Gelatin Substrate Gel Research Articles

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.

Enzymatic and protein differences between infective larvae of Trichostrongylus colubriformis conditioned or not conditioned to hypobiosis

Abstract The differences in protein and enzymatic profiles of infective larvae (L3) of Trichostrongylus colubriformis, both induced and non-induced to hypobiosis, have been evaluated by means of SDS-PAGE and densitometric analysis as well as by semiquantitative micromethod API-ZYM (Bio-Mérieux, France). Quantitative differences were identified in protein levels between the induced and non-induced larvae, where the amount of two polypeptides (200–220 kDa) decreased in range 32.3–35.4 % and the amount of six polypeptides (20–28 kDa) increased in range 20.0–27.0 % in the samples of induced larvae. In contrast to non-induced larvae, on gelatin-substrate gel in L3 in vitro released (IVR) proteases from larvae conditioned to hypobiosis, zones of proteolysis were observed between 21 and 34 kDa.

Proteolytic processing of a sea urchin, ECM‐localized protein into lower mol mass species possessing collagen‐cleavage activity

The hyaline layer is an apically located extraembryonic matrix, which blankets the sea urchin embryo. Using gelatin substrate gel zymography, we have identified a number of gelatin-cleaving activities within the hyaline layer and defined a precursor-product processing pathway which leads to the appearance of 40- and 38-kDa activities coincident with the loss of a 50-kDa species. Proteolytic processing of the precursor required the presence of both CaCl2 and NaCl at concentrations similar to those found in sea water. The cleavage activities utilized both sea urchin and rat tail tendon gelatins as substrates but demonstrated a species-specific cleavage activity towards sea urchin collagen. The gelatin-cleaving activities were refractory to inhibition by 1,10-phenanthroline but were inhibited by benzamidine. This latter result defines the serine protease nature of the cleavage activities. Both the 40- and 38-kDa activities were found to comigrate with gelatin-cleaving activities present in the sea urchin embryo.

Gnathostoma binucleatum: Excretion–secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture

The paper describes an introductory characterization of antigenic stimulation of excretion–secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO 2 RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS–PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS–PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.

Entamoeba invadens: cysteine protease inhibitors block excystation and metacystic development

We examined the effects of six cysteine protease inhibitors on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the cysteine protease inhibitors Z-Phe-Ala-DMK and E-64d in a concentration-dependent manner during incubation compared to the controls. Neither inhibitor had a significant effect on cyst viability; thus, their inhibitory effects were not due to the toxic effect on cysts. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by these protease inhibitors, because the percentage of 4-nucleate amoebae was higher than in the controls on Day 3 of incubation. Although other cysteine protease inhibitors, Z-Phe-Phe-DMK, E-64, ALLM, and cathepsin inhibitor III, had a weak or little effect on the excystation, they inhibited cysteine protease activity in the lysates of E. invadens cysts. Broad bands with gelatinase activity of metacystic amoebae, as well as cysts and trophozoites, were detected in the gelatin substrate gel electrophores and were inhibited by Z-Phe-Ala-DMK. There was a difference in the protease composition between cysts and trophozoites, and the protease composition of metacystic amoebae changed from cyst-type to trophozoite-type during development. These results strongly suggest that cysteine proteases contribute to the excystation and metacystic development of E. invadens, which leads to successful infection.

Protection studies in sheep using affinity-purified and recombinant cysteine proteinases of adult Haemonchus contortus

