Classification and Nomenclature of Plant Metallothionein-like Proteins Based on Their Cysteine Arrangement Patterns

随着植物基因组研究的进展 ,在基因文库和蛋白文库登录的植物类金属硫蛋白基因已超过 5 0个 ,接近金属硫蛋白总数的 1/ 3,而且有不断上升的趋势。鉴于目前植物类金属硫蛋白命名与分类随意性太大 ,很有必要建立一个统一合理的命名与分类法。对植物类金属硫蛋白一级结构进行详细分析后 ,发现该蛋白两端富含半胱氨酸的区域内半胱氨酸的排列方式颇具规律性 ,进而提出了以半胱氨酸排列方式为基础的分类及命名法 ,并阐述了采用这种方法的理由及其可行性。

S-Trityl-L-Cysteine诱导HL-60细胞有丝分裂阻滞和凋亡

目的研究S-三苯甲基-L-半胱氨酸(S-Trityl-L-Cysteine,STLC)对急性白血病HL-60细胞有丝分裂阻滞和凋亡的影响,初步探讨药物作用下HL-60细胞有丝分裂阻滞和凋亡间的因果关系。方法将HL-60细胞分成不加药对照组和不同剂量(1、2.5、5、10、50、100μmol/L)STLC加药组,药物作用一定时间后,台盼蓝染色观察HL-60细胞活性变化,MTT法检测其对HL-60细胞的抑制效应,免疫荧光染色观察细胞及其核的特征,流式细胞术分析细胞周期和亚二倍体峰的变化,Annexin-v/PI双染检测药物处理前后正常骨髓细胞凋亡比率。结果MTT和台盼蓝染色实验显示STLC抑制HL-60细胞生长并致其死亡;免疫荧光染色见核破裂及凋亡小体和细胞体积增大等典型有丝分裂灾变细胞凋亡现象;细胞周期和凋亡分析表明STLC致HL-60细胞凋亡比率与药物浓度和作用时间呈正相关,致G2/M期阻滞比率24h和48h随药物浓度增加而升高,而72h无明显变化,进一步研究发现STLC处理早期即可致G2/M期有丝分裂阻滞和凋亡的同时发生,撤除药物作用后有丝分裂阻滞呈可逆性恢复,STLC作用下正常骨髓细胞凋亡比率无明显变化。结论STLC诱导HL-60细胞阻滞在G2/M期并致其凋亡,显示较强的抗有丝分裂和抗肿瘤效果,药物处理早期有丝分裂阻滞和凋亡同时发生提示尚有该药物作用新机制的存在。

Inhibition of cysteine protease papain by metal ions and polysulfide complexes,especially mercuric ion

Aim Cysteine proteases are closely associated with many human and non-human pathological processes and are potential targets for metal ions especially Hg^2＋ and the related species. In the present work, on the basis of to the general study on the effects of some metal ions on the activity of papain, a well-known representative of cysteine protease family, the inhibitory effects of Hg^2＋ and polysulfide complexes were studied. Results All the metal ions tested （Hg^2＋, Cu^2＋, Ag^＋, Au^3＋, Zn^2＋, Cd^2＋, Fe^3＋, Mn^2＋, Pb^2＋, Yb^3＋） inhibit the activity of papain anda good correlation between the inhibitory potency and softness-and-hardness was observed. Among the metals, Hg^2＋ was shown to be a potent inhibitor of papain with a Kiof 2 × 10^-7 mol·L^-1 among. Excessive amounts of glutathione and cysteine could reactivate the enzyme activity of papain deactivated by Hg^2＋. These evidences supported that Hg^2＋ might bind to the catalytic site of papain. Interestingly, Hg （Ⅱ） polysulfide complexes were for the first time found to inhibit papain with a Ki of 7 × 10^-6 mol·L^-1, whose potency is close to a well known mercury compound, thimerosal （Ki=2.7 × 10^-6）. In addition, Hg （Ⅱ） polysulfide complexes exhibit good permeability （ 1.9 × 10-5 cm· s^-1） to caco-2 monolayer. Conclusion These results suggested that mercury polysulfide complexes might be potential bioactive species in the interaction with cysteine proteases and other- SH-content proteins, providing a new clue to understand the mechanism of the toxicological and pharmacological actions of cinnabar and other insoluble mercury compounds.

