An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.

Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/mL type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3-1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3-1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions.

Identification of lysosomal Npc1-binding proteins: Cathepsin D activity is regulated by NPC1.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1-binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032-1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC-MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.

Comparative proteomic analysis of hemolymph proteins from Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-sensitive or -resistant silkworm strains during infections.

We reported previously that baculovirus AcMNPV host-ranges in silkworm strains are controlled by a novel third chromosomal locus. To further isolate the potential host factor and uncover the functional pathway involved, in this study we analyzed hemolymph proteins from AcMNPV-resistant or -sensitive silkworm strains infected with baculoviruses. All the protein spots from 2D electrophoresis were characterized by MALDI-TOF MS and further systematically assessed for differentially regulated proteins at different stages of infection. Subsequently, six candidates were selected for functional analysis using Bm5 cells, where the candidates were knocked-down or overexpressed. We observed that mRNA expression levels of beta-N-acetylglucosaminidase and prophenoloxidase subunit 2 are significantly upregulated during AcMNPV infections in Bm5 cells. Ultimately, we found that RNA interference of ribosomal protein RpL34 causes serious damages to cell viability as well as abortive infection, indicating that ribosomal components are essential for productive baculovirus infection.

PO-CALC: a novel tool to correct common inconsistencies in the measurement of phenoloxidase activity.

A broad range of physiological and evolutionarily studies requires standard and robust methods to assess the strength and activity of an individual's immune defense. In insects, this goal is generally reached by spectrophotometrically measuring (pro-) phenoloxidase activity, an enzymatic and non-specific process activated after wounding and parasite infections. However, the literature surprisingly lacks a standard method to calculate these values from spectrophotometer data and thus to be able to compare results across studies. In this study, we demonstrated that nine methods commonly used to extract phenoloxidase activities (1) provide inconsistent results when tested on the same data sets, at least partly due to their specific sensitivity to the noise regularly present in enzymatic reaction curves. To circumvent this issue, we then (2) developed a novel, free and simple R-based program called PO-CALC and (3) demonstrated the robustness of its calculations for the different types of noises. Overall, we show that PO-CALC corrects overlooked though important inconsistencies in the measurement of phenoloxidase activities, and claim that its broad use would increase the significance and general validity of studies on invertebrate immunity.

Proteomic profiling of N-linked glycoproteins identifies ConA-binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma.

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N-linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty-eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high-levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA-binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, "ConA-binding proCD" (ConA-pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5-5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA-pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA-pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.

Evaluation of gelatinases, tissue inhibitor of matrix metalloproteinase-2, and myeloperoxidase protein in healthy and inflamed human dental pulp tissue.

INTRODUCTION: The aim of this study was to compare the gelatinolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9 and the expression of tissue inhibitor of matrix metalloproteinase (TIMP)-2 and myeloperoxidase protein (MPO) in clinically healthy human pulp and inflamed pulp tissue specimens. METHODS: Twenty dental pulps clinically diagnosed as inflammatory tissues and 20 healthy pulp tissues from enclosed third molars were harvested and evaluated. The gelatinolytic activity for MMP-2 and MMP-9 was assessed by using the zymography technique, TIMP-2 gene expression was evaluated using the enzyme-linked immunosorbent assay, and MPO was determined using the MPO assay. RESULTS: Data showed increased levels of MMP-9, active MMP-2, TIMP-2, and MPO in inflammatory pulp tissues compared with healthy tissues (P < .05). No statistical difference could be observed for pro-MMP-2 (P > .05). CONCLUSIONS: Although all samples were associated with MMP-2 expression, the active form of this MMP was observed only in inflamed pulps. Inflamed pulps showed an up-regulation of MMP-9, TIMP-2, and MPO.

Azaspiracid-1 inhibits the maturation of cathepsin D in mammalian cells.

