Comparison of Superoxide Dismutase Isoenzymes in Three Shrimps

[Objective] The aim of the study is to compare the activities of superoxide dismutase(SOD) isoenzymes in three species of shrimps Penaeus japonicus, Procambarus clarkia and Litopenaeus vannamei. [Method] The experimental materials were used to measure SOD activities after pretreatment, meanwhile the differences in SOD isoenzymes from different materials were assayed by polyacrylamide gel electrophoresis (PAGE). [Result] There are specific and histological differences in SOD activities of shrimps. With a similar electrophoresis pattern and migration rate, Penaeus japonicus and Litopenaeus vannamei showed remarkable differences with that of Procambarus clarkia. [Conclusion] The result showed the differences of cognation and origin of three shrimps.

Expression and kinetic changes of alkaline phosphatase and its isoenzymes in experimental rat hepatoma

AIM To explore the expression and changes of hepatoma specific alkaline phospatase (ALP) in rats during canceration.METHODS The ALPs and isoenzymes of rat livers and sera were investigated in SD hepatomas induced with 0.05% 2-fluoenylacetamide (2-FAA).RESULTS By pathological examination and biochemical analysis. ALPs were overexpressed in rat livers during canceration and then were secreted into blood. Serum total ALP activities, liver ALP specific activities (U/g) including soluble and membrane-combined ALP activities of each group were all significantly higher (P＜0.01) than those of control group. The average ratios of soluble ALP to membrane-combined ALP were increased significantly after 6 weeks. ALP isoenzymes of rat sera and livers showed 5 bands on PAGE: ALP-Ⅰ and ALP-Ⅱ were specific for normal liver and rat hepatoma tissues, the ALP-Ⅱ appeared in rat liver after 6 weeks and in sera after 8 weeks.CONCLUSIONS ALP with carcino-embryonic protein was overexpressed in hepatoma tissues; the abnormal ALP-Ⅱ of ALP isoenzymes in sera and liver of rats can be used as a tumor marker for early diagnosis of rat hepatoma.

Adenosine deaminase isoenzymes estimation - as a diagnostic tool for tuberculous pleural effusions

Objective:To assess the efficacy of ADA isoenzyme estimation over that of total ADA level in pleural fluid and serum as a more efficient diagnostic indicator in tuberculous pleural effusions in high prevalence country like India.Methods:The efficacy was analysed in total thirty four patients of pleural effusions.Total ADA was estimated by Guitsi and Galanti Calorimetric method and ADA isoenzymes with and without EHNA[Erythro-9-（2- hydroxy-3-nonyl） adenine]a potent ADA1 inhibitor using the same method.Results:The results demonstrated a statistically significant values of ADA2 in serum（P【0.001）,pleural fluid（P = 0.000） and significant value for the ratio of pleural fluid ADA2/serum ADA2（P【0.001） and pleural fluid ADA/ADA(2（P【0. 005）.The sensitivity and specificity values of pleural fluid ADA|2 is 81.8%and 91.6%（cut off value 60 IU/L for Tuberculous effusions）,serum ADA2 95.4%and 66%（cut off value 70 IU/L for tuberculous effusions）. ADA2 is an isoenzyme,which is significantly raised in tuberculous pleural effusions both in the serum and pleural fluid.Conclusion:Estimation of ADA isoenzymes is redundant as a diagnostic aid over total ADA estimation in view of the limited improvements both in specificity and sensitivity patterns and also in term of cost-benefit ratio.

UV-B irradiation and allelopathy by Sargassum thunbergii affects the activities of antioxidant enzymes and their isoenzymes in Corallina pilulifera

