Showing papers on "Proteolytic enzymes published in 1994"

Matrix Metalloproteinases (MMP-2 and MMP-9) of the Oral Cavity: Cellular Origin and Relationship to Periodontal Status:

Abstract: Proteolytic enzymes released by the host cells are associated with the tissue destruction in periodontal diseases. Matrix metalloproteinases (MMPs) have the primary role in this process, since, in concert, they can degrade most of the extracellular matrix components. In the present study, we investigated MMP-2 and MMP-9 in oral fluids of healthy subjects and periodontitis patients and the contributions of different oral cells to the enzyme production. The enzymograms revealed that the main gelatinase in oral rinses, crevicular fluid, and whole saliva migrated at 92 kDa. Activity was also detected at 200 kDa and 130 kDa and minor activity at 86 kDa, 72 kDa, and 40 kDa. Traces of gelatinolytic activity were also detected in pure parotid secretions. The 92-kDa enzyme was identified to MMP-9 and the 200-kDa gelatinase to MMP-2, by means of specific anti-72-kDa antiserum. Gingival keratinocytes produced mainly MMP-9, while gingival and granulation tissue fibroblasts expressed MMP-2. Glandular tissue contained mainly MMP-9, and mRNA for MMP-9 was also found in acinar epithelial cells. Periodontitis patients had significantly higher levels of MMP-9 than healthy subjects. Also, MMP-2 was elevated in periodontitis patients. Periodontal treatment reduced the amount of gelatinases dramatically. This study shows that gelatinases are produced by various cells in the oral cavity. The amount of gelatinases is elevated during periodontal disease, while conventional periodontal treatment efficiently reduces the levels these enzymes. We suggest that MMP-2 and MMP-9 could participate in tissue destruction in periodontitis.

Acute lung injury: the role of cytokines in the elicitation of neutrophils.

Abstract: Cytokine networks between immune and nonimmune cells of the alveolar-capillary membrane are necessary for cellular communication during pulmonary inflammation. The subsequent events of these cellular/humoral interactions are pivotal to the initiation and propagation of the inflammatory response leading to pulmonary injury. The studies cited in this paper underscore the interrelationship of early response cytokines, adhesion molecules, and the chemokine IL-8 that orchestrate the recruitment of neutrophils into the lung. The paradigm for neutrophil extravasation is likely operative in the microvasculature of the lung, and consists of four or more steps (Figure 3). First, acute lung injury results in the activation of microvascular endothelium in response to the local generation of TNF or IL-1, leading to expression of endothelial cell-derived E- and P-selectins and ICAM-1. The constitutive presence of neutrophil-derived L-selectin allows for the initial adhesive interaction of neutrophils with endothelial cell selectins leading to the "rolling" effect. Second, generation of IL-8 leads to the activation of neutrophils in the vascular compartment and expression of beta 2 integrins, while L-selectin is concomitantly shed. Third, the interaction of the neutrophil beta 2 integrin with its receptor/ligand, ICAM-1, results in the rapid arrest of neutrophils on the endothelium. Fourth, the subsequent events leading to neutrophil extravasation beyond the vascular compartment are dependent upon a combination of haplotaxis (migration in response to an insoluble gradient), the continued expression of beta 2 integrins on neutrophils and ICAM-1 on nonimmune cells, and the maintenance of a neutrophil specific (IL-8) chemotactic gradient. The participation of IL-8 and potentially other C-X-C chemokines in the inflammatory response appears to be critical for the orchestration of the directed migration of inflammatory leukocytes into the lung. After arriving in the lung, these activated leukocytes can respond to noxious stimuli or induce pulmonary injury through the release of reactive oxygen metabolites, proteolytic enzymes, and additional cytokines. Our current knowledge and future investigations regarding the mechanisms involved in neutrophil elicitation may allow us to employ clinical interventional strategies that will attenuate neutrophil-dependent acute lung injury, such as ARDS.