Azaspiracid-1 (AZA-1) inhibits endocytosis, but the consequences of this alteration on cellular processes are unknown. We hypothesized that the inhibition of endocytosis is a key step of the mode of action of AZA-1, leading to perturbation of cellular processes dependent on proper functioning of endocytic machinery. We tested this working hypothesis by probing whether AZA-1 can alter the maturation of cathepsin D in MCF-7 epithelial cells, as a model system. We found that cell treatment with AZA-1 inhibited the conversion of 52 kDa procathepsin D into the mature 30 kDa protein. The effects induced by AZA-1 were similar to those elicited by chlorpromazine and other agents preventing proper maturation of lysosomal enzymes, indicating that the inhibition of endocytic transfer of proforms to late endosomes/lysosomess is responsible for the effect induced by the toxin. By immunofluorescence microscopy, we found no colocalization of cathepsin D and the early endosomal marker EEA-1 in control cells, where most of cathepsin D resides in late endosomes/lysosomes. Co-localization of cathepsin D and EEA-1 immunoreactivity, in turn, was found in cells exposed to AZA-1, indicating that the toxin blocks protein maturation at the early steps of endocytosis, causing accumulation of procathepsin D in early endosomes. The molecular alteration induced by AZA-1 involved both secreted and intracellular pools of procathepsin D, showing that the toxin effect does not result from a general impairment of vesicular trafficking but is the outcome of a perturbed centripetal process. Furthermore, AZA-1 was found to inhibit procathepsin D maturation also in normal fibroblasts, showing that this molecular response is induced by this toxin in different cell types. The data we obtained corroborated our hypothesis and provide a unified molecular frame for the mode of action of AZAs in animal models, involving a primary alteration of endocytic processes.

Validation of BdCCp2 as a marker for Babesia divergens sexual stages in ticks.

Babesiosis is a tick-transmitted disease of mammalian hosts, caused by the intraerythrocytic protozoan parasites of the genus Babesia. Transmission of Babesia parasites from the vertebrate host to the tick is mediated by sexual stages, the gametocytes which are the only intraerythrocytic stages that survive and develop inside the vector. Very few data are available concerning these parasite stages and some markers are needed in order to refine our knowledge of Babesia life cycle inside the tick and to permit the monitoring of parasite transmission from vertebrate to vector. We previously identified some potential markers of the Babesia divergens gametocytes using an in silico post-genomic approach based on sequence identity between the available genomes of Plasmodium and Babesia spp. Here, one of the identified proteins, BdCCp2, was validated as a marker of sexual stages of B. divergens, in infected ticks challenged with antisera directed against recombinant BdCCp2 protein. The BdCCp2 protein was detected by Western blot in some infected ticks, as a discrete band of approximately 171 kDa, while no signal was detected in the laboratory-reared non-infected tick. BdCCp2 was also detected, by immunohistochemical analyses, in piriform or ovoid bodies, measuring 2.5-4.5 µm in diameter, in the gut of partially engorged ticks that were experimentally infected. This molecular marker can then be used in the future to characterize and analyze the biology of B. divergens gametocytes.

Differentiated expression of membrane type metalloproteinases (MMP-14, MMP-15) and pro-MMP2 in laryngeal squamous cell carcinoma. A novel mechanism.

