Metalloproteinase

Ontogenetic variation in venom composition was examined in the Pacific rattlesnakes Crotalus viridis helleri and C. v. oreganus. Venoms were analyzed for protease, phospholipase A2, L-amino acid oxidase, exonuclease and elastinolytic activities, and toxicity toward a native prey (Sceloporus graciosus). Protease activity increased significantly with size; L-amino acid oxidase and exonuclease activities also tended to increase. Phospholipase A2 activity decreased significantly with size, as did venom toxicity. These factors produce a highly toxic venom with low protease activity in juvenile snakes, which facilitates efficient handling of lizards and young rodents. Analysis of gut contents of museum specimens showed that lizards constitute a major fraction of prey taken by juvenile rattlesnakes. Lizards continue to be taken with high frequency until snakes reach approx. 500 mm in total length; above this size, mammals are taken exclusively. As snakes increase in size, they feed on larger mammalian prey, and a functionally different venom is produced. Venom from adult Pacific rattle-snakes is less toxic but has high protease activity, aiding in the digestion of prey in a thermally variable environment.

The Western Rattlesnake (sensu lato, now including Crotalus oreganus) is broadly distributed across the western half of the United States, northwestern Mexico and southwestern Canada, and eight subspecies are currently recognized. Although some venom characteristics have been noted for most subspecies, a systematic study of venoms from all subspecies has not been reported. Venom was extracted from snakes collected from approximate geographic range centers for all subspecies and analyzed using SDSPAGE, MALDI-TOF mass spectrometry, enzyme and toxicity assays. Electrophoretic and mass spectrometric analyses demonstrated that small myotoxins, disintegrins and PLA2
were abundant in most venoms. PIII and PI metalloproteinases (~54 kDa and 23 kDa, respectively) were common to all venoms except C. o. concolor, C. o. caliginis and C.o. helleri. Metalloproteinase activity was highest in C. o. cerberus and lowest in C. o. concolor venoms (~100-fold difference). Conversely, C. o. concolor venom was the most toxic and C. o. cerberus venom was least toxic (15-fold difference). In general, venoms with high metalloproteinase
activity were less toxic (type I venoms), while venoms which were highly toxic showed low protease activity (type II venoms). Within the C. viridis/oreganus complex, these two extremes of venom compositional phenotypes are observed, and it appears that high metalloproteinase activity and high toxicity are incompatible qualities of these venoms. The functional significance of these biochemical characteristics likely relates to characteristics of prey consumed, and venoms with low metalloproteinase activity may constrain snake prey selection or foraging activity patterns.

In immature dendritic cells (DCs) podosomes form and turn over behind the leading edge of migrating cells. The Arp2/3 complex activator Wiskott-Aldrich Syndrome Protein (WASP) localises to the actin core of forming podosomes together with WASP-Interacting Protein (WIP). A second weaker Arp2/3 activator, cortactin, is also found at podosomes where it has been proposed to participate in matrix metalloproteinase (MMP) secretion. We have previously shown that WIP−/− DCs are unable to make podosomes. WIP binds to cortactin and in this report we address whether WIP regulates cortactin-mediated MMP activity. Using DCs derived from splenic murine precursors, we found that wild-type cells were able to localise MMPs at podosomes where matrix degradation takes place. In contrast, WIP−/− DCs remain able to synthesise MMPs but do not degrade the extracellular matrix. Infection of WIP KO DCs with lentivirus expressing WIP restored both podosome formation and their ability to degrade the extracellular matrix, implicating WIP-induced podosomes as foci of functional MMP location. When WIP KO DCs were infected with a mutant form of WIP lacking the cortactin-binding domain (WIPΔ110-170) DCs were only able to elaborate disorganised podosomes that were unable to support MMP-mediated matrix degradation. Taken together, these results suggest a role for WIP not only in WASP-mediated actin polymerisation and podosome formation, but also in cortactin-mediated extracellular matrix degradation by MMPs.