Genetics and biotechnology of lactic acid bacteria

Abstract: 1 Gene transfer systems and transposition.- 1.1 Introduction.- 1.2 Conjugation.- 1.2.1 Heterologous conjugation systems.- 1.2.2 Homologous conjugation systems.- 1.3 Transduction.- 1.3.1 Transduction in Lactococcus spp..- 1.3.2 Transduction in Streptococcus thermophilus.- 1.3.3 Transduction in Lactobacillus spp..- 1.4 Transformation.- 1.4.1 Transformation systems.- 1.4.2 Protoplast transformation.- 1.4.3 Electrotransformation.- 1.4.4 Transfection.- 1.5 Protoplast fusion.- 1.6 Transposition.- 1.6.1 Insertion sequences.- 1.6.2 Heterologous transposons.- 1.6.3 Nisin transposons.- 1.7 Generalized recombination.- 1.7.1 Integration processes.- 1.7.2 Recombination genes.- 1.8 Chromosome mapping.- 1.9 Concluding remarks.- References.- 2 Gene cloning and expression systems in Lactococci.- 2.1 Introduction.- 2.2 Replicative gene cloning.- 2.2.1 Streptoccal plasmid vectors.- 2.2.2 Lactococcal plasmid vectors.- 2.2.3 Plasmid replication and stability.- 2.3 Integrative gene cloning.- 2.3.1 Integration vectors.- 2.3.2 Chromosomal gene amplification and stability.- 2.3.3 Integrative gene expression.- 2.4 Gene expression signals.- 2.4.1 Vectors for the selection of expression signals.- 2.4.2 Transcription initiation and termination.- 2.4.3 Translation initiation and codon usage.- 2.5 Control of gene expression.- 2.5.1 Lactose utilization and its control.- 2.5.2 Heat shock response.- 2.5.3 Negative regulation of transcription.- 2.5.4 Positive regulation of transcription.- 2.6 Protein secretion.- 2.6.1 Export-signal selection vectors.- 2.6.2 Sec-dependent secretion.- 2.6.3 Sec-independent secretion.- 2.7 Expression and secretion systems.- 2.7.1 Expression vectors.- 2.7.2 Secretion vectors.- 2.8 Food grade systems.- References.- 3 Bacteriophages and bacteriophage resistance.- 3.1 Introduction.- 3.2 Types and species of bacteriophages.- 3.2.1 Interactions with hosts.- 3.2.2 Temperate phages.- 3.2.3 Classification of phages of lactic acid bacteria.- 3.2.4 Characterization of bacteriophage genomes.- 3.3 Gene directed bacteriophage resistance in lactic acid bacteria.- 3.3.1 Interference with bacteriophage adsorption.- 3.3.2 Restriction and modification.- 3.3.3 Abortive infection.- 3.4 Novel phage defense mechanisms.- 3.5 Genetic strategies to construct phage-insensitive strains.- 3.6 Conclusions and perspectives.- References.- 4 The proteolytic system of lactic acid bacteria.- 4.1 Introduction.- 4.2 Proteinases.- 4.2.1 Biochemical characterization.- 4.2.2 Caseinolytic specificity and classification of proteinases.- 4.2.3 Cloning and expression of proteinase genes.- 4.2.4 Organization of prtM and prtP genes.- 4.2.5 Structural characteristics of PrtP.- 4.2.6 Proteinase maturation by PrtM.- 4.3 Endopeptidases.- 4.3.1 Endopeptidase NisP.- 4.3.2 Intracellular endopeptidases.- 4.3.3 Endopeptidase PepO.- 4.4 General aminopeptidases.- 4.4.1 Aminopeptidase A (PepA).- 4.4.2 Pyrrolidonyl carboxylyl peptidase (PCP).- 4.4.3 Aminopeptidase N (PepN).- 4.4.4 Aminopeptidase C (PepC).- 4.5 Proline-specific peptidases.- 4.5.1 Prolidases.- 4.5.2 Proline iminopeptidase.- 4.5.3 X-prolyl dipeptidyl aminopeptidase (PepXP).- 4.6 Oligo-and carboxypeptidases.- 4.6.1Dipeptidases.- 4.7 Transport of amino acid and peptides.- 4.7.1 Specific amino acid carriers.- 4.7.2 Di- and tripeptide transport system (DptT).- 4.7.3 Oligopeptide transport system (Opp).- 4.8 Cellular localization of proteolytic enzymes.- 4.9 Engineering of the proteolytic system.- 4.9.1 Construction and use of mutants.- 4.9.2 Engineering of the proteinase PrtP.- 4.9.3 Future perspectives.- References.- 5 Bacteriocins of lactic acid bacteria.- 5.1 Introduction.- 5.2 Small heat-stable bacteriocins.- 5.2.1 Genetic analysis.- 5.2.2 Common structural features.- 5.2.3 Bacteriocin translocation.- 5.2.4 Mode of action.- 5.3 Large heat-labile bacteriocins.- 5.3.1 Helvetican J.- 5.4 Lantibiotics in lactic acid bacteria.- 5.4.1 Lactocin S.- 5.4.2 Lacticin 481.- 5.4.3 Nisin structure and biosynthesis.- 5.4.4 Genetic analysis of nisin biosynthetic genes.- 5.4.5 Expression of nisin determinants.- 5.4.6 Nisin immunity resistance.- 5.4.7 Mode of action.- 5.4.8 Protein engineered nisins.- 5.4.9 Applications.- 5.5 Concluding remarks.- References.- 6 Genetic engineering of lactobacilli, leuconostocs and Streptococcus thermophilus.- 6.1 Introduction.- 6.2 Overview of taxonomy and health benefits.- 6.3 Biochemical traits.- 6.3.1 The Lac-PTS.- 6.3.2 The lac operon.- 6.3.3 Xylose metabolism.- 6.3.4 Protein metabolism.- 6.4 Plasmid biology and cloning vectors.- 6.4.1 Naturally occuring plasmids.- 6.4.2 Insertion sequences.- 6.4.3 Cloning vectors.- 6.5 Genetic transfer methods.- 6.5.1 Transformation by electroporation..- 6.5.2 Liposome-mediated protoplast transfection, fusion and transformation.- 6.5.3 Conjugation.- 6.5.4 Transduction.- 6.6 Gene expression in lactobacilli.- 6.6.1 Transcription.- 6.6.2 Translation.- 6.6.3 Regulation of transcription.- 6.6.4 Heterologous gene expression.- 6.7 Gene expression in S. thermophilus and leuconostocs.- 6.8 Chromosomal integration of genes.- 6.9 Phage and phage resistance.- 6.10 Concluding remarks.- Dedication and acknowledgements.- References.