Modeling the Cysteine Rich Domain of Plant Metallothionein-like Protein

With the progress of plant genome research, more than 50 plant metallothionein_like (MT_L) genes have been found, but only several MT_L proteins have been detected and no experimental structural information for MT_L proteins has been reported so far. Since detailed knowledge of the protein tertiary structure is required to understand its biological function, a method is needed to determine the structure of these proteins. In this study, the structural data of known mammal MT was used to determine the interatomic distance constraints of the CXC and CXXC motifs and the metal_sulfur chelating cluster. Then several possible MT conformations were predicted using a distance geometry algorithm. The statistical analysis was used to select those with much lower target function values and lower conformation energies as the predicted tertiary structural models of the cysteine_rich (CR) domains of these proteins. A suitable prediction method for modeling the CR domain of the plant MT_L protein was constructed. The accurately predicted result for the known structure of an MT protein from blue crab suggests that this method is practicable. The tertiary structures of CR domains of rape MT_L protein LSC54 was then modeled with this method.

Synergistic effects of elevated homocysteine level and abnormal blood lipids on the onset of stroke

Hyperhomocysteinemia and abnormal blood lipids are independent risk factors for stroke. However, whether both factors exert a synergistic effect in the onset of stroke remains unclear. The present study is a retrospective analysJs of 2 089 cases of stroke and 2 089 control cases of simple in- tervertebral disk protrusion using a paired multivariate logistic regression method. Adjusting for known confounding variables including the patients＇ age, gender, smoking status, alcohol con- sumption status, patient and family medical history, and clinical biochemical indices, elevated ho- mocysteine level was related to the onset of stroke. Patients with elevated homocysteJne levels and abnormal blood lipids showed a 40.9 % increase in the risk for stroke compared to patients with normal homocysteine levels and blood lipids （odds ratio 1.409; 95% confidence interval 1.127-1.761）. These results indicate that elevated homocysteine and abnormal blood lipids exert synergistic effects in the onset of stroke. Patients with elevated homocysteine levels and abnormal blood lipids are predisposed to stroke.

Cytoprotective effects of amifostine,ascorbic acid and N-acetylcysteine against methotrexate-induced hepatotoxicity in rats

AIM: To investigate the potential role of oxidative stress and the possible therapeutic effects of N-acetyl cysteine (NAC), amifostine (AMF) and ascorbic acid (ASC) in methotrexate (MTX)-induced hepatotoxicity.

Selectively colorimetric detection of cysteine with triangular silver nanoprisms

Triangular silver nanoprisms were prepared and applied to make colorimetric detection of cysteine based on our findings that cysteine could lead to the blue shift of the dipole plasmon resonance absorption, but other 19 kinds of natural amino acids could not. Cysteine with a concentration 160 nmol/L can result in a color change that can be discerned with naked eyes.

Aberrant methylation of secreted protein acidic and rich in cysteine gene and its significance in gastric cancer

