Gelatin Substrate Research Articles

Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.

In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers completely after 7days at room temperature and produced the highest levels of keratinase (446Uml(-1)). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15-140kDa range, and B. cereus produced a keratinase of ~200kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40-50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50-70°C and pH 7.0-11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate.

Proteolytic enzyme production by strains of the insect pathogen xenorhabdus and characterization of an early-log-phase-secreted protease as a potential virulence factor.

As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.

Cloning, characterisation and heterologous expression of an astacin metalloprotease, Sc-AST, from the entomoparasitic nematode Steinernema carpocapsae

Steinernema carpocapsae is a parasitic nematode that is high virulent to insects. The parasitic juvenile reaches the insect haemocoelium by passing through mid-gut barriers and develops there. During invasion, the nematode was predicted to express a large set of proteases, including metalloproteases, one of which was sequenced and expressed in this work. A 1583-nucleotide cDNA encoding a putative metalloprotease containing a 28-aa signal peptide, a 79-aa propeptide and a 311-aa mature protease with a predicted molecular mass of 35.2kDa and a theoretical pI of 5.9 was cloned from the parasitic stage of the nematode. Sequence analyses predicted signature sequences of the astacin metalloprotease family, an astacin domain, a zinc-binding motif and a methionine turn motif; therefore, this protein was identified as an astacin and designated as Sc-AST. The astacin domain of Sc-AST has an amino acid sequence homology of 46% to prototypical astacin from Astacus astacus and 82% to Caenorhabditis elegans NAS-8. Like NAS-8 of C. elegans, Sc-AST has a C-terminal ShK toxin domain. Recombinant Sc-AST was produced in an Escherichia coli system and was purified by affinity chromatography. Maldi-MS/MS analysis of purified recombinant protein matched the Sc-AST sequence with a significance score of 499. Sc-AST was produced in the correct folding conformation, showed activities against gelatin and azocasein substrates and was inhibited by divalent metal-chelating agents. Sc-AST presented an optimum pH of 7.5 and temperature of 37°C and Km, Vmax and kcat values of 1.86mM, 0.281μM/min and 27.9s−1, respectively. Expression analyses indicated that Sc-AST is up-regulated in the parasitic stage and is strongly induced in vitro by insect tissues, thus suggesting that it plays a role in the parasitic process.

CHARACTERIZATION OF THREE NOVEL ALKALINE SERINE PROTEASES FROM TOMATO (LYCOPERSICUM ESCULENTUM MILL.) FRUIT AND THEIR POTENTIAL APPLICATION

In order to quest new potential plant protease and make it industrially applicable and cost-effective, three novel proteases were detected in tomato (Lycopersicum esculentum Mill.) fruit by sodium dodecyl sulfate (SDS)–gelatin–polyacrylamide gel electrophoresis. All three proteases were isolated and partially purified by heat treatment (70C, 5 min) and ethanol precipitation (0–33%, v/v), with ninefold increase in specific activity and 31.6% recovery. These proteases exhibited the highest specificity for gelatin substrate and were indentified as serine protease. The optimum pH and temperature of proteolytic activity were pH 8–10 and 37–60C, respectively. They were stable at high pH (up to 12) and temperature (up to 70C) and in the presence of 20 mM metal ions (Na+, Ba2+, Ca2+, Mg2+, Mn2+), surfactants (SDS and 80 mM Triton X-100) and denaturants (urea and dithiothreitol). These results suggested that proteases from tomato fruit had potential application in food, detergent, tannery and medical industries. PRACTICAL APPLICATIONS Plant cysteine proteases such as papain, bromelain and ficin are extensively applied in many industries. Recently, several plant serine proteases, which were more stable under demanding conditions than cysteine proteases, were exploited for industrial applications. Tomato is one of the highest produced and consumed vegetables in the worldwide. Alkaline serine proteases from tomato fruit in this paper were stable at high pH and temperature and in the presence of metal ions and oxidation. Enzyme activity against a wide variety of nature protein substrates indicated that they would be investigated for a range of potential commercial applications especially for gelatin and casein hydrolysis in the food processing industry as well as for the dissolution of casein and bovine hemoglobin in detergent industry.

