Combination Therapy Strategy of Quorum Quenching Enzyme and Quorum Sensing Inhibitor in Suppressing Multiple Quorum Sensing Pathways of P. aeruginosa.

The threat of antibiotic resistant bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Many bacteria employ quorum sensing (QS) to govern the production of virulence factors and formation of drug-resistant biofilms. Targeting the mechanism of QS has proven to be a functional alternative to conventional antibiotic control of infections. However, the presence of multiple QS systems in individual bacterial species poses a challenge to this approach. Quorum sensing inhibitors (QSI) and quorum quenching enzymes (QQE) have been both investigated for their QS interfering capabilities. Here, we first simulated the combination effect of QQE and QSI in blocking bacterial QS. The effect was next validated by experiments using AiiA as QQE and G1 as QSI on Pseudomonas aeruginosa LasR/I and RhlR/I QS circuits. Combination of QQE and QSI almost completely blocked the P. aeruginosa las and rhl QS systems. Our findings provide a potential chemical biology application strategy for bacterial QS disruption.

Expression of recombinant staphylokinase, a fibrin-specific plasminogen activator of bacterial origin, in potato (Solanum tuberosum L.) plants.

One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the ß-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.

Inhibition of growth and caseinase production of Pseudomonas aeruginosa and Escherichia coli 28 by combination of low pH and NaCl, potassium sorbate or Thymus vulgaris extract.

Previous studies reported that, at neutral pH (e.g. 7.4), many preservatives do not have antimicrobial effect against Pseudomonas aeruginosa and some strains of E. coli . This study investigated the effect of combination of low pH and some preservatives (e.g. sodium chloride, potassium sorbate or Thymus vulgaris extract) on the growth and caseinase production of Pseudomonas aeruginosa and E. coli at 31 degrees C and 7 degrees C. At 31 degrees C, although growth of the strains was not affected by low pH (5.7) alone, caseinase production by both strains was decreased. A combination of low pH and NaCl, potassium sorbate or thyme extract significantly reduced (p < 0.05) growth and caseinase production by E. coli 28 at 31 degrees C. For P. aeruginosa , addition of NaCl or thyme extract to nutrient broth (low pH) did not affect the growth but reduced caseinase production of the tested strain. Combination of low temperature (7 degrees C) and growth in nutrient broth (low pH) plus sodium chloride or nutrient broth (low pH) plus potassium sorbate was the most effective treatment in reducing or inhibiting growth and caseinase production of the tested strains.

Panax ginseng has anti-infective activity against opportunistic pathogen Pseudomonas aeruginosa by inhibiting quorum sensing, a bacterial communication process critical for establishing infection.

Virulent factors produced by pathogens play an important role in the infectious process, which is regulated by a cell-to-cell communication mechanism called quorum sensing (QS). Pseudomonas aeruginosa is an important opportunistic human pathogen, which causes infections in patients with compromised immune systems and cystic fibrosis. The QS systems of P. aeruginosa use N-acylated homoserine lactone (AHL) as signal molecules. Previously we have demonstrated that Panax ginseng treatment allowed the animals with P. aeruginosa pneumonia to effectively clear the bacterial infection. We postulated that the ability to impact the outcome of infections is partly due to ginseng having direct effect on the production of P. aeruginosa virulence factors. The study explores the effect of ginseng on alginate, protease and AHL production. The effect of ginseng extracts on growth and expression of QS-controlled virulence factors on the prototypic P. aeruginosa PAO1 and its isogenic mucoid variant (PAOmucA22) was determined. Ginseng did not inhibit the growth of the bacteria, enhanced the extracellular protein production and stimulated the production of alginate. However, ginseng suppressed the production of LasA and LasB and down-regulated the synthesis of the AHL molecules. Ginseng has a negative effect on the QS system of P. aeruginosa, may explain the ginseng-dependent bacterial clearance from the animal lungs in vivo in our previous animal study. It is possible that enhancing and repressing activities of ginseng are mutually exclusive as it is a complex mixture, as shown with the HPLC analysis of the hot water extract. Though ginseng is a promising natural synergetic remedy, it is important to isolate and evaluate the ginseng compounds associated with the anti-QS activity.

Transcriptional regulation of ADAMTS13.

