Discovery of antibacterial cyclic peptides that inhibit the ClpXP protease

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA-tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.     

Arabidopsis XXT5 gene encodes a putative alpha-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis

The function of a putative xyloglucan xylosyltransferase from Arabidopsis thaliana (At1g74380; XXT5) was studied. The XXT5 gene is expressed in all plant tissues, with higher levels of expression in roots, stems and cauline leaves. A T-DNA insertion in the XXT5 gene generates a readily visible root hair phenotype (root hairs are shorter and form bubble-like extrusions at the tip), and also causes the alteration of the main root cellular morphology. Biochemical characterization of cell wall polysaccharides isolated from xxt5 mutant seedlings demonstrated decreased xyloglucan quantity and reduced glucan backbone substitution with xylosyl residues. Immunohistochemical analyses of xxt5 plants revealed a selective decrease in some xyloglucan epitopes, whereas the distribution patterns of epitopes characteristic for other cell wall polysaccharides remained undisturbed. Transformation of xxt5 plants with a 35S::HA-XXT5 construct resulted in complementation of the morphological, biochemical and immunological phenotypes, restoring xyloglucan content and composition to wild-type levels. These data provide evidence that XXT5 is a xyloglucan alpha-1,6-xylosyltransferase, and functions in the biosynthesis of xyloglucan.     

The response of alkaline phosphatase to osmoregulatory changes in the trout,Salmo gairdneri

Summary The relationship between alkaline phosphatase and environmental salinity was examined in the rainbow trout and the migratory rainbow (steelhead),Salmo gairdneri. The enzyme activity in tissues involved in osmoregulation was strongly correlated with the adaptation salinity and thus to the degree of salt and fluid transport in those tissues. After transfer from freshwater to seawater, the specific activity of the enzyme increased over 260% in the intestine, decreased by 50% in kidney, and was unchanged in the liver, an organ not directly involved in osmoregulation. The sea-run steelhead trout response was similar to the nonmigratory rainbow; although, the pre-migratory transformation (smoltification) had no effect on enzyme activity. Amino acid inhibitors of alkaline phosphatase significantly reduced fluid absorption in the isolated intestine of rainbow trout, reaffirming the relationship between the enzyme and fluid movement. Electrophoretic identification of trout alkaline phosphatase isozymes, clearly distinguishes the enzyme from different tissue origins. However, from the analysis of intestinal electrophoretic patterns, osmoregulatory adjustments are not associated with the induction of new alkaline phosphatase isozymes, or in the large scale preferential stimulation of one of the two existing intestinal isozymes over the other.     

Linear and non-linear impacts of a non-native plant invasion on soil microbial community structure and function

Biological invasions can alter ecosystem functions such as litter decomposition and nutrient cycling, but little is known about how invader abundance influences the impact on the ecosystem. It is often assumed that impacts are proportional to invasion density, but this assumption has never been tested and has little justification. We tested the hypothesis that the microbial community structure and function of a mixed hardwood forest soil changed after invasion by Japanese barberry (Berberis thunbergii), an invasive shrub commonly found in eastern hardwood forests, and that changes were proportional to the density of invasion. We constructed microcosms with mixtures of native and invasive leaf litter, and measured microbial community structure (phospholipid fatty acids) and function (litter decomposition). Decomposition was linearly related to the degree of invasion (R ²?=?0.945), but the ratio of bacteria to fungi exhibited a strongly non-linear, threshold response (R ²?=?0.513). These results indicate that impacts of Japanese barberry invasion are not always proportional to invasion density. This finding has implications for the study of biological invasions as well as practical implications for the management of exotic invasive species.     

Evaluating Caveolin Interactions: Do Proteins Interact with the Caveolin Scaffolding Domain through a Widespread Aromatic Residue-Rich Motif?

Caveolins are coat proteins of caveolae, small flask-shaped pits of the plasma membranes of most cells. Aside from roles in caveolae formation, caveolins recruit, retain and regulate many caveolae-associated signalling molecules. Caveolin-protein interactions are commonly considered to occur between a ∼20 amino acid region within caveolin, the caveolin scaffolding domain (CSD), and an aromatic-rich caveolin binding motif (CBM) on the binding partner (фXфXXXXф, фXXXXфXXф or фXфXXXXфXXф, where ф is an aromatic and X an unspecified amino acid). The CBM resembles a typical linear motif - a short, simple sequence independently evolved many times in different proteins for a specific function. Here we exploit recent improvements in bioinformatics tools and in our understanding of linear motifs to critically examine the role of CBMs in caveolin interactions. We find that sequences conforming to the CBM occur in 30% of human proteins, but find no evidence for their statistical enrichment in the caveolin interactome. Furthermore, sequence- and structure-based considerations suggest that CBMs do not have characteristics commonly associated with true interaction motifs. Analysis of the relative solvent accessible area of putative CBMs shows that the majority of their aromatic residues are buried within the protein and are thus unlikely to interact directly with caveolin, but may instead be important for protein structural stability. Together, these findings suggest that the canonical CBM may not be a common characteristic of caveolin-target interactions and that interfaces between caveolin and targets may be more structurally diverse than presently appreciated.     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

A “Love” Dart Allohormone Identified in the Mucous Glands of Hermaphroditic Land Snails

Animals have evolved many ways to enhance their own reproductive success. One bizarre sexual ritual is the “love” dart shooting of helicid snails, which has courted many theories regarding its precise function. Acting as a hypodermic needle, the dart transfers an allohormone that increases paternity success. Its precise physiological mechanism of action within the recipient snail is to close off the entrance to the sperm digestion organ via a contraction of the copulatory canal, thereby delaying the digestion of most donated sperm. In this study, we used the common garden snail Cornu aspersum to identify the allohormone that is responsible for this physiological change in the female system of this simultaneous hermaphrodite. The love dart allohormone (LDA) was isolated from extracts derived from mucous glands that coat the dart before it is stabbed through the partner''s body wall. We isolated LDA from extracts using bioassay-guided contractility measurement of the copulatory canal. LDA is encoded within a 235-amino acid precursor protein containing multiple cleavage sites that, when cleaved, releases multiple bioactive peptides. Synthetic LDA also stimulated copulatory canal contractility. Combined with our finding that the protein amino acid sequence resembles previously described molluscan buccalin precursors, this indicates that LDA is partially conserved in helicid snails and less in other molluscan species. In summary, our study provides the full identification of an allohormone that is hypodermically injected via a love dart. More importantly, our findings have important consequences for understanding reproductive biology and the evolution of alternative reproductive strategies.