Snakebite is a common problem especially in tropical areas all over the world includingIran. Echis carinatus as one of the most dangerous Iranian snakes is spreading in this countryexcluding central and northwest provinces. In this study gelatinase and fibrinogenolyticproperties as two disintegrating matrix metalloproteinase enzymes were evaluated by a strongclear halo between 56-72 kDa in addition to another band located 76-102 kDa for gelatinase andone major band around 38 kDa for fibrinogenolytic enzyme respectively. The electrophorectcprofile of our venom demonstrated at least one protein band between 24-31 kDa like previousreports and another two bands between 52-76 kDa and below 17 kDa stemmed probably due tothe effect of natural selection in one species. According to our results Razi institute antivenincould neutralize in-vitro effects of gelatinase enzyme comprehensively. The electrophoreticprofile of Iranian commercial antivenom as the main intravenous treatment of envenomedpatients showed impurities in addition to F (abʹ )2 weighing 96 kDa in SDS-PAGE analysis. Itproposes more efforts for refinement to avoid short and long unwanted effects in envenomedpatients.

The genus Aspergillus (Anamorph) belongs to Deutromycets and has several species. Some of the species infected human, animal, plants and nuts.  Most of these species have ability to degrade plant components by production of pectinase enzyme. The species of Aspergillus flavus and Aspergillus niger are the most popular species of this genus. Although these species could be identified using morphological characters, the interspecific and intraspecific variation could not be evaluated.  Regarding pectic enzyme serection by the species, pectic zymogram technique was used as a simple and usefull method to identify the species and to detect inter and intraspecific variation. Initially the collected samples were transferred to the laboratory and after preparation were transferred on the appropriate media. The pure cultures were obtained for the sample with Aspergillus characteristics. Then Aspergillus fungi were identified microscopically based on morphological characters.  As a result, 40 isolates of A. niger and 30 isolates of A. flavus were recognized.  Then pured sampeles were transferred to liquid media contaning citrus pectin as a sole carbon source to induce the secretion of extracellular pectinase enzymes. After incubation for an optimised period the secreted pectinase enzymes was extracted and concentrated by Cephadex G150 and then loaded on the polyacrilamide horizontal gel and electerophoresed. There were 18 zymogram patterns for A. niger and 8 for A. flavus. The results based on the comparison of the zymogram patterns showed that there is inter and intraspecific variation for studied Aspergillus species.  It seems this technique could be used not only for the species identification, but also use to study of epidemiology of the fungs.

Sheath blight caused by Rhizoctonia solani is one of the most important diseases of rice in Iran and almost in all rice-producing countries. R. solani AG1-IA is the causal agent of sheath blight in rice. It is believed that R. solani could use plant material by secretion of pectolytic enzyme such as pectinase. In this study samples were obtained during two years from diseased rice in from diseased rice in Golestan province of Iran. Seventy five isolates were identified as AG1-IA, based on the number of nuclei per cell and anastomosis reaction. The identified isolates of R. solani AG1-IA were used for pectic zymogram analyses. The pectic enzyme electrophoretic patterns were interpreted in terms of loci coding for polygalacturonase (PG) and pectic esterase (PE). Presence or absence of a band with a particular Rf value was scord as "1" and "0", respectively. The obtained zymogram electrophoretic patterns (ZPs) were divided into six groups: ZP1-ZP6. PG and PE bands were observed for all isolates tested. A total of 18 bands (15 PG and 3 PE) were identified, one PG and one PE appeared as cathodic bands. Inter-group relationships were deduced from the similarity matrices. The genetic similarity among zymogram electrophoretic phenotypes, ranged from 62% to 88% based on band frequency. The cluster analysis showed two main branches. ZP3 was clustered separately on a single branch, and the other branch was divided into two subbranches, one containing ZP5, and the other ZP1, ZP4. ZP2 and ZP6. The results indicated that there is a distinguishable diversity among different R. solani isolates of rice fields of Golestan province, and that could be detected by isozymes but not by anastomosis group. We concluded that the pectic enzyme electrophoresis could be a suitable technique to study genetic variation of AG1-IA isolates.

