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At least 685 records · Page 38Linked to original sources
PubMed1996

Soyacystatin, a novel cysteine proteinase inhibitor in soybean, is distinct in protein structure and gene organization from other cystatins of animal and plant origin.

Cystatins, cysteine proteinase inhibitors, deserve note because of their regulatory and protective functions in plant tissues. We isolated both genomic DNA and cDNA clones from soybean that encode a cystatin consisting of 245 amino acid residues (soyacystatin). It is, while basically similar in sequence to known cystatins that are generally in the range of 12-15 kDa, characterized by having extremely large extension sequences in both its amino and carboxyl termini. The genomic DNA encoding soyacystatin is also unique in that it consists of four exons with three introns in its coding regions. The mRNA for soyacystatin is distinctly expressed in soybean seeds 2 weeks after flowering. Soyacystatin purified from mature soybean seeds had a molecular mass of about 26 kDa on SDS/PAGE which suggests that it contains the extension sequences. Papain-inhibition experiments demonstrate that this endogenous soyacystatin has almost the same inhibitory activity as that of its deletion mutant (102 amino acid residues) recombinantly produced by truncation of the amino and carboxyl terminal extensions, indicating that the occurrence of the extensions does not affect the cystatin activity. Immunohistochemical experiments reveal that soyacystatin is expressed nearly uniformly in the cotyledons. These results also suggest the possible occurrence of a cysteine proteinase as the target enzyme of soyacystatin.

Amino Acid Sequence↗
PubMedDate not supplied

Regulation of cathepsin B and L expression in vitro in gastric cancer tissues by egg cystatin.

BACKGROUND: Degradation of the basement membrane and surrounding extracellular matrix is likely to represent a key step in cancer invasion and metastasis. The purpose of this study was to determine whether gastric cancer tissues demonstrate higher cysteine proteases activities: cathepsins B and L during cancer progression in compression with non-cancerous tissues. METHODS: We measured the expression of both cathepsins B and L in 30 patients with gastric cancer tissues and non-cancerous tissues activities by a fluorescence assay and immunohistochemical staining. We attempted to regulate cathepsin B and L expression using egg white cystatin. RESULTS: The activities of cathepsins B and L were significantly higher in cancerous than in non-cancerous tissues (P </= 0.0001). Activities of these enzymes decreased 18-fold after treatment of tissue homogenates with 5 nM of purified egg white cystatin. Immunohistochemical staining was significant and positive expression of low molecular weight cystatin was observed on the tumor cell surface in gastric carcinoma. It was found that non-cancerous tissues (control) were not stained with anti-egg white antibody. CONCLUSION: Our experiment demonstrated that the cysteine peptidases cathepsins B and L may be useful for the early detection of gastric cancer. The results suggest that addition of egg white cystatin reduces the activities of cathepsins B and L to that of non-cancerous values.

Adult↗
PubMed1998

Cathepsin B, alpha2-macroglobulin and cystatin levels in gingival crevicular fluid from chronic periodontitis patients.

Gingival crevicular fluid (GCF) was collected from 16 molar and premolar sites in each of 20 chronic periodontitis patients before and after periodontal therapy using filter paper strips. These were eluted individually into buffer for determination of cathepsin B and its endogenous inhibitors, alpha2-macroglobulin and cystatin. Cathepsin B activity was assayed with a fluorogenic peptide substrate, alpha2-macroglobulin by enzyme-linked immunosorbent assay and cystatin activity by inhibition of papain. Total amounts of enzyme and inhibitor per GCF sample decreased after treatment and correlated positively with pocket depth and gingival, bleeding and plaque indices. These comparisons were nearly always statistically significant for pooled site data and sometimes so for mean patient values. The amounts of alpha2-macroglobulin and cystatin were greater than those of cathepsin B and, surprisingly, enzyme and inhibitor levels correlated positively with each other. Experiments with purified reagents, however, demonstrated that the cathepsin B: alpha2-macroglobulin complex was still active against the low molecular weight substrate and that cystatin levels in GCF are probably insufficient to inhibit the enzyme substantially These factors may explain why GCF cathepsin B activity reflects the clinical status of periodontal lesions and has been identified in another study as a promising indicator of disease progression.

Adult↗
PubMed1993

Protein, albumin and cystatin concentrations in saliva of healthy subjects and of patients with gingivitis or periodontitis.

