PHA synthase from chromatium vinosum: cysteine 149 is involved in covalent catalysis

Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His)6-tagged construct. The PhaC-(His)6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His)6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His)6 to PhaC-(His)6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ([3H]-sT-CoA). After Sephadex G50 chromatography, the synthase contained approximately 0.25 equiv of the labile label per PhaC. Incubation of [3H]-sT-synthase with HB-CoA resulted in production of [3H]-polymer. Digestion of [3H]-sT-synthase with trypsin and HPLC analysis resulted in isolation of three labeled peptides. Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [3H]-sT while the other two contained, in addition to the [3H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.     

Bipolar diffusion charging characteristics of single-wall carbon nanotube aerosol particles

Bipolar diffusion charging characteristics of airborne single-wall carbon nanotube (SWCNT) agglomerates were investigated in the mobility diameter range of 100–1000 nm. Neutral fractions of three types of SWCNT aerosols following bipolar charge equilibrium in a radioactive source were experimentally measured to infer their electrical charging characteristics. Significant deviation from Boltzmann and Fuchs stationary charge equilibrium was observed, with neutral fractions of SWCNT particles lower by 30–53% compared to that of spherical particles of the same mobility. Particles with mobility diameter larger than 400 nm showed high electrical charging efficiencies compared to that of mobility-equivalent spherical particles. Higher charging efficiencies of SWCNT particles were attributed to their higher electrical capacitance resulting from complex nonspherical morphologies. Numerical calculations using idealized fiber geometries confirmed the qualitative trend in the experimental data. The electrical capacitance of nanotubes particles deduced from experimentally measured neutral fractions were also found to be higher by a factor ranging from 1.6 to 4.6 compared to that of mobility-equivalent spherical particles, indicating high charge carrying capacity. The charging-equivalent diameters of nanotube particles were computed and were found to be higher than their mobility diameter by a factor of 2.85–4.34.     

Changes in the pectic fraction of bush butter (Dacryodes edulis (G Don) HJ Lam) fruit pulp during ripening

CDTA‐soluble polysaccharides were extracted from cell wall material (prepared as alcohol‐insoluble solids) of bush butter fruit endocarp tissue at three stages of ripeness. The amount of soluble pectins remained constant but they underwent a gradual depolymerisation during ripening. The CDTA extracts were fractionated by anion exchange and the subfractions were analysed for their sugar composition and molecular weight distribution. For all degrees of ripeness the extracts were composed of three minor peaks and two major peaks. The minor peaks appeared to be composed of xyloglucan and mannan‐type polymers for the first peak and arabinogalactan‐type polymers for the other two peaks. The two main peaks were retained on the column. The first was exclusively composed of homogalacturonan polymers and the second contained principally highly branched rhamnogalacturonan polymers. During ripening, both homogalacturonan and rhamnogalacturonan populations were modified. Modifications in the rhamnogalacturonan fraction were principally marked by the accumulation of low‐molecular‐weight rhamnoglacturonan polymers in the course of ripening. © 2001 Society of Chemical Industry     

Estimates of relative binding free energies for HIV protease inhibitors using different levels of approximations

Although the free energy perturbation approach is a rigorous method for estimating the relative binding free energy between an enzyme and its inhibitors, it is computationally expensive. This paper examines the accuracy at different levels of approximations, following the series expansion of free energy derived by Aqvist et al. Level-0 calculates only the enzyme-inhibitor interaction energy at the minimum energy configuration without solvent. In Level-0MD, the inhibitor configurations are sampled by molecular dynamics. These levels assume that the second- and higher order terms in the series expansion can be neglected and that the interaction energies in the bound and unbound states are equal. Level-1 does not assume equal interaction energies in the bound and unbound states. Level-1S includes the solvent contribution but both enzyme and inhibitor are fixed. In Level-1SMD, the inhibitor configurations are sampled by molecular dynamics. Level- 2SMD retains the second-order term. We chose seven HIV-1 protease inhibitors for study: A77003, A76889, A76928, A78791, A74704, JG365 and MVT101. Level-0 and Level-0MD were found to give essentially the same relative interaction energies by using the AMBER force field, suggesting that fixing atomic positions may be a good approximation in some cases. However, as expected, Level-0 or Level-0MD gave poor predictions for the relative binding free energies between hydrophobic inhibitors (e.g. A77003) and more hydrophilic inhibitors (e.g. JG365). Level-1SMD produced a much better correlation between calculated and experimental results. Inclusion of the second-order term did not improve the accuracy.      