The metalloproteinase ADAMTS13 cleaves VWF multimers instantaneously when they are released from endothelial cells. Absent or manifestly diminished proteolytic activity of ADAMTS13 results in the appearance and accumulation of ultralarge VWF multimers (ULVWFM) in plasma, characterised by the manifestation of Thrombotic Thrombocytopenic Purpura (TTP). Despite congenital defects, infections and the actions of drugs such as cyclosporine A, doxycycline and corticosteroids apparently are involved in its development. To examine the possibility of transcriptional regulation of ADAMTS13 activity, we analyzed RNA levels in various cell culture systems and the response to known and assumed modulators of gene expression. We demonstrate the expression of ADAMTS13 in liver homogenates and a parenchyma liver cell culture system Hep3B, supporting the hypothesis that liver is an important source of plasma ADAMTS13, whereas there was no alteration in gene expression after stimulation of liver cells with proinflammatory stimuli such as endotoxin, TNF-alpha, IL-6, IL-1beta as well as immuno-suppressive agents, such as cyclosporine A, a variety of steroids as well as doxycycline. Therefore, we analysed the ADAMTS13 gene for binding sites of transcription factors in silico and compared the data with those found in two sets of 24 genes considered either as differentially regulated by prototypic inflammatory regulation or as unvaried under various conditions. On the basis of these data, the promotor of ADAMTS13 features the characteristics of a gene, which remains unvaried under a variety of conditions. To our knowledge, the current data demonstrate for the first time, that an alteration in transcriptional activity is negligible in accounting for diminished proteolytic activity as observed under various experimental and, in particular, clinical conditions.

SKI306X suppresses cartilage destruction and inhibits the production of matrix metalloproteinase in rabbit joint cartilage explant culture.

SKI306X was previously found to have cartilage protective effects in the experimental osteoarthritis (OA) model. To investigate the chondro-protective benefits of SKI306X for its capacity in altering changes in cartilage metabolism and molecular mechanisms of cartilage protective action, SKI306X is studied in rabbit cartilage explants culture. To investigate the protective effect of SKI306X on cartilage catabolism, we assessed collagen degradation in rabbit cartilage explants treated with interleukin-1alpha up to 3 weeks. To examine the reaction mechanism, matrix metalloproteinase (MMPs) were investigated by fluorimetric and Western blotting analysis. In addition, its effects on the activation process of proenzyme MMP-3 were determined by gelatin zymography. SKI306X significantly inhibited collagen degradation and inhibited the activities of several MMPs. Total MMPs activities in cultured medium were substantially increased in the third week at the time of collagen degradation with the absence of SKI306. However, the introduction of SKI306X decreased MMPs activities in cultured medium. Furthermore, Western blotting analysis proved that these inhibitory effects of this drug were the result of inhibiting MMPs expression. SKI306X also inhibited the activation of proenzyme MMP-3 to the active form of MMP-3. These results indicate that SKI306X inhibits matrix degradation by down regulating MMPs expression and secretion, inhibition of MMPs activity, and inhibiting activation of MMP-3 during the collagen breakdown process.

Regulation of TIMP-2, MT1-MMP, and MMP-2 expression during C2C12 differentiation.

Matrix metalloproteinases (MMPs) are zinc-dependent proteases capable of degrading extracellular matrix components. The activity of these proteases is tightly regulated through the actions of the tissue inhibitors of metalloproteinases (TIMPs). Although the regulation of MMPs and TIMPs during physiological and pathological remodeling has been investigated in a number of systems, almost nothing is known about their role in skeletal muscle differentiation. To investigate the role of MMP-mediated proteolysis during myogenesis, the regulation of TIMP-2, MT1-MMP, and MMP-2 expression was investigated during differentiation of the mouse myoblastic C2C12 cell line. We show that this trio is upregulated coincident with myogenesis. The more diffuse spatial distribution of TIMP-2 relative to MT1-MMP and MMP-2 suggests that TIMP-2 may exert MMP-independent functions during myogenesis. Elucidating the regulation of these molecules during muscle differentiation in vitro may lead to a better understanding of their role in pathological processes in muscle tissue in vivo.

Implication of interleukin 18 in production of matrix metalloproteinases in articular chondrocytes in arthritis: direct effect on chondrocytes may not be pivotal.