Wide Proteolytic Activity Survey Reinforces Heterogeneity Among Trypanosoma cruzi TCI and TCII Wild Populations

Chagas disease, caused by the flagellate protozoan Trypanosoma cruzi, is characterized by considerable variation in both incidence and infection severity. This variation has been attributed to a set of complex features including the host genetic background, environmental and social factors, and the genetic heterogeneity of parasite populations. Using biochemical and molecular markers these populations can be divided into two major groups (TCI and TCII). In a previous work, our group identified cysteine and metalloprotease activities as good markers for differentiating TCI from TCII wild isolates, with a higher level of heterogeneity observed among TCII isolates. In this investigation, we applied the protease activity assay to a sample of 49 sylvatic T. cruzi isolates that had been previously assessed in terms of their Swiss mice infection patterns. Protease activity profiles were determined at pH 5.5 and 10.0 and was compared with the original host species, phylogenetic lineage, and mice infection characteristics. Substantial variability, with molecular weights ranging from 35 to 220 kDa for active proteases at pH 5.5, and of 30 to 90 kDa for active proteases at pH 10.0, was observed in gelatin substrate gels, with no phenetic separation between TCI and TCII groups or original hosts. The combinatorial expression of proteases recorded among individual isolates may account for the diverse behavior observed for parasite populations in nature.

VAMP3, syntaxin-13 and SNAP23 are involved in secretion of matrix metalloproteinases, degradation of the extracellular matrix and cell invasion

Cellular remodeling of the extracellular matrix (ECM), an essential component of many physiological and pathological processes, is dependent on the trafficking and secretion of matrix metalloproteinases (MMPs). Soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic has documented roles in cell-ECM interactions and the present study specifically examines SNARE function in the trafficking of MMPs during ECM degradation. Using the invasive human fibrosarcoma cell line HT-1080, we demonstrate that a plasma membrane SNARE, SNAP23, and an endosomal v-SNARE, VAMP3 (also known as cellubrevin), partly colocalize with MMP2 and MMP9, and that inhibition of these SNAREs using dominant-negative SNARE mutants impaired secretion of the MMPs. Inhibition of VAMP3, SNAP23 or syntaxin-13 using dominant-negative SNARES, RNA interference or tetanus toxin impaired trafficking of membrane type 1 MMP to the cell surface. Consistent with these observations, we found that blocking the function of these SNAREs reduced the ability of HT-1080 cells to degrade a gelatin substrate in situ and impaired invasion of HT-1080 cells in vitro. The results reveal the importance of VAMP3, syntaxin-13 and SNAP23 in the trafficking of MMP during degradation of ECM substrates and subsequent cellular invasion.

Reduction of Lysyl Hydroxylase 3 Causes Deleterious Changes in the Deposition and Organization of Extracellular Matrix

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF(+/-)) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF(+/-), may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family.

Cleaved high molecular weight kininogen, a novel factor in the regulation of matrix metalloproteinases in vascular smooth muscle cells

We previously reported that Brown Norway Katholiek rats, which feature a deficiency of plasma kininogens, develop severe abdominal aortic aneurysm. Increased activity of matrix metalloproteinases (MMPs) in the aortic wall, leading to degradation of extracellular matrix components, is considered to play a crucial role in aneurysm formation. Using an in vitro model of vascular smooth muscle cells (VSMCs), cultured from the rat aorta, we investigated whether the cleaved form of high molecular weight kininogen, designated HKa, affects the expression of MMP-9 and MMP-2 and their tissue inhibitors (TIMPs). Treatment of VSMCs with HKa reduced in a concentration-dependent manner IL-1α-induced release of MMP-9 and MMP-2, associated with decreased MMP enzymatic activity levels in conditioned media, as demonstrated by gelatin zymography and fluorescein-labeled gelatin substrate assay, respectively. Real-time PCR revealed that HKa reduced corresponding MMP-9 mRNA levels. Further investigations showed that this effect did not result from a modified rate of MMP-9 mRNA degradation. TIMP-1 mRNA levels, already increased as a result of cytokine-stimulation, were significantly enhanced by HKa. Furthermore, we found elevated basal mRNA expression levels of MMP-2 and TIMP-2 in VSMCs derived from kininogen-deficient Brown Norway Katholiek rats. These results demonstrate for the first time that HKa affects the regulation of MMPs in VSMCs.