Intertidal macroalgae can cope with the dual effects of UV-B irradiation and allelopathy.To study the impacts of the two stressors,we co-cultured Corallina pilulifera with Sargassum thunbergii in 1:1 and 1:10 ratios under different doses of UV-B radiation.The response of the antioxidant defense system,focusing on activities of superoxide dismutase(SOD),ascorbate peroxidase(APX),peroxidase(POX)and glutathione reductase(GR),was monitored.In addition,isoenzyme patterns were analyzed using nondenaturing poly acry lamide gel electrophoresis.The results show that the activities of SOD,APX,and GR were all significantly affected by both UV-B radiation and allelopathy,and the effect of their interaction was significant.However,POX activity was only influenced by UV-B radiation.The enzymatic assay revealed four distinct bands of SOD.The SODⅢband weakened significantly when the co-cultures were exposed to extremely high dosage of UV-B irradiation under both co-culturing ratios of 1:1 and 1:10.When the coculturing ratio was 1:10,both POXⅡand APXⅡenzy me activities increased with different UV-B doses.GR activity was at its greatest when the co-culture ratio was 1:10 and exposure was to the higher UV-B doses.The activities of GRⅢand GRⅣwere elevated under all UV treatments whereas the activities of GRI and GRII were reduced under the lower UV-B treatments but were elevated under the higher UV-B treatments.However,lipid peroxidation,as indicated by the thiobarbituric acid-reacting substance(TBARS)assay,increased significantly under the dual stressors.Our data sugge st that allelopathy and UV-B radiation stre ss can each affect the antioxidant enzyme activities of C.pilulifera.Critically,the adverse effects of UV-B on C.pilulifera were intensified by the compounding effects of allelopathy.

AN ENZYMOCYTOCHEMICAL STUDY OF ALKALINE PHOSPHATASE ISOENZYMES IN GASTRIC CANCER-LIGHT AND ELECTRON MICROSCOPIC OBSERVATIONS

By using light and electron microscopic cytochemical technique, the activities and distributions of AP isoenzymes in gastric cancers and benign gastric diseases were examined.The results showed: Nagao, Regan and Kasahara isoenzymes, which were not expressed in normal gastric mucosae and non-malignant lesions, might be considered as tumor markers of gastric cancer;The epithelium of intestinal metaplasia exhibited intestinal-type AP only,which was one of the markers of well-differentiated intestinal metaplasia.In the view-point of the gene expression of AP isoenzymes, two mutation hypothesis and recessive-gene mutation hypothesis might be fit for gastric cancer.

Lactate Dehydrogenase Isoenzymes in Mammalian Cells Exposed to Isophenphos

The cytotoalcity of isophenphos, an organophosphorus insecticide that has the potential to cause delayed polyneuropathy, was evaluated in GBK and V79 ce1ls. A 72 h time course following isophenphos exposure indicated a dose-dependent growth inhibition as dctermined by cell counts. The administration of isophenphos (20g/ml) to GBK cells cultured at high densities indicated a decrease in the activities of LDH isocnzymes. Analysis of V79 cells revealed a decrease of LDH3, the only LDH isocnzyme detected in these cells.

Genetic variability of the Saudi Arabian Uromastyx aegyptia microlepis using protein and isoenzymes electrophoreses

Electrophoresis for SDS-proteins and isoenzymes were conducted to investigate the genetic variations within the agamid lizard Uromastyx aegyptia microlepis inhabiting the desert of Saudi Arabia. Samples were collected from four localities: a) Ushayrah near the town of Taif, b) Al Gwaih near Riyadh, c) Nairyah near Dammam and d) Mouileh near Tabok. A range of 7 to 14 protein bands were recorded in the patterns of the studied samples as measured by SDS-polyacrylamide gel electrophoresis. Among them, only one fraction was recorded in all samples as a common protein band. Six arbitrary chosen enzymes were examined by native-polyacrylamide gel electrophoresis. They were α and β esterase (Est), acid phosphatase (Acph), Alcohol dehydrogenase (Adh), Aldehyde oxidase (Ao) and peroxidase (Px). Seventeen heterogeneous alleles have been recorded;seven of them were fixed in all populations and 10 were polymorphic. Nearly all recorded alleles were monomeric in all samples. α-Est2, β-Est2, Acph2 and Px1 were restricted to Tabok samples and were not recorded in other localities. β-Est3, Acph3, Adh1, Adh1and Px2 were not recorded in Taif samples and the latter one was not recorded in the Dammam samples. The similarity coefficient that has been calculated according to the number of sharing bands indicated the clustering of Tabok and Dammam populations together. The constructed tree based on the sharing protein bands and isozyme alleles revealed similar results regarding the kinship of both populations. The present results indicated that the populations of this subspecies exhibits high genetic variability among its populations.