OBJECTIVE: To clarify the effect of interleukin (IL) 18 on cartilage degeneration by studying the profile of IL18 receptor (IL18R) on chondrocytes and the direct effect of IL18 on production of matrix metalloproteinases (MMPs), aggrecanases, and tissue inhibitors of metalloproteinases (TIMPs) in articular chondrocytes. METHODS: Monolayer cultured human articular chondrocytes were isolated from non-arthritic subjects and patients with rheumatoid arthritis or osteoarthritis. Gene expression of IL18, IL18Ralpha, IL18Rbeta, MMPs, and aggrecanases was detected by RT-PCR. Protein levels of IL18Ralpha were analysed by flow cytometry. Protein levels of IL18, MMPs, and TIMPs were measured by ELISA. Aggrecanase-2 mRNA expression was quantitatively analysed by real time RT-PCR. Protein levels of signalling molecules were assayed by western blotting. RESULTS: IL18 mRNA was constitutively expressed in chondrocytes, and was enhanced by IL1beta stimulation. Flow cytometric analysis showed that IL1beta, tumour necrosis factor alpha, and IL18 up regulated IL18Ralpha expression levels. The level of IL18Rbeta mRNA was much lower than that of IL18Ralpha, and was slightly up regulated by IL1beta. In chondrocytes responding to IL18, IL18 (1-100 ng/ml) slightly increased the production of MMP-1, MMP-3, and MMP-13, which was blocked by NF-kappaB inhibitor and p38 mitogen activated protein kinase inhibitor. IL18 up regulated mRNA expression of aggrecanase-2, but not aggrecanase-1. IL18 also slightly stimulated TIMP-1 production?through extracellular signal regulated kinase activation. CONCLUSION: IL18 induces production of MMPs from chondrocytes in inflammatory arthritis. Although the direct effect of IL18 on chondrocytes may not be pivotal for the induction of cartilage degeneration, IL18 seems to play some part in the degradation of articular cartilage in arthritis.

Regulation of ADAM12 cell-surface expression by protein kinase C epsilon.

The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell surface and that catalytic activity of PKCepsilon is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 microM phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCepsilon could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKCepsilon both contain a binding site for ADAM12. These studies show that PKCepsilon plays a critical role in the regulation of ADAM12 cell-surface expression.

Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1.

We demonstrate that in humans, two metalloproteases, ADAMTS-9 (1935 amino acids) and ADAMTS-20 (1911 amino acids) are orthologs of GON-1, an ADAMTS protease required for gonadal morphogenesis in Caenorhabditis elegans. ADAMTS-9 and ADAMTS-20 have an identical modular structure, are distinct in possessing 15 TSRs and a unique C-terminal domain, and have a similar gene structure, suggesting that they comprise a new subfamily of human ADAMTS proteases. ADAMTS20 is very sparingly expressed, although it is detectable in epithelial cells of the breast and lung. However, ADAMTS9 is highly expressed in embryonic and adult tissues, and therefore we characterized the ADAMTS-9 protein further. Although the ADAMTS-9 zymogen has many proprotein convertase processing sites, pulse-chase analysis, site-directed mutagenesis, and amino acid sequencing demonstrated that maturation to the active form occurs by selective proprotein convertase (e.g. furin) cleavage of the Arg(287)-Phe(288) bond. Although lacking a transmembrane sequence, ADAMTS-9 is retained near the cell surface as well as in the ECM of transiently transfected COS-1 and 293 cells. COS-1 cells transfected with ADAMTS9 (but not vector-transfected cells) proteolytically cleaved bovine versican and aggrecan core proteins at the Glu(441)-Ala(442) bond of versican V1 and the Glu(1771)-Ala(1772) bond of aggrecan, respectively. In contrast, the ADAMTS-9 catalytic domain alone was neither localized to the cell surface nor able to confer these proteolytic activities on cells, demonstrating that the ancillary domains of ADAMTS-9, including the TSRs, are required both for specific extracellular localization and for its versicanase and aggrecanase activities.

Aqueous extract from rhizoma notopterygii reduces contact sensitivity by inhibiting lymphocyte migration via down-regulating metalloproteinase activity.