The Puerto Rican Racer Alsophis portoricensis is known to use venom to subdue lizard prey, and extensive damage to specific lizard body tissues has been well documented. The
toxicity and biochemistry of the venom, however, has not been explored extensively. We employed biological assays and proteomic techniques to characterize venom from
A. portoricensis anegadae collected from Guana Island, British Virgin Islands. High metalloproteinase and gelatinase, as well as low acetylcholinesterase and phosphodiesterase activities were detected, and the venom hydrolyzed the a-subunit of human fibrinogen very rapidly. SDS-PAGE analysis of venoms revealed up to 22 protein bands, with masses of
w5–160 kDa; very little variation among individual snakes or within one snake between venom extractions was observed. Most bands were approximately 25–62 kD, but MALDITOF
analysis of crude venom indicated considerable complexity in the 1.5–13 kD mass range, including low intensity peaks in the 6.2–8.8 kD mass range (potential three-finger toxins). MALDI-TOF/TOF MS analysis of tryptic peptides confirmed that a 25 kDa band was a venom cysteine-rich secretory protein (CRiSP) with sequence homology with tigrin,
a CRiSP from the natricine colubrid Rhabdophis tigrinus. The venom was quite toxic to NSA mice (Mus musculus: LD50 ¼ 2.1 mg/g), as well as to Anolis lizards (A. carolinensis: 3.8 mg/g). Histology of the venom gland showed distinctive differences from the supralabial salivary glands (serous vs. mucosecretory), and like the Brown Treesnake (Boiga irregularis), another rear-fanged snake, serous secretory cells are arranged in densely packed secretory tubules, with little venom present in tubule lumina. These results clearly demonstrate that venom from A. portoricensis shares components with venoms of front-fanged snakes as well as with other rear-fanged species. Venom from A. portoricensis, in particular the prominent metalloproteinase activity, likely serves an important trophic function by facilitating prey handling and predigestion of prey.

Classic glaucoma treatment focuses on intraocular pressure (IOP) reduction. Better knowledge of the pathogenesis of the disease has opened up new therapeutical approaches. Whereas most of these new avenues of treatment are still in the experimental phase, others, such as magnesium, gingko, salt and fludrocortisone, are already used by some physicians. Blood pressure dips can be avoided by intake of salt or fludrocortisone. Vascular regulation can be improved locally by carbonic anhydrase inhibitors, and systemically with magnesium or with low doses of calcium channel blockers. Experimentally, glaucomatous optic neuropathy can be prevented by inhibition of astrocyte activation, either by blockage of epidermal growth factor receptor or by counteracting endothelin. Glaucomatous optic neuropathy can also be prevented by nitric oxide-2 synthase inhibition. Inhibition of matrix metalloproteinase-9 inhibits apoptosis of retinal ganglion cells and tissue remodeling. Upregulation of heat shock proteins protects the retinal ganglion cells and the optic nerve head. Reduction of oxidative stress especially at the level of mitochondria also seems to be protective. This can be achieved by gingko; dark chocolate; polyphenolic flavonoids occurring in tea, coffee, or red wine; anthocyanosides found in bilberries; as well as by ubiquinone and melatonin. (Surv Ophthalmol 52:S174--S179, 2007. Ó 2007 Elsevier Inc. All rights reserved.)

Matrix metalloproteinases (MMPs) and tissue inhibitors (TIMPs) play a key role in the pathogenesis of multiple sclerosis (MS) and have been proposed as biomarkers of response to therapy. We investigated serum levels of several MMPs and TIMPs in 43 relapsing-remitting MS (RRMS) patients undergoing interferon-beta (IFN-b) treatment and classified as responders and non-responders based on clinical criteria. Levels of MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-2 were determined by ELISA before treatment and after 3, 6, 12, and 24 months of therapy. Neutralizing antibodies were determined by the myxovirus A induction bioassay. Treatment with IFN-b induced changes in levels of MMP-9 and TIMP-1. In contrast to non-responders, IFN-b resulted in an early and sustained increase in TIMP-1 levels in MS patients who showed clinical response to IFN-b. The early and sustained increase in TIMP-1 levels could be a marker of the response to IFN-b during the first 2 years of treatment. ava i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s ev i e r. c o m / l o c a t e / yc l i m Clinical Immunology (2009) 130, 145-150

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes, and neurons. In this study, we analyzed the influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells in a species-consistent in vitro coculture model. We were able to show a barrier integrity-decreasing impact of pericytes by transendothelial electrical resistance (TEER) and 14 C-sucrose permeability measurements. The morphology analysis revealed serrated cell borders and a shift of the endothelial morphology towards a cobblestone shape under the influence of pericytes. The analysis of the two major barrier integrity modulators vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) displayed higher MMP activity and higher levels VEGF, MMP-2, and MMP-9 in the coculture, whereas VEGF levels were decreased by the MMP inhibitor GM6001, indicating a complex interplay of both. Inhibition experiments with neutralizing VEGF antibody and GM6001 increased the TEER, which proves the involvement of VEGF and MMPs in the barrier-decreasing process. Analysis of occludin yielded decreased protein content and discontinuous expression at the endothelial cell borders under the influence of pericytes. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity via MMPs and VEGF.