Vaccination with a membrane-bound thiol Sepharose-binding fraction (TSBP) of adult Haemonchus contortus has been shown to confer significant levels of protection against homologous challenge in sheep. This fraction is greatly enriched for cysteine proteinase activity. Following fractionation of TSBP by anion-exchange chromatography on MonoQ, protection was found to partition with those fractions further enriched for cysteine proteinase activity. In this study, the cysteine proteinases of adult H. contortus TSBP were specifically purified by affinity chromatography using recombinant H. contortus cystatin, a potent cysteine proteinase inhibitor. Although only 1–1.5% of total TSBP bound to cystatin-Sepharose, this fraction contained 100% of the cysteine proteinase activity, as determined by gelatin substrate gel analysis. When used to immunise sheep, less than 3 μg per dose of this cysteine proteinase fraction was found to confer a substantial and repeatable level of protection against homologous challenge infection, reducing faecal egg counts by 48 and 28% and worm burdens by 44 and 46% over two trials. Host serum immunoglobulin levels and abomasal mast cell and eosinophil numbers were evaluated, although no correlation with protection was observed. Three cathepsin B-like cysteine proteinases present in TSBP (hmcp1, 4 and 6) have been identified previously by cDNA library immunoscreening. The predicted mature forms of these three cysteine proteinases were expressed in bacteria as insoluble, GST-fusion proteins. Following solubilisation in urea/DTT, the protective capacity of a cocktail of recombinant proteins was evaluated in sheep. Although no reduction in faecal egg counts was observed, sheep vaccinated with recombinant cysteine proteinases showed a highly significant 38% reduction ( P < 0.01) in worm burdens.

Proteolytic enzyme activity and attenuation of virulence in Theileria annulata schizont-infected cells

A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.

Localization and functional role of a 41 kDa collagenase/gelatinase activity expressed in the sea urchin embryo.

The egg storage compartment of the sea urchin embryo was investigated for a protein destined for export to the extracellular matrices. Using an antiserum prepared against a 41 kDa collagenase/gelatinase localized to the extraembryonic matrices (the hyaline layer and basal lamina), the egg storage compartment was mapped for this antigen. Indirect immunofluorescence analysis revealed the 41 kDa collagenase/gelatinase in the cortical granules as well as a second compartment which was dispersed throughout the egg cytoplasm. High resolution immunogold labeling defined this cytoplasmic compartment as the yolk granule organelle. Gelatin substrate gel zymography revealed the presence of a 41 kDa gelatin cleavage activity in purified yolk granules. These results suggest a role for yolk granules in regulated protein export and challenge the traditional view of this organelle as a benign storage compartment for nutrients. In additional experiments, embryos grown in the presence of the 41 kDa cleavage activity or the anti-41 kDa antiserum had severely delayed gut formation and spicule elongation. These results demonstrate a requirement for defined levels of the 41 kDa activity in the extracellular matrices of the developing embryo.

Proteins and proteinases in the in vitro released products (IVRP) of the tissue-invasive and lung-dwelling larvae of the pentastomid Porocephalus crotali.

Pentastomids are arthropod-like parasites which inhabit the respiratory tract of tetrapods and, despite being haematophagous, cause little or no visible pathology. Nymphs (or larvae), belonging to the advanced genus Porocephalus, encyst in the tissues of rodent intermediate hosts and migrate from this site to the lung following ingestion of the intermediate host. During migration, several moults occur and instars encounter a variety of tissue barriers and environmental cues. It was therefore relevant to define the proteins and proteinases released by the different instars during this migration as they are likely to be essential for tissue penetration, feeding and immune evasion. In vitro released proteins (IVRP), commonly termed ES, were defined on the basis of molecular size and immune recognition by naturally infected rodent intermediate hosts. In addition, proteinases present in IVRP were characterized using gelatin-substrate gels and protein degradation assays. These studies showed that the protein profile is subtly different when IVRP from different instars is compared. In addition, IVRP from each instar contained metallo-proteinases with proteolysis at 48 kDa dominating in all stages. However, other higher molecular weight activities were, to an extent, released in a stage-specific manner. These proteinases degraded haemoglobin and fibrinogen as well as structural proteins such as collagen and fibronectin, suggesting roles in bloodmeal digestion and tissue penetration. Immunoglobulin digestion was not demonstrated.

Biochemical and immunological characterization of excretory-secretory products of Vesicocoelium solenophagum and plasma proteins of its bivalve host, Sinonovacula constricta.