Salivary protein, albumin and cystatin concentrations were investigated in subjects with a healthy periodontium and in patients with gingivitis or periodontitis. Protein and albumin concentrations in saliva of individuals with gingivitis or periodontitis were significantly increased compared with healthy subjects. Salivary protein and albumin appeared to be positively correlated in all the groups, which suggests that the increase in salivary protein concentration in subjects with gingivitis or periodontitis is caused by leakage of plasma proteins. Cystatin concentrations in saliva of subjects with periodontitis were significantly increased when compared with the healthy group and the gingivitis group (p < 0.01). In the gingivitis and periodontitis group, salivary cystatin was only weakly correlated with albumin concentrations, which suggests that the increased salivary cystatin activity found in subjects with gingivitis and periodontitis is derived from sources other than plasma.

Adolescent↗
PubMed1998

Cystatin A expression reduces bile salt-induced apoptosis in a rat hepatoma cell line.

We have previously demonstrated abrogation of bile salt-induced apoptosis by cathepsin B inhibitors. However, caspases have been strongly implicated in apoptosis, and the mechanistic interface between caspase and cathepsin B activation is unclear. Thus our aims were to determine the mechanistic relationship between caspases and cathepsin B in bile salt-induced apoptosis in a rat hepatoma cell line. Expression of cystatin A was used to inhibit cathepsin B, whereas Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used to inhibit caspases. Cystatin A expression prevented cathepsin B activation and apoptosis during treatment with glycochenodeoxycholate (GCDC), a toxic bile salt. Caspase N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) hydrolytic activity increased in both wild-type and cystatin A-transfected cells treated with GCDC, demonstrating caspase activation despite inhibition of cathepsin B. In contrast, Z-VAD-FMK blocked both DEVD-AMC hydrolytic activity and cathepsin B activity during GCDC treatment. Our data demonstrate that 1) bile salt-induced apoptosis can be inhibited by the cystatin A transgene and 2) caspase and cathepsin B activation are linked mechanistically with cathepsin B downstream of caspases.

Amino Acid Chloromethyl Ketones↗
PubMed1993

Localization of the human cystatin D gene (CST5) to chromosome 20p11.21 by in situ hybridization.

The gene coding for cystatin D (CST5), a cysteine proteinase inhibitor, was mapped by fluorescent in situ hybridization to human chromosome 20p11.21. This assignment, together with previous data on mapping of members of the cystatin gene family, indicates that cystatin family II genes are all clustered on the short arm of chromosome 20, whereas cystatin family I and III genes are located on the long arm of chromosome 3.

Chromosome Banding↗
PubMedDate not supplied

Intracellular delivery of cysteine protease inhibitor cystatin by polymeric nanoparticles.

Two polymers chitosan and poly(lactide-co-glycolide) copolymer (PLGA) were investigated to develop nanoparticles (NPs) for delivery of protein drug substance into tumour cells. Cystatin was selected as a model protein drug due to its high potential to inhibit cysteine proteases, known to trigger the invasive process. Ionotropic gelation of chitosan with tripolyposphate and precipitation of PLGA polymer from a double emulsion system by a solvent diffusion process were used to produce 250-300 nm in diameter NPs. The cellular uptake of NPs was tested on a transformed human breast epithelial cell line, MCF-10A neoT, characterized by an increased expression of cysteine proteases and highly invasive cell phenotype. The influence of NPs on cell viability was evaluated by MTT test showing IC50 400 microg/ml for PLGA and 5 mg/ml for chitosan NPs. As determined by fluorescence microscopy chitosan NPs did not enter the cells during 1-hour incubation whereas the same amount of PLGA NPs were in the cells already after 5 min of incubation. Cystatin delivered into MCF-10A neoT cells by PLGA NPs effectively inhibited intracellular proteolytic activity of cathepsin B, as detected by specific fluorogenic substrate Z-Arg2 cresyl violet. On the contrary, free cystatin in solution did not internalise into the cells and inhibit cathepsin B. To conclude, PLGA NPs with cystatin but not chitosan NPs were targeted through endocytosis to the lysosomal compartments that are rich of proteases enzymes. Our results suggest new strategy to inactivate intracellular tumour-associated proteases, and consequently the invasion behaviour of tumour cells, which could contribute to cancer therapy.

Cell Line, Tumor↗
PubMed1991

The effects of autonomic drugs on the concentration of kallikrein-like proteases and cysteine-proteinase inhibitor (cystatin) in rat whole saliva.

