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From the* Department of Urology, Malmö
University Hospital, Lund University, Sweden; the
Department of Clinical Chemistry, Lund
University Hospital, Lund University, Sweden; and the Department of Laboratory
Medicine, Divisions of Clinical Chemistry and
Pathology, Malmö University Hospital, Lund
University, Sweden.
| Correspondence to: Dr Thomas Jiborn, Department of Urology, Malmö University Hospital, 205 02 Malmö, Sweden (e-mail: thomas.jiborn{at}skane.se). |
| Received for publication December 5, 2003; accepted for publication January 23, 2004. |
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Abstract |
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Key words: Cysteine protease inhibitor, immunohistochemistry, in situ hybridization, quantitative real-time (QRT) PCR, seminal fluid
The cystatin protein superfamily contains 3 major types of inhibitors.
Cystatin A and B, of type 1, represent intracellular protease inhibitors, but
low levels of these are also found in some body fluids
(Järvinen, 1978;
Abrahamson et al, 1986;
Kartasova et al, 1987).
Cystatin C, D, E/M, F, S, SA, and SN are type 2 cystatins, which probably have
predominantly extracellular functions because they are synthesized with signal
peptides for secretion (Abrahamson et al,
2003). Cystatin C displays the strongest inhibitory activity of
all cystatins toward lysosomal cysteine proteases in general and has a
widespread distribution in human tissues and body fluids
(Abrahamson et al, 1986). Human
type 3 cystatins comprise L- and H-kininogen, which together with cystatin C
and 
2-macroglobulin are the most important plasma inhibitors
of cysteine proteases (Abrahamson et al,
1986).
Cystatin C inhibits family C1 cysteine proteases through tight and reversible binding in a substrate-competing mechanism resulting in an equimolar complex with the protease (Anastasi et al, 1983; Green et al, 1984; Nicklin and Barrett, 1984; Abrahamson et al, 1987a; Björk and Ylinenjarvi, 1990). The activity of cystatin C can be down-regulated in inflammatory conditions by the serine protease, neutrophil elastase (Buttle et al, 1990; Abrahamson et al, 1991). In intracellular compartments such as endosomes, regulation of cystatin C by cathepsin D might be important (Lenarcic et al, 1991).
The presence of the different cystatins in the male accessory sex glands is mainly still unknown, but cystatin A has been demonstrated in the basal epithelial cell layer in normal prostatic tissue and benign hyperplasia of the prostate (BPH; Söderström et al, 1995). Furthermore, the concentration of cystatin C in seminal plasma is higher than in any other body fluid (Abrahamson et al, 1986). Seminal plasma also contains cystatins S, A, and B in relatively high amounts, with a strikingly high total cystatin concentration of more than 5 µM as a result, strongly indicating an important need for efficient cysteine protease inhibition in the reproductive tract.
In order to further elucidate the role of cysteine proteases and their inhibitors, we now investigated the cellular localization, expression, and biochemical properties of cystatin C in tissue specimens from the male genital tract.
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Materials and Methods |
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Antibodies
A polyclonal rabbit antiserum called 8206 against human cystatin C purified
from urine (Abrahamson et al,
1986) was used for IHC, ELISA, and Western blotting. For IHC we
also used a commercially available polyclonal cystatin C antibody (code A0451;
Dako A/S, Glostrup, Denmark). Monoclonal anti-human chromogranin A (CgA)
immunoglobulins (IgGs, code M 869; DAKO) were used to identify neuroendocrine
(NE) cells in prostatic tissues.
Biotinylated secondary antibodies, anti-rabbit and anti-mouse IgGs, were included in DAKO ChemMate kit (code K5003; BioTek Solutions, Winooski, Vt). Rabbit anti-mouse IgG (code Z259, DAKO), alkaline phosphatase anti-alkaline phosphatase (APAAP) mouse monoclonal IgG (code D 0651; DAKO), and alkaline phosphatase–conjugated swine anti-rabbit IgGs (code D 0306; DAKO) were used in the APAAP method for IHC. Fab fragments of anti–digoxigenin-alkaline phosphatase (AP) (code 1093274; Roche, Mannheim, Germany) were used for ISH.