Doxycycline-Mediated Inhibition of Choroidal Neovascularization

Doxycycline, a broad-spectrum antibiotic, has certain antiangiogenic properties and can inhibit matrix metalloproteinases (MMPs/gelatinases). The authors investigated the effects of doxycycline on choroidal neovascularization (CNV) and regulation of MMP-2 and -9 and pigment epithelium-derived factor (PEDF). Doxycycline was orally administered to rats at 500, 50, 5, and 0.5 mg/kg/d; nontreated animals were used as controls. Experimental CNV was induced with laser 7 days after doxycycline treatment started. At 7 days after induction, animals were euthanatized, and eyes were collected. RPE/choroid flatmounts were labeled with isolectin IB4 to determine CNV lesion volumes using confocal microscopy and high-performance 3D imaging software. MMP-2, MMP-9, and PEDF protein levels were determined by ELISA. MMP catalytic activity was determined in solution using fluorogenic gelatin and peptide substrates, by gelatin zymography in SDS-PAGE, and by in situ fluorogenic substrate zymography in RPE/choroid sections. CNV complex lesion volumes decreased with doxycycline in a dose-response relationship. A dosage of 500 mg/kg/d caused a 70% inhibition of CNV complex volume compared with control animals. Doxycycline elevated PEDF levels in plasma and did not affect the active and pro-enzymes MMP-2 and MMP-9 levels. However, the in vitro enzymatic activities of purified MMP-2 and MMP-9 declined significantly with doxycycline. MMP-2, MMP-9, and gelatinolytic activities in situ increased early in CNV lesion development. Doxycycline treatments and exogenous additions inhibited gelatinolytic activities in CNV lesions. Doxycycline effectively hampered the progression of experimental CNV. The results suggest that orally administrated doxycycline can reach the choroid to attenuate proteolytic enzymes that remodel Bruch's membrane and promote the antiangiogenic PEDF to inhibit neovascularization.

Kinetics of inhibition of purified bovine neutrophil matrix metalloproteinase 9 by low–molecular-weight inhibitors

To measure the effects of lowmolecular-weight inhibitors on the activity of bovine neutrophil matrix metalloproteinase 9 (MMP-9). Bovine MMP-9 purified from bovine neutrophilconditioned medium. Neutrophils were degranulated by stimulation with phorbol ester. Enzyme purification was performed by use of gelatin affinity and gel-filtration chromatography. Activated enzyme was incubated with inhibitors prior to addition of substrate (gelatin fluorescein conjugate or fluorogenic peptide). Rates of enzymatic cleavage were determined by monitoring fluorescence as the reactions progressed. Values of IC(50) (molar concentration of compound that inhibits specific activity by 50%) and K(I) (in vitro inhibition constant) were determined. Rates of enzymatic activity of monomeric and dimeric bovine MMP-9 measured by use of gelatin and peptide substrates were linear with respect to time and concentrations of enzyme and substrate. The MMP-9 was potently inhibited by hydroxamic acids (IC(50) for gelatin, 29.2 to 55.7 nM; IC(50) for peptide, 4.8 to 24.6 nM; K(I), 0.2 to 0.5 nM), whereas tetracyclines (IC(50) for gelatin, 30.1 to 112.7 MM; IC(50) for peptide, 48.0 to 123.8 MM; K(I), 25.2 to 61.4 microM) and chlorhexidine (IC(50) for gelatin, 139.1MM; IC(50) for peptide, 672.5MM to 1.7 mM; K(I), 495.0 to 663.0 MM) had limited inhibition. Gelatinase-specific inhibitor SB-3CT had intermediate potency (IC(50) for peptide, 185.0 to 290.0 nM; K(I), 66.5 to 86.0 nM). Bovine MMP-9 was potently inhibited by hydroxamic acids and gelatinase inhibitor. These compounds may be useful as modulators of neutrophil-mediated protease activity in cattle.

