Zymography Research Articles

Molecular Analysis of Factors Contributing to Carotid Atherosclerotic Plaque Instability: Atheromatous versus Fibrous Plaques

P63 Introduction: The molecular events in atherosclerotic carotid disease that trigger responses within the plaque resulting in plaque instability and thromboembolic cerebrovascular sequelae are unclear. Plaque instability may be related to apoptosis and extracellular matrix degradation. We compared atheromatous and fibrous plaques in terms of apoptosis, apoptosis-related proteins, cell cycle regulatory genes, death substrates for apoptosis, proteolytic activity and infiltrating cells. Methods: Apoptosis was detected in carotid endarterectomy plaques by the TUNEL assay. Immunohistochemical studies were performed to identify macrophages and infiltrating cells, localize the expression of bcl-2, bax, cyclin D-1, caspases, Fas, PARP, MMP-2 and MMP-9. Zymography was used to compare the proteolytic activity levels in the two types of plaques. Results: There were no inflammatory cells in the fibrous plaques. Apoptotic cell death was significantly higher (p Conclusions: The greater amount of apoptosis and overexpression of mediators of apoptosis seen in atheromatous plaques suggest that deregulated apoptosis may be involved in the pathogenesis of unstable atherosclerotic plaques. Immunoreactivity to cytoplasmic death domain, Fas, and CPP-32, a prominent mediator of apoptosis, was consistent with the amount of apoptosis detected. Inflammatory cells and increased number of T-lymphocytes producing apoptotic stimuli could contribute to plaque instability. The increased levels of proteolytic activity in atheromatous plaques may make them vulnerable to rupture, and result in a cascade of thromboembolic cerebrovascular events.

Identification of Rhizoctonia solani associated with field-grown tulips using ITS rDNA polymorphism and pectic zymograms

Methods based on internal transcribed spacers (ITS) ribosomal DNA (rDNA) polymorphism and pectic zymograms (ZG) were compared for their use in routine identification of Rhizoctonia solani isolates occurring in flower bulb fields. Thirty three AG 2-t isolates, pathogenic to tulips, could be distinguished from AG 1-IC, AG 2-2IIIB and AG 2-2IV, AG 3 and AG 5 by means of ITS rDNA fragment length and after digestion with EcoR I from AG 4 and AG 5. AG 2-t isolates and two Japanese isolates, pathogenic to crucifers and tulips, had an estimated fragment size of 710 bp, whereas Dutch AG 2-1 isolates, non-pathogenic to tulips, showed an estimated fragment size of 705 bp on agarose gel. Digestion of AG 2-t and AG 2-1 isolates with EcoR I, Sau3A I, Hae III and Hinc II revealed four and five distinct ITS rDNA digestion patterns, respectively. In AG 2 isolates 2tR114, 21R14 and 21R61 a double digestion pattern, indicating different ITS sequences within an isolate, was found. The observed ITS fragment length polymorphism between isolates pathogenic and non-pathogenic to tulips were considered too small to be used in routine screening of field isolates. Sequencing of AG 2 isolates 21R01, 21R06, 2tR002 and 2tR144 showed a total ITS rDNA fragment length of 715, 713, 714, and 728 bp. As an alternative to ITS rDNA fragment length polymorphism, pectic enzyme patterns were studied using a commercially available vertical gel-electrophoresis system and non-denaturing polyacrylamide gels amended with pectin. Anastomosis tester isolates AG 1 to AG 11 revealed different ZG. Fifty AG 2-t isolates and five AG 2-1 isolates belonged to a homogeneous pectic zymogram group. We propose to assign AG 2 isolates pathogenic to crucifers and tulip to ZG5-1. AG 2-1 isolates, non-pathogenic to tulip, formed a heterogeneous group with 4 distinct ZG. Pectic zymography provides an easy, quick and unambiguous method for routine identification of large numbers of field isolates. Such a technique is needed for research on the dynamics of Rhizoctonia populations to develop environmentally friendly control measures of rhizoctonia disease in field-grown flower bulbs.

UPA Zymography

uPA Zymography

Enhanced Zymography of Proteases

Enhanced Zymography of Proteases

Immunological versatility and carbon regulation of Cellulomonas fimi endo-1,4-beta-glucanases.

More than 10 protein molecules with endo-1,4-beta-glucanase activity were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram in Cellulomonas fimi culture supernatants, grown in CMC as carbon source. These molecules are shown to belong to at least four immunologically different groups, against three of which polyclonal antibodies were raised. The protein species used as antigens showed significant differences in cross reactivity, carbon regulation, and affinity to crystalline cellulose. Three intracellular precursors of the first group were detected, two of which were under carbon catabolite control with the third apparently being synthesized constitutively. In the extracellular environment this group showed the largest versatility in protein molecules. The second group appeared to originate from two intracellular precursors both synthesized constitutively and subject to minor extracellular modifications as compared to the first group. The main extracellular protein of this group showed high affinity toward crystalline cellulose. One intracellular precursor was identified for the third group, which was subject to carbon catabolite control. Only one extracellular molecule without binding ability to crystalline cellulose corresponded to this precursor, indicating that the latter was resistant to proteolytic modifications after excretion. It appears that the C. fimi cellulases are more complex than expected and reconstitution of the whole system will be difficult.

Acid phosphatases in human plasma.

Abstract Human plasma and serum acid phosphatases are studied by biochemical methods to evaluate the total enzyme activity and by polyacrylamide gel electrophoresis to demonstrate the isoenzyme patterns. The aim is to identify the tissue or cell origin of the various isoenzymes in plasma. Normal plasma contains a tartrate-resistant isoenzyme 5 but no demonstrable isoenzymes from red blood cells or prostate. Normal serum contains an additional isoenzyme 3 which is tartrate-sensitive and is released from platelets during blood clotting. In a variety of diseases, other isoenzymes may appear in plasma. In metastatic prostate cancer, for example, the changes of plasma enzyme activity are due to the presence of isoenzyme 2. Many acid phosphatase isoenzymes are cell-specific, i.e., derived from a specific type of cell. Abnormal enzyme activity and zymogram patterns of plasma acid phosphatase frequently reflect those of tissues in disease conditions. In some of the cases with abnormal plasma acid phosphatase isoenzymes, the total enzyme activity in plasma is normal. Results of this study suggest that examination of the isoenzymes by gel electrophoresis is more sensitive than the biochemical methods in tracing the cause responsible for the enzyme changes in plasma.

Studies on isozymes (II) got isozymes in the zymogram

Studies on isozymes (II) got isozymes in the zymogram

ELECTROPHORESIS OF NONHEMOGLOBIN PROTEINS FROM CONCENTRATED HEMOGLOBIN-FREE HEMOLYSATES.

Abstract Electrophoresis of fivefold-concentrated hemolysates from which hemoglobin had been removed by substituted celluloses, Sephadex or ion-exchange resin was performed on starch gel by means of a discontinuous buffer system. Six nonhemoglobin protein (NHP) zones were stained with Amido black. These NHP zones were obscured by hemoglobin when whole hemolysates were studied and have not been previously reported by us. Zymogram techniques revealed 1 to 4 zones with glucose-6-phosphate dehydrogenase, activity, 5 zones with lactic dehydrogenase activity, and multiple esterase zones for the substrate α-naphthyl acetate.