Endotrypanum (order Kinetoplastida: family Trypanosomatidae) is a parasite of forest dwelling tree sloths (Edentata: genera Choleopus and Bradypus). Unique among the haemoflagellates, this protozoan has an intraerythrocytic phase in the mammalian host. Nevertheless, many striking similarities exist between Endotrypanum and the human pathogen Leishmania that make it a useful model for epidemiological and evolutionary aspects of the biology of trypanosomatids. Importantly, Endotrypanum species share both the insect vector and host reservoir with certain species of Leishmania (subgenus Viannia). Because mixed infections with Endotrypanum and Leishmania are common in sloths and, therefore, likely to occur in the sandfiy vector, there is a need for adequate biochemical markers to distinguish Endotrypanum from Leishmania infections. In this paper we show that Endotrypanum promastigotes possess sialidase and trans-sialidase activities, which are absent from Leishmania, and which are not closely related to the previously described trypanosomal sialidase/trans-sialidase enzyme. We also document the occurrence in Endotrypanum of homologues of the leishmanial surface metalloproteinase gp63 genes. The combined occurrence of sialidase/trans-sialidase activities and gp63 gene homologues in a unique organism has important ramifications for both field and laboratory studies on the biology of trypanosomatids, especially those related to host infection and evolution.

Objective: To detect the expression of plasma matrix metalloproteinase-2 (MMP-2) and MMP-9, and MMP-9:MMP-2 ratio in patients with pelvic inflammatory disease (PID). Design: A consecutive study with approximate 1:2 case-to-control ratio. Setting: University hospital. Patient(s): Forty-seven patients with PID and 80 healthy women. Intervention(s): Collected blood specimens of patients with PID before and after treatment. Main Outcome Measure(s): ELISA and gelatin zymography were used to measure the expression of plasma MMP-2 and MMP-9. Result(s): The level of plasma MMP-9 or MMP-9:MMP-2 ratio was elevated in patients with PID compared with healthy controls and decreased significantly after treatment. The activity of MMP-9, but not MMP-2, tended to be higher in 47 patients with PID before the treatment compared with that after the treatment. Pretreatment plasma MMP-9 or MMP-9:MMP-2 ratio was significantly correlated with white blood cell (WBC) and neutrophil counts. As prediction markers for PID, the sensitivities of MMP-9 and MMP-9:MMP-2 ratio were 76.6% and 76.6%, whereas the negative predictive values were 82.5% and 83.3%. Conclusion(s): Level of plasma MMP-9 and MMP-9:MMP-2 ratio may act as prediction markers for PID. In patients with PID, 80% have a plasma MMP-9 level higher than 115 ng/mL or a MMP-9:MMP-2 ratio higher than 2.15.

Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases.

Epithelial-mesenchymal crosstalk is essential for tissue morphogenesis, but incompletely understood. Postnatal mammary gland development requires epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AREG), which generally must be cleaved from its transmembrane form in order to function. As the transmembrane metalloproteinase ADAM17 can process AREG in culture and Adam17 −/− mice tend to phenocopy Egfr −/− mice, we examined the role of each of these molecules in mammary development. Tissue recombination and transplantation studies revealed that EGFR phosphorylation and ductal development occur only when ADAM17 and AREG are expressed on mammary epithelial cells, whereas EGFR is required stromally, and that local AREG administration can rescue Adam17 −/− transplants. Several EGFR agonists also stimulated Adam17 −/− mammary organoid growth in culture, but only AREG was expressed abundantly in the developing ductal system in vivo. Thus, ADAM17 plays a crucial role in mammary morphogenesis by releasing AREG from mammary epithelial cells, thereby eliciting paracrine activation of stromal EGFR and reciprocal responses that regulate mammary epithelial development.

