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PubMed1993

Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants.

Expression of cysteine proteinase inhibitors (cystatins) in tobacco or other plants has the potential for improving resistance against pathogens and insects that possess cysteine proteinases. A chimeric gene containing a cDNA clone of rice cystatin (oryzacystatin-I; OC-I), the cauliflower mosaic virus 35S promoter, and the nopaline synthase 3' region was introduced into tobacco plants by Agrobacterium tumefaciens. The presence of the chimeric gene in transgenic plants was detected by a polymerase chain reaction-amplified assay, and transcriptional activity was shown by RNA blot analysis. Heated extracts from transgenic tobacco plants, as well as from progeny which were obtained by selfing a primary transformant, contained protein bands that corresponded in molecular mass to OC-I and reacted with antibodies raised against rOC, a recombinant OC-I protein produced by Escherichia coli. Similar bands were absent in extracts from untransformed control plants. OC-I levels reached 0.5% and 0.6% of the total soluble proteins in leaves and roots, respectively, of some progeny. On a fresh weight basis, the OC-I content was higher in leaves (50 micrograms/g) than in roots (30 micrograms/g). OC-I was partially purified from protein extracts of rice seeds and from transgenic tobacco leaves by affinity to anti-rOC antibodies. OC-I from both sources was active against papain.

Cystatins↗
PubMed2001

Changes of skeletal muscle proteases activities during a chronic low-frequency stimulation period.

Modifications of muscular contractile patterns by chronic low-frequency stimulation induce structural, physiological, and biochemical transformations in fast-twitch fibers that cause them to act like slow-twitch muscle. During this transformation many changes in protein pattern appear and the proteolytic system may be involved in those changes. The activities of cathepsin L, B, H, D, the level of cystatin, as well as the calpain activity in rabbit fast-twitch muscle have been compared with those of slow-twitch muscle. The results show that fast-twitch muscle has lower cathepsin activities and higher calpain activities than slow-twitch muscle. Chronic low-frequency stimulation was applied for 24 days to fast-twitch muscles and changes in proteases and protease inhibitors (cystatin and calpastatin) were studied. After 7-14 days of stimulation, lysosomal cathepsin L, B, and D and cytoplasm calpain and proteosome activities increased several-fold. Involvement of the phagocyte cells in the protein fiber turnover was minimal. Although the turnover of contractile proteins during muscle electrostimulation takes place in synchrony with changes in the muscle proteolytic system, the stimulation period used did not attain the total transformation from fast- to slow-twitch muscle proteolytic pattern.

Acid Phosphatase↗
PubMed1998

Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs.

Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-I delta D86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed.

Animals↗
PubMed2004

VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens.

BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.

Apoptosis↗
PubMed2006

Renal impairment and outcomes in heart failure: systematic review and meta-analysis.

OBJECTIVES: We estimated the prevalence of renal impairment in heart failure (HF) patients and the magnitude of associated mortality risk using a systematic review of published studies. BACKGROUND: Renal impairment in HF patients is associated with excess mortality, although precise risk estimates are unclear. METHODS: A systematic search of MEDLINE (through May 2005) identified 16 studies characterizing the association between renal impairment and mortality in 80,098 hospitalized and non-hospitalized HF patients. All-cause mortality risks associated with any renal impairment (creatinine >1.0 mg/dl, creatinine clearance [CrCl] or estimated glomerular filtration rate [eGFR] <90 ml/min, or cystatin-C >1.03 mg/dl) and moderate to severe impairment (creatinine > or =1.5, CrCl or eGFR <53, or cystatin-C > or =1.56) were estimated using fixed-effects meta-analysis. RESULTS: A total of 63% of patients had any renal impairment, and 29% had moderate to severe impairment. After follow-up > or =1 year, 38% of patients with any renal impairment and 51% with moderate to severe impairment died versus 24% without impairment. Adjusted all-cause mortality was increased for patients with any impairment (hazard ratio [HR] = 1.56; 95% confidence interval [CI] 1.53 to 1.60, p < 0.001) and moderate to severe impairment (HR = 2.31; 95% CI 2.18 to 2.44, p < 0.001). Mortality worsened incrementally across the range of renal function, with 15% (95% CI 14% to 17%) increased risk for every 0.5 mg/dl increase in creatinine and 7% (95% CI 4% to 10%) increased risk for every 10 ml/min decrease in eGFR. CONCLUSIONS: Renal impairment is common among HF patients and confers excess mortality. Renal function should be considered in risk stratification and evaluation of therapeutic strategies for HF patients.

Creatinine↗
PubMed2004

Protection studies in sheep using affinity-purified and recombinant cysteine proteinases of adult Haemonchus contortus.