Isoenzymes and soluble protein in Arthrospira from alka-line lakes in Erdos Plateau, China and in exotic species

The authors compared isoenzymes of five enzymes and soluble protein in Arthrospira platensis (A3) and A. erdosensis (A4) from alkaline lakes in Erdos Plateau, Nei Monggo (Inner Mongalia), China and exotic species of A. platensis (A1) from Chad and A. maxima (A2) from Mexico by means of polyacrylamide gel electrophoresis. The results showed that the isoenzymes of EST, POD and soluble protein were polymorphic. Monomorphism and polymorphism were found in isoenzymes of AMY, CAT and SOD, and monomorphism was found only in the introduced species. The isoenzymes and soluble protein of the local species are all polymorphic. The number of bands in these species were in the order of A3>A4>A1>A2. A2 is the most primary, A1 from Chad Lake is relatively primary, A3 and A4 are ad- vanced species in evolution. Cluster analysis showed that the relation between the two introduced species are the closest to each other, and so too are the two local ones.

Purification of Active Peroxidase Isoenzymes and Their Responses to Nitrogen Fertilization and Rotation of Biomass Sorghum

Peroxidases (EC 1.11.1.7) participate in lignin biosynthesis. But peroxidation is not a tool for assaying lignocellulose metabolism because the active cannot yet be separated from the inactive peroxidases. A biochemical tool for assaying plant cell wall responses to agronomic practices is needed in the lignocellulosic feedstock renewable energy industry. Peroxidase of biomass sorghum was purified to 9 - 13 charge isomers by free solution IEF (Rotofor) technique. Free solution IEF was more effective than chromatographic purification of active peroxidase isoenzymes. Native PAGE separated each charge isomer to three anionic and three cationic isoenzymes. Hydrogen peroxide and o-dianisidine assays showed that only 20% - 30% of the isoenzymes displayed normal Michaelis-Menten kinetics. Sorghum planted without nitrogen fertilization induced the hydrogen peroxide noncompetitive inhibition of peroxidase, but 280 kg·ha–1 nitrogen fertilization and 100% sorghum mineral residue return to the soil tripled the concentration of active peroxidase and relieved the inhibition with concomitant increases of 350 kg lignin and 3532 kg·cellulose·ha–1. Nitrogen fertilization without crop rotation induced hydrogen peroxide inhibition of the peroxidase, but nitrogen fertilization and 25% sorghum rotation changed the PI of the active peroxidase from neutral to mildly acidic and relieved the inhibition with concomitant enormous increases of 690 kg lignin and 7151 kg·cellulose·ha–1. Hydrogen peroxide inhibition kinetics is consistent with the known peroxidase-substrate intermediate dead-end complex formation. Lignocellulosic yield was greatest under the agronomic management that combined 280 kg·ha–1 nitrogen fertilizer with 25% sorghum residue, which resulted in a shift of pI value of the active peroxidase due to a reduction in the Km value of the peroxidase. Therefore, up to 75% of sorghum biomass rather than only 50% can be harvested for conversion to bioenergy products.

Clinical and preclinical treatment of urologic diseases with phosphodiesterase isoenzymes 5 inhibitors： an update

Phosphodiesterase isoenzymes 5 inhibitors （PDE5-1s） are the first-line therapy for erectile dysfunction （ED）. The constant discoveries of nitric oxide （NO）/cyclic guanosine monophosphate （cGMP） cell-signaling pathway for smooth muscle （SM） control in other urogenital tracts （UGTs） make PDE5-1s promising pharmacologic agents against other benign urological diseases. This article reviews the literature and contains some previously unpublished data about characterizations and activities of PDE5 and its inhibitors in treating urological disorders. Scientific discoveries have improved our understanding of cell-signaling pathway in NO/cGMP-mediated SM relaxation in UGTs. Moreover, the clinical applications of PDE5-1s have been widely recognized. On-demand PDE5-1s are efficacious for most cases of ED, while daily-dosing and combination with testosterone are recommended for refractory cases. Soluble guanylate cyclase （sGC） stimulators also have promising role in the management of severe ED conditions. PDE5-1s are also the first rehabilitation strategy for postoperation or postradiotherapy ED for prostate cancer patients. PDE5-1s, especially combined with （z-adrenoceptor antagonists, are very effective for benign prostatic hyperplasia （BPH） except on maximum urinary flow rate （Qmax） with tadalafil recently proved for BPH with/without ED. Furthermore, PDE5-1s are currently under various phases of clinical or preclinical researches with promising potential for other urinary and genital illnesses, such as priapism, premature ejaculation, urinary tract calculi, overactive bladder, Peyronie＇s disease, and female sexual dysfunction. Inhibition of PDE5 is expected to be an effective strategy in treating benign urological diseases. However, further clinical studies and basic researches investigating mechanisms of PDE5-1s in disorders of UGTs are required.