Uroplakins Ia and Ib, two major differentiation products of bladder epithelium, belong to a family of four transmembrane domain (4TM) proteins

Abstract: The mammalian bladder epithelium elaborates, as a terminal differentiation product, a specialized plasma membrane called asymmetric unit membrane (AUM) which is believed to play a role in strengthening and stabilizing the urothelial apical surface through its interactions with an underlying cytoskeleton. Previous studies indicate that the outer leaflet of AUM is composed of crystalline patches of 12-nm protein particles, and that bovine AUMs contain three major proteins: the 27- to 28-kD uroplakin I, the 15-kD uroplakin II and the 47-kD uroplakin III. As a step towards elucidating the AUM structure and function, we have cloned the cDNAs of bovine uroplakin I (UPI). Our results established the existence of two isoforms of bovine uroplakin I: a 27-kD uroplakin Ia and a 28-kD uroplakin Ib. These two glycoproteins are closely related with 39% identity in their amino acid sequences. Hydropathy plot revealed that both have four potential transmembrane domains (TMDs) with connecting loops of similar length. Proteolytic digestion of UPIa inserted in vitro into microsomal vesicles suggested that its two main hydrophilic loops are exposed to the luminal space, possibly involved in interacting with the luminal domains of other uroplakins to form the 12-nm protein particles. The larger loop connecting TMD3 and TMD4 of both UPIa and UPIb contains six highly conserved cysteine residues; at least one centrally located cysteine doublet in UPIa is involved in forming intramolecular disulfide bridges. The sequences of UPIa and UPIb (the latter is almost identical to a hypothetical, TGF beta-inducible, TI-1 protein of mink lung epithelial cells) are homologous to members of a recently described family all possessing four transmembrane domains (the "4TM family"); members of this family include many important leukocyte differentiation markers such as CD9, CD37, CD53, and CD63. The tissue-specific and differentiation-dependent expression as well as the naturally occurring crystalline state of uroplakin I molecules make them uniquely suitable, as prototype members of the 4TM family, for studying the structure and function of these integral membrane proteins.

Molecular evolution and domain structure of plasminogen-related growth factors (HGF/SF and HGF1/MSP).

Abstract: Plasminogen-related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron-exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N-terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide-bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix-strand-helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three-dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine-binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.

Processing and metabolism of peptide-YY: pivotal roles of dipeptidylpeptidase-IV, aminopeptidase-P, and endopeptidase-24.11.

Abstract: The processing of peptide-YY (PYY) by purified membrane peptidases and brush border membrane preparations from human kidney and jejunum has been compared. Dipeptidyl peptidase-IV hydrolyzes PYY at the Pro2-Ile3 bond, producing PYY-(3-36), which is a Y2 receptor-selective ligand. Aminopeptidase-P removes the N-terminal tyrosine from PYY, but the intact peptide is not a substrate for aminopeptidase-N. Endopeptidase-24.11 (neutral endopeptidase; enkephalinase) metabolizes PYY efficiently, with a major site of hydrolysis at the Asn29-Leu30 bond, an inactivating cleavage. Angiotensin-converting enzyme does not hydrolyze PYY. In renal brush border membranes, hydrolysis of PYY is substantially (> 75%) inhibited by phosphoramidon, suggesting that endopeptidase-24.11 initiates hydrolysis of PYY in this preparation. In jejunal brush border membranes, phosphoramidon has a much smaller effect (15% inhibition), and contributions due to the actions of aminopeptidase-P and dipeptidyl peptidase-IV were evident. Few physiological substrates have yet been identified for aminopeptidase-P and dipeptidyl peptidase-IV; the pancreatic polypeptide fold family, of which PYY is a member, may well represent endogenous substrates for those cell surface ectoenzymes. Dipeptidyl peptidase-IV converts PYY to a metabolite PYY-(3-36), a highly selective agonist for the Y2 receptor type. Thus, the relative levels of the three membrane peptidases at different tissue locations and on different cell types may play a pivotal role in postsecretory processing and metabolism of PYY to receptor-selective agonists or inactive metabolites.

Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0

Abstract: Pseudomonas fluorescens strain CHA0 protects plants from various root diseases. Antibiotic metabolites synthesized by this strain play an important role in disease suppression; their production is mediated by the global activator gene gacA. Here we show by complementation that the gacA gene is also essential for the expression of two extracellular enzymes in P. fluorescens CHA0: phospholipase C and a 47-kDa metalloprotease. In contrast, the production of another exoenzyme, lipase, is not regulated by the gacA gene. Protease, phospholipase and antibiotics of P. fluorescens are all known to be optimally produced at the end of exponential growth; thus, the gacA gene appears to be a general stationary-phase regulator.

Dityrosine: a marker for oxidatively modified proteins and selective proteolysis.

Abstract: Publisher Summary Dityrosine can be found in several proteins as a product of UV irradiation, γ-irradiation, aging, exposure to oxygen free radicals, or incubation with peroxidase. In other cases, dityrosine is the product of normal posttranslational processes affecting specific structural proteins. Oxidative modification is clearly the common element involved in dityrosine formation. This chapter describes a method for measuring dityrosine release from proteins that have been preexposed to oxygen radicals. The method is based on a proteolytic digestion followed by high-performance liquid chromatography (HPLC) analysis with fluorescence detection. The formation of dityrosine in a protein exposed to oxygen free radicals can serve as a marker of oxidatively modified proteins. Indeed, dityrosine may be a useful biomarker for organismal oxidative stress. Incubation of oxidatively modified proteins with proteolytic enzymes results in increased proteolysis in which the modified proteins undergo a selective digestion by specific intracellular proteases.

Methotrexate in rheumatoid arthritis. An update.

Abstract: Methotrexate has been approved for the treatment of refractory rheumatoid arthritis by several regulatory agencies, including the Food and Drug Administration. The tendency is now to prescribe it at earlier stages of the disease. Methotrexate is a well known antifolate. Its exact mechanism of action in rheumatoid arthritis remains uncertain. The polyglutamated derivatives of methotrexate are potent inhibitors of various enzymes, including dihydrofolate reductase and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase. Inhibitory effects on cytokines, particularly interleukin-1, and on arachidonic acid metabolism, as well as effects on proteolytic enzymes, have been reported. Some of them may be linked to the antifolate properties of methotrexate. Overall, the drug appears to act in rheumatoid arthritis as an anti-inflammatory agent with subtle immunomodulating properties. Direct inhibitory effects on rapidly proliferating cells in the synovium have also been suggested. Methotrexate is usually given orally. Marked interindividual variation in its bioavailability has been found. Food intake has no significant effect on the pharmacokinetics of oral methotrexate. Methotrexate undergoes significant metabolism. The functionally important metabolites are the polyglutamated derivatives of methotrexate, which are selectively retained in the cells. Less than 10% of a dose of methotrexate is oxidised to 7-hydroxy-methotrexate, irrespective of the route of administration. This metabolite is extensively (91 to 93%) bound to plasma proteins, in contrast to the parent drug (35 to 50% bound). Methotrexate is mainly excreted by the kidneys. It undergoes tubular secretion and may thereby compete with various organic acid compounds. Early placebo-controlled trials demonstrated that weekly low dosage methotrexate produced early symptomatic improvement in most rheumatoid arthritis patients. Two meta-analyses showed that methotrexate is among the most efficacious of slow-acting antirheumatic agents, together with parenteral gold (sodium aurothiomalate), penicillamine and sulfasalazine. Furthermore, in the short term context of clinical trials, methotrexate has one of the best efficacy/toxicity ratios. There is little evidence that methotrexate, or any available slow-acting antirheumatic agent, is a true disease-modifying drug. However, the probability that a patient will continue methotrexate therapy over time appears quite favourable compared with any other slow-acting antirheumatic drug. Combination therapy with slow-acting drugs has been advised for the management of rheumatoid arthritis, but the evidence currently available does not support general use of combination therapy including methotrexate. Almost all investigations indicated that toxic effects, rather than lack of response, were the major reason for discontinuing methotrexate therapy. An analysis of more than 3000 courses of therapy with 6 slow-acting drugs indicated that the overall toxicity of methotrexate was similar to that of penicillamine and azathioprine. Hydroxychloroquine was the least toxic, followed by sodium aurothiomalate, whereas auranofin was the most toxic. Few predisposing factors to methotrexate toxicity have been clearly identified, and individual susceptibility plays a primary role in determining toxicity. Folate supplementation may decrease the incidence of common adverse effects, but whether it prevents more serious adverse reactions remains to be answered. Gastrointestinal symptoms, stomatitis, increased levels of liver enzymes and mild cytopenia are frequent adverse effects associated with methotrexate therapy. Furthermore, severe, and possibly life-threatening, complications have been reported. These include advanced liver fibrosis and cirrhosis, interstitial pneumonitis, severe neutropenia and pancytopenia, as well as opportunistic infections. Epstein-Barr virus-associated lymphomas have also been reported. Finally, a variety of other adverse events, such as central nervous system and cutaneous reactions, have been ascribed to methotrexate use. Numerous drug interactions may occur in patients receiving low dosage methotrexate. Most are probably not clinically significant. Conversely, methotrexate toxicity may be precipitated by concurrent use of cotrimoxazole (trimethoprim/sulfamethoxazole), probenecid, and possibly non-steroidal anti-inflammatory drugs (NSAIDs). On the basis of the available data, methotrexate should generally not be the first slow-acting antirheumatic drug to be used. However, unlike other cytotoxic agents, it should no longer be regarded as tertiary therapy for rheumatoid arthritis. Low dosage methotrexate may become one of the drugs considered earlier in the course of rheumatoid arthritis not controlled by NSAIDs alone.

