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Showing papers on "Proteolytic enzymes published in 2000"


Journal ArticleDOI
TL;DR: Some of the molecular and cellular events initiated by cell stress-the interrelationships between stress signaling, cell death, and oncogenesis-and chaperones as potential targets for cancer diagnosis and treatment are addressed.
Abstract: Exposure of cells to conditions of environmental stress-including heat shock, oxidative stress, heavy metals, or pathologic conditions, such as ischemia and reperfusion, inflammation, tissue damage, infection, and mutant proteins associated with genetic diseases-results in the inducible expression of heat shock proteins that function as molecular chaperones or proteases. Molecular chaperones are a class of proteins that interact with diverse protein substrates to assist in their folding, with a critical role during cell stress to prevent the appearance of folding intermediates that lead to misfolded or otherwise damaged molecules. Consequently, heat shock proteins assist in the recovery from stress either by repairing damaged proteins (protein refolding) or by degrading them, thus restoring protein homeostasis and promoting cell survival. The events of cell stress and cell death are linked, such that molecular chaperones induced in response to stress appear to function at key regulatory points in the control of apoptosis. On the basis of these observations-and on the role of molecular chaperones in the regulation of steroid aporeceptors, kinases, caspases, and other protein remodeling events involved in chromosome replication and changes in cell structure-it is not surprising that the heat shock response and molecular chaperones have been implicated in the control of cell growth. In this review, we address some of the molecular and cellular events initiated by cell stress-the interrelationships between stress signaling, cell death, and oncogenesis-and chaperones as potential targets for cancer diagnosis and treatment.

1,048 citations


Journal ArticleDOI
TL;DR: It is demonstrated that MT-MMP–expressing cells can penetrate and remodel type I collagen-rich tissues by using membrane-anchored metalloproteinases as pericellular collagenases.
Abstract: During tissue-invasive events, migrating cells penetrate type I collagen-rich interstitial tissues by mobilizing undefined proteolytic enzymes. To screen for members of the matrix metalloproteinase (MMP) family that mediate collagen-invasive activity, an in vitro model system was developed wherein MDCK cells were stably transfected to overexpress each of ten different MMPs that have been linked to matrix remodeling states. MDCK cells were then stimulated with scatter factor/hepatocyte growth factor (SF/HGF) to initiate invasion and tubulogenesis atop either type I collagen or interstitial stroma to determine the ability of MMPs to accelerate, modify, or disrupt morphogenic responses. Neither secreted collagenases (MMP-1 and MMP-13), gelatinases (gelatinase A or B), stromelysins (MMP-3 and MMP-11), or matrilysin (MMP-7) affected SF/HGF-induced responses. By contrast, the membrane-anchored metalloproteinases, membrane-type 1 MMP, membrane-type 2 MMP, and membrane-type 3 MMP (MT1-, MT2-, and MT3-MMP) each modified the morphogenic program. Of the three MT-MMPs tested, only MT1-MMP and MT2-MMP were able to directly confer invasion-incompetent cells with the ability to penetrate type I collagen matrices. MT-MMP–dependent invasion proceeded independently of proMMP-2 activation, but required the enzymes to be membrane-anchored to the cell surface. These findings demonstrate that MT-MMP–expressing cells can penetrate and remodel type I collagen-rich tissues by using membrane-anchored metalloproteinases as pericellular collagenases.

600 citations


Journal ArticleDOI
TL;DR: This review aims to bring the reader up-to-date with current research relating to MMPs, with particular emphasis on emerging mechanisms of regulation of these enzymes, and their interaction with cell adhesion molecules.

548 citations


Journal ArticleDOI
01 Jun 2000-Cytokine
TL;DR: Understanding the involvement of different growth factors and cytokines in the molecular mechanism of melanoma progression will help to provide an insight into new future therapeutic approaches for melanoma.