Vaccination with a membrane-bound thiol Sepharose-binding fraction (TSBP) of adult Haemonchus contortus has been shown to confer significant levels of protection against homologous challenge in sheep. This fraction is greatly enriched for cysteine proteinase activity. Following fractionation of TSBP by anion-exchange chromatography on MonoQ, protection was found to partition with those fractions further enriched for cysteine proteinase activity. In this study, the cysteine proteinases of adult H. contortus TSBP were specifically purified by affinity chromatography using recombinant H. contortus cystatin, a potent cysteine proteinase inhibitor. Although only 1-1.5% of total TSBP bound to cystatin-Sepharose, this fraction contained 100% of the cysteine proteinase activity, as determined by gelatin substrate gel analysis. When used to immunise sheep, less than 3microg per dose of this cysteine proteinase fraction was found to confer a substantial and repeatable level of protection against homologous challenge infection, reducing faecal egg counts by 48 and 28% and worm burdens by 44 and 46% over two trials. Host serum immunoglobulin levels and abomasal mast cell and eosinophil numbers were evaluated, although no correlation with protection was observed. Three cathepsin B-like cysteine proteinases present in TSBP (hmcp1, 4 and 6) have been identified previously by cDNA library immunoscreening. The predicted mature forms of these three cysteine proteinases were expressed in bacteria as insoluble, GST-fusion proteins. Following solubilisation in urea/DTT, the protective capacity of a cocktail of recombinant proteins was evaluated in sheep. Although no reduction in faecal egg counts was observed, sheep vaccinated with recombinant cysteine proteinases showed a highly significant 38% reduction (P <0.01) in worm burdens.

Animals↗
PubMed1998

Purification and characterization of two cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.).

Two cysteine proteinase inhibitors, designated Tromsin I and II, were purified from the skin of Atlantic salmon, using three steps of chromatography, including affinity, anion exchange and gelfiltration. The two cysteine proteinase inhibitors were both of the high molecular weight type, with apparent MW 49 and 76 kDa. The isoelectric points (pI) of Tromsin I and II were estimated to be 4.5 and 5.2, respectively. The inhibitors were both stained by the PAS reaction for carbohydrates, and showed a remarkable heat stability. Western blotting revealed that the inhibitors also could be found in significant amounts in serum. Tromsin I and II share many common features with members of the family 3 cystatins, i.e. mammalian kininogen, such as molecular weight, papain inhibition and tissue distribution. Based on N-terminal sequence from Tromsin II however, no homology with known cysteine proteinase inhibitors can be found. This does not exclude that the inhibitors belong to the family 3 cystatins, because the N-terminal amino acid sequences of known cysteine proteinase inhibitors show very low homology.

Amino Acid Sequence↗
PubMed1998

Collision-induced dissociation spectra obtained by Fourier transform ion cyclotron resonance mass spectrometry using a 13C,15N-doubly depleted protein.

Fourier transform ion cyclotron resonance mass spectra of 13C,15N-doubly depleted cystatin A M65L, produced by Escherichia coli grown on 99.9% [12C]glucose and 99.99% [14N]ammonium sulfate, showed salient monoisotopic peaks composed of 12C and 14N. Collision-induced dissociation spectra were obtained by increasing the capillary-skimmer potential for the electrospray ionization and by extending the trapping time in a radio frequency-only hexapole ion guide. Fragment ions in the spectra could be readily assigned to the amino acid sequence, owing to their markedly improved resolution and sensitivity as compared to those with the natural isotopic composition. Detailed analyses of the fragmentation patterns, facilitated by the use of 13C,15N-doubly depleted proteins, enabled the assignment of approximately 180 fragment ions to the sequence, while natural isotopic cystatin A allowed the assignment of approximately 110 fragment ions. Interestingly, no fragmentation was detected between residues 50-61 and 62-67, which are stretches known to be involved in the antiparallel beta-sheet at the center of the protein.

Amino Acid Sequence↗
PubMed2005

Biochemical and biophysical changes induced by fungicide sodium diethyl dithiocarbamate (SDD), in phytocystatin purified from Phaseolus mungo (Urd): a commonly used Indian legume.

Phytocystatins are the plant thiol protease inhibitors involved in several reaction mechanisms of the plant system like regulation of proteolytic activity and storage of proteins. Biochemical and biophysical changes induced by fungicide SDD in phytocystatin purified from Phaseolus mungo have been investigated in terms of mass spectroscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy, at pH 7.0, with varying fungicide concentrations (1-9 mM) and a time of incubation ranging from 2 to 8 h at 37 degrees C, with a fixed cystatin concentration (1.5 mM). Reactive oxygen species responsible for inhibitor damage were also investigated, and thiourea was found to scavenge the free radicals generated by SDD. FTIR analysis indicates a significant conformational transition from alpha-helix to beta-sheet structure; quenching of fluorescence is evident by fluorescence spectroscopy. The activity assay showed a decrease in inhibitory activity, as well as a fragmentation of the inhibitor was observed in electrophoresis. Results obtained implicate that exposure of phytocystatins to SDD involves physicochemical changes in cystatins leading to damage and a decrease in the activity of the inhibitor.

