Specificity and reactive loop length requirements for crmA inhibition of serine proteases

The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (approximately 10(2)-10(4) M(-1)sec(-1)), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (k(diss) approximately 10(-7) sec(-1)), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (k(cat)/K(M) of approximately 10(6) M(-1) sec(-1)). N-terminal sequencing confirmed that the P1 Arg-P1'Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1'Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases approximately 10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (10(5)-10(6) M(-1)sec(-1)), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1'Cys-P2'Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites.     

Structural model of the amino propeptide of collagen XI alpha1 chain with similarity to the LNS domains

Fibrillar collagens are the principal structural molecules of connective tissues. The assembly of collagen fibrils is regulated by quantitatively minor fibrillar collagens, types V and XI. A unique amino-terminal propeptide domain of these collagens has been attributed this regulatory role. The structure of the amino terminal propeptide has yet to be determined. Low sequence similarity necessitated a secondary structure-based method to carry out homology modeling based upon the determined structure of LNS family members, named for a common structure in the laminin LG5 domain, the neurexin 1B domain and the sex hormone binding globulin. Distribution of amino acids within the model suggested glycosaminoglycan interaction and calcium binding. These activities were tested experimentally. Sequence analyses of existing genes for collagens indicate that 16 known collagen alpha chains may contain an LNS domain. A similar approach may prove useful for structure/function studies of similar domains in other collagens with similar domains. This will provide mechanistic details of the organization and assembly of the extracellular matrix and the underlying basis of structural integrity in connective tissues. The absolute requirement for collagen XI in skeletal growth is indicated by collagen XI deficiencies such as chondrodystrophies found in the cho/cho mouse and in humans with Stickler syndrome.     

Genetic divergence does not predict change in ornament expression among populations of stalk-eyed flies

Stalk-eyed flies (Diptera: Diopsidae) possess eyes at the ends of elongated peduncles, and exhibit dramatic variation in eye span, relative to body length, among species. In some sexually dimorphic species, evidence indicates that eye span is under both intra- and intersexual selection. Theory predicts that isolated populations should evolve differences in sexually selected traits due to drift. To determine if eye span changes as a function of divergence time, 1370 flies from 10 populations of the sexually dimorphic species, Cyrtodiopsis dalmanni and Cyrtodiopsis whitei, and one population of the sexually monomorphic congener, Cyrtodiopsis quinqueguttata, were collected from Southeast Asia and measured. Genetic differentiation was used to assess divergence time by comparing mitochondrial (cytochrome oxidase II and 16S ribosomal RNA gene fragments) and nuclear (wingless gene fragment) DNA sequences for c. five individuals per population. Phylogenetic analyses indicate that most populations cluster as monophyletic units with up to 9% nucleotide substitutions between populations within a species. Analyses of molecular variance suggest a high degree of genetic structure within and among the populations; > 97% of the genetic variance occurs between populations and species while < 3% is distributed within populations, indicating that most populations have been isolated for thousands of years. Nevertheless, significant change in the allometric slope of male eye span on body length was detected for only one population of either dimorphic species. These results are not consistent with genetic drift. Rather, relative eye span appears to be under net stabilizing selection in most populations of stalk-eyed flies. Given that one population exhibited dramatic evolutionary change, selection, rather than genetic variation, appears to constrain eye span evolution.     

Beryllium-stimulated reactive oxygen species and macrophage apoptosis

Beryllium (Be), the etiologic agent of chronic beryllium disease, is a toxic metal that induces apoptosis in human alveolar macrophages. We tested the hypothesis that Be stimulates the formation of reactive oxygen species (ROS) which plays a role in Be-induced macrophage apoptosis. Mouse macrophages were exposed to 100 microM BeSO4 in the absence and presence of the catalytic antioxidant MnTBAP (100 microM). Apoptosis was measured as the percentage of TUNEL+ and caspase-8+ cells. ROS production was measured by flow cytometry using the fluorescence probes, dihydroethidine (DHE) and dichlorofluorescein diacetate (DCFH-DA). Be-exposed macrophages had increased TUNEL+ cells (15+/-1% versus controls 1+/-0.2%, P<0.05) and increased caspase-8+ cells (18.7+/-2% versus controls 1.8+/-0.4%, P<0.05). Be-induced caspase-8 activation, and a 4-fold increase in ROS formation, was ameliorated by exposure to MnTBAP. Hydrogen peroxide (30 microM) exposure potentiated Be-induced caspase-8 activation, and was also attenuated by MnTBAP. Our data are the first to demonstrate that Be stimulates macrophage ROS formation which plays an important role in Be-induced macrophage apoptosis.     

Aging reduces center-surround antagonism in visual motion processing

Discriminating the direction of motion of a low-contrast pattern becomes easier with increasing stimulus area. However, increasing the size of a high-contrast pattern makes it more difficult for observers to discriminate motion. This surprising result, termed spatial suppression, is thought to be mediated by a form of center-surround suppression found throughout the visual pathway. Here, we examine the counterintuitive hypothesis that aging alters such center-surround interactions in ways that improve performance in some tasks. We found that older observers required briefer stimulus durations than did younger observers to extract information about stimulus direction in conditions using large, high-contrast patterns. We suggest that this age-related improvement in motion discrimination may be linked to reduced GABAergic functioning in the senescent brain, which reduces center-surround suppression in motion-selective neurons.     

Abeta42 is essential for parenchymal and vascular amyloid deposition in mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.     

Use of sequence data from rainbow trout and Atlantic salmon for SNP detection in Pacific salmon

Single nucleotide polymorphisms (SNPs) are a class of genetic markers that are well suited to a broad range of research and management applications. Although advances in genotyping chemistries and analysis methods continue to increase the potential advantages of using SNPs to address molecular ecological questions, the scarcity of available DNA sequence data for most species has limited marker development. As the number and diversity of species being targeted for large-scale sequencing has increased, so has the potential for using sequence from sister taxa for marker development in species of interest. We evaluated the use of Oncorhynchus mykiss and Salmo salar sequence data to identify SNPs in three other species (Oncorhynchus tshawytscha, Oncorhynchus nerka and Oncorhynchus keta). Primers designed based on O. mykiss and S. salar alignments were more successful than primers designed based on Oncorhynchus-only alignments for sequencing target species, presumably due to the much larger number of potential targets available from the former alignments and possibly greater sequence conservation in those targets. In sequencing approximately 89 kb we observed a frequency of 4.30 x 10(-3) SNPs per base pair. Approximately half (53/101) of the subsequently designed validation assays resulted in high-throughput SNP genotyping markers. We speculate that this relatively low conversion rate may reflect the duplicated nature of the salmon genome. Our results suggest that a large number of SNPs could be developed for Pacific salmon using sequence data from other species. While the costs of DNA sequencing are still significant, these must be compared to the costs of using other marker classes for a given application.     

Life support: the alpha4 phosphatase subunit in cell survival and apoptosis

Reversible protein phosphorylation represents a cellular response to normal physiological processes as well as to cellular insults and stress. Recently, the protein phosphatase-associated alpha4 subunit was shown to be required for sustaining cell survival. Lack of alpha4 leads to apoptotic death of multiple cell types and to the death of the organism. Here, we explore how the phosphatase network might operate in controlling life-and-death decisions. We discuss the relevance of the findings for understanding the action of alpha4 in cell survival and for better discriminating between a role in maintaining cellular homeostasis, and thus survival, or actively keeping apoptotic cell death in check by targeting effectors of the cell death machinery.