Showing papers on "Proteolytic enzymes published in 2003"

Effect of pegylation on pharmaceuticals

Abstract: Protein and peptide drugs hold great promise as therapeutic agents. However, many are degraded by proteolytic enzymes, can be rapidly cleared by the kidneys, generate neutralizing antibodies and have a short circulating half-life. Pegylation, the process by which polyethylene glycol chains are attached to protein and peptide drugs, can overcome these and other shortcomings. By increasing the molecular mass of proteins and peptides and shielding them from proteolytic enzymes, pegylation improves pharmacokinetics. This article will review how PEGylation can result in drugs that are often more effective and safer, and which show improved patient convenience and compliance.

Human and mouse proteases: a comparative genomic approach

Abstract: The availability of the human and mouse genome sequences has allowed the identification and comparison of their respective degradomes--the complete repertoire of proteases that are produced by these organisms. Because of the essential roles of proteolytic enzymes in the control of cell behaviour, survival and death, degradome analysis provides a useful framework for the global exploration of these protease-mediated functions in normal and pathological conditions.

Calcium and apoptosis: facts and hypotheses.

Abstract: Although longstanding experimental evidence has associated alterations of calcium homeostasis to cell death, only in the past few years the role of calcium in the signaling of apoptosis has been extensively investigated. In this review, we will summarize the current knowledge, focusing on (i) the effect of the proteins of the Bcl-2 family on ER Ca2+ levels, (ii) the action of the proteolytic enzymes of apoptosis on the Ca2+ signaling machinery, (iii) the ensuing alterations on the signaling patterns of extracellular stimuli, and (iv) the intracellular targets of 'apoptotic' Ca2+ signals, with special emphasis on the mitochondria and cytosolic Ca2+-dependent enzymes.

Risk factors for coronary heart disease in patients treated for human immunodeficiency virus infection compared with the general population.

Abstract: The distribution of risk factors for cardiovascular disease in patients aged 35-44 years who were treated for human immunodeficiency virus type 1 (HIV-1) infection was compared with that for a population-based cohort. HIV-1-infected men treated with a protease inhibitor-containing regimen (n=223), compared with HIV-1-uninfected men (n=527), were characterized by a lower prevalence of hypertension, a lower mean high-density lipoprotein cholesterol level, a higher prevalence of smoking, a higher mean waist-to-hip ratio, and a higher mean triglyceride level. No difference was found for total plasma or low-density cholesterol levels, nor for the prevalence of diabetes. Similar trends were observed among female subjects. The predicted risk of coronary heart disease was greater among HIV-1-infected men (relative risk [RR], 1.20) and women (RR, 1.59; P<10(-6) for both), compared with the HIV-1-uninfected cohort. The estimated attributable risks due to smoking were 65% and 29% for HIV-1-infected men and women, respectively. Because most HIV-1-infected people will ultimately need antiretroviral therapy, risk factors for cardiovascular disease should be determined at the initiation of treatment, and interventions should be considered for all patients who have them.

The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy

Abstract: Cancer dissemination and metastasis is synonymous with invasive cell migration; a process in which the extracellular matrix (ECM) plays the dual role of the substratum on which the cells move as well as the physical obstacle that the cells have to surpass. To degrade the physical obstacle, which the ECM poses in the direction of migration, cells use proteolytic enzymes capable of degrading the ECM components. A major protease system responsible for ECM degradation is the plasminogen activation system, which generates the potent serine protease plasmin. The subject of this review, the urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plays an impressive range of distinct, but overlapping functions in the process of cancer invasion and metastasis: Firstly, uPA/uPAR promotes extracellular proteolysis by regulating plasminogen activation. Secondly, uPA/uPAR regulates cell/ECM interactions as an adhesion receptor for vitronectin (Vn) and through its capacity to modulate integrin function. Thirdly, uPA/uPAR regulates cell migration as a signal transduction molecule and by its intrinsic chemotactic activity. This review is focused on recent insight into the cancer related biology of the uPA/uPAR system as well as its implications for clinical cancer diagnosis, prognosis and therapy.

The polyglutamine neurodegenerative protein ataxin-3 binds polyubiquitylated proteins and has ubiquitin protease activity.