In the present paper we examined the effects of the aqueous extract from Rhizoma notopterygii (RN-ext) on picryl chloride-induced contact sensitivity (PCl-CS). The extract, administered during either induction or effector phase showed a significant inhibition on the ear swelling in mice with PCl-CS. By using the isolated spleen cells from the mice with PCl-CS, we demonstrated the inhibitory effects of RN-ext on matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. However, such inhibition was not found in those from normal mice. The inhibitory effects on MMP-2 and MMP-9 of RN-ext were also observed when it was administered in vivo. In addition, the extract significantly inhibited the migration of spleen cells from PCl-CS mice in transwell system without affecting the cell adhesion to fibronectin. These results suggest that RN-ext exerts its inhibitory activity on the contact sensitivity through decreasing the localization to the inflammation site via down-regulating MMP activities.

A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning, localization, and putative function.

Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.

Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening.

AIM: To obtain a catalytically active human macrophage elastase catalytic domain (hMECD) and to establish an efficient high-throughput method for screening macrophage elastase inhibitors. METHODS: Catalytic domain of human macrophage elastase was expressed in E coli and characterized to establish a high-throughput screening assay using a colorimetric method. A set of 8560 pure compounds and mixtures were screened. RESULTS: We have constructed an efficient E coli system for this human protein expression, and the recombinant hMECD protein was purified to homogeneity using anion-exchange chromatography after in vitro refolding from inclusion bodies. The yield of active hMECD protein was 23 mg from one liter of E coli culture after purification. Calcium and zinc ions were required both in refolding and enzymatic activity, but high concentration of zinc inhibited the refolding and activity. The hMECD cleaved several synthetic substrates including a chromogenic thiopeptolide and fluorogenic peptides with optimal activity around pH 8.0. Screening of 8560 compounds and mixtures led to identify 27 pure compounds and 14 natural products with inhibitory activity higher than 80 % at 20 mg/L. CONCLUSION: An efficient expression and purification method for hMECD protein has been established, and the assay is effective, reliable, and fast in identifying the recombinant protein inhibitors.

Effect of avocado and soybean unsaponifiables on gelatinase A (MMP-2), stromelysin 1 (MMP-3), and tissue inhibitors of matrix metalloproteinase (TIMP- 1 and TIMP-2) secretion by human fibroblasts in culture.

BACKGROUND: In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). They can also respond to growth factors and cytokines. In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable [ASF] or soy unsaponifiable [SSF]) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts. METHODS: Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0. 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta). MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis. TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis. RESULTS: In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF. The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml). A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations. Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF. As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen. ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml. CONCLUSIONS: These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.

[Effect of reduqing on HL-60 cells in secreting tumor necrosis factor alpha production and on TNF alpha converting enzyme mRNA expression].

OBJECTIVE: To explore the molecular mechanism of inhibiting effects of Reduqing on the release of tumor necrosis factor alpha (TNF alpha). METHODS: Using cytobiologic and molecular biologic technique to observe the effects of Reduqing on HL-60 cells in producing inflammatory cytokine secreting TNF alpha (sTNF alpha) and on mRNA expression of TNF alpha converting enzyme (TACE). RESULTS: (1) Reduqing, diluted in ratio 1:30, could effectively inhibit the increased HL-60 production of sTNF-alpha induced by lipopolysaccharides (LPS); (2) Although no obvious effect on TACE was shown when Reduqing was applied alone on HL-60 cells, there was evident inhibitory effect of Reduqing on TACE mRNA expression enhancement induced by LPS. CONCLUSION: Reduqing could have the double inhibitory effects both on sTNF-alpha production and on the gene expression of its key enzyme, i.e. TACE stimulated by LPS, suggesting that it might be a hopeful and excellent natural TACE inhibitor.

Coordinated expression of beta-amyloid precursor protein and the putative beta-secretase BACE and alpha-secretase ADAM10 in mouse and human brain.

To define the enzymes involved in the etiology of Alzheimer's disease, we compared in mouse and human brain the mRNA levels and cellular localization of the ubiquitous beta-amyloid precursor protein (beta-APP) with those of the putative alpha-secretases ADAM10 and ADAM17 and the beta-secretases BACE and BACE2. In situ hybridization performed in mice during prenatal and postnatal development and in adulthood revealed the coexpression of beta-APP, BACE, and ADAM10. The patterns of BACE2 and ADAM17 only partially overlapped with that of beta-APP. beta-APP, BACE, and ADAM10 mRNAs have also been detected by northern blot in human brain cortex of normal subjects and in Alzheimer's disease subjects. In situ hybridization performed using combined biotin- and radiolabeled riboprobes provided evidence for the coexpression of beta-APP with BACE and ADAM10 in human cortical neurons. Our data provide cytochemical evidence supporting the role of ADAM10 and BACE as authentic alpha- and beta-secretases.