BACKGROUND Aberrant methylation in DNA regulatory regions could downregulate tumor suppressor genes without changing the sequences.However,our knowledge of secreted protein acidic and rich in cysteine(SPARC)and its aberrant methylation in gastric cancer(GC)is still inadequate.In the present research,we performed fundamental research to clarify the precise function of methylation on SPARC and its significance in GC.AIM To investigate promoter methylation and the effects of the SPARC gene in GC cells and tissues and to evaluate its clinical significance.METHODS Plasmids that overexpressed the SPARC gene were transfected into human GC BGC-823 cells;non-transfected cells were used as a control group(NC group).Quantitative real-time polymerase chain reaction and western blotting(WB)were then used to detect the expression of SPARC.Methylation-specific polymerase chain reaction was executed to analyze the gene promoter methylation status.Cell viability was measured by the cell counting kit-8 assay.The migration and invasion ability of cells were detected by scratch assays and transwell chamber assays,respectively.Cell cycle events and apoptosis were observed with a flow cytometer.RESULTS The expression of SPARC mRNA in GC tissues and cells was significantly lower and showed differing degrees of hypermethylation,respectively,than that in normal adjacent tissues and control cells.Treatment with 5-Aza-2’-deoxycytidine(5-Aza-Cdr)was able to restore the expression of SPARC and reverse promoter hypermethylation.Overexpression of the SPARC gene significantly inhibited proliferation,migration,and invasion of GC cells,while also causing cell cycle arrest and apoptosis;the NC group exhibited the opposite effects.CONCLUSION This study demonstrated that SPARC could function as a tumor suppressor and might be silenced by promoter hypermethylation.Furthermore,in GC cells,SPARC inhibited migration,invasion,and proliferation,caused cell cycle arrest at the G0/G1 phase,and promoted apoptosis.

Characterization of Cysteine Coated Magnetite Nanoparticles as MRI Contrast Agent

In this work,a kind of stabilized ferrofluid based on magnetite nanoparticles(mean core and its coating size about 21.9 and 1.6 nm,respectively)was synthesized via coprecipitation method.Cysteine was used as surfactant due to its proper conjunction to the surface of magnetite nanoparticles.Coating density and synthesized ferrofluids were characterized by using transmission electron microscope,thermogravimetry analysis,dynamic light scattering and fourier transform infrared spectroscopy techniques.Magnetic resonance imaging studies show that the synthesized ferrofluid can be used as a potential contrast enhancement agent especially for imaging lymphatic system.

Assay of Cysteine in Human Serum with Quinine-Ce^(4+) Chemiluminescence System

A sensitive and selective chemiluminescence (CL) method was developed for the determination of cysteine. This method is based on that the weak CL of cysteine oxidized with cerium (IV) can be greatly enhanced by quinine, and the total cysteine in human serum can be detected through simply diluting with water, showing a simpler analytical characteristic.

Flow-injection Determination of Cysteine,N-Acetyl Cysteine and Glutathione in Pharmaceuticals via Potassium Ferricyanide-Fe(Ⅲ) Spectrophotometric System

A simple flow injection spectrophotometric method is reported for the determination of cysteine,N-acetyl cysteine and glutathione based on the reduction of Fe（Ⅲ）/ferricyanide,the in situ reduced ions are reacted with unreduced portion of ferricyanide/Fe（Ⅲ） to form soluble Prussian blue,which is monitored at 735 nm.The calibration graphs are linear in the concentration ranges of（1―100）×10-6 mol/L for cysteine and N-acetyl cysteine,and（1―50）×10-6 mol/L for glutathione.The relative standard deviations of 1.8%,2.5% and 1.9% were found for eleven replicate analyses of 5×10-6 mol/L cysteine,N-acetyl cysteine and glutathione.The limits of detection（3σ blank） at 5×10-7 mol/L for cysteine,and 3×10-7 mol/L for N-acetyl cysteine and glutathione were obtained.The proposed method allowed 60 injections/h.The effects of common substances present in pharmaceuticals and human physiological fluids were examined.The method was applied to determining cysteine in pharmaceutical formulations with the recoveries in a range of 97% to 106% and the results obtained are agreed well with labeled values.

A Fluorescence Ratiometric Probe for Cysteine/Homocysteine and Its Application for Living Cell Imaging

A fluorescence ratiometric probe 1 for cysteine (Cys) and homocysteine (Hcy) has been rationally constructed based on intramolecular charge transfer (ICT) mechanism. Upon treatment with Cys/Hcy, probe 1 exhibited a fluorescence ratiometric response, with the emission wavelength displaying a large shift (from 526 nm to 446 nm). When 90 μM Cys were added, the emission ratios (I446/I526) of the probe changed dramatically from 0.01797 to 4.65472. The detection limit was also measured to be 0.18 μM (S/N = 3). The theoretical calculations have confirmed that the ratiometric response of probe 1 to Cys/Hcy is due to the inhibition of ICT process upon the reaction of probe 1 with Cys/Hcy. Furthermore, the fluorescence imaging experiments in living cell demonstrated that probe 1 was favourable for intracellular Cys/Hcy imaging.

