Activities of the matrix metalloproteinase stromelysin-2 (MMP-10) in matrix degradation and keratinocyte organization in wounded skin

The matrix metalloproteinase stromelysin-2 is expressed in keratinocytes of the epithelial tongue of skin wounds, suggesting a role in keratinocyte migration. Here, we show that stromelysin-2 enhances migration of cultured keratinocytes. To gain insight into the in vivo activities of stromelysin-2 in epithelial repair, we generated transgenic mice expressing a constitutively active stromelysin-2 mutant in keratinocytes. These animals had no alterations in skin architecture, and the healing rate of skin wounds was normal. Histologically, however, we found abnormalities in the organization of the wound epithelium. Keratinocytes at the migrating epidermal tip were scattered in most sections of mice with high expression level, and there was a reduced deposition of new matrix. In particular, the staining pattern of laminin-5 at the wound site was altered. This may be due to proteolytic processing of laminin-5 by stromelysin-2, because degradation of laminin-5 by this enzyme was observed in vitro. The inappropriate matrix contact of keratinocytes was accompanied by aberrant localization of beta1-integrins and phosphorylated focal adhesion kinase, as well as by increased apoptosis of wound keratinocytes. These results suggest that a tightly regulated expression level of stromelysin-2 is required for limited matrix degradation at the wound site, thereby controlling keratinocyte migration.     

Vaccination of macaques with long-standing SIVmac251 infection lowers the viral set point after cessation of antiretroviral therapy

A cohort of rhesus macaques with long-standing SIVmac251 infection (> or =5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4(+) and CD8(+) T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4(+) and CD8(+) T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.     

Endothelial dysfunction is induced by proinflammatory oxidant hypochlorous acid

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl) plays a role in tissue injury under inflammatory conditions. The present study tests the hypothesis that HOCl decreases nitric oxide (NO) bioavailability in the vasculature of Sprague-Dawley rats. Aortic ring segments were pretreated with HOCl (1-50 microM) followed by extensive washing. Endothelium-dependent relaxation was then assessed by cumulative addition of acetylcholine (ACh) or the calcium ionophore A23187. HOCl treatment significantly impaired both ACh- and A23187-mediated relaxation. In contrast, endothelium-independent relaxation induced by sodium nitroprusside was unaffected. The inhibitory effect of HOCl on ACh-induced relaxation was reversed by exposure of ring segments to L-arginine but not D-arginine. In cellular studies, HOCl did not alter endothelial NO synthase (NOS III) protein or activity, but inhibited formation of the NO metabolites nitrate (NO3(-) and nitrite (NO2(-). The reduction in total NO metabolite production in bovine aortic endothelial cells was also reversed by addition of L-arginine. These data suggest that HOCl induces endothelial dysfunction via modification of L-arginine.     

The reactivity of plasma phospholipids with lecithin:cholesterol acyltransferase is decreased in fish oil-fed monkeys

The size of low density lipoproteins (LDL) is strongly correlated with LDL cholesteryl ester (CE) content and coronary artery atherosclerosis in monkeys fed cholesterol and saturated fat. African green monkeys fed 11% (weight) fish oil diets have smaller LDL and less CE per LDL particle than lard-fed animals. We hypothesized that this might be due to a lower plasma lecithin:cholesterol acyltransferase (LCAT) activity in fish oil-fed animals. Using recombinant particles made of egg yolk lecithin-[14C]cholesterol-apoA-I as exogenous substrate, we found no difference in plasma LCAT activity (27 versus 28 nmol CE formed per h/ml) of fish oil- versus lard-fed animals, respectively; furthermore, no diet-induced difference in immunodetectable LCAT was found. However, plasma phospholipids from fish oil-fed animals were over 4-fold enriched in n-3 fatty acids in the sn-2 position compared to those of lard-fed animals. Additionally, the proportion of n-3 fatty acid-containing CE products formed by LCAT, relative to the available n-3 fatty acid in the sn-2 position of phospholipids, was less than one-tenth of that for linoleic acid. The overall rate of LCAT-catalyzed CE formation with phospholipid substrates from fish oil-fed animals was lower (5-50%) than with phospholipid substrates from lard-fed animals. These data show that n-3 fatty acids in phospholipids are not readily utilized by LCAT for formation of CE; rather, LCAT preferentially utilizes linoleic acid for CE formation. The amount of linoleic acid in the sn-2 position of plasma phospholipids is reduced and replaced with n-3 fatty acids in fish oil-fed animals. As a result, LCAT-catalyzed plasma CE formation in vivo is likely reduced in fish oil-fed animals contributing to the decreased cholesteryl ester content and smaller size of LDL particles in the animals of this diet group.     

Evolutionary distances for protein-coding sequences: modeling site- specific residue frequencies

Estimation of evolutionary distances from coding sequences must take into account protein-level selection to avoid relative underestimation of longer evolutionary distances. Current modeling of selection via site-to-site rate heterogeneity generally neglects another aspect of selection, namely position-specific amino acid frequencies. These frequencies determine the maximum dissimilarity expected for highly diverged but functionally and structurally conserved sequences, and hence are crucial for estimating long distances. We introduce a codon- level model of coding sequence evolution in which position-specific amino acid frequencies are free parameters. In our implementation, these are estimated from an alignment using methods described previously. We use simulations to demonstrate the importance and feasibility of modeling such behavior; our model produces linear distance estimates over a wide range of distances, while several alternative models underestimate long distances relative to short distances. Site-to-site differences in rates, as well as synonymous/nonsynonymous and first/second/third-codon-position differences, arise as a natural consequence of the site-to-site differences in amino acid frequencies.      

