Presence Of Metalloproteases Research Articles

Evaluation of pathogenicity and antifungal resistance of frequently occurred Aspergillus and Fusarium species in swimming pools in selected hotels in Ekiti state

Background: Swimming pools have been reported as critical fungal reservoirs. This study was designed to investigate fungal populations in swimming pools as well as detect the presence of virulence factors in the recovered isolates. Methods: Estimation of the mycobiota in the swimming pools sampled was done and isolates recovered were identified using colonial, morphology and molecular methods. Also, virulence factors: lipase, keratinase, laccase, hydrophobicity and biofilm formation were also determined using standard methods while antifungal susceptibility was done using clinically approved methods. Also, the presence of metalloprotease and protease gene was detected using PCR. Results: Out of the 75 samples, 47 showed fungal growth, with an isolation rate of 25.5%, 34.0%, and 40.4% from the surface, bottom, and edges of the pools respectively. Six fungi species were isolated viz: Candida tropicalis, Aspergillus flavus, Aspergillus niger, Fusarium oxysporum, and Fusarium verticilloides. C. tropicalis had the highest isolation rate at 41.7%. The fungi possessed virulence factors, with keratinase and lipase being the most common. All isolates were resistant to itraconazole, while none were resistant to fluconazole. The metalloprotease gene was detected in A. flavus, F. verticilliodes, and F. oxysporum. Conclusion: Our study revealed that the fungi recovered from the swimming pool possessed virulence factors that can enhance their infectivity.

Les podosomes endothéliaux

Podosomes are highly dynamic adhesion microdomains formed at the ventral membrane of some monocyte-derived cells. Structurally, their most distinguishing feature is their two-part architecture, consisting in a core of F-actin and actin-associated proteins, surrounded by a ring structure consisting of plaque proteins as well as signalling proteins. In addition to the presence of specific markers, they are distinguished from other adhesion structures by the presence of metalloproteases, endowing them with the ability to degrade the extracellular matrix. Invadopodia are related structures, of similar molecular composition but of distinct architecture, made by fibroblasts or epithelial cells transformed by the v-src oncogene or aggressive carcinoma cells. Such membrane-associated cellular devices, now named invadosomes, are thought to have a central role in mediating polarized migration in cells that cross anatomical boundaries. Podosomes have now been shown to form in endothelial cells, non monocytic and non tumoral cells, endowed with tissue invasive activities during vascular remodelling. Here, we summarize the recent advances and developments in this field, discuss how endothelial podosomes combine specificities of monocytic podosomes and invadopodia and provide our provisional outlook into the future understanding of endothelial podosomes.

Metalloproteases and egg-hatching in Pediculus humanus, the body (clothes) louse of humans (Phthiraptera: Insecta)

To investigate the biochemical components of egg-hatch in the body louse, Pediculus humanus, egg-shell-washings (ESW) were collected during the first 2 h post-hatching and analysed by gelatin SDS-PAGE. These ESW contained proteases with molecular mass in the range of 25-100 kDa; the most abundant proteases were approximately 25 kDa. The 3 main regions of protease activity in the one-dimensional gelatin SDS-PAGE gels resolved to at least 23 distinct regions of protease activity when analysed by two-dimensional gelatin SDS-PAGE, with iso-electric points spread over the entire 3 to 10 pH range. Mechanistic characterization indicated that the ESW contained proteases of the metallo-class, inhibited by both 1,10-phenanthroline and EDTA. Several protease inhibitors were tested for their ability to inhibit louse egg-hatch in vitro. The metalloprotease inhibitor 1,10-phenanthroline and the aminopeptidase inhibitor bestatin significantly inhibited (P<0.05) louse egg-hatch (100% and 58%, respectively). The presence of metalloproteases at the time of egg-hatch and the inhibition of egg-hatch in P. humanus by metalloprotease inhibitors suggests a crucial role for these proteases in the hatching of this medically important parasite.

Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus

A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28kDa, pH and temperature optima for this protease were 10 and 60 degrees C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu(2+) ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.

Influence of Endogenous Proteases and Transglutaminase on Thermal Gelation of Salted Squid Muscle Paste

ABSTRACT: Thermal gelation of salted squid mantle muscle paste was studied in relation to endogenous proteases and transglutaminase. Myosin in the paste was preferentially degraded into 130‐kDa and 90‐kDa fragments at an optimum temperature of 30 °C. Degradation was inhibited with EDTA or 1,10 phenanthroline, suggesting the presence of metalloproteases. Myosin degradation was markedly reduced above 40 °C. Although 10 mM Ca2+ increased cross‐linking of myosin heavy chains by activating the endogenous transglutaminase, setting effect on thermal gelation of the paste was offset by degradation induced by simultaneously activated calpains. Ca2+ and the alpain inhibitor, E64, significantly improved the breaking strength and strain of thermal gels preincubated at 40 °C.