Viperid snakebite envenomation induces blistering and dermonecrosis. The pathological alterations induced by a snake venom metalloproteinase in the skin were investigated in a mouse ear model. Metalloproteinase BaP1, from Bothrops asper, induced rapid edema, hemorrhage, and blistering; the latter two effects were abrogated by preincubation with the metalloproteinase inhibitor batimastat. Neutrophils did not play a role in the pathology, as depletion of these cells resulted in a similar histological picture. Blisters are likely to result from the direct proteolytic activity of BaP1 of proteins at the dermal-epidermal junction, probably at the lamina lucida, as revealed by immunostaining for type IV collagen and laminin. Widespread apoptosis of keratinocytes was detected by the TUNEL assay, whereas no apoptosis of capillary endothelial cells was observed. BaP1 induced a drastic reduction in the microvessel density, revealed by immunostaining for the endothelial marker vascular endothelial growth factor receptor-2. This was followed by a rapid angiogenic response, leading to a partial revascularization. Skin damage was followed by inflammation and granulation tissue formation. Then, a successful re-epithelization process occurred, and the skin of the ear regained its normal structure by 2 weeks. Venom metalloproteinase-induced skin damage reproduces the pathological changes described in snakebitten patients.

metaloproteinasa (MMP) 9, enzima involucrada en inflamación, y su contraparte, el inhibidor tisular de la metaloproteinasas tipo 1 (TIMP-1), se encuentran significativamente (P < 0,01) incrementados en pacientes con asma o con enfermedad pulmonar obstructiva crónica (EPOC) en comparación con los grupos control del mismo rango etario. El incremento de ambos parámetros se hace más significativo en los pacientes severos de ambos grupos (P < 0,0001) y particularmente en los pacientes con EPOC severo en comparación con los asmáticos severos (P < 0,01). El incremento en los niveles séricos de MMP-9 en relación con severidad es mayor que lo observado para TIMP-1 en ambas patologías. Se concluye que los niveles séricos de MMP-9 y TIMP-1 pueden ser un marcador importante para determinar severidad en estas enfermedades.

Intracerebral hemorrhage (ICH) initiates an inflammatory response with secondary growth of hemorrhage and cell death. Matrix metalloproteinase (MMP) gelatinolytic activity is increased in ICH, and synthetic inhibitors to MMPs reduce edema and hemorrhage size. Recently, we found that tissue inhibitor of metalloproteinase-3 (TIMP-3) is elevated after ischemia and colocalizes with TUNEL (terminal deoxynucleotidyl transferase-mediated 2 0 -deoxyuridine 5 0 -triphosphate-biotin nick endlabeled)-labeled cells. Tissue inhibitor of metalloproteinase-3 promotes neuronal apoptosis in vitro by blocking the shedding of the tumor necrosis factor (TNF) superfamily of death receptors/ligands by stromelysin-1 (MMP-3). However, the effect of TIMP-3 and synthetic MMP inhibitors on cell death in ICH is unclear. Therefore, we used the collagenase-induced intracerebral hemorrhage (CIH) model in Timp-3 knockout and C57Bl/6 wild-type mice to study MMP expression, hemorrhage volume, and cell death. Real-time PCR showed an increase in Mmp-3 mRNA in CIH, but similar Mmp-2 and -9 mRNA expression levels in CIH and saline-injected mice. Protein levels of pro and cleaved MMP-3 were increased in CIH, and zymographic gelatinolytic activity of MMP-9 was elevated after CIH at 72 h, suggesting an exogenous source. Apoptosis was shown by increased caspase-3 levels at 2 and 72 h, and active caspase-8 by 2 and 24 h. The Timp-3 null mouse and wild types had similar hemorrhage sizes and TUNEL-labeled cells. Unexpectedly, the broad-spectrum MMP inhibitor BB-94 increased hemorrhage size and TUNEL-labeled cells. Our results fail to implicate TIMP-3 in apoptosis in CIH, but show that BB-94 increased apoptosis in CIH, possibly by blocking shedding of TNF death receptors and/or their ligands.

Vascular remodelling occurs during all stages of atherosclerotic progression. Anti-atherosclerotic drugs may function by restoring regulation of the processes involved in remodelling of the extracellular matrix. A key group of enzymes involved Ž . in these processes are the matrix metalloproteinases MMPs . Oestrogens have been demonstrated to possess anti-atherosclerotic properties at low concentrations while being associated with lesion formation at high concentrations. We examined Ž . the effect of 17b-oestradiol on MMP-2 expression in human coronary artery CAVSMC and umbilical artery vascular Ž . smooth muscle cells UAVSMC . MMP-2 expression was measured by chemiluminescent immunoblotting and quantified by laser densitometry. pro-MMP-2 was secreted by VSMCs and increasing levels of 17b-oestradiol, from physiological through supraphysiological, were associated with significant dose-dependent increases in MMP-2 levels in culture media. This effect was dependent on de novo protein synthesis and could be antagonised by the oestrogen receptor antagonist, tamoxifen, and the specific receptor antagonist ICI 182,780. 17b-Oestradiol appears to be a specific stimulator of MMP-2 release from human vascular cells. The concentration dependence of this effect suggests a basis for the differential effects of low and high oestrogen levels on vascular integrity. q 1998 Elsevier Science B.V.

