Comparative localization of cathepsin B protein and activity in colorectal cancer.

Cathepsin B is a lysosomal cysteine proteinase that may participate in cancer progression. We compared localization of its protein and activity during progression of human colorectal cancer. In adenomas and carcinomas, protein expression and, particularly, activity were elevated compared with those in normal colorectal mucosa. In normal mucosa, cathepsin B protein expression was moderate in stroma and variable in epithelium, whereas activity was mainly present in distinct areas of stroma directly underneath the surface of the colon and in epithelium at the surface of the colon. Stroma in adenomas and carcinomas contained moderate to high protein levels but little activity except for areas of angiogenesis, inflammation, and necrosis, in which activity was high. In adenomas and the majority of well-differentiated carcinomas and moderately differentiated carcinomas, cathepsin B protein and activity were found in granular form in the epithelium, close to the basement membrane. Protein and activity levels were low and diffusely distributed in cancer cells in the remainder of the well-differentiated and moderately differentiated carcinomas and in all poorly differentiated carcinomas. Invasive fronts in most cancers contained moderate protein levels but high activity. We conclude that (a) activity localization is essential to understand the role of cathepsin B in cancer progression, and (b) cathepsin B activity in human colon is associated with invasion of cancer cells, endothelial cells, and inflammatory cells, and in cell death, both apoptotic and necrotic.     

Agrobacterium-mediated transformation of five wild Solanum species using in vitro microtubers

Summary A genetic transformation method, using in vitro microtubers and Agrobacterium-mediated transformation has been developed for five wild Solanum species: S. verrucosum, S. hjertingii, S. papita, S. stoloniferum, S. demissum, which range in ploidy from diploid to hexaploid. A disarmed A. tumefaciens strain, C58 harbouring the co-integrate vector pGV3580::pKU2 with the genes of neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPTII) as selectable markers, was used. Selection of putative transformants was based on their ability to grow and produce roots on a medium containing 150 mg/l kanamycin. The transgenic nature of the putative transformants was confirmed by Polymerase Chain Reaction analysis and by NPTII dotblot assay to show the expression of the NPTII gene. Additionally, the transmission of transgenes, NPTII and HPTII in selfed-sexual progeny of some transgenic plants was also determined.Abbreviations MS Murashige and Skoog - NPTII neomycin phosphotransferase - HPTII hygromycin phosphotransferase - PCR polymerase chain reaction - GA₃ gibberellic acid - CCC chlorocholine chloride - BAP benzylaminopurine - NAA naphthalene acetic acid - ZR zeatin riboside - IAA indole acetic acid - LB Luria Broth     

Promoting glutathione synthesis after spinal cord trauma decreases secondary damage and promotes retention of function.

The study aimed to 1) quantify oxidative stress in spinal cord after crush injury at T6, 2) determine whether the administration of the procysteine compound L-2-oxothiazolidine-4-carboxylate (OTC) would up-regulate glutathione (GSH) synthesis and decrease oxidative stress, and 3) determine whether decreased oxidative stress results in better tissue and function retention. We demonstrate that spinal cord compression (5 s with a 50 g aneurysm clip) at T6 in rats results in oxidative stress that is extensive (significant increases in oxidative stress seen at C3 and L4) and rapid in onset. Indices of oxidative stress used were GSH content, protein carbonyl content, and inactivation of glutathione reductase. Administration of OTC resulted in a marked decrease in oxidative stress associated with a sparing of white matter at T6 (16+/-1.9% retained in OTC-treated animals vs. less than 1% in saline-treated). Behavioral indices in control, saline-treated, and OTC-treated animals after 6 wk were respectively: angle board scores (59 degrees, 32 degrees, and 42 degrees ), modified Tarlov score (7, 2.4, and 4.1), and Basso-Beattie-Bresnahan score (21, 5.3, and 12.9). We conclude that administration of OTC after spinal cord trauma greatly decreases oxidative stress and allows tissue preservation, thereby enabling otherwise paraplegic animals to locomote.