412 citations


Journal Article
TL;DR: The developed probes should find widespread applications for real-time in vivo imaging of a variety of clinically relevant proteases, for example, to detect endogenous protease activity in disease, to monitor the efficacy of protease inhibitors, or to image transgene expression.
Abstract: The single biggest challenge facing in vivo imaging techniques is to develop biocompatible molecular beacons that are capable of specifically and accurately measuring in vivo targets at the protein, RNA, or DNA level. Our efforts have focused on developing activatable imaging probes to measure specific enzyme activities in vivo. Using cathepsin D as a model target protease, we synthesized a long-circulating, synthetic graft copolymer bearing near-infrared (NIR) fluorochromes positioned on cleavable substrate sequences. In its native state, the reporter probe was essentially nonfluorescent at 700 nm due to energy resonance transfer among the bound fluorochromes (quenching) but became brightly fluorescent when the latter were released by cathepsin D. NIR fluorescence signal activation was linear over at least 4 orders of magnitude and specific when compared with scrambled nonsense substrates. Using matched rodent tumor models implanted into nude mice expressing or lacking the targeted protease, it could be shown that the former generated sufficient NIR signal to be directly detectable and that the signal was significantly different compared with negative control tumors. The developed probes should find widespread applications for real-time in vivo imaging of a variety of clinically relevant proteases, for example, to detect endogenous protease activity in disease, to monitor the efficacy of protease inhibitors, or to image transgene expression.

408 citations


Journal ArticleDOI
TL;DR: Gingipains seem to be key players in subverting host defense systems with, significantly, the complement and neutrophils being the main target and a strong effect on mechanisms controlling host matrix metalloproteinase activity at the level of gene expression and zymogen activation.
Abstract: Recently accumulated large bodies of evidence have strongly implicated proteolytic enzymes released by subgingival plaque bacteria in the pathogenicity of periodontal disease. With regard to proteolytic power, however, the contribution from different microbial species considered as periodontal pathogens is not equal. Two of these bacteria, P. gingivalis and T. denticola, have developed an elaborate proteolytic systems composed of several surface-located or secreted enzymes, which apparently serve a role to provide bacteria with nutrients in the form of small peptides and amino acids. Of these two species, proteinases of P. gingivalis are the most intensively studied, and during the last decade an impressive array of information has been accumulated with respect to the biochemical characterization of purified proteinases and structure of the genes encoding them, the regulation of expression and the effects of these enzymes on host systems. In addition, studies on proteinase-deficient isogenic mutants has shed light on both their housekeeping functions and potential role(s) in the pathogenicity of periodontitis. Among several proteinases produced by P. gingivalis, the cysteine proteinases, referred to as gingipains, are clearly in the spotlight. They are the subject of several recent reviews and generally considered as the major virulence factors of this periodontal pathogen (59, 105, 139, 182, 183, 186, 281, 284, 286, 289). Gingipains seem to be key players in subverting host defense systems with, significantly, the complement and neutrophils being the main target. In addition, through uncontrolled activation of kallikrein/kinin pathway and coagulation cascade they contribute to local generation of bradykinin and thrombin, two synergistically working pro-inflammatory reagents with a strongly, although indirectly, stimulatory effect on bone resorption. Furthermore, the ability to interact with the cytokine networking systems has the potential to dysregulate the local inflammatory reaction. Finally, gingipains have a strong effect on mechanisms controlling host matrix metalloproteinase activity at the level of gene expression and zymogen activation (Fig. 10). Collectively, at the periodontal lesion site, the non-restrained action of gingipains, supported by other proteinases locally produced by subgingival plaque bacteria, would dysregulate most mechanisms controlling inflammatory reaction. Although successful in limiting infection to the periodontium, the ultimate effect of uncontrolled inflammatory processes would be the destruction of periodontal connective tissue, certainly the hallmark of periodontitis.

359 citations


Journal Article
TL;DR: There is no evidence of plasma PCT binding to cellular receptors of calcitonin, and the role of PCT in calcium and phosphate metabolism during sepsis is still not clear, so other hypothetical roles of P CT (cytokine network regulation, PCT as an endogenous non-steroid antiinflammatory drug) are being considered.
Abstract: Procalcitonin (PCT), a protein of 116 amino-acids with molecular weight of 13 kDa, was discovered 25 years ago as a prohormone of calcitonin produced by C-cells of the thyroid gland and intracellularly cleaved by proteolytic enzymes into the active hormone. Circulating levels of PCT in healthy subjects are below detection limit. Since 1993 when its elevated level was found in patients with bacterial infection, PCT became an important protein in the detection and differential diagnostics of inflammatory states. The production of PCT during inflammation is linked with a bacterial endotoxin and with inflammatory cytokines (TNF, IL-6). PCT detectable in the plasma during inflammation is not produced in C-cells of the thyroid. The probable site of PCT production during inflammation are the neuroendocrine cells in the lungs or intestine. There is no evidence of plasma PCT binding to cellular receptors of calcitonin, and the role of PCT in calcium and phosphate metabolism during sepsis is still not clear. Other hypothetical roles of PCT (cytokine network regulation, PCT as an endogenous non-steroid antiinflammatory drug) are being considered.