Two plant-transformation-competent large-insert binary clone bacterial artificial chromosome (hereafter BIBAC) libraries were previously constructed for soybean cv. Forrest, using BamHI or HindIII. However, they are not well suited for clone-based genomic sequencing due to their larger ratio of vector to insert size (27.6 kbp:125 kbp). Therefore, we developed a largerinsert bacterial artificial chromosome (BAC) library for the genotype in a smaller vector (pECBAC1), using EcoRI. The BAC library contains 38,400 clones; about 99.1% of the clones have inserts; the average insert size is 157 kbp; and the ratio of vector to insert size is much smaller (7.5 kbp:157 kbp). Colony hybridization with probes derived from several chloroplast and mitochondrial genes showed that 0.89% and 0.45% of the clones were derived from the chloroplast and mitochondrial genomes, respectively. Considering these data, the library represents 5.4 haploid genomes of soybean. The library was hybridized with six RFLP marker probes, 5S rDNA and 18S-5.8S-25S rDNA, respectively. Each RFLP marker hybridized to about six clones, and the 5S and 18S-5.8S-25S rDNA probes collectively hybridized to 402 BACsabout 1.05% of the clones in the library. The BAC library complements the existing soybean Forrest BIBAC libraries by using different restriction enzymes and vector systems. Together, the BAC and BIBAC libraries encompass 13.2 haploid genomes, providing the most comprehensive clone resource for a single soybean genotype for public genome research. We show that the BAC library has enhanced the development of the soybean whole-genome physical map and use of three complementary BAC libraries improves genome physical mapping by fingerprint analysis of most of the clones of the library. The rDNA-containing clones were also fingerprinted to evaluate the feasibility of constructing contig maps of the rDNA regions. It was found that physical maps for the rDNA regions could not be readily constructed by fingerprint analysis, using one or two restriction enzymes. Additional data to fingerprints and/or different fingerprinting methods are needed to build contig maps for such highly tandem repetitive regions and thus, the physical map of the entire soybean genome.

During progression from benign nevus to vertical growth phase melanoma, melanocytes acquire the ability to invade into the dermis. This process requires rupture of the basal lamina and dissolution of dermal type I collagen. Metastases-derived human melanoma MIM cells have an invasive ability in vitro which is dependent on metalloproteinases. In the present study Ž. we analysed the role of type I collagenase MMP-1 in melanoma invasion using MIM cells in which the constitutive expression of MMP-1 was suppressed by stable transfection with a plasmid vector expressing a 777 bp antisense fragment of MMP-1 genomic DNA. Two clones were isolated in which MMP-1 mRNA expression was blocked by 90-96% with a corresponding loss in protein synthesis. In their morphological appearance and growth rate in vitro these cells were indistinguishable from wild type cells or control cells transfected with the same vector expressing the MMP-1 fragment in Ž. the sense orientation. Their mRNA and protein levels for type IV collagenase MMP-2 were unchanged as assessed by Northern and Western blot analyses and by gelatin zymography. However, when the invasive ability of the cells was measured, we found that in addition to type I collagen, invasion through type IV collagen and a reconstituted, type IV Ž. collagen-containing basement membrane Matrigel were also significantly inhibited as compared to normal or sense-transfected cells. The results indicate that despite the presence of functional MMP-2, degradation of type IV collagen matrices by the melanoma cells was dependent on expression of MMP-1.

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k a = 3.54 AE 0.03 Â 10 4 M À1 s À1 and k d = 1.16 AE 0.07 Â 10 À5 s À1 , resulting in an equilibrium dissociation constant (K D ) of 0.33 AE 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies. . Kinetic analysis of the DM43 interaction to the metalloproteinase jararhagin. Black lines correspond to the experimental data obtained by the injection of 325 to 20nM DM43 over a MAJar2/jararhagin surface. Red lines correspond to the simulated curves described by k a = 3.54 AE 0.03 Â 10 4 M À1 s À1 and k d = 1.16 AE 0.07 Â 10 À5 s À1 according to a simple bimolecular interaction model (A + B ↔ AB). Each DM43 injection was performed in duplicate. The residual plot is shown below.