Chemical Phenomena↗
PubMed1997

An extracellular insoluble inhibitor of cysteine proteinases in cell cultures and seeds of carrot.

An 18 kDa extracellular insoluble protein (EIP18) was found previously in amorphous particles suspended in the culture medium and in the interspaces of cell clusters of carrot (Daucus carota L.) callus, as well as in the extracellular spaces of carrot seeds, being located both in the embryo and at the inner edge of the endosperm. We purified EIP18 by washing the amorphous particles with the mixture of Triton X-100, NaCl and ethylenediaminetetraacetic acid (EDTA). We determined several partial amino acid sequences, and then we cloned and sequenced a cDNA for EIP18. EIP18 was found to consist of 133 amino acid residues that included a signal sequence, but it did not contain cysteine, sites for N-linked glycosylation or hydrophobic regions. Since its sequence was found to be homologous to that of inhibitors of cysteine proteinases, namely cystatins, EIP18 was renamed EICC (extracellular insoluble cystatin of carrot). EICC expressed in yeast was also found in an insoluble form in yeast cell walls. EICC prepared from the culture medium of carrot cells inhibited commercial cysteine proteinases and a proteinase extracted from germinating carrot seeds. The expression of the gene for EICC was detected in developing seeds, and the level of its transcript was markedly enhanced upon treatment of somatic embryos with abscisic acid.

Amino Acid Sequence↗
PubMed2000

Sialochemical markers of salivary gland involvement with Sjögren's syndrome secondary to rheumatoid arthritis and primary biliary cirrhosis.

Sjögren's syndrome is an autoimmune condition affecting the lacrimal and salivary glands and can be associated with rheumatoid arthritis and primary biliary cirrhosis. Parotid salivas collected from patients and normal controls were analysed for lactoferrin, IgA and beta2-microglobulin (measured by ELISA), and cystatin (measured by a enzyme inhibition assay). Output data provided less variable means, whilst expressing results as a proportion of the total protein provided greater specificity as markers for Sjögren's syndrome. Levels of specificity for IgA, lactoferrin and beta2-microglobulin were all high (100, 95 and 100%, respectively). Sensitivity levels of these markers (but not cystatin) tended to be similar for Sjögren's syndrome secondary to primary biliary cirrhosis (IgA, 25%; lactoferrin, 63%; and beta2-microglobulin, 50%), compared to Sjögren's syndrome secondary to connective tissue diseases such as rheumatoid arthritis (IgA, 50%; lactoferrin, 86%; and beta2-microglobulin; 38%).

Arthritis, Rheumatoid↗
PubMed1999

An immunohistochemical study of abnormal keratinocyte differentiation in molluscum contagiosum.

To investigate abnormalities in the keratinization process in lesional epidermis of molluscum contagiosum, production of filaggrin, loricrin, Ted-H-1 antigen, involucrin, cystatin A and CD95 ligand (CD95L) was investigated using specific antibodies. Anti-filaggrin monoclonal antibody (MoAb) did not react with keratohyalin granules (KHG), but with the substance around virus particles in the stratum corneum. KHG reacted with anti-loricrin polyclonal antibody (PoAb) and anti-Ted-H-1 MoAb. Anti-involucrin PoAb and anti-cystatin A PoAb reacted with materials in the cytoplasm of the middle stratum spinosum to the stratum granulosum. CD95L was expressed in the cell membrane region of the living cell layers in lesional epidermis. These observations suggest that the keratinization process may be altered in molluscum contagiosum.

Biomarkers↗
PubMed2003

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant.

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.

Animals↗
PubMed2003

Characterization of IgE responses in a rodent model of filariasis and the allergenic potential of filarial antigens using an in vitro assay.