Riboprobe Synthesis for in situ Hybridization
The following chemicals and reagents were purchased: T7 RNA polymerase (20
U/µL), RNase inhibitor from human placenta (40 U/µL), and DIG RNA
labeling mix from Roche Applied Science (Roche Diagnostics Scandinavia AB,
Bromma, Sweden); RNase-free DNase (1 U/µL) from Promega (Scandinavian
Diagnostic Services, Falkenberg, Sweden); Advantage 2 PCR kit from Clontech
(BD Bioscience, Stockholm, Sweden); RNA ladder, low range, from Fermentas
(Tamro Medlab AB, Gothenburg, Sweden); and JetQuick from Genomed (Saveen,
Malmö, Sweden).
Cystatin C minigenes were synthesized by PCR with the use of primers with appended T7 RNA polymerase promoters (Table 1) according to Logal et al (1992). The PCR reactions were performed with 1 µL of Advantage 2 polymerase mix in 50 µL of 0.2 mM dNTP containing buffer components provided with the Advantage 2 kit (40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mM MgCl2, 187.5 µg bovine serum albumin [BSA], and 0.005% each of Tween-20 and Nonidet-P40), 20 pmol each of PCR primers, and 10 ng of complementary DNA (cDNA) cloned in pUC18 (Abrahamson et al, 1987b). The PCR protocol consisted of an initial 1-minute denaturation at 95°C, followed by 35 cycles of 30 seconds at 95°C and 1 minute at 68°C. Finally, the reactions were incubated at 68°C for 1 minute and then chilled to 10°C. The PCR products were purified from primers and free deoxynucleotides by JetQuick and then analyzed by electrophoresis in 2% agarose gel. Concentrations were estimated by comparing the staining intensity of PCR products and size standards after staining the gel with ethidium bromide.
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RNA probes of 201 bases in length were synthesized from 200 ng of minigene with 40 U of T7 RNA polymerase in 20 µL of 40 mM Tris-HCl, pH 8.0; 6 mM MgCl2; 10 mM dithiothreitol; 2 mM spermidine; 40 U of RNase inhibitor; 0.65 mM UTP; 0.35 mM DIG-11-UTP; and 1.0 mM each of ATP, GTP, and CTP. The samples were incubated at 37°C for 2 hours, after which 2 µL of DNase was added and the incubation was allowed to continue for another 15 minutes. The reactions were stopped with 2 µL of 0.2 mM EDTA, pH 8.0, and the RNA was precipitated by addition of 2.5 µL of 4 M LiCl and 75 µL of prechilled ethanol. Following overnight storage at -20°C, the RNA precipitate was collected by centrifugation at 13,000 x g for 15 minutes. Following a wash in 70% ethanol, the material was briefly dried in vacuum and then dissolved in 100 µL H2O containing 40 U of RNase inhibitor. The RNA concentration was estimated from the staining intensity of RNA samples compared to that of components in the RNA ladder following electrophoresis in 2% agarose gel and staining with ethidium bromide. RNA probes were stored at -70°C.
Immunohistochemistry
Tissue sections 3 µm in thickness were mounted on Superfrost plus slides
(Menzel-Gläser, Braunschweig, Germany), and dried at 60°C for 20
minutes, deparaffinized in xylene, and rehydrated in decreasing ethanol
concentrations. Cystatin C immunoreactive cells were identified by the
streptavidin–horseradish peroxidase (SA-HRP) conjugate method with
diaminobenzidine as chromogen (Elias et
al, 1989). For double immunostaining, sections were first
processed with polyclonal anti-cystatin C IgGs and a DAKO ChemMate detection
kit, and subsequently by the APAAP method with monoclonal IgGs and Fast Blue
(Sigma Chemical Corp, St Louis, Mo) for detection of CgA. As a negative
control, adjacent tissue sections were processed by replacing the primary
antibody with nonimmune rabbit IgG diluted 1: 8000 (code X0936; DAKO). For
specificity test, pure recombinant human cystatin C
(Abrahamson et al, 1988) was
added to the polyclonal antiserum at 100 times molar excess and incubated
overnight before immunodetection.
