CPVL suppresses metastasis of nasopharyngeal carcinoma through inhibiting epithelial-mesenchymal transition.

PURPOSE: Distant metastasis is the main obstacle to treating nasopharyngeal carcinoma (NPC). Tumor distance metastasis is a complex process involving the jointly participation of multiple oncogenes, tumor suppressor genes, and metastasis-associated genes. Enough accurate prognostic genes for evaluating metastasis risk are lacking. We aimed to identify more precise biomarkers for NPC metastasis. METHODS: We performed weighted gene co-expression network analysis, differentially expressed gene analysis, univariate and multivariate stepwise Cox regression, and Kaplan-Meier (K-M) survival analyses, on data obtained from RNA sequencing of 10 NPC samples and the public database, to identify key genes correlated with NPC metastasis. Wound healing assays, transwell assays, and immunohistochemistry were conducted to validate our bioinformatic conclusions. Western blotting was performed to evaluate and quantify the effect of identified EMT genes on epithelial-mesenchymal transition (EMT) of NPC. RESULTS: Combined our own RNA sequencing data and public data, we determined carboxypeptidase vitellogenic-like protein (CPVL) as a tumor suppressor for NPC. Pathway enrichment analyses indicated that genes associated with CPVL are involved in EMT. NPC with low CPVL expression had high tumor purity and low levels of immune cells. Experimental results showed that CPVL protein predominantly expressed in cytoplasmic and membranous and it exhibited higher expression levels in NPC tissues without distant metastasis than those with distant metastasis. CPVL inhibits the migration and invasive capability of NPC cells. Overexpression of CPVL upregulates E-cadherin and ZO-1, whereas it downregulates vimentin, suggesting that CPVL suppresses tumor metastasis by inhibiting EMT. CONCLUSION: CPVL inhibits migration and invasion of NPC cells and is associated with tumor metastasis suppression through upregulating epithelial marker and inhibiting mesenchymal marker expression and could be a prognostic biomarker for metastasis risk evaluation in NPC.

Carboxypeptidase A6 suppresses the proliferation and invasion of colorectal cancer cells and is negatively regulated by miR-96-3p.

BACKGROUND: Colorectal cancer (CRC) is a common malignant tumor, and this study aims to explore the role and the regulatory mechanism of carboxypeptidase A6 (CPA6) in CRC cells. METHODS: Specific shRNA targeting CPA6 mRNA was transfected into NCM460 and HT29 cells to down-regulate CPA expression, and expression plasmid was transfected into HCT116 cells to exogenously overexpress CPA6. The dual luciferase assay was used to detect the direct binding of miR-96-3p to CPA6 3'UTR. Phosphorylation and activation of Akt were detected using Western blot. Cells were treated with miR-96-3p mimics, Akt inhibitor (MK-2206) or agonist (SC79) for rescue experiments. The cell functions were evaluated using CCK-8, clone formation, transwell, and Western blot assays. Xenograft tumor assay was also used to analyze the effect of altered CPA6 expression on tumor growth. RESULTS: Knockdown of CPA6 promoted the proliferation, clone formation, migration, and invasion of NCM460 and HT29 cells in vitro, and the tumor growth of nude mouse xenograft tumor in vivo. Moreover, over-expression of CPA6 significantly inhibited the malignant proliferation and invasion of HCT116 cells in vitro, and the tumor growth of xenograft tumor in vivo. Furthermore, miR-96-3p could directly regulate CPA6 expression by targeting its 3'UTR, and miR-96-3p mimics rescued the inhibitory effects of CPA6 overexpression on the malignant proliferation and invasion of CRC cells. Finally, CPA6 knockdown enhanced Akt/mTOR phosphorylation and activation, while CPA6 overexpression inhibited Akt/mTOR activation. The regulatory effect of CPA6 on Akt/mTOR signaling was naturally regulated by miR-96-3p. Akt inhibitor or agonist rescued the effects of CPA6 knockdown or overexpression on proliferation and EMT of colon cancer cells. CONCLUSION: CPA6 has a significant tumor suppressive effect on CRC by inhibiting the activation of Akt/mTOR signaling, and miR-96-3p negatively regulates the expression of CPA6.