Filarial infections are characterized by high IgE antibody responses. So far, it is not clear whether IgE antibodies are involved in protection, pathology or both. We established a bioassay to detect reactive IgE antibodies in jirds infected with the filaria Acanthocheilonema viteae. Sera of A. viteae-infected jirds were used to sensitize rat basophil leukaemia (RBL) cells and degranulation was stimulated by addition of antigens of A. viteae. Reactive IgE responses were detected from 2 weeks post infection (p.i.) and throughout the A. viteae infection. Male antigen triggered the strongest mediator release, followed by female worms, infective larvae (L3) and microfilariae. Separation of male and female antigen indicated that several antigens of both genders are potent allergens. In particular, one male specific allergen of about 550 kDa induced strongest degranulation of RBL cells. In addition, mediator release stimulated by antigen fractions of about 15 kDa was due to filarial cystatin. In conclusion, we describe a convenient in vitro assay to examine IgE mediated responses in jirds. A sex specific filarial protein with high allergenic potential is identified and cystatin is established as a potent allergen of A. viteae.

Allergens↗
PubMed2001

Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase.

Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 1010-1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pM) than parental scN, due to its increased association rate constant (2.6 +/- 0.09 x 107 M-1sec-1). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.

Base Sequence↗
PubMed2000

Antimicrobial activity of synthetic salivary peptides against voice prosthetic microorganisms.

OBJECTIVES: To investigate whether synthetic salivary antimicrobial peptides have an inhibitory effect on the growth of bacteria and yeasts isolated from used silicone rubber voice prostheses. METHODS: The antimicrobial activities of six synthetic salivary peptides (histatin 5, dhvarl, dhvar4, dhvar5, lactoferrin b 1730 [LFb 17-30], and cystatin S1-15) at concentrations of 2 and 4 mg/mL were determined against different oropharyngeal yeast (four) and bacterial (eight) strains and against a "total microflora" isolated from explanted voice prostheses using agar diffusion tests. The spectrum of susceptible microorganisms was determined qualitatively. RESULTS: Histatin 5 and cystatin S1-15 did not show any antimicrobial activity against the microorganisms involved in this study. Dhvar1 was active against some of the oropharyngeal microorganisms tested, including the yeast strains, but not against Rothia dentocariosa, Staphylococcus aureus, Escherichia coli, and the total microflora Dhvar4 was active against all microorganisms tested, including the total microflora. Dhvar5 lacked activity against E coli and the total microflora LFb 1730 did not inhibit the growth of any of the yeast strains involved and showed only minor activity against some of the bacterial strains. LFb 1730 slightly inhibited the growth of the total microflora from an explanted prosthesis. CONCLUSIONS: The synthetic salivary peptide dhvar4 has a broad antimicrobial activity against all microorganisms that are commonly isolated from explanted voice prostheses, including yeasts. Therewith, it may represent a useful drug, as an alternative for antibiotics and antimycotics employed in various ways to prolong the lifetime of voice prostheses in laryngectomees.

Bacteria↗
PubMed1995

Identification of salivary proteins inhibiting herpes simplex virus 1 replication.

Salivary proteins play an important role in the maintenance of the oral ecology. Previous studies have indicated that human submandibular-sublingual and parotid salivas can selectively suppress the in vitro infectivity of herpes simplex virus 1. The purpose of this study was to identify the salivary components in human submandibular-sublingual saliva that modulate in vitro infectivity. Assessment of the interaction of viral particles with salivary components was accomplished using an in vitro solid-phase assay. These experiments revealed that herpes simplex virus particles selectively interact with the members of the salivary proline-rich protein and cystatin families. Subsequent yield reduction assays demonstrated the ability of proline-rich proteins and salivary cystatins to inhibit the viral replication, with basic proline-rich peptides being more effective. Subsequent assays suggest that basic proline-rich peptides reduced the virus titer by interfering with penetration and/or cellular processing of virus within the target cell. Collectively, these results further suggest that salivary proteins have an important role in the host defense mechanism against recurrent herpesvirus infection.

Animals↗
PubMed2005

Normal sexual development and fertility in testatin knockout mice.

The testatin gene was previously isolated in a screen focused on finding novel signaling molecules involved in sex determination and differentiation. testatin is specifically upregulated in pre-Sertoli cells in early fetal development, immediately after the onset of Sry expression, and was therefore considered a strong candidate for involvement in early testis development. testatin expression is maintained in the adult Sertoli cell, and it can also be found in a small population of germ cells. Testatin shows homology to family 2 cystatins, a group of broadly expressed small secretory proteins that are inhibitors of cysteine proteases in vitro but whose in vivo functions are unclear. testatin belongs to a novel subfamily among the cystatins, comprising genes that all show expression patterns that are strikingly restricted to reproductive tissue. To investigate a possible role of testatin in testis development and male reproduction, we have generated a mouse with targeted disruption of the testatin gene. We found no abnormalities in the testatin knockout mice with regard to fetal and adult testis morphology, cellular ultrastructure, body and testis weight, number of offspring, spermatogenesis, or hormonal parameters (testosterone, luteinizing hormone, and follicle-stimulating hormone).

Amino Acid Sequence↗