In this study, the excretory-secretory products (ESP) of the daughter sporocysts of Vesicocoelium solenophagum (Trematoda) and plasma proteins of its host, Sinonovacula constricta were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gelatin-substrate gel analyses, and the relationships between them were analysed using immunoblotting. Proteinase activity was detected in the ESP from daughter sporocysts of V. solenophagum. Some polypeptides of the ESP were found to be recognized by antiserum, raised against plasma from non-infected S. constricta, suggesting that the ESP may mimic host molecules (molecular mimicry). In contrast, neither the obvious proteinase activity nor the binding to the antisera was observed for the soluble proteins of daughter sporocyst, indicating that the ESP may play a important role in the parasite-host relationship. Although the plasma of infected S. constricta contained polypeptides that were similar to the plasma of non-infected bivalves, increased quantities of proteins at >170 kDa, 15 kDa and decreased quantities at 60 kDa were observed in the plasma of infected bivalves. Immunoblotting analysis revealed that the plasma of infected bivalves had a faint reaction with both anti-non-infected plasma antisera and anti-sporocyst antisera. These results indicated that the structure and quantity of some polypeptides from the plasma of infected bivalves had changed because of the infection with V. solenophagum. The polypeptides between the plasma of bivalves from a non-epidemic area and that from an epidemic area were similar, but the former had more polypeptides of 170-220 kDa and much greater proteinase activity than the latter, suggesting that the increased polypeptides of 170-220 kDa and the high proteinase activity in plasma may be favourable for protecting the host from being invaded by the parasites.

Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity.

We report here the continued characterization of a 41-kDa protease expressed in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin-cleavage activity and an echinoderm-specific collagen-cleavage activity. In the experiments reported here, we have explored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10-phenanthroline (a metalloprotease inhibitor), and E-64 (a thiol protease inhibitor) were without effect on the gelatin-cleavage activity of the 41-kDa species. Using a gelatin substrate gel zymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamide appeared to partially inhibit gelatin-cleavage activity. This result was confirmed in a quantitative gelatin-cleavage assay using the water soluble, serine protease inhibitor [4-(2-aminoethyl)benzenesulfonylfluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent cations present in sea water, on the gelatin-cleavage activity. Calcium and magnesium competed for binding to the 41-kDa collagenase/gelatinase, and prebound calcium was displaced by magnesium. Cleavage activity was inhibited by magnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may regulate the proteolytic activity of the 41-kDa species.

A cysteine protease from myxosporean degrades host myofibrils in vitro

Pseudocysts of Kudoa rosenbuschi (Myxozoa, Myxosporea) contain a cysteine protease (Mr 147.2 kDa) which has a great ability to degrade in vitro constituents of whole myofibrils of the Atlantic hake, Merluccius hubbsi, skeletal muscle. The enzyme was purified to apparent homogeneity: a single peak of azocaseinolytic activity was observed after anion exchange chromatography and gel filtration. The electrophoretic patterns of this protease under non-denaturing conditions and gelatin substrate gels show a single band while the electrophoretic pattern under denaturing conditions shows a band corresponding to 25.5 kDa. The activity of the purified enzyme was maximum in the broad pH range between 4 and 7. Cysteine protease inhibitors were able to reduce the enzyme activity whereas specific inhibitors of serine proteases and aspartic proteases were ineffective. A weak activation was observed with EDTA and with 2-mercaptoethanol. The enzyme degrades M. hubbsi myofibrillar proteins, mainly titin and myosin heavy chain, at 37°C, in vitro. No degradative activity was detected on sarcoplasmic proteins. This enzyme is likely to be responsible for the proteolysis previously observed in skeletal muscle around white and black pseudocysts.

Calcium-protein interactions in the extracellular environment: calcium binding, activation, and immunolocalization of a collagenase/gelatinase activity expressed in the sea urchin embryo.