Quantitation of Cystatin C by Enzyme-Linked Immunosorbent Assay
ELISA was performed for the determination of cystatin C according to the
procedure described by Olafsson et al
(1988), slightly modified to
utilize a SA-HRP conjugate (code RPN.1231; Amersham Biosciences, Uppsala,
Sweden) for detection of biotinylated monoclonal antibody HCC3 as a second
detection antibody in the double-sandwich ELISA. For appropriate calibration
curves, recombinant human cystatin C
(Abrahamson et al, 1988) was
used. Tissue samples were analyzed at the highest possible dilution to
maximize dissociation of complexes between cystatin C and lysosomal proteases
and other possible binders in the homogenates and to minimize risks of assay
interference. To put the ELISA results for reproductive tissues in
perspective, 2 series of tissue samples obtained at autopsy within 15 hours of
death that had previously been used for cystatin C messenger RNA (mRNA)
analysis (Abrahamson et al,
1990) were analyzed by the cystatin C ELISA. The concentration of
cystatin C was related to total protein concentration in the homogenates, the
latter measured by a dye-binding assay
(Bradford, 1976).
Western Blotting
Tissue extracts containing 1–20 ng cystatin C according to ELISA were
analyzed by immunoblotting under reducing conditions as previously described
(Bjarnadottir et al, 1998).
Briefly, proteins in tissue extracts from the male reproductive system were
separated electrophoretically with 16.5% sodium dodecyl sulfate polyacrylamide
gels and electroblotted onto poly(vinilydene)difluoride membranes
(Immobilon-P; Millipore, Bedford, Mass). Subsequently, membranes were
incubated with the same polyclonal anti-cystatin C IgG preparation as used for
IHC, followed by a HRP conjugated anti-rabbit IgG secondary antibody (Dako
A/S). The immunoreactive material was visualized by enhanced chemiluminescence
and the migration positions were compared with prestained size marker proteins
(ECL Plus kit, Rainbow colored protein molecular weight markers; Amersham
Pharmacia Biotech, Uppsala, Sweden). Isolated recombinant cystatin C was
included as a reference (Abrahamson et al,
1988).
In Situ Hybridization
All reagents were purchased from Sigma or Roche. Tissue specimens were
fixed, paraffin-embedded, sectioned (4 µm), dried for 2 hours at 65°C,
and mounted on SuperFrost plus slides (Menzel-Gläser) under RNase-free
conditions.
The sections were deparaffinized in xylene and rehydrated, after which they were first treated with 0.2 M HCl to abolish endogenous enzyme activity and then digested with proteinase K (20 µg/mL in 20 mM Tris-HCl, 2 mM CaCl2, pH 7.5) for 25 minutes at 37°C. The slides were then incubated in 0.25% acetic anhydride containing 0.1 M triethanolamine and 0.15 M NaCl for 10 minutes and then equilibrated in 2x SSC (1x contains 150 mM NaCl and 15 mM sodium citrate, pH 7.0). After pre-hybridization with 40 µL of hybridization buffer containing 50% (vol/vol) formamide, 10% dextran sulfate, 1x Denhardt's solution (BSA, polyvinyl pyrrolidone, and Ficoll, all at 0.2 mg/mL), 4x SSC, and 0.4 mg/mL salmon sperm DNA at 85°C for 8 minutes and then at 42°C for 1 hour, the slides were hybridized with 40 µL of 250–500 ng/mL antisense or sense probe in hybridization buffer, first for 8 minutes at 85°C and then for 16 hours at 50°C.
