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PubMed2004

Measurement of urinary cystatin C by particle-enhanced nephelometric immunoassay: precision, interferences, stability and reference range.

BACKGROUND: Urinary excretion of the low molecular weight protein cystatin C is a marker of renal disorders and a good predictor of the severity of acute tubular necrosis. We evaluated the measurement of urinary cystatin C and determined its reference range. METHODS: Measurement of urinary cystatin C by particle-enhanced nephelometric immunoassay (PENIA) in 102 patients with various renal disorders and 133 healthy controls. We assessed the influence of pH and temperature, interferences on urinary cystatin C measurement, as well as cystatin C adsorption to plastic. RESULTS: The upper reference value for urinary cystatin C was 0.28 mg/L, independent of age and gender. Accuracy and linearity (r(2)=0.996) were excellent. Intra- and inter-assay precision were < or =4.8% and < or =5.2%, respectively. Albumin (< or =160 g/L), bilirubin (< or =500 micromol/L) and haemoglobin (< or =210 micromol/L) did not show interferences. Urinary cystatin C was stable, at urine pH> or =5, at -20 degrees C and 4 degrees C for 7 days, and at 20 degrees C for 48 h. Freezing and thawing did not influence urinary cystatin C concentration. There was no adsorption of cystatin C to plastic. CONCLUSION: Urinary cystatin C measurement by PENIA is precise. High stability and no interference add to the practicability of urinary cystatin C as a routine biochemical test.

Adult↗
PubMed1999

Cystatin C in paediatric nephrology. Present situation and prospects.

Cystatin C is a small basic protein with a MW of 13,359 Daltons, consisting of a non-glycosylated polypeptide chain containing 120 amino-acid residues. Cystatin C is produced in all the nucleated cells of the human body and its output rate is constant. The kidney is the main catabolic site of cystatin C, since the protein, by virtue of its low MW and its positive charge at normal pH, is freely filtered by the glomerulus and almost completely reabsorbed, catabolised and broken down in the cells of the proximal convoluted tubule. It is practically entirely filtered via the glomerular membrane, without any significant tubular secretion. The constant production rate of cystatin C in all the tissues, its elimination via the glomerular filter and its non-dependence on many extrinsic factors, including sex, age, diet, inflammation, are potentially ideal conditions for an endogenous biochemical marker of glomerular filtration. A recent method for determining cystatin C, is based on an immune reaction, could increase its clinical application. Not many studies have been conducted to date on cystatin C in children. The cystatin C concentration was higher during the first few days of life (range: 1.64-2.59 mg/L) with a rapid reduction during the first 4 months. Beyond the first year of life, cystatin C concentration became constant, with a reference range of 0.7-1.38 mg/L. On the basis of the data currently available, neonatal serum cystatin C would appear to derive from the newborn itself. In fact no correlations were found between maternal and neonatal serum cystatin C values. Cystatin C determination appears to be at least equivalent to serum creatinine measurement for the assessment of glomerular filtration rate in children. Further extended studies are needed to investigate these aspects more thoroughly in neonates.

Adolescent↗
PubMed1999

Evaluation of plasma cystatin C as a marker for glomerular filtration rate in patients with type 2 diabetes.