We have purified and characterized a collagenase/gelatinase activity expressed during sea urchin embryonic development. The native molecular mass was determined to be 160 kDa, while gelatin substrate gel zymography revealed an active species of 41 kDa, suggesting that the native enzyme is a tetramer of active subunits. Incubation in the presence of EGTA resulted in nearly complete loss of activity and this effect could be reversed by calcium. Calcium-induced reactivation appeared to be cooperative and occurred with an apparent kd value of 3.7 mM. Two modes of calcium binding to the 41-kDa subunit were detected; up to 80 moles of calcium bound with a kd value of 0.5 mM, while an additional 120 moles bound with a kd value of 5 mM. Amino acid analysis revealed a carboxy plus carboxyamide content of 24.3 mol/100 mol, indicating the availability of substantial numbers of weak Ca2+-binding sites. Calcium binding did not result in either secondary or quaternary structural changes in the collagenase/gelatinase, suggesting that Ca2+ may facilitate activation through directly mediating the binding of substrate to the enzyme. The collagenase/gelatinase activity was detected in blastocoelic fluid and in the hyalin fraction dissociated from 1-h-old embryos. Immunolocalization studies revealed two storage compartments in the egg; cortical granules and small granules/vesicles dispersed throughout the cytoplasm. After fertilization, the antigen was detected in both the apical and basal extracellular matrices, the hyaline layer, and basal lamina, respectively.

Exposure of human renal proximal tubular cells to glucose leads to accumulation of type IV collagen and fibronectin by decreased degradation

Thickening and reduplication of the tubular basement membrane has been reported as an early event in diabetic nephropathy. In the current study we examined the effects of elevated D-glucose concentrations on human proximal tubular (HPTC) type IV collagen and fibronectin turnover. Incubation of confluent growth arrested HPTC with 25 mM D-glucose led to accumulation of both type IV collagen and fibronectin. This effect was maximal at 48 hours and represented a sevenfold increase for fibronectin (N = 4, P = 0.04), and a threefold increase for type IV collagen (N = 3, P = 0.03) over cells exposed to 5 mM D-glucose controls. This increase was not dependent on new gene transcription for either protein. Tissue inhibitor of metalloproteinases (TIMP 1 + TIMP 2) were induced following addition of 25 mM D-glucose, but not when cells were exposed to 5 mM D-glucose. Twenty-four hours after the addition of 25 mM D-glucose there was an eightfold increase in TIMP 1 (P = 0.009, N = 4), and a tenfold increase in TIMP 2 levels (P = 0.003, N = 4), over the control values for both inhibitors. The increase in both TIMP 1 and TIMP 2 in response to 25 mM D-glucose was abrogated in a dose dependent manner by the aldose reductase inhibitor sorbinil. Gelatin-substrate gel zymography showed increased activity of gelatinase A, but not of gelatinase B in response to the addition of 25 mM D-glucose to HPTC. The induction of gelatinase A was accompanied by increased gelatinase A mRNA expression, which was inhibited both by protein kinase C (PKC) depletion using PMA pre-treatment, and by the addition of a PKC inhibitor. These data demonstrate that the glucose-induced accumulation of type IV collagen and fibronectin is unrelated to increased gene transcription, but may involve alterations in the degradative pathway of these basement membrane constituents. Furthermore, the data demonstrate that glucose may simultaneously activate two intracellular pathways (the polyol pathway and a PKC dependent activation pathway), which are involved in mediating separate, complementary effects on cell function.