After hybridization, the slides were washed in 1x SSC at room temperature (5 minutes twice), 0.1x SSC at 55°C (15 minutes 4 times), 1x SSC at room temperature (10 minutes) and then equilibrated in TBS (100 mM Tris-HCl, 0.4 M NaCl, pH 7.5). For detection of hybridization signals, tissue sections were first incubated in blocking reagent (0.5%, wt/vol, in TBS) for 1 hour at room temperature, rinsed in TBS, and subsequently incubated with anti-digoxigenin alkaline phosphatase conjugate (Roche) diluted 1:500 in TBS containing 0.5% (wt/vol) BSA for 1 hour at room temperature. After washing in TBS, the sections were equilibrated in detection buffer (100 mM Tris-HCl containing 100 mM NaCl, 50 mM MgCl2, pH 9.5) (5 minutes twice), and developed in detection buffer containing 1 mM levamisol, 0.33 mg/mL nitro blue tetrazolium chloride (NBT) and 0.167 mg/mL 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The color reaction was stopped after 20 minutes to 3 hours by incubating the sections in stop buffer (10 mM Tris-HCl containing 10 mM EDTA, 0.9% NaCl, pH 7.5) for 10 minutes. The slides were coverslipped with Faramount mounting medium (DAKO).
Quantitative Real-Time Polymerase Chain Reaction
RNA was extracted from deep-frozen tissue samples after mincing, using the
TRIzol reagent according to the instructions provided by the manufacturer
(Life Technologies, Carlsbad, Calif). RNA quantities were determined
spectrophotometrically. Complementary DNA was synthesized with TaqMan reverse
transcription reagent (Applied Biosystems, Foster City, Calif) with the use of
50 ng of RNA as template and random hexamers as primers in a total reaction
volume of 100 µL. QRT-PCR was run with TaqMan Universal Mastermix (Applied
Biosystems) in an ABI PRISM 7700 sequence detection system (Applied
Biosystems) in a total reaction volume of 25 µL. Primers and TaqMan probes
were designed with the aid of Primer Express 1.5 (Applied Biosystems) and were
purchased from DNA Technology A/S (Aarhus, Denmark). Amplification of cystatin
C was performed with a primer pair previously used for regular real-time PCR
and was shown to give a specific product: primers MA097 and MA098
(Table 1), corresponding to a
coding strand sequence in exon 1 (nt 245–264 in the cDNA sequence;
GenBank/EMBL acc. X05607; Abrahamson et al,
1987b) and a noncoding strand sequence in exon 3 (nt
464–445) of the gene, respectively. The probe, MA576
(Table 1), corresponded to a
noncoding strand sequence in exon 2 (nt 353–331 in the cDNA sequence).
As endogenous controls, human GAPD (GAPDH) mRNA and 18S rRNA were amplified
with commercially available primers/probes (GAPDH Endogenous Control and
eukaryotic 18S rRNA Endogenous Control; Applied Biosystems). Five microliters
of cDNA was used as template for the cystatin assay and 2 µL for the
endogenous control assays. All QRT-PCR experiments were performed following
the guidelines supplied by Applied Biosystems.
Complementary DNA from a pooled batch of the promyelocytic cell line U937, one of the best cystatin C producers identified to date in analyses of human cell lines (Ni et al, 1998), was used as assay calibrator, which was included on all assay plates to allow compensation for possible minor day-to-day variations of results. A 10-fold dilution series from 1 to 105 times dilution of the same cDNA was run in triplicate to generate standard curves. The input amounts of cDNA in samples were calculated from the threshold values (Ct) for the cystatin C and endogenous control assays. The standard curve also allowed calculations to compensate for interassay variations in amplification efficiency. The ratios of input cystatin C cDNA to the input amount of 18S and GAPDH cDNA were calculated. The normalized values were finally divided by the normalized values for the calibrator mRNA sample to obtain the relative levels of cystatin C mRNA in the samples.
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Results |
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The immunostaining of the prostatic epithelium was homogenous, with the exception of a few, scattered cells with more intense immunostaining. The localization and features of these cells were similar to those of NE cells. As shown by double immunostaining with polyclonal antibodies against cystatin C and monoclonal antibodies against CgA, NE immunoreactivity was demonstrated in a subpopulation of the highly immunoreactive cystatin C–positive cells. In a minor population of the CgA–positive cells, we could not detect any coexpression of cystatin C (Figure 1G and H).