AIM: To evaluate plasma cystatin C as a marker of the glomerular filtration rate in patients with type 2 diabetes and their age and sex-matched controls. MATERIALS AND METHODS: Forty-seven patients with one decade of type 2 diabetes and 51 non-diabetic control subjects were studied. Plasma cystatin C was measured by particle-enhanced turbidimetric immunoassay in a new application for the Hitachi 704 analyzer. For comparison, plasma creatinine and creatinine clearance were measured. The plasma clearance of 51Cr-EDTA by the single injection method was utilized as reference. RESULTS: In patients with type 2 diabetes the correlation coefficient between plasma cystatin C and the plasma clearance of 51Cr-EDTA was 0.774 (Spearman's coefficient) and that between plasma creatinine and the plasma clearance of 51Cr-EDTA was 0.556 (p = 0.001 for the difference). The correlation between creatinine clearance and the plasma clearance of 51Cr-EDTA was 0.411. In receiver operating characteristic (ROC) curve analysis the diagnostic accuracy of plasma cystatin C was significantly better than that of plasma creatinine (p = 0.047) or creatinine clearance (p = 0.001). The best diagnostic efficiency (98%) for cystatin C was obtained when the cut-off limit was set at 1.32 mg/l. In the control group the correlation coefficients were: between cystatin C and the plasma clearance of 51Cr-EDTA 0.627, between creatinine and the plasma clearance of 51Cr-EDTA 0.466 and between creatinine clearance and the plasma clearance of 51Cr-EDTA 0.416. The area under the ROC plot curve of cystatin C was also greatest in the control group, but the diagnostic accuracy of cystatin C was marginally better than that of either plasma creatinine (p = 0.05) or creatinine clearance (p = 0.08). Among the control subjects various non-renal causes may have interfered with cystatin C concentrations reducing the correlations. CONCLUSIONS: Cystatin C measurement is a more sensitive and specific test for GFR in patients with type 2 diabetes than plasma creatinine or its clearance, when GFR is normal or only slightly reduced. If an elevated cystatin C concentration is found, non-renal factors have to be excluded. The turbidimetric application described here can easily be applied for most clinical chemistry analyzers and is therefore useful in daily clinical practice.

Aged↗
PubMed2000

Cathepsin B and its endogenous inhibitor cystatin C in rheumatoid arthritis synovium.

OBJECTIVE: To compare the expression of cathepsin B and its endogenous inhibitor cystatin C in synovial tissue of patients with rheumatoid arthritis (RA) and to determine the cell type expressing cystatin C. METHODS: The expression of cathepsin B and cystatin C was studied by immunohistochemistry in synovial tissue of 10 patients with RA and compared to healthy controls. Applying double labeling methods, the expression of cathepsin B was compared to that of cystatin C. To determine the cell type expressing cystatin C, double labeling with anti-CD68 (PG-M1) was performed. RESULTS: Both cystatin C and cathepsin B were strongly expressed in synoviocytes of patients with RA. Furthermore, fibroproliferative tissue at the site of cartilage and bone destruction contained fibroblast-like and macrophage-like cells positive for cystatin C and cathepsin B, whereas normal synovial tissue exhibited only limited expression of these molecules. Osteoclasts revealed positive staining for CD68 and cystatin C, but not for cathepsin B. CONCLUSION: Cystatin C is a product of both macrophage-like and fibroblast-like synoviocytes. The strong expression of both the matrix degrading cysteine proteinase cathepsin B and the cysteine proteinase inhibitor cystatin C in rheumatoid synovium, particularly at the sites of bone and cartilage erosion, suggests that cystatin C--although increased--is not sufficient to prevent matrix degradation by cathepsin B.

Adult↗
PubMed2002

Cystatin C of pleural effusion as a novel diagnostic aid in pleural diseases of different aetiologies.

There has been considerable recent interest in the potential use of serum cystatin C as a diagnostic tool. Here we examined the hypothesis that the cystatin C level in the pleural effusion can differ from the corresponding serum level. We evacuated pleural effusion fluids from 47 patients by thoracentesis. Cystatin C, beta(2)-microglobulin, inorganic phosphate, creatinine and total protein were quantified in both pleural effusion fluids and corresponding sera. We determined cystatin C levels in pleural effusions and calculated the ratio of cystatin C levels in serum and effusion, to discriminate between effusions caused by severe renal impairment and other types of effusion. Extremely high concentrations of cystatin C in serum/effusion pairs were only measured in patients with renal failure (6.0 +/- 0.8/6.0 +/- 0.8 mg/l, means +/- S.D., n=11). A clearly defined region was found to correspond to pleural effusion caused by renal failure (r=0.954). The quantification of cystatin C in the effusion was justified by the discovery that there were some patients with a high serum cystatin C level but a low effusion concentration, or a low serum cystatin C but a high effusion concentration, indicating causes other than renal failure. In conclusion, the pilot data indicate a relationship between the cystatin C concentration in pleural fluid and the underlying disease. Thus cystatin C levels in pleural effusion and serum may be a valuable criterion for the differential diagnosis of pleural diseases of different aetiologies.