Cancer progression involves multiple proteolytic interactions, with metalloproteinases (MMPs) performing a crucial role. MMP-2, a major MMP, plays a key role in the degradation of basement membranes. Mechanisms underlying MMP-2 activation had to be investigated. Membrane-type matrix metalloproteinases are not only responsible for the regulation of extracellular matrix remodeling, but also involved in the activation of several inactive MMPs. The aim of this study was to evaluate the expression of pro-MMP2, MMP-14, and MMP-15 in tumor cells and tumor stroma. Immunohistochemical studies were performed on paraffin-embedded tissue sections including laryngeal squamous cell carcinoma (SCC). We found the expression of pro-MMP2 in 58% of cases, MMP-14 in 78%, and MMP-15 in 98% of cases of SCC. In all tumor cases, we revealed a higher expression of pro-MMP2 in tumor stoma than in tumor cells. The expression of MMP-14 and MMP-15 was higher in tumor cells than in the stroma. Moreover, we found a statistically significant difference between the expression of MMP-14 and MMP-15 in the tumor in comparison with the surrounding stroma (P < 0.05). An analysis of expression levels of MT-MMPs by classification trees showed that the probability of metastases was related to decreased expression of MMP-14 and increased expression of MMP-15. Our results may suggest that tumor cells with low MMP-14 expression invade tumor stroma and form metastases. Probably, in such cases, tumor progression is stimulated by MMP-15 in an MMP-14 independent pathway, a novel (alternative) mechanism.

Advances in zymography techniques and patents regarding protease analysis.

Detection of enzymatic activity on gel electrophoresis, namely zymography, is a technique that has received increasing attention in the last 10 years, according to the number of articles published. A growing amount of enzymes, mainly proteases, are now routinely detected by zymography. Detailed analytical studies are beginning to be published, as well as new patents have been developed. This new article updates the information covered in our last review, condensing the recent publications dealing with the identification of proteolytic enzymes in electrophoretic gel supports and its variations. The new advances of this method are basically focused towards two dimensional zymography and transfer zymography. Though comparatively fewer patents have been published, they basically coincide in the study of matrix metalloproteases. The tendency is foreseen to be very productive in the area of zymoproteomics, combining electrophoresis and mass spectrometry for the analysis of proteases.

Implication of MMP-9 and urokinase plasminogen activator (uPA) in the activation of pro-matrix metalloproteinase (MMP)-13.

We examined whether the expression and activation of pro-matrix metalloproteinase (MMP)-1 varies from that of pro-MMP-13 in the joint fluid of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. To do this, joint fluid was collected from 34 RA and 34 OA patients. The collagenase (pro-MMP-1 and MMP-13, total MMP-1, and MMP-13), gelatinase (total MMP-2 and MMP-9), stromelysin (total MMP-3), matrilysin (total MMP-7), uPA, and tissue inhibitor of MMP (TIMP) levels were measured by ELISA. The level of total MMP-1 in RA joint fluids was similar to that of the OA joint fluid. In contrast, the level of total MMP-13 in the RA group was significantly higher than that of the OA group. Among various MMPs (MMP-2, MMP-3, MMP-7, and MMP-9), only MMP-9 was strongly associated with total MMP-13 in both RA and OA. The level of uPA was also strongly associated with MMP-13 in RA but not OA, while the level of TIMP-1 and TIMP-2 was not significantly different between RA and OA. In conclusion, MMP-9 and uPA might be involved in the activation of pro-MMP-13 through unknown mechanisms in arthritic diseases.

Matrix metalloproteinase-2 expression induced by two different adhesive systems on human pulp fibroblasts.

INTRODUCTION: This study evaluated the expression of matrix metalloproteinase-2 (MMP-2) in primary cultures of human pulp fibroblasts (HPFs) when exposed to extracts from dentin-bonding systems. METHODS: Polymerized resin disks of the bonding agent of a 2-step self-etch adhesive (TechBond, Isasan, Rovello Porro, Italy) or of the primer/bonding agent a 2-step etch-and-rinse adhesive (Optibond Solo; Sybron-Kerr, Orange, CA) were immersed in HPF culture medium for 24 or 96 hours. HPFs were incubated in the adhesive-conditioned or control (untreated) culture medium for 24 hours. Western blot and immunofluorescence analyses were performed to assay MMP-2 expression. RESULTS: MMP-2 expression levels in HPFs cultured for 24 hours in culture medium were similar in both the control and experimental media groups showing a faint band at 67 kDa. Conversely, the HPFs incubated in the medium that contain polymerized resin disks for 96 hours showed increased MMP-2 expression compared with the untreated medium. The self-etch adhesive displayed the most pronounced induction of MMP-2 expression. These findings were confirmed by immunofluorescence analysis. CONCLUSIONS: HPFs display increased MMP-2 expression after 96 hours of conditioning of the HPF culture medium with polymerized disks of dentin bonding systems. This MMP-2 expression/activation may represent a defence mechanism exhibited by HPFs towards monomers eluted from the dentin bonding systems.

