Structural models of the snake venom factor V activators from Daboia russelli and Daboia lebetina

Blood coagulation factor V (FV) is a multifunctional protein that circulates in human plasma as a precursor molecule which can be activated by thrombin or activated factor X (FXa) in order to express its cofactor activity in prothrombin activation. FV activation is achieved by limited proteolysis after Arg709, Arg1018, and Arg1545 in the FV molecule. The venoms of Daboia russelli and Daboia lebetina contain a serine protease that specifically activates FV by a single cleavage at Arg1545. We have predicted the three-dimensional structure of these enzymes using comparative protein modeling techniques. The plasminogen activator from Agkistrodon acutus, which shows a high degree of homology with the venom FV activators and for which a high-quality crystallographic structure is available, was used as the molecular template. The RVV-V and LVV-V models provide for the first time a detailed and accurate structure of a snake venom FV activator and explain the observed sensitivity or resistance toward a number of serine protease inhibitors. Finally, electrostatic potential calculations show that two positively charged surface patches are present on opposite sides of the active site. We propose that both FV activators achieve their exquisite substrate specificity for the Arg1545 site via interactions between these exosites and FV.     

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.     

Relationship between changes in brachial artery flow-mediated dilation and basal release of nitric oxide in subjects with Type 2 diabetes

Assessment of flow-mediated dilation (FMD) after forearm ischemia is widely used as a noninvasive bioassay of stimulated nitric oxide (NO)-mediated conduit artery vasodilator function in vivo. Whether this stimulated endothelial NO function reflects basal endothelial NO function is unknown. To test this hypothesis, retrospective analysis of randomized crossover studies was undertaken in 17 subjects with Type 2 diabetes; 9 subjects undertook an exercise training or control period, whereas the remaining 8 subjects were administered an angiotensin II receptor blocker or placebo. FMD was assessed by using wall tracking of high-resolution brachial artery ultrasound images in response to reactive hyperemia. Resistance vessel basal endothelium-dependent NO function was assessed by using intrabrachial administration of NG-monomethyl-L-arginine (L-NMMA) and plethysmographic assessment of forearm blood flow (FBF). FMD was higher after intervention compared with control/placebo (6.15+/-0.53 vs. 3.81+/-0.72%, P<0.001). There were no significant changes in the FBF responses to L-NMMA. Regression analysis between FMD and L-NMMA responses at entry to the study revealed an insignificant correlation (r=-0.10, P=0.7), and improvements in FMD with the interventions were not associated with changes in the L-NMMA responses (r=-0.04, P=0.9). We conclude that conduit artery-stimulated endothelial NO function (FMD) does not reflect basal resistance vessel endothelial NO function in subjects with Type 2 diabetes.     

Age-associated Iron Accumulation in Bone: Implications for Postmenopausal Osteoporosis and a New Target for Prevention and Treatment by Chelation

Iron accumulation in tissues is believed to be a characteristic of aged humans and a risk factor for some chronic diseases. However, it is not known whether age-associated iron accumulation is part of the pathogenesis of postmenopausal osteoporosis that affects approximately one out three women worldwide. Here, we confirmed that this accumulation of iron was associated with osteopenia in ovariectomized (OVX) rats (a model of peri- and postmenopausal osteoporosis due to estrogen deficiency). To further investigate whether the increased iron level plays a causal role in the onset of bone loss, we treated OVX rats with an orally active and bone targeted chelator that prevented iron accumulation in their skeletal tissues. The results showed that this treatment mitigated the loss of bone mass and the deterioration of bone micro-architecture. We also found that one possible mechanism of the protective action of iron chelation was to significantly reduce bone resorption. Thus, these findings provide a novel target and a potentially useful therapeutic strategy for the prevention and treatment of postmenopausal osteoporosis and perhaps other age-related diseases.     