Trichuris suis:Thiol Protease Activity from Adult Worms

Trichuris suis,the whipworm of swine, causes anemia, weight loss, anorexia, mucohemorrhagic diarrhea, and death in heavy infections. A zinc metalloprotease has been suggested to play a role in the severe enteric pathology associated with infection and the infiltration of opportunistic bacteria into deeper tissues in the swine colon. In this study, a thiol protease from gut extracts of adultT. suisand from excretory/secretory components (E/S) of adult worms was characterized using fluorogenic peptide substrates and protein substrate gels. The protease cleaved the fluorogenic substrate Z-Phe-Arg-AMC, and this cleavage was completely inhibited by the thiol protease inhibitors E-64, leupeptin, Z-Phe-Ala-CH2F, and Z-Phe-Arg-CH2F. Gelatin substrate gels and fluorescence assays using both the gut and the stichosome extracts and E/S revealed enhanced activity when 2 mMdithiothreitol or 5 mMcysteine was included in the incubation buffer, and optimal activity was seen over a pH range of 5.5 to 8.5. Incubation of gut extracts or E/S material with inhibitors of aspartic, serine, or metalloproteases had no effect on the cleavage of Z-Phe-Arg-AMC. Thiol protease activity was found in extracts of gut tissue but not in the extracts of stichocytes of adult worms. N-terminal amino acid sequencing of the protease revealed sequence homologies with cathepsin B-like thiol protease identified from parasitic and free-living nematodes.

Entamoeba invadens:Characterization of Cysteine Proteinases

Cysteine proteinases have a number of important functions in the life cycle of protozoan parasites. Based on our previous studies demonstrating the role of cysteine proteinases in invasion byEntamoeba histolytica,we evaluated the cysteine proteinases ofE. invadens,a related protozoan which causes invasive disease of reptiles.E. invadensreadily encysts in axenic culture and provides a model to investigate the role of cysteine proteinases in encystation. Broad bands of ∼130–230, 55, and 35 kDa were detected on gelatin substrate gels and were inhibited with specific cysteine proteinase inhibitors. Maximal enzymatic activity was detected with peptide substrates containing arginine in the P2 position. A 567-bp fragment containing the active site of anE. invadenscysteine proteinase gene was amplified by PCR and had 37.7, 79.1, and 67.9% identity to the derived amino acid sequences of theacp 1, 2,and3genes, respectively, ofE. histolytica. The PCR product hybridized with a single band of 1.1 kb on a Southern blot ofEcoRI-restrictedE. invadensgenomic DNA. Long-term inhibition of cysteine proteinase activity during encystation resulted in significantly fewer cysts (P< 0.02); however, this effect appeared to be secondary to decreased trophozoite cell division. No difference in chitin synthase activity was detected between controls and encysting cells with inhibited cysteine proteinases, suggesting that these proteinases are not critical for activation of a zymogen form of chitin synthase. These studies demonstrate that cysteine proteinases may be critical for the survival ofE. invadens,and specific inhibition may ultimately interrupt transmission.

Relaxin modulates human cervical stromal cell activity.

The cervix undergoes profound changes in size and consistency during pregnancy which are designed to facilitate a normal delivery. These changes are under the control of a number of hormonal factors. Experimental and clinical studies suggest that relaxin, a protein hormone, plays a role in promoting cervical softening. One possible target site for the effects of relaxin on the cervix is the stroma. In the studies reported here, cultures of normal human cervical stromal cells were used to determine what direct relaxin has on cervical cells. Relaxin at concentrations ranging from 0.10-100 ng/mL had no effect on human cervical stromal cell proliferation, but was found to modulate stromal cell activity related to the remodeling of extracellular matrix. When gelatin substrate gels were used, relaxin stimulated the secretion of several cervical stromal proteinase activities, including 36,52, and 116 kDa. Inhibitor studies using ethylenediamine tetraacetate, 1,10-phenanthroline, and L-cysteine identify these gelatinases as metalloproteinases. Neither estradiol nor progesterone augmented the effects of relaxin. A relaxin-induced concentration-dependent increase in collagenase activity was observed when measured with a conventional collagen fibril assay. Finally, relaxin was found to increase glycosaminoglycan synthesis, as indicated by [3H]g-lycocyamine incorporation in human cervical stromal cell cultures. These results indicate the relaxin may regulate cervical ripening in humans, as it does in other species, by direct effects on cervical cells.