Distribution and relative activity of matrix metalloproteinase-2 in human coronal dentin.

The presence of matrix metalloproteinase-2 (MMP-2) in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP-2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner (ID), middle (MD), and outer dentin (OD) regions and demineralized. MMP-2 was extracted with 0.33 mol x L(-1) EDTA/2 mol xL(-1) guanidine-HCl, pH 7.4, and MMP-2 concentration was estimated with enzyme-linked immunoabsorbant assay (ELISA). Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP-2 per mg dentin protein in the dentin regions were significantly different (P = 0.043): 0.9 ng (ID), 0.4 ng (MD), and 2.2 ng (OD), respectively. The pattern of MMP-2 concentration was OD > ID > MD. Western blotting analysis detected -.66 and -72 kDa immunopositive proteins corresponding to pro- and mature MMP-2, respectively, in the ID and MD, and a -66 kDa protein in the OD. Gelatinolytic activity consistent with MMP-2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD > ID > OD. The concentration of MMP-2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP-2, potentially indicating region specific protein interactions.

Antioxidant diet protects against emphysema, but increases mortality in cigarette smoke-exposed mice.

Oxidative stress plays an important role in cigarette smoke-induced lung inflammation and emphysema. We produced an enriched diet by adding freeze-dried fruits and vegetables and additional supplements to the 8604 Teklad Rodent Diet, a standard rodent diet. In this study, we examined the effects of the antioxidant-enriched diet on cigarette smoke-induced lung inflammation and emphysema. CH3/HeN mice were fed either a regular diet or the supplemented diet. These mice were exposed to filtered air, a low concentration of cigarette smoke (total particulate matter: 100 mg/m3) or a high concentration of cigarette smoke (total particulate matter: 250 mg/m3) for 6 h/day, 5 days/week for total 16 weeks. Surprisingly, increased mortality (53%) was observed in the high concentration of cigarette smoke-exposed mice fed the antioxidant diet compared to the high concentration of cigarette smoke-exposed mice that were fed a regular diet (13%). The necropsy analysis revealed nasal passage obstruction due to mucous plugging in cigarette smoke-exposed mice on the antioxidant diet. However, the antioxidant diet significantly reduced neutrophilic inflammation and emphysema in the high concentration of cigarette smoke-exposed mice as compared to the regular diet /high concentration of cigarette smoke controls. The antioxidant capacity in the bronchoalveolar fluid or oxidative damage to the lung tissue was not affected by the antioxidant diet. Pro-MMP-2, MMP-2, and MMP-9 activity did not correlate with the protective effects of AOD on cigarette smoke-induced emphysema. These data suggest that the antioxidant diet reduced cigarette smoke-induced inflammation and emphysema, but increased mortality in the obligate nose-breathing mice.

Effects of cigarette smoke condensate and nicotine on human gingival fibroblast-mediated collagen degradation.

