late mature zone and have poor gas potential. Extract chromatography indicates a relatively mature product and the possible onset of hydrocarbon biodegradation, particularly in the Ballavaarkish borehole samples. Outcrop samples from near the top of the succession at Black Marble Quarry underwent thermal alteration as a result of dyke intrusion and subsequent volcanism. Although previously believed to be Brigantian (end-Dinantian) in age based on goniatites, corals and conodonts, the samples have produced a sparse palynological assemblage indicating an apparent age no older than Namurian. The samples are late- to post-mature in terms of hydrocarbon generation and have no remaining source potential. The results indicate that where Namurian sediments are preserved beneath the base-Permian unconformity, for example in the Solway Basin, hydrocarbons are likely to have been generated.     

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.     

Anionic difunctionalization with 1,1-bis(4-t-butyldimethylsiloxyphenyl) ethylene. Synthesis of ω,ω-bis(phenol)-functionalized polystyrene condensation macromonomers

ω,ω-Diphenolpolystyrenes (6) can be synthesized in quantitative yields by reacting poly(styryl)lithium with 1,1-bis(4-t-butyldimethylsiloxyphenyl)ethylene (1), followed by methanol termination and hydrolysis with dilute acid. The initially formed 1,1-bis(4-t-butyldimethylsiloxyphenyl)alkyllithium can be reacted with additional styrene monomer to form a polystyrene internally substituted with two in-chain phenol groups after methanol termination and acid hydrolysis. The diphenol-substituted polystyrene condensation macromonomers have been characterized by end-group titration, size exclusion chromatography, thin-layer chromatography, and ultraviolet-visible, ¹H and ¹³C NMR spectroscopy. Chain-extension reactions of 6 (M_n = 2.6 × 10³g mol^?1) with bis(trichloromethyl)carbonate produced the corresponding comb-type, branched polymer with estimated Mⁿ(SEC, polystyrene standards) = 1.2 × 10⁴g mol^?1 and no detectable residual condensation macromonomer. The second order rate constants for the addition reaction of excess poly(styryl)lithium with 1 and with 1-(4-t-butyldimethylsiloxy-phenyl)-1-phenylethylene (3) have been estimated to be 1.7 × 10^?3M^?1/2S^?1 and 3.2 × 10^?3M^?1/2S^?1 respectively. A sigma value (σ) of ?0.46 has been estimated for the t-butyldimethylsiloxy substituent.     

Quantitation of human cytomegalovirus glycoprotein H gene in cells using competitive PCR and a rapid fluorescence-based detection system

A technique is described for quantitation of the human cytomegalovirus (HCMV) glycoprotein H (gH) gene in cells using a quantitative-competitive polymerase chain reaction (QC-PCR). Two recombinant DNA molecules, differing in size due to a 92-bp deletion within the HCMV gH sequence, were used in co-amplification studies to construct a standard curve from which the copy number of the gH gene present in clinical samples could be interpolated. The use of primers labeled with a fluorescent dye allowed direct detection of the amplified products by measuring the amount of fluorescence emitted by each specific PCR fragment with an automated DNA sequencer coupled to a software program. This system was validated subsequently using bronchoalveolar lavage cells obtained from immunocompromised patients and found to be highly sensitive and reproducible over a range of 5-50,000 HCMV gH copies. This rapid procedure could easily be applied to study the pathogenesis of HCMV infection, identify the patients at high risk of developing HCMV disease, and monitor the effects of antiviral therapy at the molecular level.     

A wide-band high-resolution spectrum analyzer

This paper describes a two-million-channel 40-MHz-bandwidth, digital spectrum analyzer under development at the Jet Propulsion Laboratory. The analyzer system will serve as a prototype processor for the sky survey portion of NASA's Search for Extraterrestrial Intelligence program and for other applications in the Deep Space Network. The analyzer digitizes an analog input, performs a 2(21)-point, Discrete Fourier Transform, accumulates the output power, normalizes the output to remove frequency-dependent gain, and automates simple signal detection algorithms. Due to its built-in frequency-domain processing functions and configuration flexibility, the analyzer is a very powerful tool for real-time signal analysis and detection.     

Polymerization of butadiene using neodymium versatate‐based catalyst systems: preformed catalysts with SiCl4 as halide source

The ternary neodymium versatate (NdV₃)‐based catalyst system, NdV₃/SiCl₄/Al(iso‐Bu)₂H, for the stereospecific polymerization of 1,3‐butadiene (Bd) has been studied at a catalyst concentration of 0.11 mmol Nd per 100 g Bd. The effects of the concentration of SiCl₄ following in situ activation, preformed at 20 °C in the presence and absence of isoprene and the substitution of Al(iso‐Bu)₂H with AlEt₃ as alkylating agent, were established and compared to the performance of NdV₃‐based catalyst systems incorporating ethylaluminium sesquichloride (EASC), diethylaluminium chloride (DEAC) and t‐butyl chloride (t‐BuCl) as chloride sources. Comparable catalytic activity between the control catalysts based on EASC, DEAC and t‐BuCl and the studied NdV₃/SiCl₄/Al(iso‐Bu)₂H system was achieved once the optimum concentration ratios of NdV₃/SiCl₄/Al(iso‐Bu)₂H = 1:1:25 were applied in conjunction with preforming the catalyst components in the presence of isoprene for 72 h at 20 °C. Polybutadiene cis‐1,4 contents were consistently high (about 97 %) for all polymerizations. © 2000 Society of Chemical Industry