Isolation and characterization of Linocin M18, a bacteriocin produced by Brevibacterium linens

Abstract: Brevibacterium linens M18, isolated from red smear cheese, produces a substance that inhibits the growth of Listeria spp. and several coryneform and other gram-positive bacteria. No gram-negative bacteria were inhibited. The substance is heat labile, sensitive to proteolytic enzymes, and stable between pH 3 and 12. High levels of this bacteriocin, named Linocin M18, were obtained in the stationary growth phase. Linocin M18 was purified by ultrafiltration, ultracentrifugation, and gel filtration chromatography. In its native form, it is a proteinaceous aggregate with a high molecular weight. Fractions with Linocin M18 activity contained particles of 20 to 30 nm in diameter. The bacteriocin consists of a single protein subunit with a molecular mass of 31 kDa and an isoelectric point of 4.5 N-terminal sequence analysis yielded Met-Asn-Asn-Leu-Tyr-Arg-Glu-Leu-Ala-Pro-Ile-Pro-Gly-Pro-Ala-Ala-Ala-Glu- Ile. Significant homology with published sequences was lacking.

Aeromonas Species in Septicemia: Laboratory Characteristics and Clinical Observations

Abstract: We retrospectively analyzed clinical and epidemiological data on and laboratory characteristics of 53 cases of aeromonas septicemia. Only four Aeromonas genomospecies (species defined by DNA relatedness) were associated with the 53 cases, with Aeromonas hydrophila (sensu stricto) predominating (47%). Nearly 60% of all Aeromonas isolates from blood fell into one of four somatic groups: serogroups O:11, O:16, O:18, and O:34. Unlike Aeromonas-associated gastroenteritis, septicemia did not peak in frequency during the warmer months but rather was most common in January through March, when approximately 40% of cases occurred. In vitro tests of the pathogenicity of 20 selected blood isolates of Aeromonas indicated that resistance to complement-mediated lysis, elevated levels of protease and hemolysin activity, and the ability to elaborate siderophores correlated with higher virulence. Species and serogroup designations also correlated with the degree of virulence. Susceptibility studies of 50 strains indicated that A. hydrophila was the most drug-resistant species and that Aeromonas veronii was the most susceptible. Susceptibility to first- and second-generation cephalosporins and carbenicillin was species-associated.

Free radicals scavenging action and anti-enzyme activities of procyanidines from Vitis vinifera. A mechanism for their capillary protective action.