For ISH, the digoxigenin-labeled antisense and sense probes were hybridized to adjacent tissue sections. In all the investigated tissues, hybridization signals were generated by the antisense probe, whereas the sense probe did not give rise to any positive signals (Figure 2A through D). The distribution pattern in the testis, epididymis, vas deferens, and seminal vesicles was in concordance with the immunohistochemical findings. However, in the prostatic epithelium, the basal cells showed a stronger signal than the secretory epithelial cells.
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Quantitation of Cystatin C in Tissue Homogenates
Cystatin C was detected by ELISA in all examined tissue extracts from the
male reproductive system. Although we found a noticeable variation in the
levels of cystatin C between samples taken from different individuals, the
protein measurements reflected the immunohistochemical findings, with the
highest concentrations found in the epididymis and the lowest in the vas
deferens (Figure 3). The
concentration of cystatin C in tissue homogenates of male reproductive organs
is high compared with a panel of other human tissues (n = 2), with the mean
values of 15, 0.35, 5.9, 1.2, and 8.5 ng cystatin C/mg protein for kidney,
liver, muscle, spleen, and heart, respectively. Brain tissue was the only
exception, containing 289 ± 69 ng cystatin C/mg protein.
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Western blotting demonstrated a single predominant band corresponding to a size of approximately 15 kd in tissue homogenates from all investigated locations (testis, epididymis, vas deferens, seminal vesicle, and prostate; Figure 4). Besides demonstrating that the antiserum used is monospecific, this result indicates that the molecular form of cystatin C present in the reproductive tissue is biochemically intact and likely fully active. Down-regulated cystatin C with poor inhibitory activity, as is found in inflammatory conditions, would have been detected as a ca 1.5-kd smaller band at immunoblotting (Buttle et al, 1990; Abrahamson et al, 1991).
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Quantitation of Cystatin C Gene Expression in Tissue Homogenates
To determine the relative amount of cystatin C mRNA in tissue homogenates,
QRT-PCR was performed. Reverse transcriptase-generated cDNA from RNA of
freshly frozen tissue was analyzed and compared to the content of mRNA for the
housekeeping gene, GAPDH, and 18S rRNA. The expression pattern was that of a
markedly higher expression in seminal vesicle and prostate than in the vas
deferens, epididymis, and testis (Figure
5). The amounts found in seminal vesicles and prostate tissue were
high, even when compared with RNA from homogeneous cultures of the
promyelocytic cell line U937 (Figure
5).
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Discussion |
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The cystatins form a superfamily of structurally related proteins with different molecular properties and biological distribution. They are all potent, reversible, competitive inhibitors of cysteine proteases such as papain and many of the human cathepsins (Abrahamson et al, 2003). The cathepsins are mainly lysosomal proteases that can degrade extracellular matrix and basement membrane proteins (Lah et al, 1989; Werb 1989; Buck et al, 1992), and some of these enzymes are also thought to be involved in the pathogenesis of inflammatory and malignant diseases. The balance between these enzymes and their inhibitors (eg, cystatins) is thus most important for protection against endogenous proteolysis. Alterations in this balance are observed in different inflammatory diseases as well as in malignancy (Machleidt et al, 1992; Calkins and Sloane, 1995). Cystatin C is one of the main extracellular inhibitors of the papain-like cathepsins, and it is detected in many biological fluids, the highest concentrations being found in seminal plasma, cerebrospinal fluid, and synovial fluid (Abrahamson et al, 1986).
The balance between cysteine proteases and their inhibitors could also be important for protection against exogenous proteases of microbial origin. Several studies suggest that cystatins might have important functions in the protection against infections by inactivating bacterial and viral cysteine proteases. Synthetic peptide derivates of cystatin C are shown to specifically block both in vitro and in vivo growth of group A streptococci (Björck et al, 1989). Additionally, the same peptides showed strong inhibitory effects on herpes simplex type 1 replication, as does cystatin C (Björck et al, 1990). Furthermore, it has previously been shown that macrophages secrete cystatin C (Chapman et al, 1990).