Abstract: The ubiquitin-proteasome pathway is critically involved in the pathology of neurodegenerative diseases characterized by protein misfolding and aggregation. Data in the present study suggest that the polyglutamine neurodegenerative disease protein, ataxin-3 (AT3), functions in the ubiquitin-proteasome pathway. AT3 contains an ubiquitin interaction motif (UIM) domain that binds polyubiquitylated proteins with a strong preference for chains containing four or more ubiquitins. Mutating the conserved leucine in the first UIM (L229A) almost totally eliminates binding to polyubiquitin chains while a similar mutation in the second UIM (L249A) also inhibits binding to polyubiquitin chains but to a lesser extent. Both wild-type and pathological AT3 increase cellular levels of a short-lived GFP that is degraded by the ubiquitin-proteasome pathway. AT3 has several properties characteristic of ubiquitin proteases including decreasing polyubiquitylation of 125 I-lysozyme by removing ubiquitin from polyubiquitin chains, cleaving a ubiquitin protease substrate, and binding the specific ubiquitin protease inhibitor, ubiquitin-aldehyde. Mutating the predicted catalytic cysteine in AT3 inhibits each of these ubiquitin protease activities. The ability to bind and cleave ubiquitylated proteins is consistent with AT3 playing a role in the ubiquitin-proteasome system. This raises the possibility that pathological AT3, which tends to misfold and aggregate, may be exposed to aggregateprone misfolded/denatured proteins as part of its normal function.

Oxidative stress and acute lung injury.

Abstract: Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS) are frequent complications in critically ill patients and are responsible for significant morbidity and mortality (1, 2). Treatment of the underlying disease and excellent supportive care using “lung-protective” strategies of mechanical ventilation (3) contribute to successful clinical outcomes. However, aside from the use of activated protein C in the subset of ALI/ ARDS patients with sepsis (4), specific therapies are lacking, and the cascade of events leading to ALI and ARDS, once initiated, is much less amenable to specific treatment modalities. Regardless of the underlying illness, the clinical and pathologic manifestations of ALI/ARDS are very similar, indicating the existence of final common pathways that represent potential therapeutic targets (1, 5). In essence, these syndromes reflect severe injury leading to dysfunction and compromise of the barrier properties of the pulmonary endothelium and epithelium as a consequence of an unregulated acute inflammatory response (6). In this hypothetical construct, an initiating event (sepsis, shock, trauma, multiple transfusions, pancreatitis, etc.) leads to activation of the acute inflammatory response on a systemic level. One of the earliest manifestations is activation of pulmonary endothelium and macrophages (alveolar and interstitial), upregulation of adhesion molecules, and production of cytokines and chemokines that induce a massive sequestration of neutrophils within the pulmonary microvasculature. These cells transmigrate across the endothelium and epithelium into the alveolar space and release a variety of cytotoxic and proinflammatory compounds, including proteolytic enzymes, reactive oxygen species (ROS) and nitrogen species, cationic proteins, lipid mediators, and additional inflammatory

Roles of matrix metalloproteinases in tumor metastasis and angiogenesis.

Abstract: Matrix metalloproteinases (MMPs), zinc dependent proteolytic enzymes, cleave extracellular matrix (ECM: collagen, laminin, firbronectin, etc) as well as non-matrix substrates (growth factors, cell surface receptors, etc). The deregulation of MMPs is involved in many diseases, such as tumor metastasis, rheumatoid arthritis, and periodontal disease. Metastasis is the major cause of death among cancer patients. In this review, we will focus on the roles of MMPs in tumor metastasis. The process of metastasis involves a cascade of linked, sequential steps that involve multiple host-tumor interactions. Specifically, MMPs are involved in many steps of tumor metastasis. These include tumor invasion, migration, host immune escape, extravasation, angiogenesis, and tumor growth. Therefore, without MMPs, the tumor cell cannot perform successful metastasis. The activities of MMPs are tightly regulated at the gene transcription levels, zymogen activation by proteolysis, and inhibition of active forms by endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP), and RECK. The detailed regulations of MMPs are described in this review.

Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer.