There are increasing data on novel tumor markers such as gelatinase A, which play a key role in tissue invasion and metastasis. Since prostate cancer is one of the common malignancies, we designed a simple and applicable Indirect Hemagglutination (IHA) test for determination of total gelatinase A in serum samples. In this study, we have analyzed the circulating form of gelatinase A (MMP-2) in patients suffering from either benign prostate hyperplasia (n= 54) or prostate cancer (n= 26) and normal individuals as control (n= 26). The gelatinolytic activity was determined by ZYMOGRAPHY followed by densitometric analysis. PSA was quantified by using a standard ELISA technique. Correlation of densitometric analysis of gelatinase A activity and IHA titer was significant at 0.01 level (p< 0.01, r = 0.916). Correlation of PSA and IHA titer was significant at 0.01 level (p< 0.01, r = 0.746). Border line IHA titer in patients with prostate cancer was 512 ± 1 tube titer, in benign prostate hyperplasia patients was 128 ± 1 tube titer, and the titer in normal individuals was 8 ± 1 tube titer. These results demonstrate that IHA compared to ZYMOGRAPHY may be a better and simpler procedure in monitoring and screening patients with prostate cancer.

Background & Objective: Cancer metastasis is the primary cause of cancer morbidity and mortality, it accounts for about 90% of cancer deaths. Cancer treatment has improved significantly, due to early detection and inhibition of cancer growth. The ability to invade and migrate is important in malignant tumor cells. The study of cell migration is valuable in cancer diagnosis, prognosis, drug development and treatment. New methods are available to investigate the invasion, migration and the activity of enzymes involved in the invasion process in the laboratory. The effectiveness and efficacy of various anti-cancer drugs and compounds can be studied using these methods in laboratory and animal models. Conclusion: In this paper, we offer a summary of in-vitro migration assays, including the transwell migration assay, scratch wound assay, microfluidic chamber assay, exclusion zone assay, fence assay, micro carrier bead assay, spheroid migration assay and in-vivo approach, Chick chorioallantoic membrane (CAM) assay. This review also provides an overview of methods, In situ ZYMOGRAPHY, ELISA, and FRET based measurement of MMP activity and Substrate ZYMOGRAPHY for measuring the level of metalloproteinase enzyme as the major enzyme in the degradation of extracellular matrix.

Background: Streptokinase is a bacterial protein produced by different beta hemolytic streptococci and widely used in thrombolytic treatment. The main disadvantage of using streptokinase is antibody formation which causes allergic reaction to neutralize effects of streptokinase therapy. Aim of this study was investigate of recombinant mutant streptokinase fibrinolytic activity.Materials and Methods: In this study recombinant mutant streptokinase without 42 amino acids from the C terminal region was purified by affinity S-Tag column chromatography and its fibrinolytic activity was studied.Results: The concentration of expressed and purified protein was 10 mg/ml. Its enzyme activity was assayed using ZYMOGRAPHY, radial caseinolytic activity and fibrin plate test methods and estimated quantitatively by casein digestion method compared to a commercial form.Conclusion: It was found that this product had the more volume and more enzymatic activity.

Alpha-amylases are starch-degrading enzymes which catalyze the hydrolysis of internal a-1, 4-O-glycosidic bonds in polysaccharides. These enzymes have many applications in food, paper, textile and pharmaceutical industries. About 30 % of world enzyme production market is provided by this enzyme. Alpha amylases are produced by plants, animals and microorganisms. However, microbial sources are the most preferred one for large scale production. Among microorganisms, alpha-amylase is mainly derived from Bacillus genus for commercial applications. Therefore, in this study, 13 different strains of Bacillus genus were tested for their ability to hydrolyze and consume starch. Comparative studies were performed on the strains which were capable to consume starch. The curve of starch consumption by applying logul's solution was depicted for strains. The enzymes activity was assayed by using DNS (3, 5-dinitrosalicylic acid) at various times and temperatures. The best strain according to the enzymatic features was selected. Enzymatic extract was achieved by centrifugation. As alpha-amylase was precipitated by ammonium sulphate, desalting process was performed by dialysis. Then ZYMOGRAPHY and determination of molecular weight for alpha-amylase were performed. The best strain regard to enzymatic features was Bacillus amyloliquefaciens. Maximum production of this enzyme was after 36 h. The enzyme activity was at the highest level at 70oC. Molecular weight of the enzyme was about 55KD. In conclusion, the selected strain can be an appropriate candidate for industrial production of alpha-amylase.