Aged↗
PubMedDate not supplied

[Determination of blood cystatin C in pregnant women during labor and in their newborns].

INTRODUCTION: Human cystatin C is a basic low molecular mass protein (M(r) = 13,359) freely filtered by the glomerulus and almost completely reabsorbed and catabolized by the proximal tubular cells. In this study, we determined maternal and neonatal serum cystatin C levels both in a group of healthy pregnant women and in their newborns over the first five days of life. PATIENTS AND METHODS: Fifty healthy pregnant women, aged from 19 to 40 years, were selected. Newborns (31 males, 19 females) demonstrated the 1-min Apgar score ranging between 8 and 10, and the 5-min between 9 and 10. Their gestational age (GA) ranged between 37 and 43 weeks. Cystatin C was determined by using the cystatin C PET kit (Dako, Milano, Italy). We also determined serum creatinine and urea in all patients by using the Ektachem enzymatic assay (Ortho Diagnostic Division, Milano, Italy). RESULTS: In pregnant women, serum cystatin C was 1.52 +/- 0.39 mg/L, ranging from 0.69 to 2.30 mg/L. Serum creatinine was 58.9 +/- 11.5 mumol/L, and serum urea was 3.117 +/- 0.729 mmol/L. In newborns, serum cystatin C was at birth 2.29 +/- 0.52 mg/L, ranging from 1.17 to 4.84 mg/L. Subsequently, cystatin C significantly decreased over the first five days of life. Serum creatinine was at birth 80.08 +/- 14.26 mumol/L. By using analysis of variance (ANOVA) we found a statistically significant difference between maternal and neonatal cystatin C (p < 0.001) as well as between maternal and neonatal creatinine (p +/- 0.001). However, no correlation has been demonstrated by simple linear regression between maternal and neonatal cystatin C (r = 0.05), while maternal and neonatal creatinine significantly correlated (r = 0.45). CONCLUSIONS: Our preliminary findings suggest that cystatin C does not cross the placental barrier. Thus, in the neonate cystatin C serum levels may solely derived from himself.

Adult↗
PubMed2003

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry.

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.

Chromatography, High Pressure Liquid↗
PubMed2003

1,25-dihydroxyvitamin D(3) increases human cystatin A expression by inhibiting the Raf-1/MEK1/ERK signaling pathway of keratinocytes.

The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D3), regulates proliferation and differentiation of keratinocytes. Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent and a differentiation marker of keratinocytes. In the present study, we examined the effect of 1,25(OH)(2)D3 on the expression of cystatin A of cultured normal human keratinocytes (NHK). 1,25(OH)(2)D3 suppressed NHK proliferation in a dose-dependent manner with the maximal effect at 1x10(-7) M. It also stimulated cystatin A promoter activity and its expression with similar dose effects. The increased cystatin A was detected by 24 h and the effect was accompanied by the suppression of ERK activity. Cystatin A promoter activity was not affected by cotransfection of vitamin D(3) receptor or retinoid X receptor. Further analyses disclosed that the 12- o-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE), T2 (-272 to -278), in cystatin A promoter is critical for the regulation by 1,25(OH)(2)D3. Transfection of the dominant-negative form of ERK adenovirus (Ad-dnERK) increased cystatin A promoter activity and its expression, which was markedly augmented by 1,25(OH)(2)D3 treatment. Transfection of the dominant-active form of Raf-1 (Ad-daRaf-1) or MEK1 (Ad-daMEK1) inhibited 1,25(OH)(2)D3-dependent cystatin A promoter activity and its expression. Consistent with these results, the MEK1 inhibitor, PD98059, further augmented 1,25(OH)(2)D3-induced cystatin A promoter activity and its expression. The present study demonstrated that the 1,25(OH)(2)D3-responsive element in the cystatin A gene is identical to the TRE, T2 (-272 to -278), and that the suppression of Raf-1/MEK1/ERK1,2 signaling pathway increases cystatin A expression of NHK.