Abstract: Dysregulated proteolysis is a hallmark of cancer. Malignant cells require a range of proteolytic activities to enable growth, survival, and expansion. Serine proteases of the S1 or trypsin-like family have well recognized roles in the maintenance of normal homeostasis as well as in the pathology of diseases such as cancer. Recently a rapidly expanding subgroup of S1 proteases has been recognized that are directly anchored to plasma membranes. These membrane anchored serine proteases are anchored either via a carboxy-terminal transmembrane domain (Type I), a carboxy terminal hydrophobic region that functions as a signal for membrane attachment via a glycosyl-phosphatidylinositol linkage (GPI-anchored), or via an amino terminal proximal transmembrane domain (Type II or TTSP). The TTSPs also encode multiple domains in their stem regions that may function in regulatory interactions. The serine protease catalytic domains of these enzymes show high homology but also possess features indicating unique substrate specificities. It is likely that the membrane anchored serine proteases have evolved to perform complex functions in the regulation of cellular signaling events at the plasma membrane and within the extracellular matrix. Disruption or mutation of several of the genes encoding these proteases are associated with disease. Many of the membrane anchored serine proteases show restricted tissue distribution in normal cells, but their expression is widely dysregulated during tumor growth and progression. Diagnostic or therapeutic targeting of the membrane anchored serine proteases has potential as promising new approaches for the treatment of cancer and other diseases.

Characterization of bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OG1

Abstract: Lactobacillus plantarum F1 and L. brevis OG1 isolated from Nigerian fermented food products, produced bacteriocins that had broad spectrum of inhibition against both pathogenic, food spoilage organisms and various lactic acid bacteria. The test organisms exhibited activities of 6400 and 3200 AU/ml respectively against Escherichia coli NCTC10418 and Enterococcus faecalis EF1, but did not inhibit Candida albicans ATCC10231 and Klebsiella sp. UCH15. Comparison of the antimicrobial spectra and characterization of the two bacteriocins were not identical. Bacteriocin produced by L. brevis OG1 was the most heat stable at 121°C for 60 min, while that of L. plantarum F1 was stable at 121°C for 10 min. The bacteriocins produced by the test isolates maintained full stability after storage for 60 days at – 20°C; partial stability after storage for 120 days at 4°C; while activity was not detected after storage for 80 to 120 days at 37°C. Bacteriocin produced by L. brevis OG1 was stable at pH range of 2.0 to 8.0 while, that of L. plantarum F1 was found to be stable at pH 2.0 to 6.0. Their active principle was proteinaceous in nature since the bacteriocins were inactivated by proteolytic enzymes, but not by other non–proteolytic enzymes. mitomycin C and uv light did not affect the activity of the bacteriocins, while chloroform extraction completely destroyed their activity. Exposure to surfactant resulted in an increase in the bacteriocin titre, except Nonidet P-40, which led to total loss of bacteriocin activity. The bacteriocins were able to pass through cellulose membranes with 100,000 KDa and 1,000,000 KDa but could not pass through one with a 10,000 KDa and 1,000 KDa molecular weight cut off. The paper concluded that the ability of bacteriocins produced by the test isolates in inhibiting a wide-range of bacteria, is of potential interest for food safety and may have future applications as food preservative. (African Journal of Biotechnology: 2003 2(8): 219-227)

Gelatinase B/MMP-9 and neutrophil collagenase/MMP-8 process the chemokines human GCP-2/CXCL6, ENA-78/CXCL5 and mouse GCP-2/LIX and modulate their physiological activities.

