Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.
Animals ; Cystatins/pharmacology ; Cysteine Endopeptidases/metabolism ; Cysteine Proteinase Inhibitors/chemistry/*metabolism/*pharmacology ; Helminth Proteins/*metabolism/*pharmacology ; Spirometra/*metabolism

OBJECTIVETo investigate the effects of experimental left varicocele (ELV) on the cystatin-related epididymal spermatogenic (CRES) protein in the testis and epididymis of adolescent rats.

METHODSThe ELV model of Sprague-Dawley (SD) male adolescent rats was established, and the expression of CRES protein in the testis and epididymis was detected by immunohistochemistry and Western-blot at 2 and 4 weeks after surgery.

RESULTSImmunohistochemistry and Western-blot detected CRES protein in both the testis and the epididymis of the ELV rats and the control rats. Immunohistochemistry showed that within the testis, CRES protein was expressed mainly in the cytoplasm of round spermatids and elongating spermatids, sperm acrosomes and residual bodies. The expression was most intensive at Stages I-III and IX-XIV, and then decreased gradually at Stages VII-VII and IV-VI. Within the epididymis, CRES protein was expressed mainly in the cytoplasm of the principal cells of epididymal epithelia. Western-blot detected CRES protein in Mr 19,000 and 14,000, stronger in the former than in the latter. Image and statistical analyses showed that the expression of CRES protein in the 2-week and 4-week ELV groups was significantly higher than in the control group (P < 0.05, or P < 0.01).

CONCLUSIONCRES protein expressed in both the testis and epididymis of adolescent rats and the expression is stage-specific and cell-specific in the testis and segment-specific and cell-specific in the epididymis. The expression of CRES protein in the ELV rats is much stronger than in their corresponding controls. It is suggested that CRES protein may be significantly involved in the regulation of spermatogenesis and sperm maturation, and possibly associated with varicocele-related male infertility or subfertility.

Animals ; Blotting, Western ; Cystatins ; biosynthesis ; Disease Models, Animal ; Epididymis ; metabolism ; Immunohistochemistry ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; metabolism ; Varicocele ; metabolism

OBJECTIVETo investigate the expression pattern of the cystatin-related epididymal spermatogenic (Cres) gene in mouse testes and epididymis during postnatal development.

METHODSSemi-quantitative RT-PCR was used to detect the Cres mRNA level in postnatal mouse testes and epididymis on day 14, 20, 22, 28, 35, 49, 70 and 400.

RESULTSLow-level Cres mRNA was detected on day 14 in both the testes and the epididymis. The expression of the Cres gene increased gradually with the development of the mouse, and it reached the peak on day 70 in the testes and on day 400 in the epididymis.

CONCLUSIONThe time-dependent expression pattern of the Cres gene in postnatal mouse testes and epididymis suggested that the Cres gene might be involved in the regulation of spermatogenesis and sperm maturation.

Age Factors ; Animals ; Blotting, Northern ; Cystatins ; genetics ; Epididymis ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis ; Testis ; metabolism

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.
Animals ; Cystatins ; genetics ; metabolism ; pharmacology ; Cysteine Proteinase Inhibitors ; genetics ; metabolism ; pharmacology ; Enzyme Activation ; drug effects ; Fish Proteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Protein Stability ; Temperature

AIMTo investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic (Cres) gene in mouse testis and epididymis during postnatal development.

METHODSThe QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420.

RESULTS(1) In both the testis and epididymis, Cres mRNA was first detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. (2) In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. (3) The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout.

CONCLUSIONThe Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.

Aging ; genetics ; metabolism ; Animals ; Cystatins ; genetics ; metabolism ; Epididymis ; growth & development ; metabolism ; Fluorescent Antibody Technique, Indirect ; Gene Expression Regulation, Developmental ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatids ; cytology ; metabolism ; Testis ; growth & development ; metabolism

According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD86:6His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.
Cloning, Molecular ; Cystatins ; biosynthesis ; genetics ; Cysteine Endopeptidases ; metabolism ; Cysteine Proteinase Inhibitors ; genetics ; Escherichia coli ; genetics ; metabolism ; Genes, Plant ; genetics ; Mutation ; Oligonucleotides ; chemical synthesis ; genetics ; Oryza ; genetics ; Papain ; antagonists & inhibitors ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism

OBJECTIVETo clone the full-length Rcet3 gene, a novel gene related to family 2 cystatins, from mouse testis or other tissues.