A full genome scan for late onset Alzheimer's disease

We have genotyped 292 affected sibling pairs (ASPs) with Alzheimer's disease (AD) according to NINCDS-ADRDA diagnostic criteria and with onset ages of >/=65 years using 237 microsatellite markers separated by an average distance of 16.3 cM. Data were analysed by SPLINK and MAPMAKER/SIBS on the whole sample of 292 ASPs and subsets of 162 ASPs where both members possessed an apolipoprotein E (APOE)straightepsilon4 allele and 63 pairs where neither possessed anstraightepsilon4 allele. Sixteen peaks with a multipoint lod score (MLS) >1 either in the whole sample, the straightepsilon4-positive or -negative subgroups were observed on chromosomes 1 (two peaks), 2, 5, 6, 9 (two peaks), 10 (two peaks), 12, 13, 14, 19, 21 and X (two peaks). Simulation studies revealed that these findings exceeded those expected by chance, although many are likely to be false positives. The highest lod scores on chromosomes 1 (MLS 2.67), 9 (MLS 2.38), 10 (MLS 2.27) and 19 (MLS 1.79) fulfilLander and Kruglyak's definition of 'suggestive' in that they would be expected to occur by chance once or less per genome scan. Several other peaks were only marginally less significant than this, in particular those on chromosomes 14 (MLS 2.16), 5 (MLS 2.00), 12, close to alpha2-macroglobulin (MLS 1.91), and 21, close to amyloid precursor protein (MLS 1.77). This is the largest genome scan to date in AD and shows for the first time that this is a genetically complex disorder involving several, perhaps many, genes in addition to APOE. Moreover, our data will be of interest to those hoping to identify positional candidate genes using information emerging from neurobiological studies of AD.     

Development of feeding practices during the first 5 years of life

OBJECTIVE: To understand the transition from breast-and bottle-feeding to solid-feeding and factors that might affect the duration of breast- and bottle-feeding. DESIGN: Cohort followed up from birth with relatively well-educated, middle-class parents. SETTING: Community sample recruited from 3 suburban newborn nurseries (a teaching hospital, community hospital, and large health maintenance organization). PARTICIPANTS: One hundred ninety-one healthy full-term infants. MEASURES: Assessment of feeding practices through the ages of complete weaning from breast- and bottle-feeding. RESULTS: More than 90% of participants breast-fed for at least 2 weeks. Infants of older mothers were weaned from the breast later than infants of younger mothers. First-born infants were weaned from the breast earlier than later-born infants. Eighty-four percent of infants bottle-fed at some time during the first year of life. More than 40% of the cohort was still receiving bottles at 24 months of age, 16% at 36 months, and 8% at 48 months. The duration of breast- and bottle-feeding was related to maternal work status; mothers who returned to work during the first 3 months postpartum weaned sooner from the breast and later from the bottle than women who returned to work after 3 months postpartum. CONCLUSIONS: The frequency of late bottle-weaning in this well-educated, middle-class cohort was unexpected and was related to the timing of the mother's return to work. The impact of prolonged bottle-feeding on later growth and adiposity deserves further investigation.     

An optical method of measuring the temperature in a fluidised bed combustor

The paper analyses the dynamic aspects of the temperature field in a fluidised bed of solids particles (e.g., sand) in which a gaseous fuel is being burned. Such a hot bed emits electromagnetic radiation within the visible range and this can be recorded using a digital video camera. This fact has been used to develop a method for measuring the bed’s temperature in the line of sight, through the quartz sides of the reactor. A solid probe is only used for calibration.Video recordings were obtained covering different regions of the bed over three wavelength bands, red, green and blue. In the course of an experiment, the mean temperature of the bed, measured with thermocouples, was raised from ambient to 1300 K, at a rate of ∼ 1 K/s. The data collected were used for calibration, with the brightness of individual pixels converted to a temperature scale. The calibration can then be used to investigate the dynamic temperature distribution within the field of view, in individual elements of the bed. This can also help the study of heat transfer in the bed, its distribution and dissipation.Using this method, it is possible to make direct observations of the intermittent combustion of gaseous fuels in a bubbling fluidised bed. The results provide direct proof that the temperature gradients observed within such beds are associated with exothermic processes within fast moving bubbles. The method could be adapted to studying, e.g., the combustion of solid fuels.     

Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas

The MET protooncogene encodes a transmembrane tyrosine kinase identified as the receptor of a polypeptide known as hepatocyte growth factor/scatter factor. We performed PCR-based single-strand conformational polymorphism and sequencing analysis of the tyrosine kinase domain of the MET gene (exon 15-19) in 75 primary liver cancers. Three missense mutations were detected exclusively in 10 childhood hepatocellular carcinomas (HCCs), while no mutations were detected in 16 adult HCCs, 21 cholangiocarcinomas, or 28 hepatoblastomas. The extremely short incubation period from hepatitis B virus infection to the genesis of childhood HCC as compared with the adult HCC suggests that there may be an additional mechanism that accelerates the carcinogenesis of childhood HCC. Our results indicate that mutations of the tyrosine kinase domain of the MET gene may be involved in the acceleration of the carcinogenesis in childhood HCC.     

前座驾驶员一采用34.8GOPS图像识别引擎的汽车导航芯片

自己的汽车配备再多的安全保护都不为过!图像识别处理器将会被用做提高车辆和乘员的安全.但是,就像人类在学会跑之前必须学会爬和走一样,图像识别引擎目前肩负的任务是为泊车和导航提供辅助信息.     

Nanodiamonds with novel properties: A biological study

This paper discusses the prospects for using modified detonation nanodiamonds with novel properties as a new promising material for the development of nanotechnologies intended for biological and medical purposes. The authors report that nanodiamonds synthesized by detonation can be used to further purify commercial protein preparations and that enzymes adsorbed on diamond nanoparticles retain their activity. The paper presents results of in vivo experiments with nanodiamonds administered to animals via different routes.