Matrix metalloproteinases in development and disease

Matrix metalloproteinases (MMPs) are key modulators of many biological processes during pathophysiological events, such as skeletal formation, angiogenesis, cellular migration, inflammation, wound healing, coagulation, lung and cardiovascular diseases, arthritis, and cancer. Twenty-four members of the MMP family have been identified in humans, degrading many components of the extracellular matrix, cellular receptors, and cytokines. This review describes the molecular structure, activation and inhibition, and substrate specificity of MMPs, and their biological function in development and disease.     

Lysophospholipase A activity of Pseudomonas aeruginosa type III secretory toxin ExoU

Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU that is delivered directly into the eukaryotic cell via the type III secretion system. Recent studies demonstrated that ExoU has lipase activity, and that the cytotoxicity of ExoU is dependent on its patatin-like phospholipase domain. We investigated the phospholipase A (PLA) activity of ExoU. ExoU, but not non-catalytic ExoU-S142A, preincubated with the BEAS-2B cell lysate showed a weak increase of Ca(2+)-independent PLA(2) activity. When activated ExoU was mixed with secretory type PLA(2), more phospholipase activity was observed, suggesting that ExoU has lysophospholipase A (lysoPLA) activity. A significant increase in lysoPLA activity was also observed. Glycerol enhanced this activity and inhibitors of iPLA(2) suppressed ExoU's lysoPLA activity. Our results suggest that ExoU has a potent lysoPLA activity that requires the presence of the catalytically active site Ser(142) with an unknown eukaryotic cell factor(s) for its activation.     

Human immunodeficiency virus type 1 induces persistent changes in mucosal and blood gammadelta T cells despite suppressive therapy

Gammadelta T cells are primarily found in the gastrointestinal mucosa and play an important role in the first line of defense against viral, bacterial, and fungal pathogens. We sought to examine the impact of human immunodeficiency virus type 1 (HIV-1) infection on mucosal as well as peripheral blood gammadelta T-cell populations. Our results demonstrate that HIV-1 infection is associated with significant expansion of Vdelta1 and contraction of Vdelta2 cell populations in both the mucosa and peripheral blood. Such changes were observed during acute HIV-1 infection and persisted throughout the chronic phase, without apparent reversion after treatment with highly active antiretroviral therapy (HAART). Despite an increase in the expression of CCR9 and CD103 mucosal homing receptors on peripheral blood gammadelta T cells in infected individuals, mucosal and peripheral blood gammadelta T cells appeared to be distinct populations, as reflected by distinct CDR3 length polymorphisms and sequences in the two compartments. Although the underlying mechanism responsible for triggering the expansion of Vdelta1 gammadelta T cells remains unknown, HIV-1 infection appears to have a dramatic impact on gammadelta T cells, which could have important implications for HIV-1 pathogenesis.     

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.     

The Werner syndrome protein stimulates DNA polymerase beta strand displacement synthesis via its helicase activity

Werner syndrome is a hereditary premature aging disorder characterized by genomic instability. Genetic analysis and protein interaction studies indicate that the defective gene product (WRN) may play an important role in DNA replication, recombination, and repair. DNA polymerase beta (pol beta) is a central participant in both short and long-patch base excision repair (BER) pathways, which function to process most spontaneous, alkylated, and oxidative DNA damage. We report here a physical interaction between WRN and pol beta, and using purified proteins reconstitute of a portion of the long-patch BER pathway to examine a potential role for WRN in this repair response. We demonstrate that WRN stimulates pol beta strand displacement DNA synthesis and that this stimulation is dependent on the helicase activity of WRN. In addition, a truncated WRN protein, containing primarily the helicase domain, retains helicase activity and is sufficient to mediate the stimulation of pol beta. The WRN helicase also unwinds a BER substrate, providing evidence that WRN plays a role in unwinding DNA repair intermediates. Based on these findings, we propose a novel mechanism by which WRN may mediate pol beta-directed long-patch BER.     

Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13-16 A). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the PrlA4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent K(m) of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K(m) for proOmpA translocation.     

PHYLOGEOGRAPHIC STUDIES IN THE TROPICAL SEAWEED CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA,ULVOPHYCEAE) REVEAL A CRYPTIC SPECIES COMPLEX1

The seaweed Cladophoropsis membranacea (Hofman Bang ex. C. Agardh) Børgesen is a widely distributed species on coral reefs and along rocky coastlines throughout the tropics and subtropics. In a recent population‐level survey openface>1600 individuals with eight microsatellite loci, a number of isolates from biogeographically disjunct locations could not be amplified for any of the loci. Nonamplifiable and amplifiable isolates co‐occurred within the Canary Islands, Cape Verde Islands, and in the Caribbean. These unexpected results led to question whether or not C. membranacea is a single species. Phylogenetic relationships were evaluated using rDNA ITS1 and ITS2 sequence comparisons from 42 isolates sampled from a subset of 30 of the 66 locations. Four well‐supported clades were identified. Sequence divergence within clades was <1%, whereas between‐clade divergence was 2%–3%. Intraindividual variation was extremely low with no effects on the analysis. A strong, but imperfect, correspondence was found between ITS clades and amplifiable microsatellite loci. It is concluded that C. membranacea consists of three cryptic species. Using Pacific isolates as an outgroup, the most basal clade included the Central Canary Islands, Cape Verde, and Bonaire (Caribbean) isolates and thus spanned the widest latitude. Two derived sister clades consisted of a southern transtropical group stretching across the SE Caribbean to the Cape Verde Islands and African coast (but not the Canary Islands) and a NE‐Canary Island‐Mediterranean clade that also included the Red Sea. The detection of overlapping biogeographic distributions highlights the importance of ecotypic differentiation with respect to temperature and the importance of shifting sea surface isotherms that have driven periodic extinctions and recolonizations of the Canary Islands—a crossroads of marine floral exchange—since the last glacial maximum.