Abstract: On chemokine stimulation, leucocytes produce and secrete proteolytic enzymes for innate immune defence mechanisms. Some of these proteases modify the biological activity of the chemokines. For instance, neutrophils secrete gelatinase B (matrix metalloproteinase-9, MMP-9) and neutrophil collagenase (MMP-8) after stimulation with interleukin-8/CXCL8 (IL-8). Gelatinase B cleaves and potentiates IL-8, generating a positive feedback. Here, we extend these findings and compare the processing of the CXC chemokines human and mouse granulocyte chemotactic protein-2/CXCL6 (GCP-2) and the closely related human epithelial-cell derived neutrophil activating peptide-78/CXCL5 (ENA-78) with that of human IL-8. Human GCP-2 and ENA-78 are cleaved by gelatinase B at similar rates to IL-8. In addition, GCP-2 is cleaved by neutrophil collagenase, but at a lower rate. The cleavage of GCP-2 is exclusively N-terminal and does not result in any change in biological activity. In contrast, ENA-78 is cleaved by gelatinase B at eight positions at various rates, finally generating inactive fragments. Physiologically, sequential cleavage of ENA-78 may result in early potentiation and later in inactivation of the chemokine. Remarkably, in the mouse, which lacks IL-8 which is replaced by GCP-2/LIX as the most potent neutrophil activating chemokine, N-terminal clipping and twofold potentiation by gelatinase B was also observed. In addition to the similarities in the potentiation of IL-8 in humans and GCP-2 in mice, the conversion of mouse GCP-2/LIX by mouse gelatinase B is the fastest for any combination of chemokines and MMPs so far reported. This rapid conversion was also performed by crude neutrophil granule secretion under physiological conditions, extending the relevance of this proteolytic cleavage to the in vivo situation.

Regulation of myostatin in vivo by growth and differentiation factor-associated serum protein-1: a novel protein with protease inhibitor and follistatin domains.

Abstract: Myostatin, a member of the TGFbeta superfamily, is a potent and specific negative regulator of skeletal muscle mass. In serum, myostatin circulates as part of a latent complex containing myostatin propeptide and/or follistatin-related gene (FLRG). Here, we report the identification of an additional protein associated with endogenous myostatin in normal mouse and human serum, discovered by affinity purification and mass spectrometry. This protein, which we have named growth and differentiation factor-associated serum protein-1 (GASP-1), contains multiple domains associated with protease-inhibitory proteins, including a whey acidic protein domain, a Kazal domain, two Kunitz domains, and a netrin domain. GASP-1 also contains a domain homologous to the 10-cysteine repeat found in follistatin, a protein that binds and inhibits activin, another member of the TGFbeta superfamily. We have cloned mouse GASP-1 and shown that it inhibits the biological activity of mature myostatin, but not activin, in a luciferase reporter gene assay. Surprisingly, recombinant GASP-1 binds directly not only to mature myostatin, but also to the myostatin propeptide. Thus, GASP-1 represents a novel class of inhibitory TGFbeta binding proteins.

Geno2pheno: estimating phenotypic drug resistance from HIV-1 genotypes

Abstract: Therapeutic success of anti-HIV therapies is limited by the development of drug resistant viruses. These genetic variants display complex mutational patterns in their pol gene, which codes for protease and reverse transcriptase, the molecular targets of current antiretroviral therapy. Genotypic resistance testing depends on the ability to interpret such sequence data, whereas phenotypic resistance testing directly measures relative in vitro susceptibility to a drug. From a set of 650 matched genotype-phenotype pairs we construct regression models for the prediction of phenotypic drug resistance from genotypes. Since the range of resistance factors varies considerably between different drugs, two scoring functions are derived from different sets of predicted phenotypes. Firstly, we compare predicted values to those of samples derived from 178 treatment-naive patients and report the relative deviance. Secondly, estimation of the probability density of 2000 predicted phenotypes gives rise to an intrinsic definition of a susceptible and a resistant subpopulation. Thus, for a predicted phenotype, we calculate the probability of membership in the resistant subpopulation. Both scores provide standardized measures of resistance that can be calculated from the genotype and are comparable between drugs. The geno2pheno system makes these genotype interpretations available via the Internet (http://www.genafor.org/).

Proteomic Study of the Arabidopsis thaliana Chloroplastic Envelope Membrane Utilizing Alternatives to Traditional Two-Dimensional Electrophoresis

Abstract: With the completion of the sequencing of the Arabidopsis genome and with the significant increase in the amount of other plant genome and expressed sequence tags (ESTs) data, plant proteomics is rapidly becoming a very active field. We have pursued a high-throughput mass spectrometry-based proteomics approach to identify and characterize membrane proteins localized to the Arabidopsis thaliana chloroplastic envelope membrane. In this study, chloroplasts were prepared from plate- or soil-grown Arabidopsis plants using a novel isolation procedure, and “mixed” envelopes were subsequently isolated using sucrose step gradients. We applied two alternative methodologies, off-line multidimensional protein identification technology (Off-line MUDPIT) and one-dimensional (1D) gel electrophoresis followed by proteolytic digestion and liquid chromatography coupled with tandem mass spectrometry (Gel-C-MS/MS), to identify envelope membrane proteins. This proteomic study enabled us to identify 392 nonredundant proteins. K...

Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells

Abstract: To determine the effect of low culture temperature on erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells, rCHO cells producing EPO (LGE10-9-27) were cultivated at 30, 33, and 37 degrees C. At a culture temperature lower than 37 degrees C cell growth was suppressed, but cell viability remained high for a longer culture period. When the culture temperature was lowered from 37 degrees C to 33 degrees C, more than a 2.5-fold increase in the maximum EPO concentration was achieved. This enhanced EPO production at 33 degrees C was not just because of the extended culture longevity with the decreased release of proteolytic enzymes from dead cells, but mainly because of enhanced q(EPO). The q(EPO) at 33 degrees C was 0.35 +/- 0.08 microg/10(6) cells/h, which was approximately 4-fold higher than that at 37 degrees C. Although the highest q(EPO) of 0.49 +/- 0.14 micro/10(6) cells/h was obtained at 30 degrees C, the maximum EPO concentration was lowest because the detrimental effect of lowering culture temperature on cell growth outweighed its beneficial effect on q(EPO). Like q(EPO), the relative EPO mRNA content increased by lowering culture temperature, indicating that the increased transcription level of EPO was responsible in part for the enhanced q(EPO) at low culture temperature. The quality of EPO produced at 33 degrees C in regard to isoform pattern, sialic acid content, and in vivo biological activity was comparable to or even better than that produced at 37 degrees C. Taken together, the results obtained demonstrate the potential of the application of low culture temperature to the commercial EPO production in rCHO cells.

Stem Cell Mobilization

Abstract: Successful blood and marrow transplant (BMT), both autologous and allogeneic, requires the infusion of a sufficient number of hematopoietic progenitor/stem cells (HPCs) capable of homing to the marrow cavity and regenerating a full array of hematopoietic cell lineages in a timely fashion. At present, the most commonly used surrogate marker for HPCs is the cell surface marker CD34, identified in the clinical laboratory by flow cytometry. Clinical studies have shown that infusion of at least 2 x 10(6) CD34(+) cells/kg recipient body weight results in reliable engraftment as measured by recovery of adequate neutrophil and platelet counts approximately 14 days after transplant. Recruitment of HPCs from the marrow into the blood is termed mobilization, or, more commonly, stem cell mobilization. In Section I, Dr. Tsvee Lapidot and colleagues review the wide range of factors influencing stem cell mobilization. Our current understanding focuses on chemokines, proteolytic enzymes, adhesion molecules, cytokines and stromal cell-stem cell interactions. On the basis of this understanding, new approaches to mobilization have been designed and are now starting to undergo clinical testing. In Section II, Dr. Michele Cottler-Fox describes factors predicting the ability to mobilize the older patient with myeloma. In addition, clinical approaches to improving collection by individualizing the timing of apheresis and adjusting the volume of blood processed to achieve a desired product are discussed. Key to this process is the daily enumeration of blood CD34(+) cells. Newer methods of enumerating and mobilizing autologous blood HPCs are discussed. In Section III, Dr. John DiPersio and colleagues provide data on clinical results of mobilizing allogeneic donors with G-CSF, GM-CSF and the combination of both as relates to the number and type of cells collected by apheresis. Newer methods of stem cell mobilization as well as the relationship of graft composition on immune reconstitution and GVHD are discussed.

Molecular mechanisms of tumor invasion and metastasis: an integrated view.