BACKGROUND: Members of the matrix metalloproteinase (MMP) family have been shown to be involved in periodontal disease. Risk factors for periodontal disease include tobacco smoking. Cigarette smoke condensate (CSC) is comprised of thousands of chemicals. Nicotine is one of the active components in tobacco. This study compares the effects of CSC and nicotine at the level in CSC on the collagen-degrading ability of human gingival fibroblasts (HGFs) and the expression of selected MMPs and tissue inhibitors of metalloproteinases (TIMPs). METHODS: HGFs were seeded in six-well collagen-coated plates, exposed to 100 µg/mL (2.4 µg/mL nicotine) of CSC or 2.4 µg/mL nicotine for 3 days, and then collagen degradation was analyzed. After 3 days exposure to CSC or nicotine, the conditioned media from HGFs was collected and the membrane proteins were extracted for gelatin zymography and Western blot analyses. The mRNA levels of MMP-2, MMP-14, and TIMP-2 were measured by reverse transcription-polymerase chain reaction. RESULTS: The CSC increased collagen degradation, and increased the levels of TIMP-2, MMP-14, and the active MMP-2 in the membrane extracts, and their mRNA levels. CSC also increased the level of active MMP-2 in the conditioned media. Nicotine at the level in CSC (2.4 µg/mL) had little influence on collagen degradation, as well as on the protein and mRNA levels of MMP-2, MMP-14, and TIMP-2. CONCLUSIONS: CSC may increase HGF-mediated collagen degradation by affecting membrane-associated MMPs and TIMPs.

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages.

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.

'Young' oral fibroblasts are geno/phenotypically distinct.

Wound healing within the oral mucosa results in minimal scar formation compared with wounds within the skin. We have recently demonstrated distinct differences in the aging profiles of cells (oral mucosal and patient-matched skin fibroblasts) isolated from these tissues. We hypothesized that the increased replicative potential of oral mucosal fibroblasts may confer upon them preferential wound-healing capacities. Passage-matched early cultures of oral mucosal fibroblasts and skin fibroblasts demonstrated distinct gene expression profiles, with several genes linked to wound healing/tissue repair. This was related to an increased ability of the 'replicatively younger' oral mucosal fibroblasts to repopulate a wound space and reorganize their surrounding extracellular matrix environment, key activities during the wound-healing process. We conclude that oral mucosal fibroblasts exhibit a preferential healing response in vivo, due to their 'replicatively younger' phenotype when compared with that of patient-matched skin fibroblasts.

Effects of tobacco and P. gingivalis on gingival fibroblasts.

Cigarette smoke condensate (CSC) is the particulate matter of cigarette smoke. Porphyromonas gingivalis (P. gingivalis) is an opportunistic pathogen involved in periodontitis. It was hypothesized that the combination of CSC and P. gingivalis would increase the collagen-degrading ability of human gingival fibroblasts (HGFs). In this study, HGFs were exposed to CSC, P. gingivalis supernatant, and CSC plus P. gingivalis supernatant. The collagen-degrading ability and protein/mRNA levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) of HGFs were examined. The combined treatment increased collagen degradation, protein levels of active forms of MMP-1, MMP-2, MMP-3, and MMP-14 in conditioned media, and the low-molecular-weight fragment of MMP-14 in membrane extracts, as well as mRNA levels of MMP-1, MMP-2, and MMP-14. In conclusion, the combined effects of CSC and P. gingivalis increased HGF-mediated collagen degradation by destroying the balance between MMPs and TIMPs at multiple levels.

Tissue localization and the establishment of a sensitive immunoassay of the newly discovered human phospholipase B-precursor (PLB-P).

Human phospholipase B-precursor (PLB-P) is a newly identified and purified protein from human neutrophils. The precise function of PLB-P in vivo is not yet known. Its existence in neutrophils and the enzymatic activity against phospholipids imply a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. We describe here the generation of specific antibodies against PLB-P, the tissue localizations of PLB-P and the establishment of an accurate, specific, and reproducible radioimmunoassay (RIA). A survey of normal and malignant tissues showed strong immunostaining of PLB-P in neuronal and myeloid cells and in adrenal glands. Elevated levels were found in sera of patients with influenza A infection i.e. >1 microg/L and in gut fluids of patients with inflammatory bowel disease i.e. >20 microg/L. The levels correlated to markers of neutrophil activation, suggesting a neutrophil origin of PLB-P in these conditions. The antibodies and the assay will be useful in the future basic and clinical investigations of PLB-P.