Sperm acrosin: liberation from the acrosome and activity of the free proteinase in the presence of nonoxinol-9.

The effects of the non-ionic detergent nonoxinol-9 and a vaginal contraceptive containing nonoxinol-9 on the liberation of the cellular proteinase acrosin from human spermatozoa and on the activity of the solubilized proteinase were investigated. Detachment of acrosin from the acrosome was observed in the presence of 0.01 to 1% nonoxinol-9 as assessed by activity assays and the gelatin substrate film technique. Activity of the free proteinase was modulated by nonoxinol-9 in a biphasic manner, i.e. lower concentrations of the detergent (0.001 to 0.1%) stimulated the activity of acrosin up to 180%, while higher concentrations of the detergent (1%) resulted in an extensive inhibition of acrosin (30% residual activity). It is demonstrated that local intravaginal nonoxinol-9 concentrations of 1% equalling the post-coital concentration of the intravaginal suppository Patentex oval when properly applied will result in an effective inhibition of the liberated sperm proteinase acrosin thus preventing unspecific proteolysis of the vaginal epithelia.

On the Teratogenesis of Round-Headed Spermatozoa: Investigations with Antibodies Against Acrosin, an Intraacrosomally Located Acrosin-Inhibitor, and the Outer Acrosomal Membrane

Acrosin, the outer acrosomal membrane (OAM) and an acrosin inhibitor were studied in testicular cells and ejaculated spermatozoa of fertile men and in those of an infertile patient with exclusively round-headed spermatozoa in his ejaculates. The investigations were performed with the aid of immunohistochemical techniques using specific antibodies against the three acrosomal markers isolated from boar spermatozoa. The spermatozoa of fertile men exhibit staining for acrosin, the OAM and the acrosin inhibitor in the cap region while the round-headed spermatozoa of the patient are totally negative for the three markers, clearly supporting the conclusions of other authors that round-headed spermatozoa lack acrosomes. The lack of the acrosin system was further substantiated by the gelatin substrate film technique. In the course of normal human spermatogenesis acrosin, the OAM and the acrosin inhibitor we first demonstrable in early round spermatids, namely in identical compartments adjacent to the cell nucleus. During spermatid differentiation the staining for the three markers becomes flattened over the nucleus, resulting in a cap-like structure in testicular spermatozoa. In contrast to the ejaculated round-headed spermatozoa, the early round spermatids in the testis of the infertile patient exhibit fluorescent staining for the three markers in the region adjacent to the nuclear membrane. In the course of further spermiogenesis, the staining did not extend over the nuclear membrane, as was observed during normal spermiogenesis, but became separated from the nuclear membrane, as was observed during normal spermiogenesis, but became separated from the nuclear membrane, was translocated at various locations in the cytoplasm and was finally eliminated with the loss of the cytoplasm. These results are in accordance with the results of electron microscopically investigations on the teratogenesis of round-headed spermatozoa. Furthermore, the developmental pattern of the acrosin inhibitor during normal and abnormal spermiogenesis supports the intraacrosomal location of the acrosin inhibitor recently described by Tschesche et al. (1982).

Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss)

Here, we describe optimal conditions for the culture of rainbow trout (Oncorhynchus mykiss) pre-adipocytes obtained from adipose tissue and their differentiation into mature adipocytes, in order to study the endocrine control of adipogenesis. Pre-adipocytes were isolated by collagenase digestion and cultured on laminin or 1% gelatin substrate. The expression of proliferating cell nuclear antigen was used as a marker of cell proliferation on various days of culture. Insulin growth factor-I stimulated cell proliferation especially on days 5 and 7 of culture. Tumor necrosis factor alpha (TNFalpha) slightly enhanced cell proliferation only at a low dose. We verified the differentiation of cells grown in specific medium into mature adipocytes by oil red O (ORO) staining. Quantification of ORO showed an increase in triglycerides throughout culture. Immunofluorescence staining of cells at day 11 revealed the expression of CCAAT/enhancer-binding protein and peroxisome proliferator-activator receptor gamma, suggesting that these transcriptional factors are involved in adipocyte differentiation in trout. We also examined the effect of TNFalpha on the differentiation of these adipocytes in primary culture. TNFalpha inhibited the differentiation of these cells, as indicated by a decrease in glycerol-3-phosphate dehydrogenase activity, an established marker of adipocyte differentiation. In conclusion, the culture system described here for trout pre-adipocytes is a powerful tool to study the endocrine regulation of adipogenesis in this species.