Calcitriol↗
PubMed2006

Molecular cloning, sequencing, expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity.

Cystatin, a superfamily of cysteine proteinase inhibitor of cathepsins and other cysteine proteinases, is widely distributed in animal tissues and body fluids. Although considerable attention has been given to mammalian and avian cystatins, little is known about cystatins from other vertebrates. In this study, a cDNA coding for Chinese sturgeon (Acipenser sinensis) cystatin was isolated and characterized. The corresponding mature cystatin peptide cDNA is 336 nucleotides long and encodes a protein of 112 amino acids. Sequence comparison showed that the cloned cystatin was a homolog of the mammalian Family II cystatin. The cystatin cDNA of Chinese sturgeon was subcloned into yeast expression vector pPICZalphaA and transformed into Pichia pastoris GS115 strain. After methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215 mg/l medium supernatant in shaking-flask fermentation medium, accounting for 73.6% of the total supernatant secreted proteins. Our data also showed that the recombinant cystatin is active in inhibiting the protease activity of papain and cathepsin B. Heat stability of the recombinant cystatin was also measured.

Amino Acid Sequence↗
PubMedDate not supplied

Homology model and molecular dynamics simulation of carp ovum cystatin.

In this study, a homology model of carp ovum cystatin was constructed based on the crystal structure of chicken egg white cystatin. The results of amino acid sequence alignment indicate that these two proteins exhibit 36.11% of sequence identity. The resultant homology model reveals that carp ovum cystatin shares similar folds as chicken egg white cystatin, particularly in the conserved regions of Q48-V49-G52 and P98-W99 and the locations of two disulfide bonds, C67-C76 and C90-C110. However, the results of 1 ns molecular dynamics simulations show that carp ovum cystatin exhibits less structural integrity than chicken egg white cystatin in explicit water at 300 K. The relatively hydrophilic Met62 of carp ovum cystatin, corresponding to the hydrophobic Leu68 of human cystatin C and Ile66 of chicken egg white cystatin, may destabilize the hydrophobic core and form a dimeric structure more easily through domain swapping. A total of 16 positively charged residues are equally distributed on the surface of carp ovum cystatin, resulting in agglutination with the negatively charged spermatozoa via electrostatic interaction. Thus, carp ovum cystatin is considered to be important in preventing carp eggs from polyspermy.

Agglutination↗
PubMed1991

Human salivary cystatin S. Cloning, sequence analysis, hybridization in situ and immunocytochemistry.

A human submandibular-gland (SMG) cDNA library was constructed in a lambda was constructed in a lambda gt11 Sfi-Not orientation-specific expression vector and then screened with antibody generated against human salivary cystatins. The clone C4-4 encoded an N-terminally truncated cystatin S, whereas the others encoded cystatin SN. The library was then rescreened with the C4-4, and the inserts of several positive clones were directly amplified from the eluted plaques by linear PCR and the PCR products analysed by Southern blotting and direct DNA sequencing. Two clones (C3 and C12) encoded a full-length secreted cystatin S and its leader peptide and included 5'- and 3'-untranslated regions. These clones showed a high degree of sequence similarity to cDNA clones encoding human salivary cystatin SN and genomic clones encoding cystatin SN and SA. Hybridization in situ of normal human SMG and parotid-gland (PG) tissue sections localized the cystatin-gene transcripts to the cytoplasm of serous acinar cells of both glands, with a much higher concentration of cystatin mRNA in the SMG. Immunocytochemistry localized the salivary cystatin gene products also to the serous cells, and the levels of cystatin protein correlated with the amount of cystatin mRNA, with a much stronger signal in the SMG than in the PG.