Bothrops jararaca venom (Bjv) is known to induce local inflammation and severe pain. Since, mast cells are able to secrete mediators involved in algesic processes, in this study we examined the putative role of these cells in the hyperalgesia triggered by Bjv in the rat paw. We noted that treatment with mast cell stabilizer sodium cromoglicate as well as with histamine and 5hydroxytriptamine receptor antagonists meclizine and methysergide, respectively, inhibited the Bjv-induced hyperalgesia. In addition, we showed that stimulation of isolated rat peritoneal mast cells with Bjv in vitro resulted in the release of stored and neo-generated inflammatory mediators such as histamine and leukotriene C 4 , respectively. Bjv-induced histamine secretion was clearly sensitive to treatment with sodium cromoglicate and sodium nedocromil. We further observed that metalloproteinase inhibitors 1,10-phenantroline and DM43 inhibited mast cell degranulation in vitro, under conditions where inhibitors of phospholipase A 2 as well as of serine-and cysteine-proteinases were inactive. Altogether, our findings indicate that mast cells seem to contribute to the hyperalgesia caused by Bjv in the rat paw, and also provide evidence that this response might be dependent on the ability of the Bjv to activate directly mast cells.

. The expression of matrix metalloproteinases MMP with gelatinase activity was found in the whole hemolymph of the marine mussel Mytilus gallopro¨incialis Lam. Cleavage activity was specific for gelatin; very little activity towards human type-IV collagen, and no activity for cold fish gelatin, casein or bovine serum albumin were detected. EDTA and 1,10-phenanthroline were inhibitory, suggesting that mussel MMPs require divalent cations for their proteolytic activity; in fact, the presence of exogenously added divalent ions significantly protected the MMPs from inhibition. No inhibition was detected with serine or cysteine proteinase inhibitors. The specific vertebrate inhibitors as well as the classical vertebrate activator of MMPs were without effect, whereas sulphydryl reducing agents had a strong inhibitory effect. Mussel MMPs showed an exponential curve of thermal-dependent decay that was not protected by the presence of metal ions. Overall the results indicate both similarities and differences between invertebrate and vertebrate gelatinases, providing information for understanding the biological role of these ancient proteinases. ᮊ F. Mannello .

Abbreviations: ADAM, a disintegrin and metalloproteinase; a.m.u., atomic mass unit; MHD, minimum hemorrhagic dose; MMP, matrix metalloproteinase; SVMP, snake venom metalloproteinase. Enzymes: atrolysin C (EC 3.4.24.42); jararhagin (EC 3.4.24.73). q FEBS 2001 The snake venom metalloproteinase inhibitor BJ46a (Eur. J. Biochem. 268) 3043 q FEBS 2001 The snake venom metalloproteinase inhibitor BJ46a (Eur. J. Biochem. 268) 3045 q FEBS 2001 The snake venom metalloproteinase inhibitor BJ46a (Eur. J. Biochem. 268) 3049 q FEBS 2001 The snake venom metalloproteinase inhibitor BJ46a (Eur. J. Biochem. 268) 3051

In some animals, such as mice and guinea pigs, a hormonally controlled mechanism increases the flexibility of the pubic symphysis and enhances the cervical remodeling necessary for safe delivery. Cervical ripening during pregnancy is associated with a paradoxical influx of leukocytes. However, the changes in cell metabolism during relaxation of the mouse pubic symphysis for delivery have not been extensively studied. In this work, we used light microscopy and transmission and scanning electron microcopy, as well as immunohistochemistry and Western blotting for MMP-8, to investigate the involvement of granulocytes or resident stromal cells in the relaxation of the virgin pubic symphysis during late pregnancy (days 18 and 19, before delivery) in vivo and in explanted joints. MMP-8 was studied because this collagenase is a hallmark for cervical ripening associated with the influx of granulocytes during late pregnancy. Extensive dissolution and disorganization of the extracellular matrix was seen around fibroblastic-like cells in late pregnancy. In contrast to the cervix (positive control), morphological and immunohistochemical analyses revealed that there was no characteristic cellular inflammatory response in the interpubic tissue. Staining for MMP-8 was observed in chondroid and fibroblastic-like cells of virgin and relaxed interpubic ligament, respectively. However, no granulocytes were seen during the extensive remodeling of the pubic joint in late pregnancy. These results indicate that constitutive stromal cells may have an important role in tissue relaxation during remodeling of the pubic symphysis in pregnancy. Microsc. Res. Tech. 00:000-000, 2007.

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases.