Background: Tetracyclines are antimicrobial agents that possess anti-inflammatory and matrix metalloproteinase (MMP) inhibitory effects. Tetracycline, doxycycline, and minocycline have shown different potencies in inhibiting various MMPs. MMPs are a conserved family of zinc-dependent endopeptidases, which are involved in various physiologic and pathologic conditions. MMP-9 is among the most important proteases involved in the development of cardiovascular disease, cancer, and inflammatory disease Objectives: Considering the central role of MMP-9 in the above-mentioned diseases and the variable inhibitory potentials of routine tetracyclines, including tetracycline, doxycycline, and minocycline, this study measured the inhibitory effect of these routine tetracycline agents on MMP-9 activity obtained from U-937 cell cultures using the ZYMOGRAPHY method.MaterialsandMethods: AnMMP-9-rich culturemediumof U-937 cellswas used as the source of the enzyme. Serial dilutions of 5, 20, 100, 200, and 500mM of each drug were placed in ZYMOGRAPHY incubation buffer. Gelatin ZYMOGRAPHY was run, and densitometry of the bands was performed using Image J software. The resulting data were used to draw dose-response curves using GraphPad Prism software.Results: Our results showed that tetracyclines inhibited MMP-9 activity with IC50 values of 40.0, 10.7, and 608.0 mM for tetracycline, minocycline, and doxycycline, respectively. Minocycline showed the highest potency in MMP-9 inhibition (P<0.0001), while tetracycline was found to be more potent than doxycycline in MMP-9inhibition based on the ZYMOGRAPHY experiments (P<0.0001).Conclusions: Our results suggest that tetracyclines dose-dependently inhibitMMP-9activity, andamongthecommonclinical tetracyclines, minocycline provides the most potent inhibition of excess MMP-9 activity and its consequences.

One hundred and three Aspergillus isolates belonging to 12 species including: A. alliaceus, A. candidus, A. carneus, A. flavus, A. fumigatus, A. niger var. niger, A. niger var. awamori, A. niveus, A. ochraceous, A. sydowii, A. terreus, A. ustus, andA. versicolor based on morphological characters were obtained from various sources. The isolates were subjected to pectic zymogram electrophoresis. Based on the similarity of isozyme electrophoretic patterns, 45 zymogram patterns were identified. Analyses of the electrophoretic patterns revealed 26 isozyme loci corresponding to the polygalacturonase and pectin estrase. Although the examined species had a considerable intraspecific variation they were finely distinguished using pectic zymogram electrophoresis and no common zymogram pattern was observed among the species. The results imply that pectic zymogram electrophoresis may be used as a helpful approach for delimitation of Aspergillus species.

Background and Aim: Multiple sclerosis (MS) is considered as a common inflammatory disease of the human central nervous system (CNS). 4 amino pyridine (4-AP), a potassium channel blocker, is used widely in MS treatment to reduce fatigue and cachexia often experienced by the patients. The objective of this study was to get a better understanding of the mechanism of action of this drug, using the cell culture model. More specifically, we attempted to determine the effects of the drug on 1. The profileration of, and its cytotoxicity on, the neurons, and 2. The activity of MMP-9 in the neurons.Materials and Methods: Considering the available reports on the wide range of 4-AP effects, we designed this study to investigate its possible role in proliferation or cytotoxicity of the MS cellular model, i.e. astrocytoma U373-MG, using the MTT assay technique. We also analyzed the effect of 4-AP on cell functionality as assessed by matrix metalloproteinase-9 (MMP-9) ZYMOGRAPHY.Results: The results showed that while 4-AP at a concentration of 0.1 and 1 mM has no significant cytotoxic effects on the treated cells, it has remarkable MMP-9 inhibitory effects (p<0.01). The proliferation analyses confirmed the stability of 4-AP effects on cell functionality.Conclusion: On the whole, the results of the present study show the desirable effect of 4-AP on MMP-9 activity at non-cytotoxic doses, promising its further therapeutic applications.