The influence of isoproterenol, phenylephrine, propranolol, and reserpine on the salivary concentration of kallikrein-like proteases and cysteine-proteinase inhibitor (cystatin) was investigated. The protease activities in saliva from treated rats were studied by means of five different chromogenic substrates. In the isoproterenol- and phenylephrine-treated groups, a significant decrease in protease activity was found, compared with the control group. The protease activity of saliva was found to be elevated by about 25-50% after chronic administration of reserpine (0.5 mg/kg). Specific polyclonal antibodies to rat glandular kallikrein and cystatin were utilized to determine the salivary concentrations of these proteins. Results from the use of anti-kallikrein antibodies in Western blot analysis and crossed immuno-electrophoresis indicated that differences observed in the kallikrein-like protease levels of saliva from treated animals were due to altered immunoreactive protein levels. The salivary concentrations of kallikrein and cystatin were measured by direct radio-immunoassays with specific antibodies. The concentration of cystatin in the saliva of normal animals or animals treated with reserpine or propranolol was very low, but was increased about 100-fold in phenylephrine-treated animals and more than 5000-fold in isoproterenol-treated animals. Western blot analysis with antibodies to submandibular gland mucin, glutamine/glutamic-acid-rich protein (GRP), and proline-rich proteins (PRP) were also utilized to compare the effects of autonomic drugs on these salivary proteins. The salivary mucin showed an increase in reactivity and increased mobility in saliva from both isoproterenol- and phenylephrine-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

The N-terminal part of recombinant human tear lipocalin/von Ebner's gland protein confers cysteine proteinase inhibition depending on the presence of the entire cystatin-like sequence motifs.

Human Tear Lipocalin/von Ebner's gland protein (TL) is a member of the lipocalin superfamily. The protein is secreted by a number of serous glands and tissues and is overproduced under conditions of stress, infection and inflammation. In addition to its typical affinity for lipophilic ligands it was recently found to be able to inhibit cysteine proteinases [van't Hof et al., J. Biol. Chem. 272 (1997), 1837-1841], probably due to the presence of amino acid motifs resembling the papain binding domains of family 2 cystatins. In this work we have used a recombinant protein to confirm the results obtained with native TL. The inhibitory activity of the recombinant protein against papain was dependent on the ratio of papain and TL. At higher papain concentrations, the N-terminal sequence of TL was cleaved off by the protease, indicating that it can act in an inhibitor- or a substrate-like mode. This behaviour resembles that observed with certain chicken cystatin mutants. Using a recombinant TL mutant we found that the two Leu residues (Leu4-Leu5) contained within the first cystatin-like motif are absolutely essential for the inhibitory activity. These results were supported by experiments using a recombinant form of the corresponding pig von Ebner's gland protein (VEGp). This protein, which does not possess a fully conserved first cystatin-like motif, is unable to inhibit papain.

Amino Acid Motifs↗
PubMed2000

Cystatin capture enzyme-linked immunosorbent assay for serodiagnosis of human schistosomiasis mansoni.

To diagnose human schistosomiasis mansoni, the current study tested an ELISA system that employs indirectly, through a chicken cystatin capture agent, the S. mansoni cysteine proteinases as antigens to detect IgG anticysteine proteinases. The study examined 40 patients with S. mansoni, 20 patients with parasitic infections other than schistosomiasis mansoni, and 20 apparently healthy individuals with no parasitic infections. The results showed that the cystatin capture ELISA gave a high sensitivity of 100% even in patients with low egg count. A similar high sensitivity was also recorded with conventional ELISA using SWAP alone without presensitization of ELISA plate with cystatin. Moreover, the capture ELISA showed no cross reactions with patients with other parasitic infections or normal individuals giving a 100% specificity, while the conventional ELISA using SWAP alone gave a lower specificity of 85%. The cystatin capture ELISA was found to be an easy, highly sensitive and specific serological method for the diagnosis of human schistosomiasis mansoni without the need of purified proteinases antigens.

Adult↗
PubMed1990

Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II.

