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At least 883 records · Page 49Linked to original sources
PubMed1999

CMAP: a novel cystatin-like gene involved in liver metastasis.

A novel metastasis-associated gene was identified with a differential display system in murine carcinoma cells showing a high rate of metastasis to the liver. A human homologue was also identified using a PCR-based strategy. The protein coded by this gene was named cystatin-like metastasis-associated protein (CMAP) and showed 22.1-28.1% homology to human family 2 cystatins. CMAP mRNA was selectively overexpressed in all murine liver metastatic tumors but not in any pulmonary metastatic tumors examined. Transfection of CMAP antisense DNA into highly metastatic liver cells greatly decreased their metastatic potential and CMAP expression, indicating that CMAP is involved in liver metastatic ability after intravasation of malignant cells. The human homologue of CMAP was found to be expressed in various human cancer cell lines established from malignant tumors. Our discovery of this novel liver metastasis-related gene indicates a new approach to the diagnosis and/or prevention of liver metastasis of human cancer.

Amino Acid Sequence↗
PubMed1997

Expression of cysteine protease inhibitors stefin A, stefin B, and cystatin C in human lung tumor tissue.

In human lung tumor tissue specimen (n = 73) concentrations of stefins A and B were found to be increased 2.0-fold (p < 0.01) and 1.3-fold (p < 0.01), respectively, as compared to matched normal tissue. Stefin A and B concentrations were higher in primary tumors than in secondary tumors, i.e. metastases from other organs to the lung (p < 0.01; p < 0.05, respectively). Cystatin C concentrations were rather low and did not differ between tumor and normal tissue. Both concentrations of stefins did not correlate with TNM stages. Stefin A was higher in squamous cell carcinoma than in adenocarcinoma (p < 0.01), while stefin B did not show such a difference. At investigation of a relationship between survival probability of patients with primary tumors it was found that increased stefin B concentrations and total cysteine-protease-inhibitory activities but not stefin A concentrations were positively correlated with survival probability. It is concluded that stefins A and B are major contributors to the cysteine protease inhibitory activity in primary lung tumors. Stefin B proved to be a prognostic factor, especially in squamous cell carcinoma.

Adolescent↗
PubMed1993

A new, sensitive fluorogenic substrate for papain based on the sequence of the cystatin inhibitory site.

We have designed and tested a new papain substrate with intramolecularly quenched fluorescence. It is based on a highly conserved sequence in all members of the cystatin superfamily that participates in the inhibition of cysteine proteinases. This substrate, O-aminobenzoyl (Abz)-QVVAGA-ethylenediamine-2-4-dinitrophenyl (EDDnp) is very sensitive to papain with a second-order rate constant kcat/Km of 3.1 10(7) M-1S-1. It is also efficiently hydrolyzed by cathepsin L, although the kcat/Km for this proteinase is about 60-fold lower than that for papain. This change is due to a decrease in kcat, the Km's are almost identical. This allows clear functional discrimination between these two proteinases, and may lead to the development of selective inhibitors for individual cysteine proteinases. Unlike most commonly used papain substrates, Abz-QVVAGA-EDDnp is not hydrolyzed by trypsin. The papain cleavage site was identified as the A-G bond by N-terminal amino acid sequencing. The use of sensitive and specific substrates such as the one described here will prove invaluable for investigating cysteine proteinase activities in parasite infections. The close interaction between papain or cathepsin L with Abz-QVVAGA-EDDnp is compared to that with cystatin inhibitors, which all include a QxVxG consensus segment in their structure.

Amino Acid Sequence↗
PubMed1988

Diversity of rat brain cysteine proteinase inhibitors: isolation of low-molecular-weight cystatins and a higher-molecular weight T-kininogen-like glycoprotein.