Aged↗
PubMed1992

Cystatin activity in gingival crevicular fluid from periodontal disease patients measured by a new quantitative analysis method.

Cystatins are protein inhibitors of cysteine proteinases, which are believed to play an important role in the pathogenesis of periodontal disease. In this study, we report a new sensitive method for the quantitative analysis of cystatin activity in a small amount of crude sample such as gingival crevicular fluid. Cystatin activity in the crude sample was determined by using active site-titrated papain, which is a cysteine proteinase from the plant Carica papaya. Crude samples usually contain endogenous cysteine proteinases. These competed with the added papain for the active sites of the cystatins. The cystatin-cysteine proteinase complex was able to be dissociated by the addition of papain. This competition and dissociation could interfere with the determination of cystatin activity, since some of the cysteine proteinases, such as cathepsin B, hydrolyzed the specific substrate for papain during titration with the papain. In order to exclude this interference and measure total cystatin activity, the crude sample must be alkalinized (pH 11.0) for 5 min at 4 degrees C followed by 10 min at 40 degrees C before titration with papain. The minimum detectable amount of cystatins was 20 fmol/assay when it was calculated per mole of papain inhibitory sites. Using this method, significant levels of cystatin activity were detected in all the samples of gingival crevicular fluid taken from periodontal disease patients. These results suggest that cystatins could regulate the cysteine proteinases in gingival crevicular fluid and that this new method could be useful to clarify the role of cystatins in the pathogenesis of periodontal disease.

Alkalies↗
PubMed2006

Two secreted cystatins of the soft tick Ornithodoros moubata: differential expression pattern and inhibitory specificity.

Two genes coding for cysteine peptidase inhibitors of the cystatin family (Om-cystatin 1 and 2) were isolated from a gut-specific cDNA library of the soft tick Ornithodoros moubata. Both cystatins were clearly down-regulated after a blood meal. Om-cystatin 1 is mainly expressed in the tick gut, while Om-cystatin 2 mRNA was also found in other tick tissues. Authentic Om-cystatin 2 was significantly more abundant than Om-cystatin 1 in the gut contents of fasting ticks and was associated with hemosome-derived residual bodies accumulated in the gut lumen. Om-cystatin 2 was also expressed by type 2 secretory cells in the salivary glands of unfed ticks. The inhibitory specificity of recombinant Om-cystatins 1 and 2 was tested with mammalian cysteine peptidases, as well as endogenous cysteine peptidases present in the tick gut. Both cystatins efficiently inhibited papain-like peptidases, including cathepsin B and H, but differed significantly in their affinity towards cathepsin C and failed to block asparaginyl endopeptidase. Our results suggest that the secreted cystatin isoinhibitors are involved in the regulation of multiple proteolytic targets in the tick digestive system and tick-host interaction.

Amino Acid Sequence↗
PubMed1997

Cystatin C, and inhibitor of bone resorption produced by osteoblasts.