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

The antennal system and cockroach evasive behavior. II. Stimulus identification and localization are separable antennal functions

Cockroaches ( Periplaneta americana) orient their antennae toward moving objects based on visual cues. Presumably, this allows exploration of novel objects by the antennal flagellum. We used videographic and electrophysiological methods to determine if receptors on the flagellum are essential for triggering escape, or if they enable cockroaches to discriminate threatening from non-threatening objects that are encountered. When a flagellum was removed, and replaced with a plastic fiber, deflection of a "prosthetic flagellum" still activated the descending mechanosensory interneurons associated with escape and produced typical escape responses. However, escape was essentially eliminated by constraining the movement of the scape and pedicel at the antennal base. When cockroaches approached and briefly explored the surface of a spider or another cockroach with the flagellum, they produced escape significantly more often in response to subsequent controlled contact from a spider than from a cockroach. This discrimination did not depend on visual or wind-sensory input, but required flagellar palpation of the surface. The crucial sensory cues appear to involve texture rather than surface chemicals. These results indicate that cockroaches acquire basic information on stimulus identity during exploration of surfaces with flagellar receptors, but that basal receptors are triggers for escape behavior.     

Redox state regulates binding of p53 to sequence-specific DNA, but not to non-specific or mismatched DNA.

Redox modulation of wild-type p53 plays a role in sequence-specific DNA binding in vitro . Reduction produces a DNA-binding form of the protein while oxidation produces a non-DNA-binding form. Primer extension analysis reveals that increasing concentrations of reduced p53 result in enhanced protection of the consensus sequence, while increasing concentrations of oxidized p53 confer minimal protection of the consensus sequence. DNA binding by oxidized p53 is, therefore, not sequence-specific. In contrast, there is no observable difference in the binding of oxidized p53 and reduced p53 to double-stranded non-specific or mismatched DNA in gel mobility shift assays. Both forms of p53 bind equally well, suggesting that redox modulation of p53 does not play a role in its binding to non-specific or mismatched DNA. In view of the in vitro evidence that redox state influences the sequence-specific DNA-binding of p53, we have examined the effect of oxidative stress on the in vivo ability of p53 to bind to and transactivate PG13-CAT, a reporter construct containing multiple copies of the p53 consensus binding site linked to the chloramphenicol acetyltransferase gene. Hydrogen peroxide treatment of cells cotransfected with p53 results in a marked decrease in CAT activity, suggesting that oxidation of p53 decreases the ability of the protein to bind to consensus DNA and transactivate target genes in vivo.     

Performance of the Coriolis air sampler,a high-volume aerosol-collection system for quantification of airborne spores and pollen grains

The Coriolis δ air sampler manufactured by Bertin Technologies (France) is a continuous air sampler, dedicated to outdoor monitoring of airborne spores and pollen grains. This high-volume sampler is based on patented Coriolis technology delivering a liquid sample. The air is drawn into a conical vial in a whirling type motion using suction; particles are pulled against the wall by centrifugal force. Airborne particles are separated from the air and collected in a liquid medium. This innovative solution allows rapid analysis by several techniques including PCR assay and serological assay in order to measure the antigenicity/allergenicity of pollen grains and fungal spores. Also, traditional counting of pollen grains or taxa identification by optical microscopy can be done. A study has been carried out by the Health Protection Agency (HPA), Porton Down, UK, to measure the physical efficiency of the Coriolis air sampler. The physical efficiency of the sampler for collection of micro-organism-laden particles of various sizes has been compared with that of membrane filter samplers using the techniques described by ISO 14698-1. The Coriolis was operated simultaneously with membrane filter samplers in a controlled room where they were challenged with uniform-sized particles of different diameters containing bacterial spores. For the larger particle sizes, it was found that the physical efficiency of the Coriolis was 92% for 10-μm particles. The biological performance of the Coriolis in the collection of airborne fungal spores and pollen grains was evaluated in comparison with a Hirst spore trap (one-week tape-on-drum type sampler) which is one of the most frequently used traps in the measurement of outdoor pollen grain concentrations. The advantages and limitations of both technologies are discussed. The Coriolis was operated simultaneously with a Hirst spore trap in the sampling station of Réseau National de Surveillance Aérobiologique, France (RNSA); the pollen grain and fungal spore counts were analysed by optical microscopy. The pollen grain count m⁻³ collected was compared for both devices. The dispersion values were obtained and statistical analysis was carried out. This study shows that the Coriolis air sampler provided equivalent recovery of pollen grain and fungal spores compared with the volumetric trap standard method (not significantly different, W test, α = 0.05). Nowadays, the French-led project, acronym MONALISA, with financial support from the European Commission––Life-Environment (LIFE05 ENV/F/000068), is testing this innovative air sampler in order to measure the antigenicity/allergenicity of the main aeroallergen particles, i.e. Betula (birch), Poaceae (grasses), Parietaria (pellitory), Olea spp (olive tree), and Artemisia (mugwort) pollen grains, and Alternaria (fungal spores) to validate a new approach of monitoring instead of quantifying pollen grains by their morphology. The robustness and efficiency of the MONALISA system is being demonstrated at a national level throughout Europe in eight different countries with different bio-climatic and topography characteristics: France, UK, Finland, Poland, Spain, Portugal, Switzerland, and Italy.