Abstract: As tumors progress to increased malignancy, cells within them develop the ability to invade into surrounding normal tissues and through tissue boundaries to form new growths (metastases) at sites distinct from the primary tumor. The molecular mechanisms involved in this process are incompletely understood but those associated with cell-cell and cell-matrix adhesion, with the degradation of extracellular matrix, and with the initiation and maintenance of early growth at the new site are generally accepted to be critical. This article discusses current knowledge of molecular events involved in these various processes. The potential role of adhesion molecules (eg. integrins and cadherins) has undergone a major transition over the last ten years, as it has become apparent that such molecules play a major role in signaling from outside to inside a cell, thereby controlling how a cell is able (or not) to sense and interact with its local environment. Similarly the roles of proteolytic enzymes and their inhibitors (eg. matrix metalloproteinases and TIMPs) have also expanded as it has become apparent that they not only have the abilities to break down the components of the extracellular matrix but also are involved in the release of factors which can affect the growth of the tumor cells positively or negatively. Recent work has highlighted the importance of the later, post-extravasational stages of metastasis, where adhesion and proteolysis are now known to play a role along with other processes such as apoptosis, dormancy, growth factor-receptor interactions and signal transduction. Recent work has also demonstrated that not only the immediate cellular microenvironment, in terms of specific cell-cell and cell-matrix interactions, but also the extended cellular microenvironment, in terms of vascular insufficiency and hypoxia in the primary tumor, can modify cellular gene expression and enhance metastasis. Mechanisms of metastasis appear to involve a complex array of genetic and epigenetic changes many of which appear to be specific both for different types of tumors and for different sites of metastasis. Our improved understanding of the expanded roles of the individual molecules involved has resulted in a mechanistic blurring of the previously described discrete stages of the metastatic process.

Candida albicans binds human plasminogen: identification of eight plasminogen‐binding proteins

Abstract: Several microbial pathogens augment their invasive potential by binding and activating human plasminogen to generate the proteolytic enzyme plasmin. Yeast cells and cell wall proteins (CWP) of the human pathogenic fungus Candida albicans bound plasminogen with a K(d) of 70 +/- 11 nM and 112 +/- 20 nM respectively. Bound plasminogen could be activated to plasmin by mammalian plasminogen activators; no C. albicans plasminogen activator was detected. Binding of plasminogen to CWP and whole cells was inhibited by epsilon ACA, indicating that binding was predominantly to lysine residues. Candida albicans mutant strains defective in protein glycosylation did not show altered plasminogen binding, suggesting that binding was not mediated via a surface lectin. Binding was sensitive to digestion by basic carboxypeptidase, implicating C-terminal lysine residues in binding. Proteomic analysis identified eight major plasminogen-binding proteins in isolated CWP. Five of these (phosphoglycerate mutase, alcohol dehydrogenase, thioredoxin peroxidase, catalase, transcription elongation factor) had C-terminal lysine residues and three (glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and fructose bisphosphate aldolase) did not. Activation of plasminogen could potentially increase the capacity of this pathogenic fungus for tissue invasion and necrosis. Although surface-bound plasmin(ogen) degraded fibrin, no direct evidence for a role in invasion of endothelial matrix or in penetration and damage of endothelial cells was found.

Glutathione S-transferase omega-1 modifies age-at-onset of Alzheimer disease and Parkinson disease

Abstract: We previously reported genetic linkage of loci controlling age-at-onset in Alzheimer disease (AD) and Parkinson's disease (PD) to a 15 cM region on chromosome 10q. Given the large number of genes in this initial starting region, we applied the process of ‘genomic convergence’ to prioritize and reduce the number of candidate genes for further analysis. As our second convergence factor we performed gene expression studies on hippocampus obtained from AD patients and controls. Analysis revealed that four of the genes [stearoyl-CoA desaturase; NADH-ubiquinone oxidoreductase 1 beta subcomplex 8; protease, serine 11; and glutathione S-transferase, omega-1 (GSTO1)] were significantly different in their expression between AD and controls and mapped to the 10q age-at-onset linkage region, the first convergence factor. Using 2814 samples from our AD dataset (1773 AD patients) and 1362 samples from our PD dataset (635 PD patients), allelic association studies for age-at-onset effects in AD and PD revealed no association for three of the candidates, but a significant association was found for GSTO1 (P=0.007) and a second transcribed member of the GST omega class, GSTO2 (P=0.005), located next to GSTO1. The functions of GSTO1 and GSTO2 are not well understood, but recent data suggest that GSTO1 maybe involved in the post-translational modification of the inflammatory cytokine interleukin-1β. This is provocative given reports of the possible role of inflammation in these two neurodegenerative disorders.

Degradation of extracellular matrix components by defined proteinases from the greenbottle larva Lucilia sericata used for the clinical debridement of non-healing wounds.