The effects of human cystatin C on bone resorption, enzyme release, osteoclast generation, bone cell proliferation and bone matrix protein biosynthesis have been examined in different in vitro systems. The effects of cystatin C were compared with those of calcitonin and E 64 (trans-Epoxysuccinyl-L-leucyl-amido-(4-guanidino)butane). Recombinant human cystatin C and E 64 dose dependently inhibited the mobilization of 45Ca and the release of 3H (from [3H]-proline-labelled bones) in mouse calvariae stimulated to resorb by parathyroid hormone (PTH) or 1,25(OH)2-vitamin D3. Cystatin C and E 64 also inhibited the release of 45Ca from bones stimulated by thrombin, interleukin-1 and prostaglandin E2. In PTH-stimulated bones, the inhibitory action of cystatin C and E 64 on 45Ca release was observed after 6-9 h, whereas the inhibitory effect on 3H release was seen after just 2 h. In contrast, calcitonin caused an inhibition of both 45Ca and 3H release which was seen after 2 h. The PTH-stimulated release of the lysosomal enzymes was not affected by cystatin C and E 64, whereas calcitonin caused a significant inhibition. In contrast to calcitonin, cystatin C did not affect PTH-stimulated enhancement of osteoclast generation in the mouse calvariae. Using Western blot analysis and radioimmunoassay, we demonstrated that mouse calvarial bones and MC3T3-E1 cells produce cystatin C. These data show that cystatin C is synthesized by bone cells and that recombinant human cystatin C inhibits bone resorption in vitro without affecting bone cell proliferation, bone matrix formation or osteoclast generation. The mechanism seems to be due primarily to inhibition of the activity of osteoclastic proteolytic enzymes released into the resorption lacunae.

Acetylglucosaminidase↗
PubMed1986

The cystatins: a diverse superfamily of cysteine peptidase inhibitors.

The cystatins comprise a homologous group of protein inhibitors of peptidases, of which the first to be studied in detail was chicken cystatin from egg-white. Three distinct protein families or "types" are recognized within the cystatin superfamily. The type 1 cystatins (also called "stefins") are the simplest in structure, being single chains of about 100 amino acids, with no disulphide bonds or carbohydrate. The Type 2 cystatins, which include the egg-white protein, have about 115 amino acids, and two disulphide loops, but still no carbohydrate. The Type 3 cystatins are the plasma kininogens in which each molecule contains three divergent copies of the typical cystatin sequence differing in activity as well as structure; these complex inhibitor molecules contain disulphide bonds and also carbohydrate groups. The cystatins inhibit most cysteine endopeptidases of the papain type, and also the exopeptidase dipeptidyl peptidase I. Each cystatin molecule has a single reactive site for all the peptidases it inhibits, but there are large differences in K(i) values for different combinations of cystatin and enzyme, and calpains are inhibited only by one of the segments of the kininogens. The cystatins have many important characteristics in common, but their differences in molecular structure imply different routes of biosynthesis, are associated with different in vivo distributions, and suggest a variety of physiological functions.

Animals↗
PubMed1992

Cystatin C and cathepsin B in human colon carcinoma: expression by cell lines and matrix degradation.

Expression of the cysteine proteinase cathepsin B and its physiological inhibitor cystatin C was analyzed in vitro in 1 human fibrosarcoma and 4 human colon carcinoma cell lines. Cystatin C antigen as well as cathepsin B activity were detected in the conditioned media of the 5 cell lines. The corresponding cell extracts expressed high levels of cathepsin B activity, whereas only trace amounts of cystatin C antigen could be found. Northern-blot analysis revealed the presence in the 5 cell lines of a 0.8-kb cystatin C mRNA transcript and 2 cathepsin B transcripts of 2.3 and 4.3 kb. Pepsin treatment of tumor-cell-released cathepsin B induced an average 7.3-fold increase in activity, indicating that the enzyme was mainly present as a latent form in conditioned medium. The pepsin-activated cathepsin B from one colon carcinoma cell line was further characterized using the cysteine proteinase inhibitors E-64, recombinant cystatin C, a cystatin-C-derived peptidyl inhibitor (Z-LVG-CHN2), and cathepsin-B-specific diazomethyl ketone inhibitors (Z-FT(OBzl)-CHN2, Z-FS(OBzl)-CHN2). This activity was totally neutralized by recombinant cystatin C, suggesting a potential for interaction between released extracellular cathepsin B and cystatin C. In vitro assays of degradation of extracellular matrix showed that cysteine proteinase inhibitors could decrease matrix degradation induced by pepsin-activated conditioned media. With colon cells, this inhibition was not observed, indicating a requirement for an extracellular activation of latent cathepsin B. Our data provide evidence that cystatin C and latent cathepsin B are both released extracellularly by colon carcinoma cells in vitro. They suggest that cystatin C and cathepsin B interactions may participate, in an as yet unelucidated way, in the modulation of the invasive phenotype of human colonic tumors.

