Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

Plasmodium falciparum falcilysin: a metalloprotease with dual specificity

The malaria parasite Plasmodium falciparum degrades host cell hemoglobin within an acidic food vacuole. The metalloprotease falcilysin has previously been identified as an important component of this catabolic process. Using random peptide substrate analysis, we confirm that recombinant falcilysin is highly active at acidic pH, consistent with its role in hemoglobin degradation. Unexpectedly, the enzyme is also robustly active at neutral pH, but with a substantially different substrate specificity. Imaging studies confirm the location of falcilysin in the food vacuole and reveal association with vesicular structures elsewhere within the parasite. These data suggest that falcilysin may have an expanded role beyond globin catabolism and may function as two different proteases in its two locations.     

Opposite modulatory roles for adenosine A1 and A2A receptors on glutamate and dopamine release in the shell of the nucleus accumbens. Effects of chronic caffeine exposure

Previous studies have demonstrated opposing roles for adenosine A1 and A2A receptors in the modulation of extracellular levels of glutamate and dopamine in the striatum. In the present study, acute systemic administration of motor-activating doses of the A2A receptor antagonist MSX-3 significantly decreased extracellular levels of dopamine and glutamate in the shell of the rat nucleus accumbens (NAc) and counteracted both dopamine and glutamate release induced by systemic administration of motor-activating doses of either the A1 receptor antagonist CPT or caffeine. Furthermore, exposure to caffeine in the drinking water (1 mg/mL, 14 days) resulted in tolerance to the effects of systemic injection of CPT or caffeine, but not MSX-3, on extracellular levels of dopamine and glutamate in the NAc shell. The present results show: first, the existence of opposite tonic effects of adenosine on extracellular levels of dopamine and glutamate in the shell of the NAc mediated by A1 and A2A receptors; second, that complete tolerance to caffeine's dopamine- and glutamate-releasing effects which develops after chronic caffeine exposure is attributable to an A1 receptor-mediated mechanism. Development of tolerance to the dopamine-releasing effects of caffeine in the shell of the NAc may explain its weak addictive properties and atypical psychostimulant profile.     

Mitochondrial dysfunction and oxidative damage in parkin-deficient mice

Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease.     

‘Gelatinase-like’ activity from articular chondrocytes in monolayer culture

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TC_A and TC_B fragments of human Type II collagen into smaller peptides at 37°C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1,10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.     

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.     

Pectin immunolocalization and calcium visualization in differentiating derivatives from poplar cambium

Summary Calcium distribution and pectin esterification patterns in the cambial zone of poplar branches were studied with ionic microscopy and immunological tools respectively. Dynamic changes correlating with cell growth and cell differentiation were observed both on the xylem and on the phloem sides. In expanding cell walls of xylem derivatives, unesterified pectins were restricted to cell junctions and middle lamellae, occasionally accompanied by calcium ions. In contrast, in differentiating and mature phloem cells, acidic pectins and Ca²⁺ were present all over the walls leading to early stiffening of the polysaccharide network. Significant labelling was detected with JIM5 antibodies in some dictyosomes suggesting exocytosis of low methylated polymers towards the cell walls. At cell junctions, unesterified pectins might originate from the activity of pectinmethylesterases localized in these areas. Thus un- and deesterified pectins might be located in different cell wall domains whose distribution, varying with cell type, will confer specific extensibility to the wall matrix.Abbreviations BSA bovine serum albumin - DM degree of methylation - FITC fluorescein isothiocyanate - HM highly methylated pectins - LM low methylated pectins - PME pectin methylesterase - SIMS secondary ion mass spectrometry - TBS tris-buffered saline     

Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production.