Possible genetic reproductive isolation between two tilapiine genera and species:Oreochromis niloticus and Sarotherodon melanotheron

Successful crossbreeding between Oreochromis niloticus and Sarotherodon melanotheron to produce a commercial hybrid has been difficult.The karyotypes and isoenzyme of these two species and their reciprocal hybrids（O.niloticus ♀ × S.melanotheron ♂,S.melanotheron ♀ × O.niloticus ♂,the last not included in the isoenzyme study） were investigated via metaphase chromosomes obtained from head kidney cells and electropherogram of lactate dehydrogenase（LDH） isoenzymes from the liver,kidney,white muscle,heart,and eye balls.The diploid chromosome number（2n=44） and the fundamental number（NF=50） of the four tilapia genotypes were the same.However,the karyotype of O.niloticus had three pairs of sub-metacentric（sm）,twelve pairs of sub-telocentric（st）,and seven pairs of telocentric（t） chromosomes,while S.melanotheron had one pair of metacentric（m）,two pairs of sm,12 pairs of st,and seven pairs of t chromosomes.The reciprocal hybrids both showed a mixed karyotype range between their parents：0.5 pair of m,2.5 pairs of sm,12 pairs of st,and seven pairs of t chromosomes.In view of the electropherogram of isozymes,only the LDH of the kidney showed significant clear bands,with five bands in O.niloticus,three bands in S.melanotheron,and duplicated six bands in the hybrids.The bands varied depending on their activities and mobilities.We considered that the differences in karyotype and isoenzyme were related to the genetic mechanism for post-mating isolation,and provided some additional basic genetic background of their taxonomy.

Effects of Manganese Stress on POD Activities and Isozymes of Soybean

[Objective]The aim was to discuss the internal reason of some inherited characteristics and genetic differences of different varieties of poisoned soybeans in molecule level.[Method]Soybeans were cultivated by water culture and treated for 10 days under natural light conditions.The influence of increasing concentration of manganese（0-120 mg/L） on the POD activities and isoenzyme of roots stems and leaves of soybean were studied.[Result]Results showed that the POD activities in roots,stems and leaves firstly increased and then decreased with increasing concentration of manganese.There were differences in the response of POD isoenzyme zymograms to manganese stress in different organs,the POD isoenzyme activity and quantities of soybean roots changed apparently when the manganese concentration increased,while the POD isoenzyme activity and quantities of soybean stems and leaves kept stable.[Conclusion]The isoenzyme zymograms changes of different organs of soybean were related to the resistance of soybean to manganese stress.

Analysis of Antioxidant Characteristics in Seedling Roots of Sweetpotato(Ipomoea batatas(L.) Lam.) with Different Drought Tolerance under Simulated Drought Stress

[Objective] Jishu 21 with strong drought tolerance and Jizishu 1 with weak drought tolerance were chosen to reveal antioxidant characteristics in seedling roots of sweetpotato with different drought tolerance. [Method] Active oxygen, antioxidant substances and antioxidant enzymes in seedling roots of sweetpotato with different drought tolerance were analyzed by hydroponics with PEG-6000 for simulating drought stress in the experiment. [Result] The results showed that under drought stress, H202 content and formation rate of 02_ in seedling roots of Jizishul were higher than Jishu 21 by 1.9% and 102.6%, respectively, the contents of Vc in seed- ing roots of Jizishu 1 and Jizishu 1 increased by 25.3% and 81.1%, respectively, and the contents of polyphenols increased by 24.8% and 37.6% in the two vari- eties, respectively. The increase amplitudes of Jizishu 1 were higher than Jishu 21 in the above indexes. Antioxidant enzyme activities were higher in seedling roots of Jisl^u 21 and Jizishul under drought stress than normal treatments; and among them, POD activity was the highest, while PPO activity was the lowest in all an- tioxidant enzymes. The increases of SOD, POD, APX and PPO activities in Jishu 21 were twice of those in Jizishul. The bands of SOD3, POD2, CAT1, APX2 and PPO2 changed obviously between the drought treatments of the two varieties. [Con- clusion] In conclusion, stronger drought tolerance in sweetpotato results in lower in- crease amplitudes of H202 content, formation rate of 02- and contents of Vc and polyphenols under drought stress, and the activities of antioxidant enzymes are stronger in seeding roots under drought stress; and the differences in antioxidant enzymes are mainly related to the changes of isoenzymes including SOD3, POD2, CAT1. APX2 and PPO2.