An Elevated Dietary Cysteine to Methionine Ratio Does Not Impact on Dietary Methionine Efficiency and the Derived Optimal Methionine to Lysine Ratio in Diets for Meat Type Chicken

Optimal dietary methionine (Met) to lysine (Lys) ratio in presence of elevated dietary cysteine (Cys) levels was derived for meat type growing chicken. Twelve averaged weighed Ross 308 birds (each 50% of male and female per dietary treatment) were utilized in N balance trials. During starter (d10 - 20) and grower period (d25 - 35) five dietary treatments were used. Diets based on uniform mixtures of maize, wheat, soybean meal, potato protein and fish meal were supplemented with crystalline amino acids (AA). In diets 1 - 3, the dietary Cys to Met ratio was set as 85, 95 and 105 to 100, respectively. Diet 4, at a Cys to Met ratio of 105 to 100, was additionally supplemented with betaine (BET) as methyl group donor. Diets 1 - 4 were limiting in Met, diet 5 without L-Lys·HCl addition was limiting in Lys. Individual N-balance data per treatment were utilized for assessing protein quality and efficiency of dietary Met (Diets 1 - 4) or Lys (Diet 5) based on “Goettingen approach”. Elevated dietary Cys supply and supplemented BET failed to improve both dietary protein quality and Met efficiency. The established optimal Met to Lys ratio was on average 34 to 100 for growing chicken during starter and grower period, respectively.

Contribution of lysosomal cysteine proteases in cardiac and renal diseases

Cardiac and renal diseases(CRDs) are characterized by extensive remodeling of the extracellular matrix(ECM)architecture of the cardiorenal system. Among the many extracellular proteolytic enzymes present in cardiorenal cells and involved in ECM remodeling, members of the matrix metalloproteinase family and serine protease family have received the most attention. However, recent findings from laboratory and clinical studies have indicated that cysteine protease cathepsins also participate in pathogenesis of the heart and kidney.Deficiency and pharmacological inhibition of cathepsins have allowed their in vivo evaluation in the setting of pathological conditions. Furthermore, recent studiesevaluating the feasibility of cathepsins as a diagnostic tool have suggested that the serum levels of cathepsins L, S and K and their endogenous inhibitor cystatin C have predictive value as biomarkers in patients with coronary artery disease and heart and renal failure. The goal of this review is to highlight recent discoveries regarding the contributions of cathepsins in CRDs, particularly hypertensive heart failure and proteinuric kidney disease.

Effect of vitamin E and human placenta cysteine peptidase inhibitor on expression of cathepsins B and L in implanted hepatoma Morris 5123 tumor model in Wistar rats

AIM: To examine the effectiveness of human placental inhibitors, by injecting vitamin E to rats with transplanted Morris-5123 hepatoma, on the expression of cathepsins B and L in tumor, liver, lung and blood sera after transplantation of Morris 5123 hepatoma. METHODS: Animals were divided into 10 groups receiving three different concentrations of vitamin E and inhibitors along or in combination and compared with negative control (healthy rats) and positive control (tumor rats). Effectiveness of treatment was evaluated with regard to survival time, tumor response and determination of the activities of proteolytic enzymes and their inhibitors using flurogenic substrates. RESULTS: Cathepsins B and L activities were elevated by 16-fold in comparison with negative control tissues, and their endogenous inhibitor activity decreased by 1.2-fold before treatment. In several cases, tumors completely disappeared following vitamin E plus human placental cyteine protease inhibitor (CPI) compared with controls. The number of complete tumor responses was higher when 20 m/kg vitamin E plus 400 μg of CPI was used, i.e. 7/10 rats survived more than two mo. Cathepsins B and L were expressed significantly in tumor, liver, lung tissues and sera in parallel to the increasing of the endogenous inhibitor activity compared with the controls after treatment(P<0.0001) CONCLUSION: The data indicate formation of metastasis significantly reduced in treated rats, which might provide a therapeutic basis for anti-cancer therapy.

Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation

Rat protein DE is an androgen-dependent cysteine-rich secretory protein （CRISP） synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 （S2）. The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx- 1 （CRISP-2） was capable of binding to rodent eggs, human epididymal AEG-related protein （ARP） and helothermine （from lizard saliva） were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx- 1 and anti-Tpx- 1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx- 1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods. （Asian J Androl 2007 July; 9： 528-532）

Cysteine peptidase and its inhibitor activity levels and vitamin E concentration in normal human serum and colorectal carcinomas

AIM: Cysteine peptidase (CP) and its inhibitor (CPI) are a matrix protease that may be associated with colorectal carcinoma invasion and progression, and vitamin E is also a stimulator of the immunological system. Our purpose was to determine the correlation between the expression of cysteine peptidases and their endogenous inhibitors,and the level of vitamin E in sera of patients with colorectal cancer in comparison with healthy individuals.METHODS: The levels of cysteine peptidases and their inhibitors were determined in the sera of patients with primary and metastatic colorectal carcinoma and healthy individuals using fluorogenic substrate, and the level of vitamin E was determined by HPLC.RESULTS: The levels of cysteine peptidases and their inhibitors were significantly higher in the metastatic colorectal cancer patients than that in the healthy controls (P＜0.05).The activity of CP increased 2.2-fold, CPI 2.8-fold and vitamin E decreased 3.4-fold in sera of patients with metastasis in comparison with controls. The level of vitamin E in healthy individuals was higher, whereas the activity of cysteine peptidases and their inhibitors associated with complexes was lower than that in patients with cancer of the digestive tract.CONCLUSION: These results suggest that the serum levels of CP and their inhibitors could be an indicator of the prognosis for patients with metastatic colorectal cancer. Vitamin E can be administered prophylactically to prevent digestive tract neoplasmas.

Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk （LEY） extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L^-1 （group Ⅰ, control）, 5 mmol L^-1 （group Ⅱ）, 10 mmol L^-1 （group Ⅲ） and 15 mmol L^-1 （group Ⅳ）. Semen suspensions were loaded in straws （0.5 mL） and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher （P 〈 0.01） percentage of progressive motility, viability and acrosomal integrity in two L-cysteine-supplemented groups （group Ⅱ and group Ⅲ） compared with the control. There was a biphasic effect of L-cysteine, with the highest percentage of progressive motility, viability and acrosomal integrity in group Ⅲ. In conclusion, 5 or 10 mmol L^-1 was the optimum concentration of L-cysteine to be added to the LEY extender for improving the quality of frozen-thawed boar semen.

Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen

The objective of the present study was to determine the effects of docosahexaenoic acid （DHA）-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk （group Ⅰ）, DHA-enriched hen egg yolk （group Ⅱ）, normal hen egg yolk with 5 mmol L^-1 of cysteine supplementation （group Ⅲ） and DHA-enriched hen egg yolk with 5 mmol L1 of cysteine supplementation （group Ⅳ）. The semen was cryopreserved using controlled rate freezer and was thawed at 50℃ for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone （group Ⅲ） improved progressive motility （P 〈 0.05）, and the supplementation of L-cysteine in combination with DHA-enriched hen egg yolk （group Ⅳ） improved both progressive motility （P 〈 0.05） and acrosome integrity （P 〈 0.01）. The use of DHA-enriched hen egg yolk alone （group Ⅱ） did not enhance any of the post-thawed semen qualities （P 〉 0.05）. In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity.

Structure and function of epididymal protein cysteine-rich secretory protein-1

Cysteine-rich secretory protein-1 （CRISP-1） is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP- 1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract. （Asian J Androl 2007 July; 9： 508-514）