In this study, we found strong immunostaining for cystatin C in tissue specimens from the epididymis, seminal vesicle, and prostate. In the testis, Sertoli cells and Leydig cells showed strong and intermediate immunostaining, respectively. The spermatogonia were negative, whereas a weak immunoreactivity was detected in the rest of the germinal epithelium. In the majority of the tissue specimens, especially in the prostate, we found a few scattered cystatin C immunoreactive stromal cells. On the basis of their size and shape, they are most likely identified as macrophages, which is in concordance with previous studies showing that human macrophages express and release cystatin C (Chapman et al, 1990). ELISA showed high tissue levels of cystatin C in all investigated locations, the highest concentrations being found in the epididymis, indicating that cystatin C could have an important function in the protection of the spermatozoa from bacterial proteolytic activity.
Our findings support other data suggesting that cystatin C is one of the major protease inhibitors in the male reproductive system that might play an important role in the protection against infections, as well as being involved in the pathogenesis of inflammatory and malignant diseases.
An interesting finding in the present study was that cystatin C immunoreactivity in benign prostatic tissue showed 2 different patterns. In addition to the general cystatin C immunostaining that was seen throughout the prostatic epithelium, we found scattered, strongly cystatin C–positive intraepithelial cells with a distribution and appearance like NE cells. A subpopulation of these cells also expressed the NE cell marker CgA. NE cells containing neurosecretory granules rich in various peptide hormones and biogenic amines are dendritic intraepithelial regulatory cells in the human prostate gland. These cells are thought to be involved in the regulation of both prostatic growth and differentiation, as well as the homeostasis of exocrine secretory activity (Tutton and Barkla, 1987; Zachary et al, 1987; Bologna et al, 1989; Dalsgård, 1989; Seuwen and Pouyssegur, 1990; di Sant'Agnese, 1992). Previous studies have shown cystatin C immunoreactivity in human brain neurons and astrocytes, as well as in NE cells in the pancreas, gastrointestinal tract, adrenal medulla, thyroid and pituitary (Löfberg et al, 1981; Grubb and Löfberg, 1985; Möller et al, 1985; Lignelid and Jacobsson, 1992; Lignelid et al, 1997). Interestingly, the nonendocrine epithelial cells of the pancreatic ducts and gastrointestinal mucosa showed weaker immunoreactivity for cystatin C than the adjacent NE cells, which resembles the staining pattern that we found in the secretory epithelium of the prostate. Lignelid et al (1997) have demonstrated the presence of cystatin C mRNA in human brain tissue and pituitary, indicating that cystatin C is produced in these locations. It has been suggested that cystatin C might be located in the secretory vesicles of the NE cells and that cystatin C could be involved in the intracellular regulation of the peptide hormones.
Cystatin C is widely expressed in the male reproductive system. Our data suggest that the presence of this potent protease inhibitor might be of significant importance in the regulation of normal physiological proteolysis, as well as in inflammatory diseases and malignancies in the male genital tract. Further studies are urgently needed to explore these suggestions, and the relevance of cystatin C in prostatic NE cells remains to be elucidated.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abrahamson M, Barrett AJ, Salvesen G, Grubb A. Isolation of six
cysteine proteinase inhibitors from human urine. J Biol
Chem. 1986;261: 11282
-11289.
Abrahamson M, Buttle DJ, Mason RW, Hansson H, Grubb A, Lilja H, Ohlsson K. Regulation of cystatin C activity by serine proteinases. Biomed Biochim Acta. 1991; 50: 4-6, 587–593.
Abrahamson M, Dalbøge H, Olafsson I, Carlsen S, Grubb A. Efficient production of native, biologically active human cystatin C by Escherichia coli. FEBS Lett. 1988; 236: 14 -18.[Medline]
Abrahamson M, Grubb A, Olafsson I, Lundwall Å. Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C. FEBS Lett. 1987b; 216: 229 -233.[Medline]
Abrahamson M, Olafsson I, Palsdottir A, Ulvsbäck M, Lundwall Å, Jensson O, Grubb A. Structure and expression of the human cystatin C gene. Biochem J. 1990; 268: 287 -294.[Medline]
Abrahamson M, Ritonja A, Brown MA, Grubb A, Machleidt W, Barrett
AJ. Identification of the probable inhibitor reactive sites of the cysteine
proteinase inhibitors human cystatin C and chicken cystatin. J Biol
Chem. 1987a;262: 9688
-9694.