326 citations


Journal ArticleDOI
TL;DR: Results showed that supplementation of the adherent Lactobacillus cultures to chickens, either as a single strain of L. acidophilus or as a mixture of 12 LactOBacillus strains, increased significantly (P < 0.05) the levels of amylase in the small intestine, however, the proteolytic and lipolytic activities in theSmall intestine were not affected by addition of either of the adherents.

294 citations


Journal ArticleDOI
TL;DR: The relationship between plasma human immunodeficiency virus (HIV) RNA levels and peripheral CD4+ T cell counts was examined in 380 HIV-infected adults receiving long-term protease inhibitor therapy as mentioned in this paper.
Abstract: The relationship between plasma human immunodeficiency virus (HIV) RNA levels and peripheral CD4+ T cell counts was examined in 380 HIV-infected adults receiving long-term protease inhibitor therapy. Patients experiencing virologic failure (persistent HIV RNA >500 copies RNA/mL) generally had CD4+ T cell counts that remained greater than pretherapy baseline levels, at least through 96 weeks of follow-up. The CD4+ T cell response was directly and independently related to degree of viral suppression below the pretreatment baseline. For any given HIV RNA level measured 12 weeks after virologic failure, subsequent CD4+ T cell decline was slower in patients receiving a protease inhibitor-based regimen than in a historical control group of untreated patients. These observations suggest that transient or partial declines in plasma HIV RNA levels can have sustained effects on CD4+ T cell levels.

288 citations


Journal ArticleDOI
TL;DR: The area of bio-adhesion in drug delivery had started some 20 years ago by using so-called muco-adhesive polymers Many of these polymers were already used as excipients in pharmaceutical formulations This has facilitated the development of the first bioadhesive drug products, which are now commercially available as discussed by the authors.

287 citations


Journal Article
TL;DR: There is accumulating evidence that administration of C1-Inh may have a beneficial effect as well in other clinical conditions such as sepsis, cytokine-induced vascular leak syndrome, acute myocardial infarction, or other diseases.
Abstract: C1-esterase inhibitor (C1-Inh) therapy was introduced in clinical medicine about 25 years ago as a replacement therapy for patients with hereditary angioedema caused by a deficiency of C1-Inh. There is now accumulating evidence, obtained from studies in animals and observations in patients, that administration of C1-Inh may have a beneficial effect as well in other clinical conditions such as sepsis, cytokine-induced vascular leak syndrome, acute myocardial infarction, or other diseases. Activation of the complement system, the contact activation system, and the coagulation system has been observed in these diseases. A typical feature of the contact and complement system is that on activation they give rise to vasoactive peptides such as bradykinin or the anaphylatoxins, which in part explains the proinflammatory effects of either system. C1-Inh, belonging to the superfamily of serine proteinase inhibitors (serpins), is a major inhibitor of the classical complement pathway, the contact activation system, and the intrinsic pathway of coagulation, respectively. It is, therefore, endowed with anti-inflammatory properties. However, inactivation of C1-Inh occurs locally in inflamed tissues by proteolytic enzymes (e.g., elastase) released from activated neutrophils or bacteria thereby leading to increased local activation of the various host defense systems. Here we will give an overview on the biochemistry and biology of C1-Inh. We will discuss studies addressing therapeutic administration of C1-Inh in experimental and clinical conditions. Finally, we will provide an explanation for the therapeutic benefit of C1-Inh in so many different diseases.