Matrix Metalloproteinase Expression During Mouse Peri‐implantation Development

The purpose of this study was to define the temporal expression and to quantitate the mRNA levels of collagenase, 72 kDa, 92 kDa, and membrane-type matrix metalloproteinase during the peri-implantation period of pregnancy in the mouse uterus. Embryonic expression of 72 kDa and 92 kDa matrix metalloproteinases, as well as interleukin 1 alpha, was also investigated. Uterine matrix metalloproteinases were detected using gelatin substrate gel electrophoresis (zymography) and reverse-transcription polymerase chain reaction methodology was used to detect and quantitate different mRNA species in the mouse uterus and blastocyst. Collagenase, 72 kDa, and 92 kDa matrix metalloproteinases are developmentally regulated during the peri-implantation period of pregnancy, but membrane-type matrix metalloproteinase appears to be expressed constitutively. Matrix metalloproteinase mRNA levels have been quantitated and confirm the observed developmental expression patterns. Prominent expression of bot 92 kDa matrix metalloproteinase and interleukin 1 alpha was observed in blastocysts during outgrowth while weak expression of the 72 kDa matrix metalloproteinase was detected. The date provide evidence of matrix metalloproteinase expression in vivo and substantiate their potential role in tissue remodeling prior to and during blastocyst implantation. Expression of interleukin 1 alpha, 92 kDa, and 72 kDa matrix metalloproteinases suggests that these proteins are important for trophoblast invasion associated with implantation of the early embryo.

Chondrocyte Matrix Metalloproteinase-8: HUMAN ARTICULAR CHONDROCYTES EXPRESS NEUTROPHIL COLLAGENASE

This study confirms that normal human articular chondrocytes express neutrophil collagenase or matrix metalloproteinase-8 (MMP-8), a gene product previously thought to be expressed exclusively by neutrophil leukocytes. Both MMP-8 protein and mRNA were present in articular cartilages collected from normal human donors. Cartilage extracts were assayed by immunoblotting and by analysis of enzymatic activity on gelatin-substrate gels. Latent MMP-8 extracted from cartilage has a molecular mass of 55 kDa; active MMP-8 was identified at 46 and 42 kDa. In the absence of a reducing agent, MMP-8 migrated in a high molecular mass complex above 200 kDa. Northern blotting results demonstrated the expression of MMP-8 in chondrocytes, which could be up-regulated by stimulation with interleukin-1 beta. In addition, reverse transcription-polymerase chain reaction using nested primers and in situ hybridization revealed the presence of MMP-8 mRNA in chondrocytes. The presence of both MMP-8 protein and message in cartilage supports the concept that neutrophil collagenase could be the enzyme described as "aggrecanase".

Protease repertoires of cells adherent to membranes recovered after guided tissue regeneration.

Guided tissue regeneration is a clinical procedure used to restore mineralized tissue that has been lost to periodontal disease or after tooth extraction. The procedure makes use of Gore-tex membranes or Gore-tex augmentation membranes (GTAM) to prevent migration of keratinocytes and gingival fibroblasts into healing wounds. To begin to characterize the regenerative cells associated with these membranes, human cells have been rescued from membranes retrieved after bone-inductive procedures. Cell lines were established from tissue adherent to Gore-tex membranes used to regenerate bone around periodontally compromised teeth, and from tissue adherent to GTAM used in edentulous ridge augmentation procedures or in conjunction with implant placement. Cell lines were screened for mineralized nodule formation in vitro prior to their subsequent analysis. All but one of the lines selected for this study formed mineralized nodules in vitro with cells from GTAM tending to form nodules more quickly than cells from Gore-tex. Zymograms and Western blots were used to compare protease profiles of these cells with those of human gingival fibroblasts, keratinocytes and periodontal ligament (PDL) cells. All cell types except for keratinocytes produced a 72 kD protease. In contrast, keratinocytes were the only cells that produced 92 kD gelatinase. In some cell lines, notably those removed from patients after short periods of regeneration, collagenase was the major protease detected on gelatin substrate gels. Some of these cell lines also produced additional proteases including a low molecular weight protease (30 kD) not seen in gingival fibroblasts, PDL cells or keratinocytes.