Synthesis of potent pyrrolidine influenza neuraminidase inhibitors

The synthesis of several pyrrolidine inhibitor analogs is described that possess nanomolar in vitro potencies against the neuraminidase enzymes expressed by the B/Memphis/3/89 and A/N1/PR/8/34 influenza strains.     

Butterfly,spider, and plant communities in different land-use types in Sardinia,Italy

Butterfly, spider, and plant species richness and diversity were investigated in five different land-use types in Sardinia. In 16 one-hectare plots we measured a set of 15 environmental variables to detect the most important factors determining patterns of variation in species richness, particularly endemicity. The studied land-use types encompassed homogeneous and heterogeneous shrublands, shrublands with tree-overstorey, Quercus forest and agricultural land. A total of 30 butterfly species, among which 10 endemics, and 50 spider (morpho)species, were recorded. Butterfly and spider community composition differed according to land-use type. The main environmental factors determining diversity patterns in butterflies were the presence of flowers and trees. Spiders reacted mainly to habitat heterogeneity and land-use type. Traditional land-use did not have adverse effects on the diversity of butterflies, spiders, or plants. The number of endemic butterfly species per treatment increased with total species richness and altitude. Butterfly and spider richness did not co-vary across the five land-use types. Butterflies were, however, positively associated with plant species richness and elevation, whereas spiders were not. Conclusively, butterflies did not appear to be good indicators for spider diversity and species richness at the studied sites.     

Investigating the origin of the slow-binding inhibition of HCV NS3 serine protease by a novel substrate based inhibitor

Indandiones were identified as a novel class of small molecule inhibitors of hepatitis C virus NS3 serine protease from high throughput screening. We further studied the structure activity relationships and the mechanisms of inhibition for this class of compounds. Our studies revealed two similar, yet different, mechanisms accounting for the apparent indandione inhibition of HCV NS3 protease. In one case, the apparent inhibition results from the chemical breakdown of the parent compound and the subsequent redox chemistry of the compound. Oxidation of the cysteine containing substrate A to a disulfide-linked dimer converts this substrate to a potent, slow-binding inhibitor with a K(i) value of 170 nM. The second class of indandiones appears to react directly with the substrate to form an S-phenyl disulfide adduct with the P1 cysteine. This modification converts the substrate to a slow-binding inhibitor with a K(i) value of 110 nM, a k(on) = 2370 M(-1) s(-1), and k(off) = 2.5 x 10(-4) s(-1). A stable analogue of this latter compound was synthesized that contained a CH(2)-S linkage instead of the S-S linkage. The CH(2)-S compound showed no inhibition at concentrations as high as 40 microM, which suggests an important role for the S-S linkage in the inhibitory mechanism. Cysteine 159, which lies near the active site of the HCV protease, was mutated to serine. The C159S mutant displayed wild-type catalytic activity and susceptibility to inhibition by the S-S linked inhibitor. This result argues against a mechanism involving disulfide exchange between the inhibitor and the sulfhydryl group of C159. The mechanism of inhibition for this S-S linked substrate based inhibitor is likely due to oxidation of cysteines involved in chelation of the structural zinc atom.     

Cdc42/Rac1-mediated activation primes PAK2 for superactivation by tyrosine phosphorylation

The involvement of p21-activated kinases (PAKs) in important cellular processes such as regulation of the actin skeleton morphology, transduction of signals controlling gene expression, and execution of programmed cell death has directed attention to the regulation of the activity of these kinases. Here we report that activation of PAK2 by p21 GTPases can be strongly potentiated by cellular tyrosine kinases. PAK2 became tyrosine phosphorylated in its N-terminal regulatory domain, where Y130 was identified as the major phosphoacceptor site. Tyrosine phosphorylation-mediated superactivation of PAK2 could be induced by overexpression of different Src kinases or by inhibiting cellular tyrosine phosphatases with pervanadate and could be blocked by the Src kinase inhibitor PP1 or by mutating the Y130 residue. Analysis of PAK2 mutants activated by amino acid changes in the autoinhibitory domain or the catalytic domain indicated that GTPase-induced conformational changes, rather than catalytic activation per se, rendered PAK2 a target for tyrosine phosphorylation. Thus, PAK activation represents a potentially important point of convergence of tyrosine kinase- and p21 GTPase-dependent signaling pathways.     

Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages

The metabolite profiles of 20 Stachybotrys spp. isolates from Finnish water-damaged buildings were compared with their biological activities. Effects of purified compounds on cytotoxicity and production of inflammatory mediators such as nitric oxide, IL-6 and TNFalpha in murine RAW264.7 macrophage cells were studied. The 11 isolates belonging to the satratoxin-producing chemotype were highly cytotoxic to the macrophages. The isolates inducing inflammatory mediators all belonged to the atranone-producing chemotype, but pure atranones B, and D did not elicit a response in the bioassay. Altogether, cytotoxicity of Stachybotrys sp. isolates appear to be related to satratoxin production whereas the specific component inducing inflammatory responses in atranone-producing isolates remains obscure.     

ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism

Oxysterol binding protein (OSBP) related proteins (ORPs) constitute a family that has at least 12 members in humans. In the present study we characterize one of the novel OSBP homologs, ORP2, which we show to be expressed ubiquitously in mammalian tissues. The ORP2 cDNA encodes a deduced 55 kDa protein that lacks a pleckstrin homology (PH) domain, a feature found in the other family members. Sucrose gradient centrifugation analysis of Chinese hamster ovary (CHO) cell post-nuclear supernatant demonstrated that ORP2 is distributed in soluble and membrane-bound fractions. Immunofluorescence microscopy of the endogenous and overexpressed ORP2 in CHO cells suggested that the membrane-bound fraction of the protein localizes to the Golgi apparatus. Stably transfected CHO cells that overexpress ORP2 showed an increase in [14C]cholesterol efflux to serum, apolipoprotein A-I (apoA-I), and phosphatidyl choline vesicles. The proportion of cellular [14C]cholesterol that is esterified and the ACAT activity measured as [14C]oleyl-CoA conversion into cholesteryl [14C]oleate by the cellular membranes, were markedly decreased in the ORP2 expressing cells. Transient high level overexpression of ORP2 interfered with the clearance of a secretory pathway protein marker from the Golgi complex. The results implicate ORP2 as a novel regulator of cellular sterol homeostasis and intracellular membrane trafficking.     

Effect of 6-month athletic training on motor abilities in seven-year-old schoolgirls

The effects of six-month athletic training on improving motor abilities in 7-year-old schoolgirls were assessed. Analysis of the results of 12 motor tests showed significant improvement in the study group (n = 38) in comparison with control group (n = 140) subjected to conventional physical education classes only. The improvement referred to the variables of aerobic endurance (3-min run), flexibility (forward bow), explosive strength (ball throwing and 20-m run), keeping balance (bench standing), static strength (bent arm hang), and repetitive strength (sit-ups). These are probably adaptive changes brought up by discriminant functions. The varimax factor and discriminative function correlations indicated that all four factors of changes contributed significantly to the explanation of discriminative function. An almost equally high correlation of varimax factors and discriminative function was obtained on the basis of differences in the third factor responsible for changes in the frequency of movements and in the explosive strength of the jump type; in the second factor responsible for changes in coordination with changes in the repetitive strength of the body; and in the fourth factor responsible for changes in the explosive strength of the throw and sprint types with changes and endurance.     

Bovine distal pulmonary arterial media is composed of a uniform population of well-differentiated smooth muscle cells with low proliferative capabilities

The media of the normal bovine main pulmonary artery (MPA) is composed of phenotypically heterogeneous smooth muscle cells (SMC) with markedly different proliferative capabilities in response to serum, mitogens, and hypoxia. Little, however, is known of the SMC phenotype in distal pulmonary arteries (PA), particularly in arterioles, which regulate the pulmonary circulation. With a panel of muscle-specific antibodies against alpha-smooth muscle (SM)-actin, SM-myosin heavy chains (SM-MHC), SM-MHC-B isoform, desmin, and meta-vinculin, we demonstrate a progressive increase in phenotypic uniformity and level of differentiation of SMC along the proximal-to-distal axis of normal adult bovine pulmonary circulation so that the media of distal PA (1,500- to 100-microm diameter) is composed of a phenotypically uniform population of "well-differentiated" SMC. Similarly, when isolated and assessed in vitro, distal PA-SMC is composed of a single, uniform population of differentiated SMC that exhibited minimal growth responses to a variety of mitogens while their cell size increased substantially in response to serum. Their growth was inhibited by hypoxic exposure under all conditions tested. Distal PA-SMC also differed from MPA-SMC by exhibiting a distinct pattern of DNA synthesis in response to serum and mitogens. Thus, in contrast to the MPA, distal PA media is composed of an apparently uniform population of well-differentiated SMC that are proliferation resistant and have a substantial capacity to hypertrophy in response to growth-promoting stimuli. We thus speculate that distinct SMC phenotypes present in distal vs. proximal PA may confer different response mechanisms during remodeling in conditions such as hypertension.     

Monoclonal antibody to fibronectin which inhibits extracellular matrix assembly

A monoclonal antibody L8 specific to fibronectin was shown to inhibit fibronectin incorporation into the fibroblast extracellular matrix. Antibody L8 could not interact with fibronectin complexed with gelatin. The results suggest the existence of a specific site on the fibronectin molecule playing a critical role in the assembly of the fibronectin extracellular matrix. This site is located near the collagen-binding domain.     

Diversity, ecological structure, and conservation of the landbird community of Dadia reserve, Greece

Birds are integral to many environmental monitoring schemes. However, there has been little research on the ecological basis of utilizing bird species as indicators of their respective communities and habitats. We used point counts to survey 72 landbird species, 16 of conservation concern, in the Dadia Nature Reserve, Greece, in order to understand the ecology of bird diversity patterns, analyse community composition, identify species characteristic of major vegetation types, and improve long-term management and monitoring protocols. We sampled 36 sites representing 21 vegetation types. Highly heterogeneous sites were the most species rich and rural mosaics (small fields and pastures of low intensity land use, separated by thick hedgerows and tree lines) were twice as rich as intensified crop monocultures. Using multivariate analysis, we found that vegetation cover and height affected the composition of the avifauna. Twenty-one predefined vegetation categories clustered into eight distinct bird habitat types: field crops, rural mosaics, mosaic sites, poplar trees, broadleaved woods, pinewoods, shrubs, and heaths. Ten bird species were highly characteristic of the main bird habitat types in the study area. Our results emphasize the importance of conserving rural mosaics, hedgerows, and openings within forests for landbird conservation in the Mediterranean countryside. We also provide evidence in support of maintaining horizontal heterogeneity at a local scale. Finally, we suggest that monitoring populations of certain indicator bird species can be a cost-effective and efficient way to monitor the state and habitat quality of the entire landbird community, thereby integrating the knowledge of community structure into conservation decision-making.