Journal ArticleDOI
TL;DR: Evidence supporting the concept that menstruation occurs as a result of an inflammatory process is examined, with evidence that endometrial granular lymphocytes may also play a role, although their increase in numbers is somewhat earlier during the menstrual cycle than that of the others, suggesting perhaps a primary role in embryo implantation.
Abstract: This review examines evidence supporting the concept that menstruation occurs as a result of an inflammatory process. In the endometrium, leukocyte numbers rise in the late secretory phase following the fall in serum progesterone concentrations. It is postulated that products released following activation of these leukocytes are critically important for menstruation. Mast cells, eosinophils, neutrophils and macrophages in particular are involved. Endometrial granular lymphocytes may also play a role, although their increase in numbers is somewhat earlier during the menstrual cycle than that of the others, suggesting perhaps a primary role in embryo implantation. Leukocyte products include a range of proteases, chemokines and cytokines which in concert result in focal production and activation of matrix metalloproteinases by endometrial cells and the subsequent breakdown of tissue that characterizes menstruation. Regulation of leukocyte entry, proliferation, differentiation and activation within the endometrium is not yet well understood, although both chemokines and cytokines produced locally by endometrial cells are clearly implicated. The role of progesterone in regulating these events is still not understood although the lack of progesterone receptors on endometrial leukocytes suggests indirect actions.

Journal ArticleDOI
TL;DR: Three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases.

Journal ArticleDOI
TL;DR: A tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized was synthesized, which was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin.
Abstract: We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.

Journal ArticleDOI
TL;DR: The role of proteinases in plant cell death is investigated in this paper, where characteristics of the best known proteinases from plants including subtilisin-type and papain-type enzymes, phytepsins, metalloproteinases and the 26S proteasome are summarized.
Abstract: Proteolytic enzymes are known to be associated with developmentally programmed cell death during organ senescence and tracheary element differentiation. Recent evidence also links proteinases with some types of pathogen- and stress-induced cell suicide. The precise roles of proteinases in these and other plant programmed cell death processes are not understood, however. To provide a framework for consideration of the importance of proteinases during plant cell suicide, characteristics of the best-known proteinases from plants including subtilisin-type and papain-type enzymes, phytepsins, metalloproteinases and the 26S proteasome are summarized. Examples of serine, cysteine, aspartic, metallo- and threonine proteinases linked to animal programmed cell death are cited and the potential for plant proteinases to act as mediators of signal transduction and as effectors of programmed cell death is discussed.

Journal ArticleDOI
TL;DR: Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method and conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin.
Abstract: Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (μLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyamidomethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interf...

Journal ArticleDOI
TL;DR: Omi protein consists of 458 amino acids and has homology to bacterial HtrA endoprotease, which acts as a chaperone at low temperatures and as a proteolytic enzyme that removes denatured or damaged substrates at elevated temperatures.

Journal ArticleDOI
TL;DR: The data are consistent with the hypothesis that proteasome is progressively inhibited by small accumulations of oxidized and cross‐linked proteins during proliferative senescence until late proliferation stages, when so much proteasomes activity has been lost that oxidized proteins accumulate at ever‐increasing rates.
Abstract: Oxidized and cross-linked proteins tend to accumulate in aging cells Declining activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibition of the proteasome by oxidized and cross-linked proteins has been demonstrated in vitro We have further examined this hypothesis during both proliferative senescence (this paper) and postmitotic senescence (see the accompanying paper, ref 1⤻) of human BJ fibroblasts During proliferative senescence, we found a marked decline in all proteasome activities (trypsin-like activity, chymotrypsin-like activity, and peptidyl-glutamyl-hydrolyzing activity) and in lysosomal cathepsin activity Despite the loss of proteasome activity, there was no concomitant change in cellular levels of actual proteasome protein (immunoassays) or in the steady-state levels of mRNAs for essential proteasome subunits The decline in proteasome activities and lysosomal cathepsin activities was accompanied by d

Journal ArticleDOI
TL;DR: There is a marked increase globally in the reporting of invasive infections caused by Streptococcus pyogenes, Lancefield group A streptococci, and the re-emergence of more 'virulent' strains, such as the M1 serotype, which in earlier decades was primarily seen in cases of either superficial disease or scarlet fever.
Abstract: The last decade has witnessed a remarkable change in the epidemiology of group A streptococcal infections. There has been a marked increase globally in the reporting of invasive infections caused by Streptococcus pyogenes, Lancefield group A streptococci. Many of these cases were deep-seated infections associated with shock and multi-organ failure and are defined as streptococcal toxic shock syndrome. In addition, reports of streptococcal sequelae, in particular, acute rheumatic fever, have re-emerged and remain a serious health threat in developed countries. It appears that these infections are related to the type distributions of the organism among the general population, with the re-emergence of more 'virulent' strains, such as the M1 serotype which in earlier decades was primarily seen in cases of either superficial disease or scarlet fever. Population-based surveillance studies have clearly indicated the importance and relevance of type identification for epidemiological purposes. There have also been suggestions that certain extracellular products and toxins play a major role in the socalled 'increased virulence' of the organism; these include cell surface molecules such as the M protein, opacity factor, the hyaluronic acid capsule, C5a peptidase and streptococcal inhibitor of complement (SIC), in addition to secreted proteins, pyrogenic exotoxins, cysteine proteinase, streptolysins O and S, hyaluronidase, streptokinase and other enzymes. All these factors, and events during the last decade, strongly emphasize the need for a better understanding of the epidemiology, pathogenesis, treatment and prevention of group A streptococcal infections.