Silencing of adipocyte enhancer-binding protein 1 (AEBP1) alleviates renal fibrosis in vivo and in vitro via inhibition of the ß-catenin signaling pathway.

Renal fibrosis is the common final pathway in many renal diseases regardless of the underlying etiology. Adipocyte enhancer-binding protein 1 (AEBP1) was reported to play a vital role in the development of organ fibrosis, but its role in renal fibrosis has not been reported. Thus, the aim of this study was to investigate the possible function of AEBP1 in renal fibrosis and the mechanism associated with the ß-catenin signaling pathway. A total of 83 genes upregulated after unilateral ureteral obstruction (UUO) were screened from two Gene Expression Omnibus (GEO) datasets and subjected to Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Among them, AEBP1 was enriched in collagen binding and the regulation of collagen fibril organization and was confirmed to be upregulated in UUO kidneys and TGF-ß1-induced cells. Knockdown of AEBP1 ameliorated renal fibrosis via reducing collagen accumulation, inhibiting epithelial-mesenchymal transition and fibroblast transformation, as evidenced by decreases in the expression of collagen I and III, Col1a1, Col3a1, fibronectin, Snail, α-SMA, as well as collagen-specific staining of kidney tissues, whereas the E-cadherin was increased. Besides, AEBP1 silencing inhibited the expression of ß-catenin in nucleus and ß-catenin downstream proteins (Axin2, Myc, and Ccnd1). Continuously active ß-catenin-S33Y further restored the inhibitory effect of AEBP1 silencing on renal fibrosis. These findings indicate that knockdown of AEBP1 could potentially slow down renal fibrosis by blocking the ß-catenin signaling pathway, highlighting the potential of AEBP1 as a therapeutic target for renal fibrosis.

Carboxypeptidase vitellogenic like facilitates resistance to CDK4/6 inhibitors in breast cancer.

OBJECTIVE: Inhibitors of cyclin-dependent kinase 4 and 6 (CDK4/6) are targeted therapeutic drugs for breast cancer treatment. The mechanism of resistance to these inhibitors requires further investigation. METHODS: We used bioinformatics to screen differentially expressed genes between cells that were susceptible and resistant to CDK4/6 inhibitors. Quantitative real-time PCR (qRT-PCR) was used to identify gene expressions in different cell lines. Cell viability, colony formation, cell cycle, and apoptosis assays were used to evaluate the effect of carboxypeptidase vitellogenic like (CPVL) on breast cancer cells under the condition of CDK4/6 inhibitors. Gene set enrichment analysis (GSEA) suggested the potential regulatory pathway of CPVL in breast cancer. Xenograft formation assay was conducted in nude mice to study the role of CPVL in vivo. RESULTS: Based on bioinformatics analysis and qRT-PCR, CPVL was identified more abundantly in cells that were resistant than sensitive to CDK4/6 inhibitors. Overexpressed or knocked down CPVL regulated the effects of CDK4/6 inhibitors in resistant cell lines. GSEA showed that resistance might be induced by CPVL through altered phosphatase and tensin homolog (PTEN)-related pathways. Our findings showed that CPVL negatively regulates PTEN to impact the anticancer effects of CDK4/6 inhibitors in vitro and in vivo. CONCLUSION: CPVL might be a key factor in regulating breast cancer resistance to CDK4/6 inhibitors.

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

A newly characterized vacuolar serine carboxypeptidase, Atg42/Ybr139w, is required for normal vacuole function and the terminal steps of autophagy in the yeast Saccharomyces cerevisiae.