Oryzacystatin (oryzacystatin-I) is a proteinaceous cysteine proteinase inhibitor (cystatin) in rice seeds and is the first well defined cystatin of plant origin. In this study we isolated cDNA clones for a new type of cystatin (oryzacystatin-II) in rice seeds by screening with the oryzacystatin-I cDNA probe. The newly isolated cDNA clone encodes 107 amino acid residues whose sequence is similar to that of oryzacystatin-I (approximately 55% of identity). These oryzacystatins have no disulfide bonds, and so could be classified as family-I cystatins; however, the amino acid sequences resemble those of family-II members more than family-I members. Oryzacystatin-I and -II are remarkably distinct in two respects: 1) their specificities against cysteine proteinases; and 2) the expression patterns of their mRNAs in the ripening stage of rice seeds. Oryzacystatin-I inhibits papain more effectively (Ki 3.0 x 10(-8) M) than cathepsin H (Ki 0.79 x 10(-6) M), while oryzacystatin-II inhibits cathepsin H (Ki 1.0 x 10(-8) M) better than papain (Ki 0.83 x 10(-6) M). The mRNA for oryzacystatin-I is expressed maximally at 2 weeks after flowering and is not detected in mature seeds, whereas the mRNA for oryzacystatin-II is constantly expressed throughout the maturation stages and is clearly detected in mature seeds. Western blotting analysis using antibody to oryzacystatin-II showed that, as is the case with oryzacystatin-I, oryzacystatin-II occurs in mature rice seeds. Thus, these two oryzacystatin species are believed to be involved in the regulation of proteolysis caused by different proteinases.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). Homology with animal cystatins and transient expression in the ripening process of rice seeds.

A cDNA clone for a cysteine proteinase inhibitor of rice (oryzacystatin) was isolated from a lambda gt10 cDNA library of rice immature seeds by screening with synthesized oligonucleotide probes based on partial amino acid sequences of oryzacystatin. A nearly full-length cDNA clone was obtained which encoded 102-amino acid residues. The amino acid sequence of oryzacystatin deduced from the cDNA sequence was significantly homologous to those of mammalian cystatins, especially family 2 cystatins. Oryzacystatin contained the sequence Gln-Val-Val-Ala-Gly conserved among most members of the cystatin superfamily. The gene for oryzacystatin was transcribed into a single mRNA species of about 700 nucleotides. The content of mRNA reached its highest level 2 weeks after flowering and then gradually decreased to undetectable levels at 10 weeks. This feature of transient expression is coordinate with that of glutelin (a major storage protein), although the expression of oryzacystatin precedes that of glutelin by about 1 week.

Amino Acid Sequence↗
PubMed1985

Genealogy of mammalian cysteine proteinase inhibitors. Common evolutionary origin of stefins, cystatins and kininogens.

A model for the evolution of mammalian cysteine proteinase inhibitors has been constructed on the basis of sequence homology. This model suggests that the diversity of cysteine proteinase inhibitors has evolved from two ancestral units forming the building blocks of stefin and cystatin. Gene triplication of the archetypal inhibitor generated the kininogen heavy chain which contains three cystatin-like copies. Hence, the superfamily of mammalian cysteine proteinase inhibitors is constituted by at least three distinct families, with stefin, cystatin and kininogen as their prototypes.

Amino Acid Sequence↗
PubMedDate not supplied

Cathepsins B and L and their inhibitors stefin B and cystatin C as markers for malignant progression of benign meningiomas.

Meningiomas are, in general, slowly growing benign tumors attached to the dura mater and composed of neoplastic meningothelial (arachnoidal) cells. They have a wide range of histopathological appearances and are classified, according to the aggressiveness of their growth and the risk of recurrence, as WHO grade I (benign) meningiomas, WHO grade II (atypical) meningiomas and WHO grade III anaplastic (malignant) meningiomas. As invasion of normal tissue may occur in all grades, independent biological markers are needed to identify the more aggressive and recurrent meningiomas. The lysosomal cysteine proteinases, cathepsins B and L, have been associated with tumor invasiveness and the aim of this study was therefore to evaluate them, together with their endogenous inhibitors stefin B and cystatin C, as potential markers for the aggressiveness of meningiomas. The expression of cathepsins B and L and their inhibitors stefin B and cystatin C in 21 benign (grade I) and 9 atypical (grade II) meningiomas has been compared by immunohistochemical staining, QRT-PCR and Northern blot analysis. The protein levels of cathepsins B (p=0.050) and L (p=0.019) were found to be significantly higher in atypical than in benign meningiomas. In contrast, their mRNA levels did not differ, indicating that the synthesis of cathepsins was accelerated at the translational level. Protein and mRNA levels of stefin B (p= 0.007), but not cystatin C, were significantly lower in atypical compared with benign meningiomas. The expression of cathepsins and inhibitors was not different between central and peripheral meningioma tissue or between histological subtypes of meningiomas, with the exception of cathepsin L, the level of which was significantly lower in transitional meningiomas. We conclude that higher protein levels of cathepsins B and L and lower mRNA levels of stefin B are potential diagnostic markers for invasive and aggressive behavior of meningiomas. The diagnostic and prognostic value for relapse of meningioma needs to be confirmed in a larger population of patients.