Conditions for extraction of rat brain soluble and particulate cysteine proteinase inhibitors (CPIs) were compared and an optimal one was selected to isolate low- and high-molecular-weight forms active toward papain or brain cathepsins B/L. The different forms were purified by affinity chromatography on alkylated papain, and identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by use of Schiff's reagent, or by immunoblots using antisera to monomer or polymeric forms of human urinary cystatin c, to a human plasma histidine-rich glycoprotein (HRG), or to rat plasma T-kininogen. In particulates containing nuclei (P1) or synaptosomes (P2) the predominant CPI was an 80-kDa glycoprotein cross-reacting to anti-HRG and shown to be a T-kininogen by treatment with TPCK-trypsin, and subsequent bioassay of the released kinins. The levels found in rat brain were approximately 0.5 nmol/g wet weight. The higher-molecular-weight CPI potently inhibited cathepsin L hydrolysis of Leu-enkephalin at the Gly2-Gly3 bond with a Ki 10(-10) M. In contrast the low-molecular-weight CPIs were present in postmicrosomal fractions (S3) and cross-reacted with anti-cystatin c, but not with anti-HRG, anti-lysozyme, anti-beta protein amyloid peptide, or anti-T-kininogen. The low-molecular-weight forms were present at approximately 1-1.5 nmol/g wet weight and resembled "cerebrocystatin" purified previously from rat brain cytosol by M. Kopitar, F. Stern, and N. Marks [1983) Biochem. Biophys. Res. Commun. 112, 1000-1006.).

Animals↗
PubMed2003

Cloning, functional expression in Pichia pastoris, and purification of potato cystatin and multicystatin.

In the tubers and leaves of potato, Solanum tuberosum, cysteine protease inhibitors are thought to play roles in the defence against herbivores and in regulating physiological processes like senescence and cell death. The cDNAs for two such inhibitors, potato multicystatin (PMC) with 8 cystatin domains and potato cystatin (PC) with a single domain, were cloned and expressed in the yeast Pichia pastoris. PC yielded on average 100 mg of purified active protein from 1l of culture supernatant. Purification to homogeneity was done in one step by cation exchange. The apparent equilibrium dissociation constant (K(i)) for papain was 0.1 nM. Cloning of the PMC cDNA was successful despite apparent toxicity for Escherichia coli and a high frequency of recombination events in RecA- strains of E. coli. In yeast, the expression of the cloned full length PMC gene was poor compared to that of the single domain.

Journal Article↗
PubMed2001

Temporal and tissue localization of a cowpea (Vigna unguiculata) cystatin.

A cowpea (Vigna unguiculata L. Walpers cv. Pitiúba) cystatin was analysed to determine its localization during development and germination of cowpea seeds, using western blotting with a specific antiserum. The pattern of immunoreactive proteins changed during development, with the major reactive bands present in dried seeds being mobilized after a 62-h period of imbibition. Immunohistochemical analysis revealed that cowpea cystatin is distributed in both embryonic axes and cotyledons with the highest level being present in the outer cell walls of the adaxial surface of the cotyledons.

Journal Article↗
PubMed1984

Cystatin-like cysteine proteinase inhibitors from human liver.

Cysteine proteinase inhibitor (CPI) forms from human liver were purified from the tissue homogenate by alkaline denaturation of cysteine proteinases with which they are complexed, acetone fractionation, affinity chromatography on S-carboxymethyl-papain-Sepharose and chromatofocusing. The multiple forms of CPI were shown immunologically to be forms of two proteins, referred to as CPI-A (comprising the forms of relatively acidic pI) and CPI-B (comprising the more basic forms). CPI-A and CPI-B are similar in their Mr of about 12400, considerable stability to pH2, pH11 and 80 degrees C, and tight-binding inhibition of papain, several related cysteine proteinases and dipeptidyl peptidase I. Ki values were determined for papain, human cathepsins B, H and L, and dipeptidyl peptidase I. The affinity of CPI-A for cathepsin B was about 10-fold greater than that of CPI-B, whereas CBI-B showed about 100-fold stronger inhibition of dipeptidyl peptidase I. For all the cysteine proteinases the liver inhibitors were somewhat less tight binding than cystatin. The resemblance of both CPI-A and CPI-B in several respects to egg-white cystatin is discussed. CPI-A seems to correspond to the epithelial inhibitor described previously, and CPI-B to the inhibitor from other cell types [Järvinen & Rinne (1982) Biochim. Biophys. Acta 708, 210-217].

Amino Acid Sequence↗
PubMed1996

Preliminary X-ray crystallographic analysis of human salivary cystatin.

Human salivary cystatin, a thiol proteinase inhibitor, has been implicated in potential antimicrobial and antiviral functions of saliva. A variant of human salivary cystatin SN expressed and purified in an Escherichia coli expression system lacking residues 12-16 near the N-terminus (Delta12-16) has been crystallized by the vapor-diffusion technique. The crystals are of the hexagonal space group P622 and have cell constants of a = 85.41, b = 85.41, c = 131.6 A, alpha = beta = 90, gamma = 120 degrees, and contain two molecules of molecular weight 13 500 per asymmetric unit. The crystals diffract up to a resolution of 2.2 A and are suitable for X-ray diffraction analysis.

