The epididymal lumen represents a unique extracellular environment because of the active sperm maturation process that takes place within its confines. Although much focus has been placed on the interaction of epididymal secretory proteins with spermatozoa in the lumen, very little is known regarding how the complex epididymal milieu as a whole is maintained, including mechanisms to prevent or control proteins that may not stay in their native folded state following secretion. Because some misfolded proteins can form cytotoxic aggregate structures known as amyloid, it is likely that control/surveillance mechanisms exist within the epididymis to protect against this process and allow sperm maturation to occur. To study protein aggregation and to identify extracellular quality control mechanisms in the epididymis, we used the cystatin family of cysteine protease inhibitors, including cystatin-related epididymal spermatogenic and cystatin C as molecular models because both proteins have inherent properties to aggregate and form amyloid. In this chapter, we present a brief summary of protein aggregation by the amyloid pathway based on what is known from other organ systems and describe quality control mechanisms that exist intracellularly to control protein misfolding and aggregation. We then present a summary of our studies of cystatin-related epididymal spermatogenic (CRES) oligomerization within the epididymal lumen, including studies suggesting that transglutaminase cross-linking may be one mechanism of extracellular quality control within the epididymis.
Amino Acid Substitution ; Amyloid ; physiology ; standards ; Cystatin C ; Cystatins ; genetics ; Dimerization ; Epididymis ; physiology ; Humans ; Male ; Mutation ; Protein Folding ; Proteins ; standards ; Quality Control ; Sperm Maturation ; physiology ; Transglutaminases ; physiology

Biomarkers ; urine ; Child ; Child, Preschool ; Cystatin C ; Cystatins ; urine ; Cytomegalovirus Infections ; pathology ; urine ; Female ; Humans ; Kidney ; pathology ; Male ; TATA Box Binding Protein-Like Proteins ; urine ; alpha-Macroglobulins ; urine

BACKGROUND: Cystatin C (cysC) is said to be an ideal marker for glomerular filtration rate (GFR), independent of external factors such as age, nutrition and inflammation. The authors compared the accuracy and precision of cysC-based and creatinine (Cr)-based GFR estimates using Cr51-EDTA GFR method as a reference. METHODS: Serum concentrations of cysC and Cr were measured in adults over 17 yr (n=170) and children below 17 yr (n=79) who had had GFR estimated by Cr51-EDTA method. CysC-based GFR was estimated by the formula of Thierry [CysC-based GFR estimates (mL/min/1.73 m2)=78 x (1/cysC, in mg/L)+4] and Cr-based GFR by the formula of modified Modification of Diet in Renal Disease [MDRD II, Cr-based GFR estimates (mL/min/1.73 m2)=186 x (Scr)(-1.154) x (Age)(-0.203) x 0.742 (for a female patient) x 1.212 (for a black patient). RESULTS: In comparison with Cr51-EDTA GFR, in children below 17 yr, the bias +/- standard deviation (SD) of cysC-based and Cr-based GFR estimates were 7.5 +/- 6.1 and 106.5 +/- 98.2, respectively, in the range of below 90 of Cr51-EDTA GFR (mL/min/1.73 m2), and 33.7 +/- 33.0 and 174.4 +/- 18.8 in the range of over 90. In adults over 17 yr, the respective figures were 13.1 +/- 11.0 and 17.4 +/- 29.8 in below 90, and 21.2 +/- 20.1 and 83.6 +/- 108.8 in over 90 of Cr51-EDTA GFR. CONCLUSIONS: CysC-based GFR estimates show acceptable ranges of biases over the whole age and GFR ranges. CysC-based GFR estimates is considered to be the marker for GFR, which could be used without limitation of age and GFR ranges.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biological Markers/urine ; Child ; Chromium Radioisotopes/diagnostic use ; Creatinine/*urine ; Cystatin C ; Cystatins/*urine ; Edetic Acid/diagnostic use ; Female ; Glomerular Filtration Rate/*physiology ; Humans ; Male ; Middle Aged ; Organometallic Compounds/diagnostic use

The principal aims of this study were to identify the composition of salivary pellicles formed on various orthodontic brackets and to obtain a detailed information about the protein adsorption profiles from whole whole saliva and two major glandular salivas. Four different types of orthodontic brackets were used. All were upper bicuspid brackets with a 022 x 028 slot Roth prescription; stainless steel metal, monocrystalline sapphire, polycrystalline alumina, and plastic brackets. Bracket pellicles were formed by the incubation of orthodontic brackets with whole saliva, submandibular-sublingual saliva, and parotid saliva for 2 hours. The bracket pellicles were extracted and confirmed by employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western transfer methods, and immunodetection. The results showed that low-molecular weight salivary mucin, alpha-amylase, secretory IgA (sIgA), acidic proline-rich proteins, and cystatins were attached to all of these brackets regardless of the bracket types. High-molecular weight mucin, which promotes the adhesion of Streptococcus mutans, did not adhere to any orthodontic brackets. Though the same components were detected in all bracket pellicles, however, the gel profiles showed qualitatively and quantitatively different pellicles, according to the origins of saliva and the bracket types. In particular, the binding of sIgA was more prominent in the pellicles from parotid saliva and the binding of cystatins was prominent in the pellicles from the form plastic brackets. This study indicates that numerous salivary proteins adhere to the orthodontic brackets and these salivary proteins adhere selectively according to bracket types and the types of the saliva.
Adsorption ; alpha-Amylases ; Aluminum Oxide ; Bicuspid ; Cystatins ; Electrophoresis ; Immunoglobulin A, Secretory ; Mucins ; Orthodontic Brackets* ; Plastics ; Prescriptions ; Saliva ; Salivary Proteins and Peptides* ; Sodium ; Stainless Steel ; Streptococcus mutans

In this study, we evaluated the performance of a recently developed immunoassay analyser, the VISTA 500 (Siemens, Germany). Precision, linearity, and comparison studies were performed according to the Clinical and Laboratory Standards Institute guidelines. The test items evaluated included IgG, IgA, IgM, C3, C4, ceruloplasmin, prealbumin, transferrin, haptoglobin, rheumatoid factor, anti-streptolysin O, and cystatin C. Commercial control materials (BioRad Laboratories, USA), commercial linearity validation materials (Maine Standards, USA), and patient samples were used for the evaluation. For the correlation study, analysis with a BN-II nephelometer (Siemens) was used as a comparative method. Total coefficients of variation of analytes were found to be between 1.9% and 5.5%. Results of the linearity evaluation were also acceptable for the range tested. Correlations with comparative methods were acceptable. The VISTA 500 analyser showed satisfactory analytical performance with respect to precision, linearity, and comparison. We conclude that the VISTA 500 is likely a good candidate as an immunology analyser.
Allergy and Immunology ; Ceruloplasmin ; Cystatin C ; Evaluation Studies as Topic ; Haptoglobins ; Humans ; Immunoassay ; Immunoglobulin A ; Immunoglobulin G ; Immunoglobulin M ; Prealbumin ; Rheumatoid Factor ; Statistics as Topic ; Transferrin

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

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

<b>METHODSb>The 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.

<b>RESULTSb>Immunohistochemistry 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).

<b>CONCLUSIONb>CRES 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

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

<b>OBJECTIVEb>To investigate the expression pattern of the cystatin-related epididymal spermatogenic (Cres) gene in mouse testes and epididymis during postnatal development.

<b>METHODSb>Semi-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.

<b>RESULTSb>Low-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.

<b>CONCLUSIONb>The 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