Enzymatically‐Modified Melt‐Extruded Guides for Peripheral Nerve Repair

AbstractIn this work, melt‐extruded guides for peripheral nerve repair were produced, based on blends between poly‐(ϵ‐caprolactone) and gelatin with a low gelatin amount (10 wt. %), with the aim of combining the good melt process ability of the synthetic polymer with the optimal biocompatibility of the natural polymer. In one case, a blend was produced between poly‐(ϵ‐caprolactone) and gelatin previously crosslinked by transglutaminase, using a solution mixing technique. The blend was then melt‐extruded obtaining nerve guidance channels. In another case, a blend between poly‐(ϵ‐caprolactone) and uncrosslinked gelatin was first produced, then melt‐extruded into tube‐shaped manufacts. Finally, poly‐L‐lysine was grafted on the gelatin domains exposed on the inner tube surface using transglutaminase catalysis, with the aim to confer to the channel guide a specific signaling for nerve cells attachment, proliferation and migration. Scanning electron microscopy (SEM) and Fourier transform infrared‐attenuated total reflectance spectroscopy (FTIR‐ATR) coupled with Chemical Imaging analysis showed that the obtained binary blends were poor compatible, however, appropriate mixing techniques might lead to an homogeneous distribution of gelatin domains within the poly‐(ϵ‐caprolactone) matrix. Confocal microscopy (CM) was applied as a powerful tool to study the accessibility of mTGase towards gelatin substrates, using suitable model lysine‐rich peptides (FITC‐labeled KKKKGY). Confocal microscopy also gave a confirmation of poly‐L‐lysine enzymatic grafting on the exposed gelatin domains on the inner blend tube surface. In vitro cell tests using S5Y5 neuroblastoma cells showed that the produced nerve guides were biocompatible, however, gelatin was confirmed to be a non‐specific protein for the attachment and proliferation of nerve cells. On the other hand, poly‐L‐lysine functionalization of the inner tube surface greatly improved the in‐vitro cell response.

Investigation of the effects and mechanisms in exogenous Rac1 gene expression on HT1080fibrosarcoma cell invasion across collagen barrier

Objective To investigate the effects and relevant mechanisms of exogenous Rac1 gene expression on HT1080 fibrosarcoma cell invasion across collagen barrier. Methods HT1080 fibrosarcoma cell lines that stably expressed transfected dominant negative [Rac1V12N17(HN)], constitutively active Rac1 [Rac1V12(HV)] and vector(HW) respectively were used. Structure of actin cytoskeleton was stained with Texas Red-conjugated phalloidin to show the morphological characters of the cells cultured in 3D medium containing collagen protein. Assay of cell invasion across collagen barrier was performed on a thin layer of collagen gel covered the membrane of transwell chamber, and two kinds of protease inhibitor were used to observe their effects on above-mentioned invasive assay. Gelatin substrate zymography were used to detect secreted MMP activity in the medium of cells cultured in 3D matrix. Results HT1080 cells stably expressing Racl mutant exhibited distinct morphological and invasive properties, and the increased invasive ability could be eradicated after using MMP inhibitor. Exogenous Rac1 gene expression on HT1080 fibrosarcoma cell could facilitate the activation of MMP-2 secreted in the medium of either collagen or fibrin 3D cell culture system.Conclusion The stable expression of exogenous Rac1 gene in HT1080 fibrosarcoma cells could induce aggregation of actin fiber and promote invasivc property. The enhancement of MMP-2 activation by exogenous Rac1 gene expression may be one of the relevant mechanisms. Key words: Gene, Rac1; Fibrosarcoma; Matrix metalloproteinase-2; Collagen

Social bacteria and asocial eukaryotes

The end of 2007 brought us draft genome sequences of two eukaryotic microorganisms, Babesia bovis and Malassezia globosa, as well as complete genomes of the ammonia-oxidizing archaeal chemoautotroph Nitrosopumilus maritimus, and several environmental bacteria (Table 1). In terms of genome size, this list covers both sides of the spectrum: the 245 kb genome of an obligate insect symbiont Sulcia muelleri is the second smallest microbial genome sequenced so far, whereas the 13 033 kb chromosome of the social myxobacterium Sorangium cellulosum breaks the record as the largest bacterial genome – and the largest DNA molecule in the prokaryotic world.