Journal ArticleDOI
TL;DR: In this paper, the shell waste from shrimp Crangon crangon processing is used to recover chitin and protein hydrolysate from the shells of crangons.

Journal ArticleDOI
01 Nov 2000-Bone
TL;DR: The view that TRAP, like several other hydrolases, is synthesized as a relatively inactive proen enzyme, and cleavage is the physiological mechanism of proenzyme activation in osteoclasts is put forth.

Journal ArticleDOI
TL;DR: It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles and remains in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput.
Abstract: A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.

Journal Article
TL;DR: It is proposed that MT6-MMP may facilitate tumor progression through its ability to activate progelatinase A at the membrane of cells from colon carcinomas or brain tumors.
Abstract: The localization of proteolytic enzymes at the cell surface is a widely used strategy for facilitating tumor invasion. In this study, we have cloned a new member of the membrane-type subfamily of matrix metalloproteinases (MT-MMPs), a group of enzymes associated with tumor progression. The cloned cDNA encodes a protein of 562 amino acids with a domain organization similar to that of other MT-MMPs, including a prodomain with a cysteine switch, a catalytic domain with the zinc-binding site, a hemopexin-like domain, and a COOH-terminal extension rich in hydrophobic residues. The predicted protein sequence also contains a short insertion of basic residues located between the propeptide and the catalytic domain and involved in the proteolytic activation of MT-MMPs by furin-like enzymes. Furthermore, immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA revealed that the encoded protein is localized at the cell surface. Based on these properties, this novel human matrix metalloproteinase has been called MT6-MMP because it is the sixth identified member of this subfamily of matrix metalloproteinase. Cotransfection of expression plasmids encoding MT6-MMP and progelatinase A resulted in activation of COS-7-secreted progelatinase A, as demonstrated by gelatin zymography. In contrast, transfection of progelatinase A cDNA alone did not lead to the activation of the proenzyme. Northern blot analysis of polyadenylated RNAs isolated from human tissues demonstrated that MT6-MMP is predominantly expressed in leukocytes, lung, and spleen. MT6-MMP was also detected at high levels in SW480 colon carcinoma cells as well as in some anaplastic astrocytomas and glioblastomas, but not in normal colon or brain or in meningiomas. On the basis of these results, we propose that MT6-MMP may facilitate tumor progression through its ability to activate progelatinase A at the membrane of cells from colon carcinomas or brain tumors.

Journal ArticleDOI
TL;DR: The root-hypocotyl of Arabidopsis produces a relatively large amount of secondary vascular tissue when senescence is delayed by the removal of inflorescences, and plants are grown at low population density.
Abstract: The root-hypocotyl of Arabidopsis produces a relatively large amount of secondary vascular tissue when senescence is delayed by the removal of inflorescences, and plants are grown at low population density. Peptidase zymograms prepared from isolated xylem and phloem revealed the existence of distinct proteolytic enzyme profiles within these tissues. cDNA libraries were constructed from isolated xylem and bark of the root-hypocotyl and screened for cDNAs coding for cysteine, serine, and aspartic peptidases. Three cDNAs, two putative papain-type cysteine peptidases (XCP1 and XCP2) and one putative subtilisin-type serine peptidase (XSP1), were identified from the xylem library for further analysis. Using RNA gel blots it was determined that these peptidases were expressed in the xylem and not in the bark. Quantitative reverse transcriptase-polymerase chain reaction confirmed the RNA gel-blot results and revealed high levels of XCP1 and XCP2 mRNA in stems and flowers of the infloresence. A poly-histidine-tagged version of XCP1 was purified from Escherichia coli by denaturing metal-chelate chromatography. Following renaturation, the 40-kD recombinant XCP1 was not proteolytically active. Activation was achieved by incubation of recombinant XCP1 at pH 5.5 and was dependent on proteolytic processing of the 40-kD inactive polypeptide to a 26-kD active peptidase.