Abstract: Summary Background Larvae of the greenbottle fly Lucilia sericata are used routinely for the clinical treatment of difficult necrotic and infected wounds. Degradation by proteinases contained in larval excretory/secretory (ES) products is thought to contribute to wound debridement by removal of dead tissue. However, proteinase activity may also affect host tissue remodelling processes. Objectives To identify proteolytic enzymes derived from L. sericata ES products with activities against fibrin and extracellular matrix (ECM) components. Methods Larval proteinase activities were assayed in vitro using class-specific substrates and inhibitors. Their action against fibrin and ECM components was examined using sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Results Three classes of proteolytic enzyme were detected in the secretions using fluorescein isothiocyanate-labelled casein as a model substrate. The predominant activity belonged to serine proteinases (pH optima 8–9) of two different subclasses (trypsin-like and chymotrypsin-like), with a weaker aspartyl proteinase (pH 5) and a metalloproteinase (pH 9) with exopeptidase characteristics also present. Using skin-relevant ECM components as substrates L. sericata ES products solubilized fibrin clots and degraded fibronectin, laminin and acid-solubilized collagen types I and III. Hydrolysis of ECM macromolecules was inhibited by preincubating ES products with phenylmethylsulphonyl fluoride but not 4-amidinophenylmethylsulphonyl fluoride, indicating that degradation was due to the ‘chymotrypsin-like’ serine proteinase. Conclusions These data suggest that a combination of L. sericata ES proteinases involving chymotrypsin-like and trypsin-like activities could potentially influence wound healing events when maggots are introduced into necrotic and infected wounds, with the chymotrypsin-like activity involved in the remodelling of ECM components.

Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasite-infected erythrocytes.

Abstract: The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of antigenically diverse proteins is expressed on the surface of human erythrocytes infected with the malaria parasite P. falciparum, and mediates cytoadherence to the host vascular endothelium. In this report, we show that export of PfEMP1 is slow and inefficient as it takes several hours to traffic newly synthesized proteins to the erythrocyte membrane. Upon removal by trypsin treatment, the surface-exposed population of PfEMP1 is not replenished during subsequent culture indicating that there is no cycling of PfEMP1 between the erythrocyte surface and an intracellular compartment. The role of Maurer's clefts as an intermediate sorting compartment in trafficking of PfEMP1 was investigated using immunoelectron microscopy and proteolytic digestion of streptolysin O-permeabilized parasitized erythrocytes. We show that PfEMP1 is inserted into the Maurer's cleft membrane with the C-terminal domain exposed to the erythrocyte cytoplasm, whereas the N-terminal domain is buried inside the cleft. Transfer of PfEMP1 to the erythrocyte surface appears to involve electron-lucent extensions of the Maurer's clefts. Thus, we have delineated some important aspects of the unusual trafficking mechanism for delivery of this critical parasite virulence factor to the erythrocyte surface.

Exploitation of Astrocytes by Glioma Cells to Facilitate Invasiveness: A Mechanism Involving Matrix Metalloproteinase-2 and the Urokinase-Type Plasminogen Activator–Plasmin Cascade

Abstract: The presence of reactive astrocytes around glioma cells in the CNS suggests the possibility that these two cell types could be interacting. We addressed whether glioma cells use the astrocyte environment to modulate matrix metalloproteinase-2 (MMP-2), a proteolytic enzyme implicated in the invasiveness of glioma cells. We found that astrocytes in culture produce significant amounts of the pro-form of MMP-2 but undetectable levels of active MMP-2. However, after coculture with the U251N glioma line, astrocyte pro-MMP-2 was converted to the active form. The mechanism of pro-MMP-2 activation in glioma-astrocyte coculture was investigated and was found to involve the urokinase-type plasminogen activator (uPA)-plasmin cascade whereby uPA bound to uPA receptor (uPAR), leading to the conversion of plasminogen to plasmin. The latter cleaved pro-MMP-2 to generate its active form. Furthermore, key components (i.e., uPAR, uPA, and pro-MMP-2) were contributed principally by astrocytes, whereas the U251N glioma cells provided plasminogen. In correspondence with this biochemical cascade, the transmigration of U251N cells through Boyden invasion chambers coated with an extracellular matrix barrier was increased significantly in the presence of astrocytes, and this was inhibited by agents that disrupted the uPA-plasmin cascade. Finally, using resected human glioblastoma specimens, we found that tumor cells, but not astrocytes, expressed plasminogen in situ. We conclude that glioma cells exploit their astrocyte environment to activate MMP-2 and that this leads to the increased invasiveness of glioma cells.