Macroautophagy (hereafter autophagy) is a cellular recycling pathway essential for cell survival during nutrient deprivation that culminates in the degradation of cargo within the vacuole in yeast and the lysosome in mammals, followed by efflux of the resultant macromolecules back into the cytosol. The yeast vacuole is home to many different hydrolytic proteins and while few have established roles in autophagy, the involvement of others remains unclear. The vacuolar serine carboxypeptidase Y (Prc1) has not been previously shown to have a role in vacuolar zymogen activation and has not been directly implicated in the terminal degradation steps of autophagy. Through a combination of molecular genetic, cell biological, and biochemical approaches, we have shown that Prc1 has a functional homologue, Ybr139w, and that cells deficient in both Prc1 and Ybr139w have defects in autophagy-dependent protein synthesis, vacuolar zymogen activation, and autophagic body breakdown. Thus, we have demonstrated that Ybr139w and Prc1 have important roles in proteolytic processing in the vacuole and the terminal steps of autophagy.

Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10.

Bacillus licheniformis HS10 is a good biocontrol agent against Pseudoperonospora cubensis which caused cucumber downy disease. To identify and characterize the antifungal proteins produced by B.licheniformis HS10, the proteins from HS10 were isolated by using 30-60% ammonium sulfate precipitation, and purified with column chromatography on DEAE Sepharose Fast Flow, RESOURCE Q and Sephadex G-75. And the SDS-PAGE and MALDI-TOF/TOF-MS analysis results demonstrated that the antifungal protein was a monomer with molecular weight of about 55 kDa, identified as carboxypeptidase. Our experiments also showed that the antifungal protein from B. licheniformis HS10 had significantly inhibition on eight different kinds of plant pathogenic fungi, and it was stable with good biological activity at as high as 100°C for 30 min and in pH value ranged from 6 to 10. The biological activity was negatively affected by protease K and 10mM metal cations except Ca(2+).

Mitrecin A, an endolysin-like bacteriolytic enzyme from a newly isolated soil streptomycete.

UNLABELLED: An open reading frame with homology to known endolysin genes was identified in the genome of Streptomyces sp. strain 212, which is a newly isolated soil bacterium. The heterologously expressed gene product of this endolysin-like gene, called Mitrecin A, demonstrated bacteriolytic activity against several Gram-negative bacteria. The genome of the bacterial strain was sequenced to draft quality using pyrosequencing followed by genome assembly and gene annotation. Within the sequence, a chromosomally located endolysin-like open reading frame was predicted. The gene product, designated Mitrecin A, was heterologously expressed and isolated from contaminating proteins as a fusion protein to a 6-histidine tag. Mitrecin A consists of 127 amino acids arranged in modular domains of activity. It has an estimated molecular weight of 14.3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases. The heat-labile purified recombinant protein has an overall positive charge, has optimal catalytic activities at 26°C in solution of pH 9 with 1% saline and has bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. SIGNIFICANCE AND IMPACT OF THE STUDY: The gene of a new protein antimicrobial, Mitrecin A, was discovered in the genome of a soil bacterium. The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. Characterization of enzymes such as Mitrecin A from previously uncharacterized bacteria provides potential options for new biocontrol agents in medically and economically important applications like therapeutics, disinfectants, food preservatives, agricultural livestock antimicrobials, and inhibitors of biofilm production.

Effect of collagen I and aortic carboxypeptidase-like protein on 3T3-L1 adipocyte differentiation.