The Metalloproteinase (Mep) gene of Puccinia helianthi was expressed in Escherichia coli in order to produce the mature protein. The hydrolytic activity of this enzyme was tested, and the subcellular localization of Mep was detected. The mature peptide cDNA of P. helianthi Mep, when introduced into E. coli, expressed a protein with a size, amino acids sequence identical to the putative fungal enzyme. The recombinant expression products of Mep gene could hydrolyze azocasein and the hydrolytic activity was suppressed by metal ion chelating agents such as 1,10-phenanthroline and ethylene diamine tetraacetic acid (EDTA). Confocal laser scanning microscopy of the leaves from a 2-day-old Nicotiana benthamiana seedlings expressed Mep-GFP fusion that showed green fluorescent protein (GFP) signal accumulated in the cell wall, or the plasma membrane of epithelial cells. Mep may serve as a pathogenicity factor contributing to P. helianthi penetration and infection.

Keywords: Metalloproteinase, Puccinia helianthi, Prokaryotic expression, Hydrolytic activity, Subcellular localization

Two homologous P-III hemorrhagic metalloproteinases were purified from Russell's viper venoms from Myanmar and Kolkata (eastern India), and designated as daborhagin-M and daborhagin-K, respectively. They induced severe dermal hemorrhage in mice at a minimum hemorrhagic dose of 0.8e0.9 mg. Daborhagin-M specifically hydrolyzed an Aa-chain of fibrinogen, fibronectin, and type IV collagen in vitro. Analyses of its cleavage sites on insulin chain B and kinetic specificities toward oligopeptides suggested that daborhagin-M prefers hydrophobic residues at the P 1 , P 1 0 , and P 2 0 positions on the substrates. Of the eight Daboia geographic venom samples analyzed by Western blotting, only those from Myanmar and eastern India showed a strong positive band at 65 kDa, which correlated with the high risk of systemic hemorrhagic symptoms elicited by Daboia envenoming in both regions. The full sequence of daborhagin-K was determined by cDNA cloning and sequencing, and then confirmed by peptide mass fingerprinting. Furthermore, molecular phylogenetic analyses based on 27 P-IIIs revealed the co-evolution of two major P-III classes with distinct hemorrhagic potencies, and daborhagin-K belongs to the most hemorrhagic subclass. By comparing the absolute complexity profiles between these two classes, we identified four structural motifs probably responsible for the phylogenetic subtyping and hemorrhagic potencies of P-III SVMPs.

Leucurolysin-a (leuc-a), a 23 kDa non-hemorrhagic metalloproteinase, is found in venom of the viper Bothrops leucurus. Here, we examine the biological consequences of leuc-a, including thrombolytic activity, direct effects on endothelial cells in culture and edematogenic activity in vivo. We demonstrate fibrinolytic activity of leuc-a, in which the protease specifically degrades a, b, and g2g chains. While not causing hemorrhaging, leuc-a does cause thrombolytic activities in whole blood clots. Endothelial cells are highly resistant to leuc-a in culture. Cell viability suffered only when cells were exposed to large quantities of the protease. Nevertheless, leuc-a induces changes in cell morphology. The impact of leuc-a on cell adhesion was confirmed by an adhesion assay, in which cell adhesion to fibronectin decreased due to leuc-a. This mild cellular impact is unlike that of crude venom, where lower concentrations triggered cell death and a greater reduction in cell adhesion. Also, leuc-a increased microvessel permeability with marked edema in mice peritoneum and foot pads. These effects are similar to those of other P-I class SVPMs. These in vivo effects were weaker when crude venom was tested. In conclusion, albeit not showing significant hemorrhagic activity, leuc-a can induce a prominent edema which appears to be significant in the local effects observed after B. leucurus venom accidents. r

metaloproteinasa (MMP) 9, enzima involucrada en inflamación, y su contraparte, el inhibidor tisular de la metaloproteinasas tipo 1 (TIMP-1), se encuentran significativamente (P < 0,01) incrementados en pacientes con asma o con enfermedad pulmonar obstructiva crónica (EPOC) en comparación con los grupos control del mismo rango etario. El incremento de ambos parámetros se hace más significativo en los pacientes severos de ambos grupos (P < 0,0001) y particularmente en los pacientes con EPOC severo en comparación con los asmáticos severos (P < 0,01). El incremento en los niveles séricos de MMP-9 en relación con severidad es mayor que lo observado para TIMP-1 en ambas patologías. Se concluye que los niveles séricos de MMP-9 y TIMP-1 pueden ser un marcador importante para determinar severidad en estas enfermedades.