Anastasi A, Brown MA, Kembhavi AA, Nicklin MJ, Sayers CA, Sunter DC, Barret AJ. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem J. 1983; 211: 129 -138.[Medline]
Barrett AJ. The cystatins: a new class of peptidase inhibitors. Trends Biochem Sci. 1987; 12: 193 -196.
Bjarnadottir M, Wulff BS, Sameni M, et al. Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells. Mol Pathol. 1998; 51: 317 -326.[Abstract]
Björck L, Åkesson P, Bohus M, Trojnar J, Abrahamson M, Olafsson I, Grubb A. Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor. Nature. 1989; 337: 385 -386.[Medline]
Björck L, Grubb A, Kjellen L. Cystatin C, a human proteinase
inhibitor, blocks replication of Herpes simplex virus. J
Virol. 1990;64: 941
-943.
Björk I, Ylinenjarvi K. Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin. Biochemistry. 1990; 29: 1770 -1776.[Medline]
Bologna M, Festuccia C, Muzi P, Biordi L, Ciomei M. Bombesin stimulates growth of human prostatic cancer cells in vitro. Cancer. 1989;63: 1714 -1720.[Medline]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem. 1976; 72: 248 -254.[Medline]
Buck MR, Karustis DG, Day NA, Hohn KV, Sloane BF. Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues. Biochem J. 1992; 282: 273 -278.
Buttle DJ, Burnett D, Abrahamson M. Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation. Scand J Clin Lab Invest. 1990; 50: 509 -516.[Medline]
Calkins CC, Sloane BF. Mammalian cysteine protease inhibitors: biochemical properties and possible roles in tumor progression. Biol Chem Hoppe-Seyler. 1995; 376: 71 -80.[Medline]
Chapman HA Jr, Reilly JJ Jr, Yee R, Grubb A. Identification of cystatin C, a cysteine proteinase inhibitor, as a major secretory product of human alveolar macrophages in vitro. Am Rev Respir Dis. 1990;141: 698 -705.[Medline]
Dalsgård CJ, Hultgårdh A, Hägerstrand A, Nilsson J. Neuropeptides as growth factors. Possible roles in human diseases. Regul Pept. 1989; 25: 1 -9.[Medline]
di Sant'Agnese PA. Neuroendocrine differentiation in carcinoma of the prostate. Cancer. 1992; 70: 254 -268.[Medline]
Elias JM, Margiotta M, Gaborc D. Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin peroxidase complex (ABC), and peroxidase-labeled avidin-biotin (LAB) methods. Am J Clin Pathol. 1989; 92: 62 -67.[Medline]
Gacko M, Bankowska A, Chyczewska E, Worowska A. Lysosomal cysteine proteinases and their significance in pathology. Rocz Akad Med. 1997;42(suppl 1): 60 -71.
Green GD, Kembhavi AA, Davies ME, Barrett AJ. Cystatin-like cysteine proteinase inhibitors from human liver. Biochem J. 1984;218: 939 -946.[Medline]
Grubb A, Löfberg H. Human 
-trace structure, function
and clinical use of concentration measurements. Scand J Clin Lab
Invest. 1985;45(suppl 177): 7
-13.[Medline]
Grubb AO, Weiber H, Löfberg H. The -trace concentration
of human seminal plasma is thirty-six times that of normal human blood plasma.
Scan J Clin Lab Invest. 1983; 43: 421
-425.[Medline]
Järvinen M. Purification and some characteristics of the human epidermal SH-protease inhibitor. J Invest Dermatol. 1978; 72: 114 -118.
Kartasova T, Cornelissen BJC, Belt P, van de Putte P. Effects of
UV, 4NQO and TPA on gene expression in cultured human epidermal keratinocytes.