Aortic carboxypeptidase-like protein (ACLP) is a secreted protein expressed in preadipocytes and down-regulated during adipogenesis. Results from previous studies on the influence of ACLP overexpression on adipogenesis vary from no effect to complete inhibition. We hypothesized that ACLP may modulate adipogenesis in the presence of collagen I, a protein to which it binds. We compared control (pLXSN) 3T3-L1 preadipocytes with 3T3-L1 preadipocytes stably overexpressing ACLP (pLXSN-ACLP) that were grown in standard vs collagen I-coated dishes. Aortic carboxypeptidase-like protein overexpression, via retroviral transduction, resulted in a 3.2-fold increase in ACLP cellular levels and a 2.1-fold increase in ACLP levels released into medium. Aortic carboxypeptidase-like protein overexpression did not inhibit differentiation in standard dishes. In collagen I-coated dishes compared with standard dishes, control preadipocytes, when induced to differentiate, exhibited the same increase in triacylglycerol accumulation, but showed a significantly higher induction of fatty acid synthase (1.6-fold more), peroxisome proliferator-activated receptor γ (1.4-fold more), and CCAAT/enhancer-binding protein α (1.4-fold more). Aortic carboxypeptidase-like protein overexpression significantly reduced this enhanced induction of fatty acid synthase, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer-binding protein α by 65%, 59%, and 66%, respectively, but had no effect on the accumulation of triacylglycerol during differentiation. Finally, studies on proadipogenic insulin signaling in ACLP-overexpressing preadipocytes demonstrated that insulin-stimulated Akt phosphorylation was significantly decreased by 27% in cells cultured in collagen I-coated dishes vs standard dishes. Our data suggest that ACLP inhibits certain aspects of 3T3-L1 adipogenesis in a collagen I-rich environment.

Inhibitory effects of aloe carboxypeptidase fraction on streptozotocin-induced enhancement of vascular permeability in the pancreatic islets.

The protective actions of components isolated from Aloe arborescens Miller var. natalensis Berger (Kidachi aloe in Japanese) on streptozotocin (Sz)-induced necrosis of B cells in the pancreatic islets of the mouse were investigated to clarify its action mechanism involved in anti-diabetic effects. In this experiment, phenol low molecular weight components of aloin and aloin A that were anti-oxidants and derived from the leaf skin or pulp extract, an aloe carboxypeptidase fraction that is a inhibitor of enhanced vascular permeability and a glycoprotein component that decreases blood glucose were tested with mice precedently administered with Sz which is known as a cytotoxin specific to B cells. The results showed that the treatment group receiving Sz followed by the aloe carboxypeptidase fraction increased the inhibition of dye leakage by 75.8% (p<0.001) in the extract of whole pancreas in comparison to the control group and the aloe carboxypeptidase fraction group also increased the inhibition effect by 68.4% (p<0.001) in the extract of pancreatic islets as compared to the control group. The carboxypeptidase is an aloe-derived protease known to inhibit the acetic acid-related enhancement of intraperitoneal vascular permeability in mice. Further, the elevation of blood glucose in Sz-induced diabetic mice intraperitoneally given the aloe carboxypeptitase fraction was significantly (p<0.01-0.001) restrained at 3, 7 and 14 days after the injection as compared to the control group given solvent only. The results of this experiment suggested that the inhibitory effect on the enhancement of vascular permeability related to the vascular acute inflammatory response at Sz-induced lesions of pancreatic islets was involved in the action mechanism of this enzyme.

Carboxypeptidase A-catalyzed direct conversion of leukotriene C4 to leukotriene F4.

Leukotrienes (LTs) are 5-lipoxygenase (5-LO)-derived arachidonic metabolites that constitute a potent set of lipid mediators produced by inflammatory cells. Leukotriene A(4), a labile allylic epoxide formed from arachidonic acid by dual 5-LO activity, is the precursor for LTB(4) and LTC(4) synthesis. LTC(4) is further transformed enzymatically by the sequential action of gamma-glutamyltranspeptidase and dipeptidase to LTD(4) and LTE(4), respectively. In this report, we present evidence that bovine pancreatic carboxypeptidase A (CPA), which shares significant sequence homology with CPA in mast cell granules, catalyzes the conversion of LTC(4) to LTF(4) via the hydrolysis of an amide bond. The identity of CPA-catalyzed LTC(4) hydrolysis product as LTF(4) was confirmed by several analytical criteria, including enzymatic conversion to conjugated tetraene by soybean LO, conversion to LTE(4) by gamma-glutamyltranspeptidase, cochromatography with the standard LTF(4) and positive-ion fast-atom bombardment mass spectral analysis. Thus, it appears that the physiological significance of this single-step transformation may point toward a major cellular homeostatic mechanism of metabolizing LTC(4), a potent bronco- and vasoconstrictor, to a less potent form of cysteinyl LTs.