Presence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in threatened corroboree frog populations in the Australian Alps

Since the early 1980s, the southern corroboree frog Pseudophryne corroboree and northern corroboree frog P. pengilleyi have been in a state of decline from their sub-alpine and high montane bog environments on the southern tablelands of New South Wales, Australia. To date, there has been no adequate explanation as to what is causing the decline of these species. We investigated the possibility that a pathogen associated with other recent frog declines in Australia, the amphibian chytrid fungus Batrachochytrium dendrobatidis, may have been implicated in the decline of the corroboree frogs. We used histology of toe material and real-time PCR of skin swabs to investigate the presence and infection rates with B. dendrobatidis in historic and extant populations of both corroboree frog species. Using histology, we did not detect any B. dendrobatidis infections in corroboree frog populations prior to their decline. However, using the same technique, high rates of infection were observed in populations of both species after the onset of substantial population declines. The real-time PCR screening of skin swabs identified high overall infection rates in extant populations of P. corroboree (between 44 and 59%), while significantly lower rates of infection were observed in low-altitude P. pengilleyi populations (14%). These results suggest that the initial and continued decline of the corroboree frogs may well be attributed to the emergence of B. dendrobatidis in populations of these species.     

Predictive testing for hereditary nonpolyposis colorectal cancer: subjective perception regarding colorectal and endometrial cancer, distress, and health-related behavior at one year post-test

This study evaluated illness representations, distress, and health-related behavior one year after disclosure of a predictive genetic test result for hereditary nonpolyposis colorectal cancer (HNPCC) in 36 carriers and 36 noncarriers. Post-test, no significant differences between carriers and noncarriers were found in perceived risk and perceived seriousness of colorectal and endometrial cancer or in perceived control over endometrial cancer. Confidence in the controllability of colorectal cancer by means of medical examinations was higher for carriers than noncarriers post-test. Mean levels of distress (cancer-specific distress, state anxiety, psychoneuroticism) were within normal ranges and none of the participants had an overall pattern (on all scales) of clinically elevated levels of distress. Carriers had significantly higher cancer-related distress one year posttest than noncarriers. In both groups, colorectal cancer-related distress decreased. Noncarriers additionally showed decreased endometrial cancer-related distress and state anxiety. Within the year after testing, none of the noncarriers had a colonoscopy and all carriers where adherent to the recommendations regarding colorectal cancer screening. Although interview data delineated individually different problems specifically related to predictive testing (e.g., worry, difficulties in relation to other relatives, burden of regular follow-up), generally, predictive testing for HNPCC does not seem to induce major psychological problems. Moreover, the presented data are promising regarding the impact on health-related behavior.     

Neurodevelopment on route p63

All known members of the p53 gene family, including the two homologs p73 and p63, have multiple biological functions. In neurons, p53 and p73 are known to regulate cell death in the developing and adult nervous system. A report by Jacobs et al. in this issue of Neuron shows that the more ancestral member of this gene family, p63, is an essential proapoptotic protein during neuronal development.     

Foliar N application reduces soil NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-N leaching loss in apple orchards

A comparison of the effects of foliar and soil N application was made in field-grown mature fruiting Gala/M9 apple trees (Malus domestica Borkh) in 2001 and 2002 growing seasons under Pacific Northwest growing conditions in southern British Columbia, Canada. The trees, six years old at the start of the experiment, were treated: (1) with 5 g/l urea sprays supplied every two weeks (7 times) from mid May to mid August (total about 50 g N/tree/year), (2) with the same amount of N applied to the soil with the same timing and quantity as for the foliar treatment, and (3) with no N (control). Leaf color (as SPAD readings) and N concentrations (mg/g), and soil NH₄⁺-N and NO₃^–-N were measured periodically throughout the two seasons. Leached NO₃^–-N was monitored monthly via an anion exchange probe from June to October in 2001 and from May to November in 2002. Shoot length was measured in October and N concentration of one-year-old wood and roots was determined in December of each growing s eason. Soil N application significantly increased shoot length relative to control or foliar N application. Leaf color, leaf N, and N concentration of one-year-old wood and roots were similarly increased relative to control by both soil and foliar N application. These treatments also increased fruit yield relative to control. There was no significant difference in yield and fruit quality between soil and foliar N applications. Soil N application increased soil NH₄⁺-N and NO₃^–-N content in the root zone, and also increased the NO₃^– leaching loss below the root zone especially late in the growing season. Our results suggested that tree N status and yield and fruit quality could be maintained by multiple urea sprays during the growing season in apple orchards, and foliar N application will reduce the risk of soil NO₃^–-N leaching.