Adult↗
PubMed1989

Demonstration of proteinase inhibitors cystatin A, B and C in breast cancer and in cell lines MCF-7 and ZR-75-1.

Polyclonal rabbit antibodies to endogenous natural inhibitors of cysteine proteinases (cystatins) were used for their immunohistochemical demonstration in samples of breast cancers and in two breast cancer derived cell lines (MCF-7 and ZR-75-1). The influence of estrogen stimulation on the expression of these inhibitors was also studied. The results showed that all cystatins tested can be found equally inside carcinomatous cells or in surrounding connective tissue, as well as in both cell lines examined. Further, the expression of inhibitors can be regulated by estrogen, and a certain role of cystatins in the regulation of the mitotic activity and differentiation of mammary cells can be supposed.

Breast Neoplasms↗
PubMed1988

Cystatin domains in alpha-2-HS-glycoprotein and fetuin.

We have found that chain A of alpha-2-HS-glycoprotein contains two cystatin domains that show closest similarity to those of kininogen. Most likely, the two proteins diverged after the primary duplication of a single cystatin domain as the two cystatin domains of alpha-2-HS-glycoprotein are more similar, especially in disulfide bonding, to the corresponding domains of kininogen than to each other. We also propose that the carboxyl-terminal (non-cystatin) parts of kininogen and alpha-2-HS-glycoprotein contain homologous segments. We suggest that alpha-2-HS-glycoprotein may act as an inhibitor of the cysteine proteinases responsible for bone resorption. We have also found that fetuin is closely related to alpha-2-HS-glycoprotein.

Amino Acid Sequence↗
PubMed2001

Preliminary evaluation of a particle-enhanced turbidimetric immunoassay (PETIA) for the determination of serum cystatin C-like immunoreactivity in dogs.

Serum cystatin C often is used in humans as a rapid and more sensitive marker than serum creatinine for glomerular filtration rate. The purpose of the present study was to evaluate whether cystatin C-like immunoreactivity (CLI) could be measured reliably in canine serum and to investigate whether dogs with clinical renal insufficiency had higher CLI levels than did clinically healthy dogs and dogs with nonrenal diseases. A commercially available particle-enhanced turbidimetric immunoassay (PETIA) for human serum cystatin C was used to measure canine serum CLI in a linear and proportional manner, with a mean recovery of 104% +/- 7.5% and coefficients of variation of 1.7 to 9.6%. The assay was then applied to serum samples from 17 clinically healthy dogs, 12 dogs with nonrenal diseases, and 8 dogs with renal insufficiency. Serum CLI was significantly higher in dogs with renal insufficiency (median serum CLI = 5.01 mg/L) than in clinically healthy dogs and dogs with nonrenal diseases (median serum CLI = 1.06 mg/L and 1.62 mg/L, respectively). Thus, canine serum CLI could be reliably measured using a commercially available PETIA designed for human serum cystatin C, and dogs with clinical renal insufficiency had, as expected, significantly higher serum CLI levels.

Journal Article↗
PubMed1988

Immunocytochemical localization of cathepsins B, H, and their endogenous inhibitor, cystatin beta, in islet endocrine cells of rat pancreas.

To determine the characteristics of lysosomes in rat islet endocrine cells, we examined the precise localization of cathepsins B, H, and L and their specific inhibitors, cystatins alpha and beta, using immunocytochemical techniques. By use of serial semi-thin sections, we detected immunoreactivity for cathepsin B in insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-positive (PP) cells. Strong immunoreactivity for cathepsin H was seen in A-cells and weak immunoreactivity in PP cells, but none in others. Immunodeposits for cystatin beta were demonstrated in B-cells. Brief dipping of thin sections in 1% sodium methoxide before the following immunocytochemical reaction enhanced specific deposits of immunogold particles on the target organelles. Use of a double-immunostaining technique showed co-localization of insulin with cystatin beta in many secretory granules. This suggests that cystatin beta may regulate converting enzymes participating in the maturation process of insulin. By use of an immunogold technique, heterogeneous localization of cathepsins B and H in lysosomes was also found among islet cells at the light microscopic level. This may be due to the difference in peptides degraded in lysosomes among the cells.

Animals↗