Perturbation of the wing color pattern of a swallowtail butterfly, Papilio xuthus, induced by acid carboxypeptidase.

Hypodermal injection of Toughmac-E, a digestive mixture composed of nine digestive components, or Molsin induced perturbation of the wing color pattern in 0- to 2-day-old pupae of Papilio xuthus, but had no effect on prepupae or 3- to 4-day-old pupae. The effective component in Toughmac-E was identified as Molsin, an acid carboxypeptidase of Aspergillus saitoi which specifically liberates tyrosine and phenylalanine from the C-terminal residues of proteins. The pattern perturbation occurred in either side of the fore- and hindwings of both sexes. When this enzyme was administered, stronger melanization than in the normal wings was found in the whole wings of most butterflies, but in other butterflies, the yellow region was enlarged. These findings suggest that the pattern perturbation was caused by changes in the levels of melanin and papiliochromes in scales. Melanin is a black pigment and papiliochromes are yellow pigments; their common precursor is dopamine. The normal pattern is formed by a predetermined balance of melanin and papiliochromes, whereas the deposit of an excess amount of tyrosine and/or phenylalanine disturbs this balance and results in perturbation of the color pattern. Administration of L-dopa or dopamine had no effect on the wing pattern. When the activity of an endogenous acid carboxypeptidase similar to Molsin appears in the early pupa, the summed activities of the endogenous and exogenous acid carboxypeptidases must induce a pattern perturbation. The relations between the endogenous acid carboxypeptidase and its probable substrate, the reserve protein, and the physiological roles of these relations in the regulation, utilization and excretion of amino acids are discussed.

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

Anti-tumor effects of toxins targeted to the prostate specific membrane antigen.

BACKGROUND: There is presently no effective therapy for relapsing, metastatic, androgen-independent prostate cancer. Immunotherapy with monoclonal antibody-vehicled toxins (Immunotoxins, ITs) may be a promising novel treatment option for the management of prostate cancer in these cases. METHODS: Three anti-prostate specific membrane antigen (anti-PSMA) monoclonals (J591, PEQ226.5, and PM2P079.1) were cross-linked to ricin A-chain (RTA; native or recombinant), and their cytotoxic effects were investigated in monolayer and three-dimensional (3-D) cell cultures of prostate carcinoma cells (LNCaP). RESULTS: The various Immunotoxins showed effects in the nanomolar range (IC(50s) of 1.6-99 ng/ml) against PSMA+ cells (IC(50) being the concentration inhibiting 50% cell proliferation or protein synthesis). PSMA(-) cell lines were 62- to 277-fold less sensitive to anti-PSMA ITs, evidencing an appreciable therapeutic window. Treatment with J591-smpt-nRTA (0.35-31.7ng/ml) resulted in complete eradication of 3-D tumor micromasses or in 1.46- to 0.35-log reduction of target cells number, depending on the dose. CONCLUSION: Anti-PSMA ITs appear to be promising for use in the eradication of small prostate tumor cell aggregates present in tissues and in the bone marrow.

Rapid microscale enzymic semisynthesis of epidermal growth factor (EGF) analogues.