METHODSRcet3 gene was cloned using digital differential display (DDD) and RT-PCR was performed for cloning the full-length Rcet3 gene from adult mouse testis cDNA library with sequence analysis.

RESULTSRcet3 cDNA was 610 bp in length, consisting of 4 exons to encode a protein with 140 amino acid residues. The encoded protein contained a potential signal peptide and a cystatin domain, but lacked critical consensus site important for cysteine protease inhibition. These characteristics could be seen in the Cres subgroup related to the family 2 cystatins. Rcet3 was specifically expressed in adult mouse testis, epididymis and the cerebrum, but at higher levels in the testis than in the epididymis and cerebrum.

CONCLUSIONRcet3 may be a new member of Cres subgroup of family 2 cystatins.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Cystatins ; genetics ; DNA, Complementary ; chemistry ; genetics ; Gene Expression Profiling ; Male ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Testis ; metabolism

The CRES (cystatin-related epididymal spermatogenic) protein defines a new subgroup in the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, unlike the ubiquitous expression of cystatin C, the Cres gene is preferentialy expressed in postmeiotic germ cells, the proximal caput epididymidis, and anterior pituitary gonadotrophs. Furthermore, CRES protein lacks two of the three consensus sites necessary for the cystatin inhibition of C1 cysteine proteases. Therefore, CRES may perform unique and tissue-specific functions in the reproductive and neuroendocrine systems. In the present review, we describe our studies on: 1. the Cres gene promoter and the transcriptional regulatory protein and their associated DNA binding sites that may be important for tissue-specific expression; and 2. the biochemical function of CRES protein. In brief, Northern blot, gel shift analyses, and transient transfection assays demonstrated that the C/EBP beta (CCAAT/enhancer binding protein) transcription factor is the predominant C/EBP family member expressed in the epididymis and gonadotroph cells and is necessary for high levels of Cres expression in these two tissues. In other studies, analyses of transgenic mice expressing a CAT reporter gene driven by 1.6 kb of Cres promoter revealed CAT mRNA and protein only in the germ cells. These studies suggest that the 1.6 kb of Cres 5' flanking sequence contains the required DNA elements for expression in the testis, but lacks the elements to correctly target expression of the reporter gene in the epididymis. Alternatively, repressor elements may be present. Finally, in vitro protease assays were performed to determine if CRES functions as a protease inhibitor. In contrast to cystain C, CRES did not inhibit the C1 cysteine protease papain but rather inhibited at nanomolar concentrations the serine protease PC2, a prohormone processing enzyme. Therefore, CRES is a new cross-class inhibitor that may regulate PC2 of PC2-like proteases and suggests a role for CRES in the regulation of prohormone and proprotein processing.
Amino Acid Sequence ; Animals ; CCAAT-Enhancer-Binding Protein-beta ; biosynthesis ; Cystatins ; classification ; genetics ; physiology ; Epididymis ; metabolism ; Gene Expression Regulation ; Humans ; Male ; Mice ; Mice, Transgenic ; Promoter Regions, Genetic ; genetics ; Sequence Homology, Amino Acid ; Spermatogenesis ; physiology

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-gamma and TNF-alpha in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-alpha in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.
Animals ; Antigens, Differentiation/analysis ; Clonorchis sinensis/*enzymology ; Colon/pathology ; Cystatins/*metabolism ; Cytokines/secretion ; Dextran Sulfate/toxicity ; Female ; Helminth Proteins/*metabolism ; Immunologic Factors/*metabolism ; Inflammation/chemically induced/*pathology ; Interleukin-10/analysis ; Intestines/*drug effects/pathology ; Lymph Nodes/immunology ; Macrophages/chemistry/*immunology ; Mice ; Mice, Inbred C57BL ; Severity of Illness Index ; Spleen/immunology

Antigens, Neoplasm ; genetics ; Apoprotein(a) ; genetics ; Carrier Proteins ; genetics ; Endometriosis ; metabolism ; pathology ; Endometrium ; metabolism ; pathology ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Membrane Proteins ; genetics ; Mutation, Missense ; genetics ; Nuclear Proteins ; genetics ; Ovarian Neoplasms ; metabolism ; pathology ; Proprotein Convertase 5 ; genetics ; Salivary Cystatins ; genetics ; Ubiquitin-Protein Ligases ; genetics ; Whole Exome Sequencing