Carcinoma↗
PubMed1992

Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide of malignancy.

The effect of human recombinant cystatin C, a cysteine proteinase inhibitor, on bone resorption in vitro was evaluated. Bone resorption was assessed by analyzing the release of 45Ca and 3H from mouse calvarial bones prelabeled in vivo by injections with 45Ca or [3H]proline, respectively. In 24 h cultures, cystatin C (50 micrograms/ml) significantly inhibited the release of 45Ca and 3H stimulated by parathyroid hormone (PTH, 15 nmol/liter) or parathyroid hormone-related peptide of malignancy (PTHrP, 15 nmol/liter). The degree of inhibition caused by cystatin C in these 24 h cultures was similar to that caused by calcitonin (30 ng/ml). The inhibitory effect of cystatin C on 45Ca release induced by PTH was sustained in 96 h cultures, whereas the initial inhibition caused by calcitonin was transient. Cystatin C, 10-100 micrograms/ml, caused a dose-dependent inhibition of PTH (15 nmol/liter), and PTHrP (15 nmol/liter) stimulated 45Ca release. Addition of 50 micrograms/ml of cystatin C to mouse bone cultures inhibited the release of 45Ca induced by PTH and PTHrP at a wide range of submaximal and maximal concentrations of hormones (0.01-10 nmol/liter). No effect of cystatin C on 45Ca release in dead bones could be observed, nor did the inhibitor decrease the release of calcium in control bones. The inhibition by cystatin C on PTH-induced mineral mobilization was reversible. Cystatin C (1-100 micrograms/ml) did not affect protein synthesis or mitotic activities in mouse calvarial bones as assessed by the incorporation of [3H]proline and [3H]thymidine, respectively. These data show that cystatin C is a potent inhibitor of mineral mobilization and matrix degradation in cultured bones stimulated to resorb by PTH and PTHrP and that this effect is not due to general cytotoxicity.

Bone Resorption↗
PubMed2003

Expression of cystatins, high molecular weight cytokeratin, and proliferation markers in prostatic adenocarcinoma and hyperplasia.

BACKGROUND: Prostatic adenocarcinoma is the most common malignancy among men in the western world but the diagnostic and prognostic criteria for it are still not clearly defined. Additional means for its diagnosis and prognosis are clearly needed. Previously it has been shown that cystatin A is expressed in the basal cells of normal prostate and the expression disappears in prostatic carcinoma. METHODS: We have now studied the expression of both cystatins A and B in benign prostatic hyperplasias (BPH), prostatic intraepithelial neoplasias (PIN) and carcinomas of the prostatic epithelium and compared it with the expression of high molecular weight (HMW) cytokeratin as well as the proliferation markers cyclin A and Ki-67. The expression of the proteins was immunohistochemically assessed using 33 total prostatectomy specimens. RESULTS: Cystatin A was expressed in the basal cells in all cases of BPH, low-grade PIN, and high-grade PIN whereas carcinomas showed no staining of cystatin A. The 34 beta E12 cytokeratin expression was similar to basal cystatin A staining and was not seen in carcinoma foci. Cystatin B showed both nuclear and cytoplasmic expression in the columnar epithelial cells. The decrease in median cytoplasmic staining of cystatin B in carcinomas compared to other lesions was significant, but there was a significant increase in expression with dedifferentiation of carcinoma. Also cyclin A and Ki-67 staining were significantly different in non-carcinomatous foci compared to carcinoma foci and had a remarkably similar negative correlations with basal cystatin A and 34 beta E12 staining. CONCLUSIONS: The results show that cystatin expression can be used as an aid in the diagnosis of prostatic adenocarcinoma and especially cystatin A in the distinction between high grade PIN and grade I carcinoma.

Adenocarcinoma↗