Epidermal Growth Factor (EGF) is a small growth factor containing 53 amino acid residues capable of stimulating the proliferation of both mesenchymal and epithelial cells. Comparison of the amino acid sequences of EGF from several species, and related proteins that can bind to the EGF receptor (e.g. TGFalpha, VGF, heparin-binding EGF, and betacellulin), suggests that Leu47, which is highly conserved, is important for biological function. Additionally, we have shown previously, using a combination of trypsin and carboxypeptidase Y digestion of native murine EGF, that removal of Leu47 results in more than 100-fold decrease in both receptor binding and mitogenic activity. We now describe a micromethod for the rapid generation of C-terminally modified EGFs to investigate further the role of C-terminal residues in determining functional activity. These analogues have been generated by digesting native murine EGF with trypsin, purifying the biologically inactive, but structurally intact, EGF1-45 core by micropreparative RP-HPLC, and then reversing the action of trypsin to couple synthetic peptides (e.g. DL, DI, DF, EL, DLLW) onto the C-terminus of the EGF1-45 core. This enzymic semisynthesis method allows multiple derivatives to be generated rapidly from microgram quantities of EGF1-45 in sufficient quantities for sensitive biological and physicochemical analysis. We have validated the method by regenerating EGF1-47 from EGF1-45 with equivalent mitogenic and receptor binding activity to EGF1-47 generated from wild type EGF by digestion with trypsin and carboxypeptidase Y. We have also investigated the effect of substituting alternative normal or nonphysiological amino acids (e.g. allo-Ile) for Asp46, Leu47 or Arg48. Even small changes in these C-terminal residues reduce the mitogenic potency of the analogue.

Biosynthesis and post-translational processing of site-directed endoproteolytic cleavage mutants of Pro-CCK in AtT-20 cells.

Site-directed mutagenesis in which individual cleavage site P1 amino acids were changed to Ala was performed to delineate their importance in the processing of pro-CCK in mouse pituitary tumor AtT-20 cells. Individual substitution of cleavage sites on pro-CCK, viz., CCK 58 cleavage site R/A to A/A, CCK 33 cleavage site R/K to A/K, CCK 22 cleavage site K/N to A/N, and CCK 8 cleavage site R/D to A/D, did not inhibit pro-CCK expression or the production of some form of amidated CCK. Wild-type CCK cDNA expression in these cells results in production and secretion of CCK 8 and CCK 22. Substitution of the 58R/A cleavage site with A/A produces only CCK 33; 33A/K and 22A/N produce only CCK 8, whereas 8A/D produces CCK 12 and some CCK 22. Where the GRR residues on the C-terminus of CCK 8 were mutated to GAA, no amidated CCK was produced. Significant amounts of the pro-CCK, C-terminal peptide S9S was found in the medium of cells transfected with GAA mutant cDNA, indicating that this pro-CCK was cleaved at the GAA site probably by a nonprohormone convertase enzyme. Further analysis of the cells expressing the GAA mutant demonstrated that it is not extensively cleaved at other sites to produce CCK 8 GAA or larger peptides. In the mutant where the entire pro-CCK, C-terminal S9S was deleted, CCK 8 is processed and secreted normally. Thus, the cleavage at the C-terminal GRR site is essential for subsequent cleavages, and modification of other cleavage sites (58, 33, 22, and 8) has a major impact on pro-CCK processing. These results suggest that there is a temporal order of cleavages, and the structure of pro-CCK has a strong influence on where and whether pro-CCK is processed.

Trypsin mediates growth phase-dependent transcriptional tegulation of genes involved in biosynthesis of ruminococcin A, a lantibiotic produced by a Ruminococcus gnavus strain from a human intestinal microbiota.

Ruminococcin A (RumA) is a trypsin-dependent lantibiotic produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from a human intestinal microbiota. A 12.8-kb region from R. gnavus E1 chromosome, containing the biosynthetic gene cluster of RumA, has been cloned and sequenced. It consisted of 13 open reading frames, organized in three operons with predicted functions in lantibiotic biosynthesis, signal transduction regulation, and immunity. One unusual feature of the locus is the presence of three almost identical structural genes, all of them encoding the RumA precursor. In order to determine the role of trypsin in RumA production, the transcription of the rum genes has been investigated under inducing and noninducing conditions. Trypsin activity is needed for the growth phase-dependent transcriptional activation of RumA operons. Our results suggest that bacteriocin production by R. gnavus E1 is controlled through a complex signaling mechanism involving the proteolytic processing of a putative extracellular inducer-peptide by trypsin, a specific environmental cue of the digestive ecosystem.