Journal ArticleDOI
TL;DR: A simple mathematical model based on the theory of reinforced random walks coupled with Michaelis-Menten mechanisms which view endothelial cell receptors as the catalysts for transforming both tumor and macrophage derived angiogenic factors into proteolytic enzyme which in turn degrade the basal lamina is extended.
Abstract: In this paper, a simple mathematical model developed in H.A. Levine, B.D. Sleeman, M. Nilsen-Hamilton [J. Math. Biol., in press] to describe the initiation of capillary formation in tumor angiogenesis is extended to include the roles of pericytes and macrophages in regulating angiogenesis. The model also allows for the presence of anti-angiogenic (angiostatic) factors. The model is based on the observation that angiostatin can prevent the degradation of fibronectin in the basal lamina by inhibiting the catalytic action of active proteolytic enzyme. That is, it is proposed that the inhibitor 'deactivates' the protease but that it does not reduce the over all concentration of the protease. It consequently explores the possibility of preventing neovascular capillaries from migrating through the extra-cellular matrix toward the tumor by inhibiting protease action. The model is based on the theory of reinforced random walks coupled with Michaelis-Menten mechanisms which view endothelial cell receptors as the catalysts for transforming both tumor and macrophage derived angiogenic factors into proteolytic enzyme which in turn degrade the basal lamina. A simple catalytic reaction is proposed for the degradation of the basal lamina by the active proteases. A mechanism, in which the angiostatin acts as a protease inhibitor is discussed which has been substantiated experimentally. A second mechanism for the production of protease inhibitor from angiostatin by endothelial cells is proposed to be of Michaelis-Menten type. Mathematically, this mechanism includes the former as a subcase.

Journal ArticleDOI
TL;DR: None of the reports demonstrates conclusively a decisive role for any of the virulence factors described thus far, but it is conceivable that proteolytic enzyme activities such as those expressed by AFAlp are one of a number of factors that combine to facilitate disease progression.
Abstract: Various putative virulence factors of Aspergillus fumigatus have been studied over the past decades. A. fumigatus gliotoxin is a potent inhibitor of the mucociliary system. Several fungal metabolites interfere with phagocytosis and opsonization including toxins, 'conidial inhibitory factor', 'A. fumigatus diffusible product' and 'complement inhibitory factor'. A. fumigatus can bind specifically to different host tissues components, whereas toxins give a general and significant immunosuppressive effect on host defences. Circumstantial evidence links the production of elastinolytic proteases with the ability to cause disease. However, none of the reports demonstrates conclusively a decisive role for any of the virulence factors described thus far. It is conceivable that proteolytic enzyme activities such as those expressed by AFAlp are one of a number of factors, each with a minor effect, that combine to facilitate disease progression.

Journal ArticleDOI
TL;DR: Proteolytic enzymes called secretases are involved in generating Aβ, and one of these may have been identified as presenilin — a discovery that paves the way for a more complete understanding of presenILin structure and function.
Abstract: Many neurodegenerative diseases involve the deposition of insoluble amyloid molecules. In Alzheimer's disease, for example, the amyloid beta-peptide (A beta) is the main component of the characteristic senile plaques. Proteolytic enzymes called secretases are involved in generating A beta, and one of these may have been identified as presenilin--a discovery that paves the way for a more complete understanding of presenilin structure and function.