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC-7721 cells

AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma:Correlation with microvessel density

AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.

Effects of melatonin on the expression of iNOS and COX-2 in rat models of colitis

AIM: To investigate the effects of melatonin (MT) on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in rat models of colitis.METHODS: Healthy adult Sprague-Dawlay (SD) rats of both sexes, weighing 280±30 g, were employed in the present study. The rat models of colitis were induced by either acetic acid or 2,4,6-trinitrobenzene sulfonic acid (TNBS) enemas. The experimental animals were randomly divided into melatonin treatment and model control group that were intracolicly treated daily with melatonin at doses of 2.5, 5.0, 10.0 mg.kg-1 and equal amount of saline respectively from 24 h following induction of colitis in rats inflicted with acetic acid enema and the seventh day in rats with TNBS to the end of study. A normal control group of rats treated with neither acetic acid nor TNBS but saline enema was also included in the study. On the 28th day of the experiment, the rat colon mucosal damage index (CDMI) was calculated, and the colonic prostaglandin E2(PGE2), nitric oxide (NO), as well as the iNOS and COX-2expression were also determined biochemically or immunohistochemically.RESULTS: CDMI increased to 2.87±0.64 and 3.12±1.12respectively in rats treated with acetic acid and TNBS enema,which was in accordance with the significantly elevated colonic NO and PGE2 contents, as well as the up-regulated colonic iNOS and COX-2 expression in both of the two rat models of colitis. With treatment by melatonin at the doses of 5.0 and 10.0 mg@kg-1, CDMI in both models of rat colitis was significantly decreased (P＜0.05-0.01), which accorded synchronously and unanimously with the reduced colonic NO and PGE2 content, as well as the down-regulated expression of colonic iNOS and COX-2.CONCLUSION: Melatonin has a protective effect on colonic injury induced by both acetic acid and TNBS enemas, which is probably via a mechanism of local inhibition of iNOS and COX-2 expression in colonic mucosa.

Analysis of LDH activities and its isoenzyme patterns in colorectal cancer tissues

AIM To investigate the relationship between lactate dehydrogenase (LDH) activity or LDH isoenzyme pattern and the pathogenesis of colorectal cancer.

Expression of 5α-Reductase Type 2 Gene in Human Testis, Epididymis and Vas Deferens

Objectives To study the expression pattern of 5α-reductase type 2 gene in human male reproductive organsMethods The expression level of 5α-reductase type 2 gene inhuman testis, epididymis and vas deferens tissues was determined by in situ hybridization using Digoxin labeled 5α-reductase type 2 cRNA probe.Results The brown granules of hybridizing signals distributed in the cytoplasm of Sertoli and Leydig cells of the testis, the principle cells of epididymis and the epithelial cells of vas deferens, but there was no positive signal in the nuclei of above-mentioned cells. No positive signal was observed in germ cells, basement of the testis, interstium of epididymis and basement, as well as smooth muscle of vas deferens. Conclusion This study confirmed that the 5α-reductase type 2 gene expressed in Sertoli , Leydig cells of the testis, and the principle cells of epididymis. The expression pattern of the gene in these cells in human was similar to that of rat and monkey. The presence of 5α-reductase type 2 gene in epithelial cells of the vas deferens suggested it might possess an important physiological role in human reproduction.

Tissue-Specific Isoenzyme Variations in Tilapia Fish, <i>Oreochromis niloticus</i>

Native polyacrylamide gel electrophoresis have been used to analyze malate dehydrogenase (MDH), acid phosphatase (Acph) and peroxidase (Px) isoenzymes in different tissues (liver, kidney, muscle and heart) of the tilapia fish, Oreo- chromis niloticus in order to study the tissue specificity of these isoenzymes. Three, two and one fractions have been recorded respectively for the three isoenzymes in different studied tissues. The MDH-1 and MDH-2 have been ex- pressed only in muscle and heart while MDH-3 has been expressed in all studied tissues. The percentage amount of MDH in general varied significantly between muscle and different studied tissues. With respect to acid phosphatase, the percentage amount of the total enzyme showed significant difference between liver and muscle and that this variation may be due to higher gene activity in liver. Peroxidase isoenzyme was recorded in liver and heart only with significant increase in liver. The kidney was the least among the studied tissues in showing gene expression for the studied isoenzymes and therefore, liver, heart and muscle tissues are better applicable in studying the isoenzymatic profiles for fish physiology and systematics.