Inactivation of the 7B2 inhibitory CT peptide depends on a functional furin cleavage site.

The eukaryotic subtilisin prohormone convertase 2 (PC2) is known to require in vivo exposure to the neuroendocrine protein 7B2 in order to produce an enzymatically active species capable of proteolytic action on prohormone substrates. In the present study, we examined the role of the pentabasic site within 27-kDa 7B2 in this process. We prepared two His-tagged recombinant 7B2s by overexpression in bacteria: 7B2-Ser-Ser (SS), with an inactivating mutation in the CT peptide from Lys171-Lys172 (KK) to SS, rendering the CT peptide non-inhibitory; blockade-SS, a double mutant of both the CT peptide as well as of the pentabasic furin cleavage site. These purified proteins were used in a cell-free proPC2 activation assay. Both 7B2-SS as well as blockade-SS were able to facilitate the activation of proPC2 (as judged by efficient production of enzyme activity), suggesting that cleavage at the furin site is not required for 7B2s lacking inhibitory CT peptides. Plasmids encoding proPC2 and various 7B2s were transiently transfected into human embryonic kidney (HEK293) cells and PC2 enzymatic activity and CT forms in each overnight conditioned medium were measured. Cells transfected with proPC2 and wild-type 7B2 secreted CT peptide cleavage products, but cells transfected with proPC2 and the blockade mutant overwhelmingly secreted intact, 27-kDa, blockaded 7B2. Medium obtained from HEK293 cells transfected with proPC2 and either wild-type 7B2, 7B2-SS, or blockade-SS exhibited PC2 activity, but medium from cells expressing the 7B2 blockade mutant did not. We conclude that cleavage at the 7B2 furin consensus site is required to produce PC2 capable of efficient proteolytic inactivation of the CT peptide.

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins.

Primary structure analysis of the four river buffalo alpha-globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two alpha-globin chains, Ialpha1 and IIalpha3, which differ at positions 129 and 131: Ialpha1 has 64 Ala, 129 Phe, 131 Asn; IIalpha3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two alpha-globin chains, Ialpha2 and IIalpha4, which differ at positions 10 and 11: Ialpha2 has 10 I1e, 11 Gln, 64 Asn; IIalpha4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic alpha-globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo alpha-globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.

Thrombin-activatable fibrinolysis inhibitor deficiency in cirrhosis is not associated with increased plasma fibrinolysis.

BACKGROUND AND AIMS: The bleeding tendency of patients suffering from cirrhosis is in part ascribed to accelerated fibrinolysis. In this study, the role of the recently discovered inhibitor of fibrinolysis, thrombin-activatable fibrinolysis inhibitor (TAFI) in cirrhosis was examined. METHODS: In 64 patients with cirrhosis of varying severity, TAFI antigen levels were measured by enzyme-linked immunosorbent assay and compared with TAFI levels in control subjects. Furthermore, a plasma-based fibrinolysis assay was performed in the presence and absence of a specific inhibitor of activated TAFI. RESULTS: TAFI levels were decreased in cirrhosis. Mean TAFI levels were 66% in Child's A, 55% in Child's B, 47% in Child's C cirrhosis, and 26% in acute liver failure. Decreased TAFI antigen levels were highly correlated with antithrombin and alpha(2)-antiplasmin activity levels. Clot lysis times and clot lysis ratio (defined as ratio between clot lysis time in the absence and presence of a specific inhibitor of activated TAFI) of cirrhotics were not significantly different from healthy controls. CONCLUSIONS: Despite decreased levels of TAFI and other components of the fibrinolytic system, no evidence of increased plasma fibrinolytic potential in cirrhosis is observed using the plasma-based assay of this study. The reduction of antifibrinolytic factors in cirrhosis is compensated by the concomitant reduction in profibrinolytics.