Journal Article↗
PubMed1989

Cystatins of human placenta.

Two low-molecular protein fractions inhibiting cysteine proteases were isolated from human placenta by alkalization to pH 11, acetone fractionation, affinity chromatography on CM-papain-Sepharose 4B, and Sephadex G-75 gel filtration. The results of polyacrylamide gel electrophoresis, isoelectric focusing indicate that one of there fractions in a dimer of cystatins B, and another is a mixture of cystatins A and B.

Cathepsin H↗
PubMed1988

Interactions between a viral protease and cystatins.

The interactions of two cystatins with a viral cysteine protease were studied using several types of assays. Complex formation between the protease and inhibitor was directly demonstrated using a gel retardation assay. It was also shown that the formation of enzyme inhibitor complexes could occur after first binding either the enzyme or the inhibitor to filter paper, and ultimately decorating the complex with antibody and radio-labelled protein A or by preparing one of the protein ligands with an internal radiolabel. The procedure can be adapted to provide a method for screening expression libraries for protease or inhibitor genes. The inhibition of a cysteine protease by a cystatin was shown not to directly involve binding to the active site thiol of the enzyme, but rather to be the result of a steric block in the active site region which prevents large affinity labels and protein substrates from reaching the active site.

Animals↗
PubMed1988

The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli.

Oryzacystatin, a proteinaceous cysteine proteinase inhibitor (cystatin) in rice, is comprised of 102 residues (Met1-Ala102) (Abe, K., Emori, Y., Kondo, H., Suzuki, K., and Arai, S. (1987) J. Biol. Chem. 262, 16793-16797). We constructed an expression plasmid containing a full length oryzacystatin cDNA at the multi-cloning site of pUC18 and produced a lacZ'-oryzacystatin fusion protein in Escherichia coli. The partially purified expressed protein efficiently inhibits papain activity assayed using N-benzoyl-DL-arginine-2-naphthylamide as a substrate. We also constructed expression plasmids lacking the 5'- and 3'-regions of cDNAs that encode NH2- and COOH-terminally truncated oryzacystatins. An N-truncated oryzacystatin lacking Gly5 and retaining Gln53-Val54-Val55-Ala56-Gly57 inhibited papain as efficiently as the full length oryzacystatin, although both Gly5 and Gln53-Gly57 (oryzacystatin numbering) are conserved among members of most cystatin superfamilies. However, another N-truncated oryzacystatin lacking the NH2-terminal 38 residues was almost completely inactive. On the other hand, a COOH-terminally truncated oryzacystatin lacking the COOH-terminal 11 residues possesses potent papain-inhibitory activity, whereas another COOH-terminally truncated oryzacystatin lacking 35 residues shows much less inhibitory activity, although it retains the two well conserved features Gly5 and Gln53-Gly57. These results indicate that the NH2-terminal 21 residues containing Gly5 and the COOH-terminal 11 residues are not essential, suggesting that a portion of the polypeptide segment containing Gln53-Gly57 is necessary for oryzacystatin to elicite its papain-inhibitory activity efficiently.

Amino Acid Sequence↗
PubMed1990

Refolding of chicken cystatin and human stefin A studied by fast protein liquid chromatography (FPLC).

Denaturant gradient gel-chromatography [Endo, S. et al. (1983) Anal. Biochem. 131, 108-120] has been used to follow renaturation of chicken cystatin and human stefin A. Slow gradient of GuHCl was applied to the size-exclusion Superose 12 column. The column was maintained at 5 degrees C by circulating water from a cryostate. When the denatured protein has been injected it started to refold on the column. By the simplified equation, according to [Hanai, R. et al. (1986) Biophys. Chem. 25, 27-36] approximate kinetics of folding was obtained. No intermediate species in folding could be detected from 6M to 2M GuHCl, only the close-to-denatured state and the close-to-native state.

Animals↗
PubMed2000

Genetic association of a cystatin C gene polymorphism with late-onset Alzheimer disease.