Identification of ocular matrix metalloproteinases present within the aqueous humor and iridocorneal drainage angle tissue of normal and glaucomatous canine eyes

The purpose of this study was to identify the most active matrix metalloproteinases (MMP) within the aqueous humor and iridocorneal angle tissue involved in the normal canine eye, and to compare these results to the MMP activity in dogs with glaucoma. Aqueous humor samples from 32 normal eyes and 26 glaucomatous eyes were obtained through aqueous centesis and analyzed for MMP activity. Iridocorneal angle tissue from 16 enucleated normal canine eyes and 5 enucleated glaucomatous eyes were dissected and homogenized into solution. Bradford total protein assays were determined for aqueous humor samples and iridocorneal angle tissue samples. Substrate gelatin zymography was performed using 0.2 microL volumes of an MMP-2/MMP-9 control, 2 microL volumes of aqueous humor samples, and 10 microg weights of total protein from iridocorneal angle tissue. The presence of MMP gel bands were identified visually and measured quantitatively by densitometry technique. A statistical analysis was performed on the data using Student's t-test, multiple logistic Wald's chi-squared regression, Pearson correlations, and repeated measures analysis. Within the aqueous humor of canine eyes, MMP-2 latent form was found to be the most relevant MMP. The quantity of latent MMP-2 within the aqueous humor of the glaucoma samples was significantly increased compared to the normal aqueous samples (P < 0.0001). Glaucoma occurrence was associated with elevated aqueous humor latent MMP-2 (P = 0.0002). Within the canine iridocorneal angle tissue, MMP-9 latent form and MMP-2 active form were found to be the most relevant MMPs. MMP-2 active form was found to be significantly increased in the glaucoma tissue samples when compared to the normal tissue samples (P = 0.0044). MMP-9 latent form was also found to be significantly increased in glaucomatous tissue when compared to the normal eyes (P = 0.0002). Tissue MMP-9 latent form was found to be associated with glaucoma status (P = 0.042). Glaucoma aqueous humor samples expressed a statistically increased latent MMP-2 when compared to normal eyes. Iridocorneal angle tissue from glaucomatous eyes expressed a statistically significant increase in active MMP-2 and latent MMP-9 when compared to normal eyes. These data demonstrate that there is an association between elevated levels of intraocular MMP-2 and MMP-9 and the presence of glaucoma.

Inhibition of MMP-2 gelatinolysis by targeting exodomain–substrate interactions

MMP-2 (matrix metalloproteinase 2) contains a CBD (collagen-binding domain), which is essential for positioning gelatin substrate molecules relative to the catalytic site for cleavage. Deletion of the CBD or disruption of CBD-mediated gelatin binding inhibits gelatinolysis by MMP-2. To identify CBD-binding sites on type I collagen and collagen peptides with the capacity to compete CBD binding of gelatin and thereby inhibit gelatinolysis by MMP-2, we screened a one-bead one-peptide combinatorial peptide library with recombinant CBD as bait. Analyses of sequences from the CBD-binding peptides pointed to residues 715-721 in human alpha1(I) collagen chain as a binding site for CBD. A peptide (P713) including this collagen segment was synthesized for analyses. In SPR (surface plasmon resonance) assays, the CBD and MMP-2(E404A), a catalytically inactive MMP-2 mutant, both bound immobilized P713 in a concentration-dependent manner, but not a scrambled control peptide. Furthermore, P713 competed gelatin binding by the CBD and MMP-2(E404A). In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2DeltaCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited approximately 90% of the MMP-2 gelatin cleavage, but less than 20% of the MMP-2 activity on a peptide substrate (NFF-1) which does not require the CBD for cleavage. Confirming the specificity of the inhibition, P713 did not alter MMP-2DeltaCBD or MMP-8 activities. These experiments identified a CBD-binding site on type I collagen and demonstrated that a corresponding synthetic peptide can inhibit hydrolysis of type I and IV collagens by competing CBD-mediated gelatin binding to MMP-2.