A fibrino(geno)lytic nonhemorrhagic metalloproteinase (BleucMP) was purified from Bothrops leucurus snake venom by two chromatographic steps procedure on DEAE-Sephadex A-25 followed by CM-Sepharose Fast Flow column. BleucMP represented 1.75% (w/w) of the crude venom and was homogeneous on SDS-PAGE. BleucMP analyzed by MALDI TOF/TOF, showed a molecular mass of 23,057.54Da and when alkylated and reduced, the mass is 23,830.40Da. Their peptides analyzed in MS (MALDI TOF\TOF) showed significant score when compared with those of other proteins by NCBI-BLAST2 alignment display. As regards their proteolytic activities, BleucMP efficiently acted on fibrinogen, fibrin, and was inhibited by EDTA and 1.10-phenanthroline. This enzyme was also able to decrease significantly the plasma fibrinogen level provoking blood incoagulability, however was devoid of hemorrhagic activity when tested in the mice skin and did not induce relevant biochemical, hematological and histopathological alterations in ...

Untimely rupture of the fetal membranes (FMs) is a major precipitant of preterm birth. Although the mechanism of FM weakening leading to rupture is not completely understood, proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 beta (IL1B), have been shown to weaken FMs concomitant with the induction of reactive oxygen species, collagen remodeling, and prostaglandin release. We hypothesized that alpha-lipoic acid, a dietary antioxidant, may block the effect of inflammatory mediators and thereby inhibit FM weakening. Full-thickness FM fragments were incubated with control media or TNF, with or without alpha-lipoic acid pretreatment. Fetal membrane rupture strength and the release of matrix metalloproteinase 9 (MMP9) and prostaglandin E 2 (PGE 2 ) from the full-thickness FM fragments were determined. The two constituent cell populations in amnion, the mechanically strongest FM component, were similarly examined. Amnion epithelial and mesenchymal cells were treated with TNF or IL1B, with or without alpha-lipoic acid pretreatment. MMP9 and PGE 2 were analyzed by ELISA, Western blot, and zymography. TNF decreased FM rupture strength 50% while increasing MMP9 and PGE 2 release. Lipoic acid inhibited these TNF-induced effects. Lipoic acid pretreatment also inhibited TNF-and IL1B-induced increases in MMP9 protein activity and release in amnion epithelial cells, as well as PGE 2 increases in both amnion epithelial and mesenchymal cells. In summary, lipoic acid pretreatment inhibited TNF-induced weakening of FM and cytokine-induced MMP9 and PGE 2 in both intact FM and amnion cells. We speculate that dietary supplementation with alpha-lipoic acid might prove clinically useful in prevention of preterm premature rupture of fetal membranes.

Endothelin converting enzyme (ECE) from intact cells of a permanent human endothelial cell line, EA.hy926, was studied by examining the effects of phosphoramidon, an endothelin converting enzyme inhibitor, on the levels of secreted endothelin-1 and big endothelin-1. The specific ECE activity was demonstrated by a phosphoramidon dose-dependent decrease in ET-1 level with a concomitant increase in big ET-1 level. By using a specific neutral endopeptidase 24.11 (NEP 24.11) inhibitor, thiorphan, it was also shown that the phosphoramidon-sensitive ET-1 degrading activity in this cell line is due to the NEP 24.11 activity. Other serine, acid, and cysteine protease inhibitors had no effect on the endogenous synthesis of ET-1 and big ET-1 supporting the evidence that ECE is insensitive to these protease inhibitors as has been demonstrated with the isolated enzyme.

Expression of telomerase (hTERT) in certain cell types has been shown to extend cellular life span without malignant transformation. We studied the phenotype of 26 telomerase-transduced fibroblast clones (TTFC) generated from a mass culture of hTERT retrovirally transduced MRC-5 cells. About two-thirds of the transduced clones senesced at the expected time or shortly thereafter, despite high levels of expression of telomerase and telomere length maintenance. The remaining one-third of the clones were "immortalized" (followed for over 200 cumulative population doublings ). All clones maintained a nontransformed phenotype: contact inhibition, anchorage dependency, lack of tumor formation in nude mice, dose dependency to serum and growth factors, low expression of a matrix metalloproteinase associated with metastatic invasion (MMP-9) and high expression of its inhibitor TIMP-1, and no cytogenetic abnormalities by G-banding. In addition, fibroblast-specific biological parameters, such as colony size, production of collagenase, and response to MMC and gamma radiation were tightly regulated at the clonal and subclonal levels.