The availability of a technique for site-directed mutagenesis by gene replacement provides a powerful tool for genetic analysis in any bacterial species. We report here a general technique for gene replacement in Pseudomonas aeruginosa. Genes on fragments of cloned P. aeruginosa DNA, altered by transposon mutagenesis, can be transduced into a recipient strain and can replace homologous genes in the P. aeruginosa genome. In this study we applied this technique to the construction of recA mutants of P. aeruginosa. A cloned segment of P. aeruginosa FRD1 DNA was isolated which encoded a protein analogous to the recA gene product of Escherichia coli. The P. aeruginosa recA gene was able to complement several defects associated with recA mutation in E. coli. Transposon Tn1 and Tn501 insertions in the cloned recA gene of P. aeruginosa were used to generate chromosomal recA mutants by gene replacement. These recA strains of P. aeruginosa were more sensitive to UV irradiation and methyl methane sulfonate and showed reduced recombination proficiency compared with the wild type. Also examined was the effect of recA mutations on the expression of alginate, a virulence trait. Alginate is a capsulelike polysaccharide associated with certain pulmonary infections, and its expression is typically unstable. The genetic mechanism responsible for the instability of alginate biosynthesis was shown to be recA independent.     

Impacts and Recovery from Severe Tropical Cyclone Yasi on the Great Barrier Reef

Full recovery of coral reefs from tropical cyclone (TC) damage can take decades, making cyclones a major driver of habitat condition where they occur regularly. Since 1985, 44 TCs generated gale force winds (≥17 metres/second) within the Great Barrier Reef Marine Park (GBRMP). Of the hurricane strength TCs (≥H1—Saffir Simpson scale; ≥ category 3 Australian scale), TC Yasi (February, 2011) was the largest. In the weeks after TC Yasi crossed the GBRMP, participating researchers, managers and rangers assessed the extent and severity of reef damage via 841 Reef Health and Impact Surveys at 70 reefs. Records were scaled into five damage levels representing increasingly widespread colony-level damage (1, 2, 3) and reef structural damage (4, 5). Average damage severity was significantly affected by direction (north vs south of the cyclone track), reef shelf position (mid-shelf vs outer-shelf) and habitat type. More outer-shelf reefs suffered structural damage than mid-shelf reefs within 150 km of the track. Structural damage spanned a greater latitudinal range for mid-shelf reefs than outer-shelf reefs (400 vs 300 km). Structural damage was patchily distributed at all distances, but more so as distance from the track increased. Damage extended much further from the track than during other recent intense cyclones that had smaller circulation sizes. Just over 15% (3,834 km²) of the total reef area of the GBRMP is estimated to have sustained some level of coral damage, with ~4% (949 km²) sustaining a degree of structural damage. TC Yasi likely caused the greatest loss of coral cover on the GBR in a 24-hour period since 1985. Severely impacted reefs have started to recover; coral cover increased an average of 4% between 2011 and 2013 at re-surveyed reefs. The in situ assessment of impacts described here is the largest in scale ever conducted on the Great Barrier Reef following a reef health disturbance.     

Transforming Growth Factor-β, Macrophage Colony-Stimulating Factor and C-Reactive Protein Levels Correlate with CD14highCD16+ Monocyte Induction and Activation in Trauma Patients

Severe injury remains a leading cause of death and morbidity in patients under 40, with the number of annual trauma-related deaths approaching 160,000 in the United States. Patients who survive the initial trauma and post-traumatic resuscitation are at risk for immune dysregulation, which contributes to late mortality and accounts for approximately 20% of deaths after traumatic injury. This post-traumatic immunosuppressed state has been attributed to over-expression of anti-inflammatory mediators in an effort to restore host homeostasis. We measured a panel of monocyte markers and cytokines in 50 severely injured trauma patients for 3 days following admission. We made the novel observation that the subpopulation of monocytes expressing high levels of CD14 and CD16 was expanded in the majority of patients. These cells also expressed CD163 consistent with differentiation into alternatively activated macrophages with potential regulatory or wound-healing activity. We examined factors in trauma plasma that may contribute to the generation and activation of these cells. The percentage of CD14^highCD16⁺ monocytes after trauma correlated strongly with plasma C-reactive protein (CRP) transforming growth factor-β (TGF-β), and macrophage colony-stimulating factor (M-CSF) levels. We demonstrate a role for TGF-β and M-CSF, but not CRP in generating these cells using monocytes from healthy volunteers incubated with plasma from trauma patients. CD16 is a receptor for CRP and IgG, and we showed that monocytes differentiated to the CD14^highCD16⁺ phenotype produced anti-inflammatory cytokines in response to acute phase concentrations of CRP. The role of these cells in immunosuppression following trauma is an area of ongoing investigation.