Characterization of the fetuin-binding fraction ofNeospora caninumtachyzoites and its potential involvement in host-parasite interactions

Terminal sialic acid residues on surface-associated glycoconjugates mediate host cell interactions of many pathogens. Addition of sialic acid-rich fetuin enhanced, and the presence of the sialidiase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reduced, the physical interaction of Neospora caninum tachyzoites and bradyzoites with Vero cell monolayers. Thus, Neospora extracts were subjected to fetuin-agarose affinity chromatography in order to isolate components potentially interacting with sialic acid residues. SDS-PAGE and silver staining of the fetuin binding fraction revealed the presence of a single protein band of approximately 65 kDa, subsequently named NcFBP (Neospora caninum fetuin-binding protein), which was localized at the apical tip of the tachyzoites and was continuously released into the surrounding medium in a temperature-independent manner. NcFBP readily interacted with Vero cells and bound to chondroitin sulfate A and C, and anti-NcFBP antibodies interfered in tachyzoite adhesion to host cell monolayers. In additon, analysis of the fetuin binding fraction by gelatin substrate zymography was performed, and demonstrated the presence of two bands of 96 and 140 kDa exhibiting metalloprotease-activity. The metalloprotease activity readily degraded glycosylated proteins such as fetuin and bovine immunoglobulin G heavy chain, whereas non-glycosylated proteins such as bovine serum albumin and immunoglobulin G light chain were not affected. These findings suggest that the fetuin-binding fraction of Neospora caninum tachyzoites contains components that could be potentially involved in host-parasite interactions.

Atividade proteolítica de isolados de Metarhizium anisopliae sobre substratos cuticulares e não-cuticulares

Cigarrinhas são as principais pragas da pastagem e da cana-de-açúcar, destacando-se os gêneros Deois e Mahanarva. Essas pragas vêm causando sérios danos a estas culturas na região de Tangará da Serra, MT, Brasil. O fungo Metarhizium anisopliae é considerado um eficiente agente para o controle biológico de insetos, possuindo vários fatores de virulência, incluindo a produção de proteases, consideradas fundamentais no processo de penetração através da cutícula do inseto. Este trabalho tem como objetivos analisar a produção de proteases extracelulares a partir de isolados de M. anisopliae, utilizando substratos cuticulares e não-cuticulares, e verificar a influência desses substratos na expressão dessas enzimas. Foram utilizados 4 isolados de M. anisopliae obtidos na região de Tangará da Serra e o isolado R3 BB 40, empregado para o controle biológico das cigarrinhas da cana-de-açúcar pelas Usinas Itamarati (Nova Olímpia / MT). A atividade enzimática foi determinada através do Índice de Relação Enzimática (IRE) em Meio Mínimo (MM), sem glicose, acrescido de caseína, gelatina e cutícula de adultos de Mahanarva fimbriolata e Deois flavopicta. Os isolados UNEMAT 03, UNEMAT 04, UNEMAT 05, UNEMAT 06 e R3-BB-40 apresentaram valores de IRE que variaram de 1,82 ± 0,062 a 2,16 ± 0,027 frente ao substrato gelatina e de 1,60 ± 0,076 a 2,08 ± 0,063 em caseína. Para os substratos cuticulares, houve crescimento das colônias, porém não apresentaram revelação de halo (IRE.= 1). Todos os isolados apresentaram IRE frente ao substrato gelatina&gt;caseína&gt;cutícula, destacando-se os isolados R3-BB-40 e UNEMAT 05. Existe uma variabilidade genética entre os isolados quanto à produção de proteases extracelulares e, sob tais condições, as cutículas não demonstraram ser substrato adequado para a detecção de atividade proteolítica.