OBJECTIVE: To determine whether the cystatin C gene (CST3) is genetically associated with late-onset Alzheimer disease (AD). DESIGN: A case-control study with 2 independent study populations of patients with AD and age-matched, cognitively normal control subjects. SETTING: The Alzheimer's Disease Research Unit at the University Hospital Hamburg-Eppendorf, Hamburg, Germany, for the initial study (n = 260). For the independent multicenter study (n = 647), an international consortium that included the Massachusetts Alzheimer's Disease Research Center at the Massachusetts General Hospital, Boston; the Scientific Institute for Research and Patient Care, Brescia, Italy; and Alzheimer's research units at the Universities of Basel and Zurich, Switzerland, and Bonn, Goettingen, and Hamburg, Germany. PARTICIPANTS: Five hundred seventeen patients with AD and 390 control subjects. MEASURES: Molecular testing of the KspI polymorphisms in the 5' flanking region and exon 1 of CST3 and the apolipoprotein E (APOE) genotype. Mini-Mental State Examination scores for both patients with AD and control subjects. RESULTS: Homozygosity for haplotype B of CST3 was significantly associated with late-onset AD in both study populations, with an odds ratio of 3.8 (95% confidence interval, 1.56-9.25) in the combined data set; heterozygosity was not associated with an increased risk. The odds ratios for CST3 B/B increased from 2.6 in those younger than 75 years to 8.8 for those aged 75 years and older. The association of CST3 B/B with AD was independent of APOE epsilon4; both genotypes independently reduced disease-free survival. CONCLUSIONS: CST3 is a susceptibility gene for late-onset AD, especially in patients aged 75 years and older. To our knowledge, CST3 B is the first autosomal recessive risk allele in late-onset AD.

Age of Onset↗
PubMed2007

Cystatin C in cerebrospinal fluid and multiple sclerosis.

OBJECTIVE: A recent study using surface-enhanced laser desorption/ionization time-of-flight analysis of cerebrospinal fluid identified a 12.5 kDa truncated isoform of cystatin C (CysC) as a specific biomarker for multiple sclerosis (MS). METHODS: Surface-enhanced laser desorption/ionization time-of-flight analysis of cerebrospinal fluid samples from 43 MS patients and 46 healthy control subjects. RESULTS: Full-length CysC (13.4 kDa) concentration was similar in MS and control samples. The 12.5 kDa CysC protein was produced from full-length CysC by N-terminal cleavage during storage at -20 degrees C. INTERPRETATION: The 12.5 kDa CysC isoform is a storage-related artifact and is not useful as a diagnostic marker for MS.

Amino Acid Sequence↗
PubMed2003

DNA deamination enables direct PCR amplification of the cystatin B (CSTB) gene-associated dodecamer repeat expansion in myoclonus epilepsy type Unverricht-Lundborg.

The Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1) is an autosomal recessive disorder that is caused by the dysfunction of the cystatin B (CSTB) gene product. In the vast majority of affected cases, mRNA transcription is impaired by a biallelic expansion of a dodecamer repeat within the 5'-untranslated region of the respective gene. Since this minisatellite contains exclusively G and C nucleotides, direct PCR analysis of allele expansion is extremely difficult and error prone. To circumvent these problems, we have developed a PCR assay that is based on the deamination of the DNA prior to amplification. We have developed a method based on PCR after DNA deamination of the GC-rich repeat region, which improves the PCR condition to such an extent that we were not only able to reliably amplify expanded alleles of affected individuals (homozygotes and compound heterozygotes), but also the two alleles of full mutation carriers, whose analysis is particularly difficult because of PCR bias and heteroduplex formation between the two alleles. We used promoter- and repeat-specific primer combinations to investigate whether dodecamer repeat expansion concurs with de novo methylation of the CSTB gene promoter in a similar fashion to other repeat expansion syndromes. We confirmed previous evidence obtained by HpaII digestion and Southern blot analysis that both the promoter and the repeat regions are unmethylated, in both healthy and affected individuals. Thus, in contrast to certain trinucleotide repeat expansion-associated diseases, such as fragile X syndrome (FRAXA) and myotonic dystrophy, methylation analyses can not be utilized for indirect diagnostic testing.

Cystatin B↗
PubMed2006

Estimation of glomerular filtration rates after orthotopic liver transplantation: Evaluation of cystatin C-based equations.