Nucleic Acid Res. 1987; 15: 5945
-5962.
Lah TT, Buck MR, Honn KV, Crissman JD, Rao NC, Liotta LA, Sloane BF. Degradation of laminin by human tumor cathepsin B. Clin Exp Metastasis. 1989;7: 461 -468.[Medline]
Lenarcic B, Krasovec M, Ritonja A, Olafsson I, Turk V. Inactivation of human cystatin C and kininogen by human cathepsin D. FEBS Lett. 1991;280: 211 -215.[Medline]
Lignelid H, Collins VP, Jacobsson B. Cystatin C and transthyretin expression in normal and neoplastic tissues of the human brain and pituitary. Acta Neuropathol. 1997; 93: 494 -500.[Medline]
Lignelid H, Jacobsson B. Cystatin C in the human pancreas and gut: an immunohistochemical study of normal and neoplastic tissues. Virchows Arch A. 1992; 421: 491 -495.
Löfberg H, Grubb A, Brun A. Human brain cortical neurons
contain -trace. Rapid isolation, immunohistochemical and physiological
characterization of human -trace. Biomed Res. 1981; 2: 298
-306.
Logal J, Dill D, Leonard S. Synthesis of cRNA probes from PCR-generated DNA. Biotechniques. 1992; 13: 604 -660.[Medline]
Machleidt W, Assfalg-Machleidt I, Jochum M, Jänicke F, Schmitt M. Lysosomal cysteine proteinases as mediators of inflammation and tumor spread: control of their extracellular proteolytic activity. Fibrinolysis. 1992; 6(suppl 4): 125 -129.
Merz GS, Benedikz E, Schwenk V, Johansen TE, Vogel LK, Rushbrook JI, Wisniewski HM. Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells. J Cell Physiol. 1997;173: 423 -432.[Medline]
Möller C, Löfberg H, Grubb A, Olsson S, Davies E, Barrett A. Distribution of cystatin C (gamma-trace), an inhibitor of lysosomal cysteine proteinases, in the anterior lobe of simian and human pituitary glands. Neuroendocrinology. 1985; 41: 400 -404.[Medline]
Nathanson C-M, Wassélius J, Wallin H, Abrahamson M. Regulated expression and intracellular localization of cystatin F in human U937 cells. Eur J Biochem. 2002; 269: 5502 -5511.[Medline]
Ni J, Alvarez Fernandez M, Danielsson L, et al. Cystatin F is a
glycosylated human low molecular weight cysteine proteinase inhibitor.
J Biol Chem. 1998; 273: 24797
-24804.
Nicklin MJ, Barrett AJ. Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg white cystatin. Biochem J. 1984;223: 245 -253.[Medline]
Olafsson I, Löfberg H, Abrahamson M, Grubb A. Production, characterization and use of monoclonal antibodies against the major extracellular human cysteine proteinase inhibitors cystatin C and kininogen. Scand J Clin Lab Invest. 1988; 48: 573 -582.[Medline]
Seuwen K, Pouyssegur J. Serotonin as a growth factor. Biochem Pharmacol. 1990; 39: 985 -990.[Medline]
Söderström K-O, Laato M, Wu P, Hopsu-Havu VK, Nurmi M, Rinne A. Expression of acid cysteine proteinase inhibitor (ACPI) in the normal human prostate, benign prostatic hyperplasia and adenocarcinoma. Int J Cancer. 1995; 62: 1 -4.[Medline]
Tutton PJM, Barkla DH. Biogenic amines as regulators of the proliferative activity of normal and neoplastic intestinal epithelial cells [review.] Anticancer Res. 1987; 7: 1 -12.[Medline]
Werb Z. Proteinases and matrix degradation. In: Kelly WN, Harris ED Jr, Ruddy S, Sledge CB, eds. Textbook of Rheumatology. New York, NY: Saunders; 1989: 300 -321.
Zachary I, Woll PJ, Rozengurt E. A role for neuropeptides in the control of cell proliferation. Dev Biol. 1987; 124: 295 -308.[Medline]
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