Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project

As a part of the Multinational Genome Sequencing Project of Brassica rapa,linkage group R9 and R3 were sequenced using a bacterial artificial chromosome(BAC) by BAC strategy.The current physical contigs are expected to cover approximately 90%euchromatins of both chromosomes.As the project progresses,BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific.To support the project,a random sheared fosmid library was constructed.The library consists of 9...     

Trp(359) regulates flavin thermodynamics and coenzyme selectivity in Mycobacterium tuberculosis FprA

Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp(359), proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp(359) is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp(359). W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (-168/-181 mV respectively, versus the standard hydrogen electrode, compared with -230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase k(cat) and NAD(P)H K(m) values, but this led to improvements in catalytic efficiency (k(cat)/K(m)) with NADH as reducing coenzyme (9.6/18.8 muM(-1).min(-1) respectively, compared with 5.7 muM(-1).min(-1) for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s(-1) for wild-type FprA and 4.8 s(-1)/13.4 s(-1) for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp(359) for flavin stability. The results demonstrate that the conserved Trp(359) is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.     

ARP2/3-mediated junction-associated lamellipodia control VE-cadherin–based cell junction dynamics and maintain monolayer integrity

Maintenance and remodeling of endothelial cell junctions critically depend on the VE-cadherin/catenin complex and its interaction with the actin filament cytoskeleton. Here we demonstrate that local lack of vascular endothelial (VE)-cadherin at established cell junctions causes actin-driven and actin-related protein 2/3 complex (ARP2/3)–controlled lamellipodia to appear intermittently at those sites. Lamellipodia overlap the VE-cadherin–free adjacent plasma membranes and facilitate formation of new VE-cadherin adhesion sites, which quickly move into the junctions, driving VE-cadherin dynamics and remodeling. Inhibition of the ARP2/3 complex by expression of the N-WASP (V)CA domain or application of two ARP2/3 inhibitors, CK-548 and CK-666, blocks VE-cadherin dynamics and causes intercellular gaps. Furthermore, expression of carboxy-terminal–truncated VE-cadherin increases the number of ARP2/3-controlled lamellipodia, whereas overexpression of wild-type VE-cadherin largely blocks it and decreases cell motility. The data demonstrate a functional interrelationship between VE-cadherin–mediated cell adhesion and actin-driven, ARP2/3-controlled formation of new VE-cadherin adhesion sites via intermittently appearing lamellipodia at established cell junctions. This coordinated mechanism controls VE-cadherin dynamics and cell motility and maintains monolayer integrity, thus potentially being relevant in disease and angiogenesis.     

Use of local knowledge for assessing vegetation changes in the Southern Gadarif Region,Sudan

The vegetation cover in Gadarif Region, Sudan, underwent drastic changes within the last 60 years because of the combined effect of anthropogenic and climatic factors. As a consequence, a large number of plant species are threatened or even became extinct, and vast areas are now deforested. The purpose of this study is to use the local knowledge as a tool to identify and describe patterns of vegetation changes associated with the introduction of mechanized rain‐fed agriculture in the southern Gadarif Region. Data were collected by interviewing 10 key informants and 41 large‐scale farmers. The results provided rich ethnobotanical knowledge and a clear historical perspective of vegetation changes including plant species which have disappeared, decreased and increased in the area. The main factors leading to changes in plant species composition and abundance were vegetation clearance, charcoal and firewood production, wood cuttings, grazing and recently herbicides application. Respondents acknowledged that grazing, duration of abandonment, length of cultivation period, amount and variability of rainfall, fire events and weed composition are the major factors determining succession on fallow land. Farmers showed interest to preserve some multi‐purpose woody species (e.g. Acacia senegal, Balanites aegyptiaca) in their land. Such species should be given priority in rehabilitation activities.     

Effect of Guanosine 5''-O-(3-Thiotriphosphate) and Calcium on γ-Aminobutyric AcidB Binding as a Function of Postnatal Development

We have examined the development of gamma-aminobutyric acidB (GABAB) receptors in rat cerebrum using a binding assay that has achieved specific binding levels of approximately 50% with the GABAB ligand (-)-[3H]baclofen. As early as postnatal day 1, GABAB receptors are present and are linked to both calcium- and guanosine triphosphate-binding protein (G protein)-regulatory sites, as indicated by the stimulation of binding by calcium and the inhibition of binding by the guanine nucleotide guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). However, whereas the EC50 for the calcium effect was at a mature value in the neonate, the IC50 for the inhibition of binding by GTP gamma S was not, and declined more than two orders of magnitude by adulthood. Moreover, while many previous studies had shown that manipulation of G proteins by guanine nucleotides affects receptors affinity rather than density, our saturation analysis of binding suggests that calcium affected GABAB receptor density rather than affinity. The results therefore suggest that calcium and the manipulation of G proteins by GTP gamma S may affect the GABAB receptor by different mechanisms.     

Distribution of Anthropometric Variables and the Prevalence of Obesity in Populations of West African Origin: The International Collaborative Study on Hypertension in Blacks (ICSHIB)

A survey of the prevalence of hypertension and associated risk factors including obesity was carried out among persons of West African heritage currently living in societies at different stages of social, economic and technological development. We present here the distribution of several anthropometric variables and the prevalence of obesity in these populations. Using a standard protocol with centralized training of field staff, 7 439 men and women aged 24 to 75 from six multinational sites were recruited and examined. Although men were taller, women were more obese across sites. Body mass index (BMI) and consequently the prevalence of overweight and obesity increased with westernization from rural African subsistence farming communities to suburban Chicago. Average BMI increased with age until about age 54, and then began to decline or at least level off. The mean BMI for African-American men and women was 27.1 kg/m² and 30.8 kg/m², respectively. Men displayed high levels of centripetal fatness, measured as the waist-to-hip ratio (WHR), compared to the women across site. Based on the US Department of Agriculture guidelines, 22.6% and 56.9% of the African-American men and women had elevated WHR. Although account must be taken of the important contribution of an individual's genetic background, this multinational study of persons with similar heritage clearly shows the potent impact of current environmental factors on the distribution and level of obesity.