Topographically graded postischemic presence of metalloproteinases is inhibited by hypothermia

To test the hypothesis that presence of metalloproteases (MMPs), their inhibitors (TIMPs) and their substrate laminin-5 differs between the ischemic core and the surrounding tissue, we examined the impact of middle cerebral artery occlusion/reperfusion (MCA:O/R) on these molecules in different regions of the infarct. We also investigated the influence of hypothermia on the progression of the ischemic lesion and MMP activity. Brain sections from 64 Wistar rats subjected to MCA:O/R were examined by means of cytohistochemistry and zymography. The artery was occluded for 2 h followed by 3, 5, 8 and 12 h of reperfusion. Well characterized antibodies against laminin-5, MMPs and TIMP-2 were used. A total of 32 rats were treated with hypothermia. The presence of each antigen was related to the following regions of interest: ischemic core with BBB breakdown (I c), surrounding ischemic tissue without BBB breakdown (I r), and the contralateral non-ischemic region (N). Regions of interest were defined by MRI. The I c increased over time at the cost of the I r. BBB breakdown occurred early in the ischemic core and increased over time. Hypothermia reduced the BBB breakdown at all time points. A graded decreased presence of laminin-5 was observed with 16.5±3.7(N)>10±2.8(I r)>4±1.4(I c) immunopositive microvessels/mm 2 at 3 h of reperfusion. MMP-9 showed a reverse pattern with 0 (N)<4±0.8(I r)<10±1.5(I c) immunopositive microvessels/mm 2. Hypothermia decreased the MMP activity measured by zymography. Laminin-5 and MMP presence relate directly to the degree of postischemic injury. Hypothermia reduces the conversion from the I r to ischemic core and the degree of BBB as well as MMP abundance.

Endogenous meltrin alpha is ubiquitously expressed and associated with the plasma membrane but exogenous meltrin alpha is retained in the endoplasmic reticulum.

Meltrin alpha (ADAM12) is a member of the ADAM (MDC) protein family characterized by the presence of metalloprotease and disintegrin domains. ADAM proteins contain single transmembrane domains, and the processed mature proteins are postulated to span the plasma membrane. It has been reported that transfection of a truncated meltrin alpha cDNA lacking the prodomain and metalloprotease domain promotes skeletal muscle cell fusion. We show here that meltrin alpha was constitutively expressed in both undifferentiated and differentiated C2 skeletal muscle cells and also in fibroblasts. Both its precursor and processed mature forms were present in these cells. Thus, meltrin alpha may play general roles in addition to its roles in myogenesis. Since endogenous meltrin alpha cannot be detected by immunofluorescence microscopy, we examined the location of the exogenously expressed protein by transfection. Unexpectedly, the exogenously expressed meltrin alpha was located to a network structure of the endoplasmic reticulum (ER) but not to the plasma membrane. Cell fractionation revealed that the intrinsic mature protein was associated with the plasma membrane. However, the exogenously expressed protein remained unprocessed. These results seem to imply that the exogenously expressed meltrin alpha is not translocated from the ER to the trans-Golgi network, where a processing enzyme resides, and that it is consequently not converted to the mature form. Thus, the transfected meltrin alpha is unlikely to exert its physiological functions. Conversely, the ER may serve as a reservoir of the latent form of intrinsic meltrin alpha.

Meltrin alpha cytoplasmic domain interacts with SH3 domains of Src and Grb2 and is phosphorylated by v-Src.

Meltrin alpha/ADAM12 is a member of the ADAM/MDC family proteins characterized by the presence of metalloprotease and disintegrin domains. This protein also contains a single transmembrane domain and a relatively long cytoplasmic domain containing several proline-rich sequences. These sequences are compatible with the consensus sequences for binding the Src homology 3 (SH3) domains. To determine whether the proline-rich sequences interact with SH3 domains in several proteins, binding of recombinant SH3 domains to the meltrin alpha cytoplasmic domain was analysed by pull-down assays. The SH3 domains of Src and Yes bound strongly, but that of Abl or phosphatidylinositol 3-kinase p85 subunit did not. Full-length Grb2/Ash bound strongly, whereas its N-terminal SH3 domain alone did less strongly. Src and Grb2 in bovine brain extracts also bound to meltrin alpha cytoplasmic domain on affinity resin. Furthermore, immunoprecipitation with a monoclonal antibody to meltrin alpha resulted in coprecipitation of Src and Grb2 with meltrin alpha in cell extracts, suggesting that Src and Grb2 are associated in vivo with meltrin alpha cytoplasmic domain. This notion was also supported by the findings that exogenously expressed meltrin cytoplasmic domain coexisted with Src and Grb2 on the membrane ruffles. The C-terminal Tyr901 of meltrin alpha was phosphorylated both in vitro and in cultured cells by v-Src. These results may imply that meltrin alpha cytoplasmic domain is involved in a signal transduction for some biological function through the interaction with SH3-containing proteins.

Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis.

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.

DNA methylation by wheat cytosine DNA methyltransferase: modulation by protease inhibitor E-64.

Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent K(m) for AdoMet and [I]50 for AdoHcy values are about 6 microM and 12 microM, respectively. The enzyme is active in wide pH range (pH 5.5-8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E-64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH-groups but is not due to proteolytic enzyme cleavage.