Abstract: The scavenging by procyanidines (polyphenol oligomers from Vitis vinifera seeds, CAS 85594-37-2) of reactive oxygen species (ROS) involved in the onset (HO degrees) and the maintenance of microvascular injury (lipid radicals R degrees, RO degrees, ROO degrees) has been studied in phosphatidylcholine liposomes (PCL), using two different models of free radical generation: a) iron-promoted and b) ultrasound-induced lipid peroxidation. In a) lipid peroxidation was assessed by determination of thiobarbituric acid-reactive substances (TBARS); in b) by determination of conjugated dienes, formation of breakdown carbonyl products (as 2,4-dinitrophenylhydrazones) and loss of native phosphatidylcholine. In the iron-promoted (Fenton-driven) model, procyanidines had a remarkable, dose-dependent antilipoperoxidant activity (IC50 = 2.5 mumol/l), more than one order of magnitude greater than that of the monomeric unit catechin (IC50 = 50 mumol/l), activity which is due, at least in part, to their metal-chelating properties. In the more specific model b), which discriminates between the initiator (hydroxyl radical from water sonolysis) and the propagator species of lipid peroxidation (the peroxyl radical, from autooxidation of C-centered radicals), procyanidines are highly effective in preventing conjugated diene formation in both the induction (IC50 = 0.1 mumol/l) and propagation (IC50 = 0.05 mumol/l) phases (the scavenging effect of alpha-tocopherol was weaker, with IC50 of 1.5 and 1.25 mumol/l). In addition, procyanidines at 0.5 mumol/l markedly delayed the onset of the breakdown phase (48 h), totally inhibiting during this time the formation of degradation products (the lag-time induced by alpha-tocopherol was only of 24 h at 10 mumol/l concentration). The HO degrees entrapping capacity of these compounds was further confirmed by UV studies and by electron spin resonance (ESR) spectroscopy, using DMPO as spin trapper: procyanidines markedly reduced, in a dose-dependent fashion, the signal intensity of the DMPO-OH radical spin adduct (100% inhibition at 40 mumol/l). The results of the second part of this study show that procyanidines, in addition to free radical scavenging action, strongly and non-competitively, inhibit xanthine oxidase activity, the enzyme which triggers the oxy radical cascade (IC50 = 2.4 mumol/l). In addition procyanidines non-competitively inhibit the activities of the proteolytic enzymes collagenase (IC50 = 38 mumol/l) and elastase (IC50 = 4.24 mumol/l) and of the glycosidases hyaluronidase and beta-glucuronidase (IC50 = 80 mumol/l and 1.1 mumol/l), involved in the turnover of the main structural components of the extravascular matrix collagen, elastin and hyaluronic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Bioadhesion technologies for the delivery of peptide and protein drugs to the gastrointestinal tract.

Abstract: For the efficient delivery of peptides, proteins, and other biopharmaceuticals by nonparenteral routes, in particular via the gastrointestinal, or GI, tract, novel concepts are needed to overcome significant enzymatic and diffusional barriers. In this context, bioadhesion technologies offer some new perspectives. The original idea of oral bioadhesive drug delivery systems was to prolong and/or to intensify the contact between controlled-release dosage forms and the stomach or gut mucosa. However, the results obtained during the past decade using existing pharmaceutical polymers for such purposes were rather disappointing. The encountered difficulties were mainly related to the physiological peculiarities of GI mucus. Nevertheless, research in this area has also shed new light on the potential of mucoadhesive polymers. First, one important class of mucoadhesive polymers, poly(acrylic acid), could be identified as a potent inhibitor of proteolytic enzymes. Second, there is increasing evidence that the interaction between various types of bio(muco)adhesive polymers and epithelial cells has direct influence on the permeability of mucosal epithelia. Rather than being just adhesives, mucoadhesive polymers may therefore be considered as a novel class of multifunctional macromolecules with a number of desirable properties for their use as biologically active drug delivery adjuvants. To overcome the problems related to GI mucus and to allow longer lasting fixation within the GI lumen, bioadhesion probably may be better achieved using specific bioadhesive molecules. Ideally, these bind to surface structures of the epithelial cells themselves rather than to mucus by receptor-ligand-like interactions. Such compounds possibly can be found in the future among plant lectins, novel synthetic polymers, and bacterial or viral adhesion/invasion factors. Apart from the plain fixation of drug carriers within the GI lumen, direct bioadhesive contact to the apical cell membrane possibly can be used to induce active transport processes by membrane-derived vesicles (endo- and transcytosis). The nonspecific interaction between epithelia and some mucoadhesive polymers induces a temporary loosening of the tight intercellular junctions, which is suitable for the rapid absorption of smaller peptide drugs along the paracellular pathway. In contrast, specific endo- and transcytosis may ultimately allow the selectively enhanced transport of very large bioactive molecules (polypeptides, polysaccharides, or polynucleotides) or drug carriers across tight clusters of polarized epi- or endothelial cells, whereas the formidable barrier function of such tissues against all other solutes remains intact.

Prognostic value of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in breast carcinomas.