CA-MMP: a matrix metalloproteinase with a novel cysteine array, but without the classic cysteine switch.

A matrix metalloproteinase (MMP)-like gene was identified in mouse to contain a conserved MMP catalytic domain and an RRRR motif. It lacks a classic cysteine switch, but it possesses two novel motifs: a cysteine array (Cys-X(6)-Cys-X(8)-Cys-X(10)-Cys-X(3)-Cys-X(2)-Cys), and a novel Ig-fold. It is named CA-MMP after the distinct cysteine array motif, and little is known about its biochemical function. In an attempt to characterize CA-MMP activity, the full-length sequence was expressed in mammalian cells and its product found to be cell-associated without detectable secretion. In light of this unusual finding, a chimera combining the catalytic domain of CA-MMP with the prodomain of stromelysin-3 was constructed to express a fully active enzyme in mammalian cells. Purified CA-MMP catalytic domain expresses proteolytic activity against protein substrates in an MMP inhibitor sensitive fashion. Taken together, it is concluded that CA-MMP is an MMP with distinct structure, biochemical properties and evolutionary history that may define a new subclass of the MMP superfamily.

Characterization of the cDNA and gene for mouse tumour necrosis factor alpha converting enzyme (TACE/ADAM17) and its location to mouse chromosome 12 and human chromosome 2p25.

Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.

Inhibition of gelatinase A (MMP-2) by batimastat and captopril reduces tumor growth and lung metastases in mice bearing Lewis lung carcinoma.

We have examined the effects of the synthetic matrix metalloproteinase inhibitor, batimastat (BB-94) and the angiotensin-converting enzyme inhibitor, captopril, on metalloproteinase activity of murine Lewis-lung-carcinoma cells (3LL) in vitro, and on local growth and lung metastasis of the same tumor implanted intramuscularly in syngeneic C57BL/6 mice. The effect of BB-94 and captopril on the survival of the 3LL-tumor-bearing mice was also examined. Here we report that captopril treatment resulted in decreased transcription and protein levels of gelatinase A by 3LL cells. Both BB-94 and captopril also prevented substrate degradation by gelatinase A and B released in conditioned medium by cultured cells. Treatment of tumor-bearing animals with BB-94 (i.p.) or captopril (in drinking water) resulted in significant inhibition of the mean tumor volume (25 and 33% respectively) and of the mean lung metastasis number (26 and 29% respectively). When both agents were given, they acted in synergy, resulting in 51 and 80% inhibition of tumor growth and metastasis. The survival time of the mice treated with both BB-94 and captopril was also significantly longer compared with the groups treated with each agent alone or with the vehicle. Our data support the hypothesis of an essential role of metalloproteinase(s) in the metastatic process. Moreover, blockade of invasion, angiogenesis and other processes mediated by metalloproteinases may underlie the anti-tumor and anti-metastatic effect of BB-94 and captopril and their combination. It is conceivable that this combination could be tested in selected clinical conditions as an adjuvant modality to cytotoxic therapy.

Identification of a human cDNA encoding a novel protein structurally related to the yeast membrane-associated metalloprotease, Ste24p.

Recently, a novel membrane-associated metalloprotease, designated Ste24p, has been identified in Saccharomyces cerevisiae [K. Fujimura-Kamada, F.J. Nouvet, S. Michaelis, J. Cell Biol. 27 (1997) 271-285]. We cloned a human brain cDNA encoding a protein homologous to Ste24p (designated Hs Ste24p). The predicted 475-amino acid product of its open reading frame exhibited 62% similarity to Ste24p, and contained a zinc metalloprotease motif (HEXXH) and multiple predicted membrane spans. Northern blot analysis showed that this gene was expressed in most tissues. Immunofluorescence analysis of epitope-tagged Hs Ste24p constructs suggested that it is localized in the ER and possibly also in the Golgi compartment. A search of the expression sequence tag database identified a fragment of DNA encoding a segment homologous to the segment of Hs Ste24p containing the HEXXH motif in insects and nematodes. Thus, Hs Ste24p could be a member of a new family of Ste24p-like membrane-associated metalloproteases which are widely conserved in eukaryotes.