Journal ArticleDOI
TL;DR: It is demonstrated that TNF-α inhibits transcription of the gene coding for the α2 chain of type I collagen [α2(I) collagen] in cultured fibroblasts by stimulating the synthesis and binding of repressive CCAAT/enhancer proteins (C/EBPs) to a previously identified T NF-α-responsive element.
Abstract: Extracellular matrix (ECM) formation and remodeling are critical processes for proper morphogenesis, organogenesis, and tissue repair. The proinflammatory cytokine tumor necrosis factor alpha (TNF-a) inhibits ECM accumulation by stimulating the expression of matrix proteolytic enzymes and by downregulating the deposition of structural macromolecules such as type I collagen. Stimulation of ECM degradation has been linked to prolonged activation of jun gene expression by the cytokine. Here we demonstrate that TNF-a inhibits transcription of the gene coding for the a2 chain of type I collagen [a2(I) collagen] in cultured fibroblasts by stimulating the synthesis and binding of repressive CCAAT/enhancer proteins (C/EBPs) to a previously identified TNF-a-responsive element. This conclusion was based on the concomitant identification of C/EBPb and C/EBPd as TNF-a-induced factors by biochemical purification and expression library screening. It was further supported by the ability of the C/EBP-specific dominant-negative (DN) protein to block TNF-a inhibition of a2(I) collagen but not TNF-a stimulation of the MMP-13 protease. The DN protein also blocked TNF-a downregulation of the gene coding for the a1 chain of type I collagen. The study therefore implicates repressive C/EBPs in the TNF-a-induced signaling pathway that controls ECM formation and remodeling. Tumor necrosis factor alpha (TNF-a) is a 17-kDa cytokine released by activated macrophages that locally elicits a wide spectrum of metabolic responses and cellular activities (20). The pleiotropic properties of TNF-a are signaled through alternative routes that culminate in diversified responses, such as cell proliferation, differentiation, or death (20). These ill-defined intracellular pathways in part influence gene expression by modulating the activity and synthesis of several transcription factors. Relevant examples include the activation of AP1 and NF-kB in the regulation of cell death and cell survival, the induction of IRF1 in the stimulation of the alpha and beta interferon genes, and the undefined pathway that inhibits C/EBP expression in an experimental model of inflammationinduced atrophy of adipose tissue (3, 6, 17). Aside from cachexia, TNF-a is believed to be a key player in inflammatory disorders such as rheumatoid arthritis and osteoarthritis (20). Extracellular matrix (ECM) degradation is the hallmark of these conditions and an important component in morphogenesis, organogenesis, and tissue remodeling, as well as in wound healing and tissue repair. TNF-a stimulates ECM degradation by inducing the production of stromal collagenases. The stimulation is the result of TNF-a prolonged activation of jun gene expression with the consequent binding of the AP1 complex to the responsive elements in the collagenase promoters (4). TNF-a reduces ECM deposition by inhibiting the synthesis of structural components. They include elastin, osteocalcin, and type I collagen, the major structural

Journal ArticleDOI
TL;DR: The purpose of this article is to provide an overview of the current knowledge of genome structure and gene expression in the enteric caliciviruses.
Abstract: The application of molecular techniques to the characterization of caliciviruses has resulted in an extensive database of sequence information. This information has led to the identification of 4 distinct genera. The human enteric caliciviruses have been assigned to 2 of these genera. This division is reflected not only in sequence diversity but in a fundamental difference in genome organization. Complete genome sequences are now available for 5 enteric caliciviruses and demonstrate that human and animal enteric caliciviruses are phylogenetically closely related. Currently, there is no cell culture system for the human viruses; therefore, studies have relied on heterologous expression and in vitro systems. These studies have shown that in both human and animal viruses the viral nonstructural proteins are produced from a polyprotein precursor that is cleaved by a single viral protease. The purpose of this article is to provide an overview of the current knowledge of genome structure and gene expression in the enteric caliciviruses.

Journal ArticleDOI
TL;DR: Low-molecular-mass beta-(2,6)-linked fructose-oligosaccharides were examined as a new carbohydrate source for growth of bifidobacteria and showed the best growth, produced the highest amounts of organic acids and metabolized both short- and long-chain beta-FOS.
Abstract: Low-molecular-mass beta-(2,6)-linked fructose-oligosaccharides (beta-(2,6)-FOS) were examined as a new carbohydrate source for growth of bifidobacteria. beta-(2,6)-FOS were prepared from microbial high-molecular-mass levan by acid hydrolysis and refined by cation-exchange chromatography. (13)C-NMR spectroscopy confirmed the presence of predominantly beta-(2,6)-fructosyl linkages in the oligosaccharides. More than 80% beta-(2,6)-FOS was recovered after in vitro incubation with amylolytic and proteolytic enzymes, implying resistance to degradation in the upper intestinal tract. Bifidobacterium adolescentis, B. longum, B. breve, and B. pseudocatenulatum were studied in vitro for their ability to metabolize beta-(2,6)-FOS. Growth, decrease in pH, formation of short- chain fatty acids (lactate, acetate, formate) and degradation of beta-(2,6)-FOS were markedly different among species. B. adolescentis showed the best growth, produced the highest amounts of organic acids and metabolized both short- and long-chain beta-(2, 6)-FOS.