Abstract: It is now clearly established that proteolytic enzymes, including plasminogen activator (uPA), play an important role in breaking down the extracellular matrix, which is considered to be a step in metastasis formation. Plasminogen activators are controlled at various levels. Two inhibitors, PAI-1 and PAI-2, have been identified, the latter being more specific for uPA. In attempts to determine their prognostic value, it is essential to investigate the relative importance of these parameters and their interactions. We used an immunoenzymatic method to assay uPA, PAI-1 and PAI-2 antigens in cytosols prepared from 314 primary breast tumours. The patients were followed up for a minimum of 6 years and all relevant clinical and laboratory findings were recorded. Univariate analysis confirmed the poor outcome of patients whose tumours contained large amounts of uPA and PAI-1. In addition, low levels of PAI-2 correlated with shorter disease-free survival in the overall population (P = 0.02), post-menopausal women (P = 0.02) and women without lymph node involvement (P = 0.02). Multivariate analysis in the 'main effects' Cox model identified node involvement, macroscopic tumour size and PAI-2 as significant variables. The 'interactive' model, taking into account interactions between uPA and its two inhibitors, identified a first subgroup with a very poor prognosis associating either high levels of PAI-1 with low levels of PAI-2 in the overall population and the women with no node involvement or high levels of uPA with low levels of PAI-2 in the group of menopausal women. We conclude that PAI-1 provides the same prognostic information as uPA, and does not appear to play a role as an inhibitor. In contrast, PAI-2 increases the prognostic value of uPA, particularly in post-menopausal women, and PAI-1 in patients with no node involvement.

Protein identification by peptide mass fingerprinting.

Abstract: A mass spectrum of the peptide mixture resulting from the digestion of a protein by an enzyme can provide a fingerprint of great specificity--so specific, in fact, that it is often possible to identify the protein from this information alone, without ambiguity. The general approach is to take a small sample of the protein of interest and digest it with a proteolytic enzyme, such as trypsin. The resulting digest mixture is analyzed by mass spectrometry, the ionization techniques of choice being matrix-assisted laser desorption or electrospray ionization. The experimental mass values are then compared with a database of peptide mass values, calculated by applying the enzyme cleavage rules to the entries in one of the major collections of sequence data, such as SwissProt or PIR. By using an appropriate scoring algorithm, the closest match or matches can be identified. If the "unknown" protein was present in the sequence database, then the goal is to identify that precise entry. If the sequence database does not contain the unknown protein, then a successful search will identify those entries that exhibit the closest sequence homology, often equivalent proteins from related species. An inverted strategy, in which experimental peptide mass fingerprints are accumulated in a database, has significant potential for identifying coding regions within sequence data from genomic DNA and, in doing so, correlating the genes with their expressed proteins. This review aims to provide an overview of the technique, compare the different database matching algorithms which have been described in the literature, and discuss the practical and theoretical factors which influence identification accuracy.

Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping.

Abstract: The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.

Evidence that human bone cells in culture produce insulin-like growth factor-binding protein-4 and -5 proteases.

Abstract: Previous studies have shown that the actions of insulin-like growth factor-II (IGF-II) in bone are determined by both its concentration and the concentrations of the IGF-binding proteins (IGFBPs). As IGFBP concentrations may be regulated not only at the level of production, but also at the level of degradation, IGFBP proteases may be an important component of the IGF-II regulatory system. In this study, we have identified IGFBP-4 and IGFBP-5 protease activity in the conditioned medium (CM) of the human osteosarcoma U2 cell line (U2OS) and untransformed normal human bone cell (HBC) derived from skull. Proteolysis of the 29-kilodalton (kDa) [125I]IGFBP-5 produced an 18- to 20-kDa fragment of IGFBP-5, and 25 kDa [125I]IGFBP-4 yielded two lower mol wt fragments in the presence of IGF-II. CM from IGF-II-treated U2OS and normal HBC cultures exhibited decreased IGFBP-5 proteolytic activity compared to control cultures. In contrast, CM from IGF-II-treated HBC cultures had increased proteolytic activity against IG...

Physiology and molecular biology of the lignin peroxidases of Phanerochaete chrysosporium.

Abstract: The white-rot basidiomycete Phanerochaete chrysosporium produces lignin peroxidases (LiPs), a family of extracellular glycosylated heme proteins, as major components of its lignin-degrading system. Upto 15 LiP isozymes, ranging in Mr values from 38000 to 43000, are produced depending on culture conditions and strains employed. Manganese-dependent peroxidases (MnPs) are a second family of extracellular heme proteins produced by P. chrysosporium that are also believed to be important in lignin degradation by this organism. LiP and MnP production is seen during secondary metabolism and is completely suppressed under conditions of excess nitrogen and carbon. Excess Mn(II) in the medium, on the other hand, suppresses LiP production but enhances MnP production. Nitrogen regulation of LiP and MnP production is independent of carbon and Mn(II) regulation. LiP activity is also affected by idiophasic extracetlular proteases. Intracellular cAMP levels appear to be important in regulating the production of LiPs and MnPs, although LiP production is affected more than MnP production. Studies on the sequencing and characterization of lip cDNAs and genes of P. chrysosporium have shown that the major LiP isozymes are each encoded by a separate gene. Each lip gene encodes a mature protein that is 343–344 amino acids long, contains 1 putative N-glycosylation site, a number of putative O-glycosylation sites, and is preceded by a 27–28-amino acid leader peptide ending in a Lys-Arg cleavage site. The coding region of each lip gene is interrupted by 8–9 introns (50–63 bp), and the positions of the last two introns appear to be highly conserved. There are substantial differences in the temporal transcription patterns of the major lip genes. The sequence data suggest the presence of three lip gene subfamilies. The genomic DNA of P. chrysosporium strain BKMF-1767 was resolved into 10 chromosomes (genome size of 29 Mb), and that of strain ME-446 into 11 chromosomes (genome size of 32 Mb). The lip genes have been localized to five chromosomes in BKMF-1767 and to four chromosomes in ME-446. DNA transformation studies have reported both integrative and non-integrative transformation in P. chrysosporium.