Early detection of renal dysfunction in patients after orthotopic liver transplantation is important. Creatinine-based equations to estimate glomerular filtration rate (GFR) were found to be less accurate in liver transplant recipients than in their original populations. Since cystatin C (CysC) is independent from muscle mass and hepatic biosynthesis, we evaluated the diagnostic accuracy of 3 CysC-based equations (Larson, Hoek, and Filler formulae) that are based on the same CysC method as that of our center in comparison to the abbreviated creatinine-based modification of diet in renal disease (MDRD) formula in 59 liver transplant recipients. "True GFR" was measured by 99mTc-diethylene triamine pentaacetic acid ((99m)Tc-DTPA) clearance. Neither correlation with the GFR (correlation coefficients: 0.594-0.640) nor precision (root mean square error: 15.7-18.17 mL/min/1.73 m(2)) differed significantly between the tested formulae. The biases of the Hoek and Larsson formulae were significantly smaller than those of the MDRD and Filler equations (-0.1 and -2.3 vs. 10.1 and 7.9 mL/min/1.73 m(2), respectively; P </= 0.0023). Mean estimates of MDRD (61.9 +/- 21.4 mL/min/1.73 m(2)) and Filler (61.2 +/- 22.1 mL/min/1.73 m(2)) differed significantly from the measured GFR (52.3 +/- 17.5 mL/min/1.73 m(2); P < 0.005), whereas Larsson and Hoek did not (49.5 +/- 20.2 and 51.4 +/- 17.9 mL/min/1.73 m(2), respectively). Accuracy within 30% and 50% of the true GFR was best for the Hoek (76.3% and 93.2%) formula, albeit not significantly different from MDRD (64.4% and 83.1%). Taken together, these data show the best overall performance for GFR estimates derived from the Hoek equation with respect to bias, precision, and accuracy.

Adult↗
PubMed2005

VAP1, with cystatin C motif, an oocyte protein encoded by a novel ovarian-specific gene during oogenesis in the common brushtail possum (Trichosurus vulpecula).

In the brushtail possum oocyte, vesicles accumulate in a polarized fashion at the vegetal pole and cytoplasm rich in mitochondria and containing the germinal vesicle comprise the animal pole. During cleavage to early blastocyst stages, animal pole cytoplasm locates to the cells of the embryonic hemisphere (pluriblast) and vegetal pole vesicular cytoplasm to cells of the abembryonic hemisphere (trophoblast). Previously identified 16 amino acid residues, associated with the vesicle-rich cytoplasm were used for molecular cloning and characterization of a vesicle associated protein, VAP1. The degenerate primer was used in a 3'RACE for vap1 gene cloning. The cDNA encoding VAP1 was 516 bp in length with no significant homologies and coded for 172 amino acid residues for the mature protein. The N-terminal domain of VAP1 showed a structural homology to the cysteine protease inhibitor, Cystatin. Gene expression studies during oogenesis revealed that vap1 had an ovary-specific, possibly oocyte-specific expression, which occurs during follicle formation and growth and in adult ovaries. Recombinant VAP1 fusion protein generated polyclonal antibodies in the mouse and in the brushtail possum.

Aging↗
PubMed2006

Serum cystatin C is a suitable marker for routine monitoring of renal function in pediatric cancer patients, especially of very young age.

BACKGROUND: Monitoring renal function is crucial in children undergoing chemotherapy. To date, a combination of routine serum creatinine (SCR) monitoring with occasional determination of creatinine clearance ratio (CCR) is widely used as clinical standard for this purpose. Both methods have their limitations regarding diagnostic value (SCR) or practicability (CCR), especially in young children. Diagnostic alternatives, such as glomerular filtration rate (GFR) estimation formulas have not been proved to be superior. The aim of the study was to evaluate whether serum cystatin C (CysC) may have a diagnostic impact on pediatric patients. PROCEDURE: CysC, SCR, several GFR estimation formulas (Counahan-Barratt, Ghazali-Barratt, Schwartz, Shull, Traub), and CCR were studied in 80 pediatric cancer patients (age range: 0.17-17.9 years) during their chemotherapy. Special attention was given to children under the age of 3 in whom accurate urine collection for CCR is difficult. RESULTS: All parameters correlated similarly well with CCR. Total accuracy was 66% and 67% for CysC and SCR, respectively. In very young children (<3 years), correlation with CCR was for CysC r = -0.74 with an area under the curve (AUC) of 0.646, and for SCR r = -0.27 with AUC = 0.594. Total accuracy was 60% for CysC, 50% for SCR. CONCLUSIONS: CysC represents a suitable marker for monitoring renal function in pediatric cancer patients. In young children (<3 years), CysC may have a better diagnostic value than SCR. Future studies should show if CysC can improve renal monitoring by replacing SCR, especially in very young children.

Adolescent↗