The Staphylococcus aureus cidAB Operon: Evaluation of Its Role in Regulation of Murein Hydrolase Activity and Penicillin Tolerance

Abstract: Bacterial murein hydrolases, or autolysins, comprise a broad and diverse family of enzymes that specifically cleave structural components of the bacterial cell wall. These enzymes have been shown previously to participate in a number of important biological functions during cell growth and division, including daughter cell separation, cell wall growth, and peptidoglycan recycling and turnover (14, 15, 17, 23, 38, 42, 55). Murein hydrolases also appear to play an important role in several bacterial developmental processes, such as spore formation, swarming motility, and competence (10, 24, 29, 40). Because of their potentially lethal capacity to hydrolyze the cell wall, it is critical that tight control be exercised over the expression and activity of murein hydrolases. For example, compartment-specific and temporal expression of the complement of murein hydrolases involved during Bacillus subtilis sporulation is dependent on several sporulation-specific sigma factors (11, 30, 32, 51), as well as the late-growth regulator Sin (30, 47). At the posttranscriptional level, murein hydrolase activity has been shown elsewhere to be modulated by several mechanisms, such as substrate modification, selective transport, interactions with lipoteichoic acids and cationic peptides, and cleavage by proteolytic enzymes (7, 13, 17, 23, 34, 36, 49, 54). Recent evidence has suggested that the proton motive force (PMF) and its effect on the cell wall pH also regulate murein hydrolase activity in B. subtilis (4, 5). In the case of Staphylococcus aureus, Mani et al. (31) have provided initial evidence for a murein hydrolase regulatory locus. Two Tn917-lacZ mutants were identified that displayed negligible rates of autolysis and exhibited normal cell division (31). Furthermore, our group had previously identified a second regulatory locus, designated lytSR, whose mutation resulted in a significantly increased rate of autolysis (2). The lytSR gene products are members of the bacterial two-component regulatory family of proteins and positively regulate expression of the lrgAB operon that is located immediately downstream of lytSR (3). Recent studies in our laboratory have demonstrated that lrgAB expression inhibits extracellular murein hydrolase activity and promotes penicillin tolerance (19). Interestingly, the lrgA gene product shares many structural similarities to the bacteriophage holin family of proteins (19). These small membrane proteins control the timing of bacteriophage-induced lysis by regulating access of the bacteriophage-encoded murein hydrolase, or endolysin, to the cell wall peptidoglycan (53). The activity of a holin is usually inhibited by the presence of a homologous protein, called the antiholin (53). Since the lrgAB locus inhibits murein hydrolase activity, it was hypothesized that LrgA functions in a manner analogous to an antiholin that, accordingly, functions by inhibiting an unidentified holin (19). In the present study, we have identified an operon homologous to lrgAB, designated cidAB, within the S. aureus genome. Like LrgA, the cidA gene product shared many holin-like characteristics. Analysis of a cidA mutant indicated that this gene enhances extracellular murein hydrolase activity and promotes sensitivity to penicillin. Therefore, we propose that the proteins encoded by the cidAB locus may indeed function in a holin-like manner that is inhibited by the products of the lrgAB operon.

Cheesemaking with vegetable coagulants—the use of Cynara L. for the production of ovine milk cheeses

Abstract: This paper reviews the use of plant extracts as vegetable coagulants for cheesemaking. It covers the plants used as sources of coagulants, with a historical overview and particular emphasis on Cynara species. The genus Cynara L., its composition, milk clotting and proteolytic enzymes (cardosins) and their specificity towards peptide linkages are also described. Cheeses produced in the Iberian Peninsula using Cynara L. as coagulant are documented. Cynara L. is still the most used vegetable coagulant in cheesemaking, and also the most investigated. However, much work remains to be done to understand its action during cheese maturation and further characterization.