Involvement of the lysosomal system in yolk protein deposit and degradation during vitellogenesis and embryonic development in trout

Abstract: In adult female rainbow trout (Oncorhynchus mykiss), an immunocytochemical study of the oocyte has shown that a proteolytic enzyme, cathepsin D, is localized in multivesicular bodies (MVB) which begin to differentiate before the phase of vitellogenesis. Estrogens cause the liver to synthesize the protein vitellogenin (VTG), which then enters the systemic circulation. During vitellogenesis, endocytosed VTG is co-localized with cathepsin D in the MVB. Assays of oocyte cathepsin activities have shown that the only proteolytic activity of note is that of cathepsin D. Along with the yolk proteins derived from VTG, this enzyme will be included in the central coalescent yolk mass. With the installation of the yolk syncytial layer and the surrounding yolk vascular system, embryonic development is characterized by high lysosomal activity, especially in the syncytial layer. At this level, proteolytic activity concerns a cathepsin L, secreted as a proenzyme. We propose the hypothesis that cathepsin D, along with the yolk proteins in the yolk globules that break away from the yolk mass in the vitellolysis zone, activates the proenzyme and leads to protein degradation, which then becomes very rapid. © Wiley-Liss, Inc.

Increased proteolysis of insulin-like growth factor-binding protein-3 (IGFBP-3) in noninsulin-dependent diabetes mellitus serum, with elevation of a 29-kilodalton (kDa) glycosylated IGFBP-3 fragment contained in the approximately 130- to 150-kDa ternary complex.

Abstract: Insulin-like growth factor-I (IGF-I) in serum is predominantly bound in a ternary complex, consisting of IGF peptide, IGF-binding protein-3 (IGFBP-3), and an acid-labile subunit, or a binary complex, consisting of IGF peptide and any of the six IGFBPs. In the binary complex, IGF-I is more bioavailable and has a faster turnover rate. Proteolysis of IGFBP-3 may alter the distribution of IGF-I between these complexes by reducing IGFBP-3 affinity for IGF-I and/or acid-labile subunit and may offer an additional mechanism for regulation of IGF availability. In the present study, sera from patients with noninsulin-dependent diabetes mellitus (NIDDM) were found to have significantly higher IGFBP-3 proteolytic activity than sera from age-matched healthy subjects (188 +/- 12% vs. 104 +/- 6% of a control serum pool; P < 0.001). The mean (+/- SE) of serum IGFBP-3 levels determined by Western ligand blotting was lower in NIDDM patients than in healthy control subjects (61.5 +/- 5% and 79 +/- 5% of a control serum pool, respectively; P < 0.01). However, IGFBP-3 concentrations determined by RIA did not differ. This discrepancy could be explained by IGFBP-3 proteolysis, resulting in IGFBP-3 fragments that are detectable by RIA, but not by Western ligand blotting. Western immunoblotting of sera with or without prior treatment with endoglycosidase-F demonstrated that a glycosylated 29-kilodalton (kDa) IGFBP-3 form with a protein core of 20 kDa was present in sera from healthy controls, and this fragment was increased in NIDDM and term pregnancy sera, suggesting that it is produced by endogenous proteolysis. The presence of the 29-kDa IGFBP-3 proteolytic fragment at about 130-150 kDa after neutral size chromatography of pooled sera may suggest that 29-kDa IGFBP-3 participates in ternary complex formation. Further studies are required to determine whether the avidity of ternary complex formation with the 29-kDa IGFBP-3 fragment is reduced and whether the resulting increased IGF turnover can explain the reduced IGF-I levels (z scores) observed in NIDDM patients compared to healthy subjects (-0.81 +/- 0.32 SD vs. +0.26 +/- 0.17 SD; P < 0.001). Neutral size-chromatography of sera demonstrated that IGFBP-3 protease activity in the approximately 130- to 150-kDa mol wt range is regulated by NIDDM and pregnancy in parallel with that of unfractionated sera.(ABSTRACT TRUNCATED AT 400 WORDS)

Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin".

Abstract: Elafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gln and Lys residues. The prosegment, termed "cementoin," exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminase-mediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher M(r) species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant.(ABSTRACT TRUNCATED AT 250 WORDS)