To investigate the mechanisms by which androgens regulate ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) in mouse kidney, a cDNA clone encoding OrnDCase mRNA was prepared. Purification of OrnDCase mRNA from kidneys of androgen-treated mice was accomplished by immunoadsorption of renal polysomes to a protein A-Sepharose column and enrichment for poly(A)-containing RNA by oligo(dT)-cellulose. Double-stranded cDNA synthesized from this mRNA was inserted into the Pst I site of plasmid pBR322 by using oligo(dGdC)-tailing and was propagated in Escherichia coli. Plasmids containing cDNA sequences coding for OrnDCase were identified by differential colony hybridization, by radioimmunological detection of OrnDCase-like antigens in bacterial cultures, and by cell-free translation of hybrid-selected mRNA followed by imununoprecipitation with monospecific OrnDCase antiserun%. A restriction endonuclease fragment of the selected plasmid DNA (pODCS4) was labeled by nick-translation and used to study changes in OrnDCase mRNA concentration. After a single dose of testosterone, renal OrnDCase mRNA concentration increased as soon as 6 hr and peaked 24 hr after steroid injection, as measured by RNA blot hybridization. Continuous androgen treatment for 4 days resulted in a 10-to 20-fold increase in OrnDCase mRNA concentration in normal animals, but no induction of this mRNA was detected in mice that have an inherent defect of the androgen receptor (testicular feminization). These results indicate that androgens regulate OrnDCase synthesis in mouse kidney, at least in part, by increasing OrnDCase mRNA accumulation.Ornithine decarboxylase (OrnDCase; L-ornithine carboxylyase, EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine and is the first and apparently rate-limiting enzyme in polyamine biosynthesis. A striking feature of this enzyme is that it has extremely rapid induction kinetics and a high turnover rate (for review, see refs. 1-4). Several studies (1-6) have suggested that regulation of OrnDCase activity occurs through modulation of the amount of the enzyme protein, although other factors such as inhibitors (7-10) or activators (11) of OrnDCase and post-translational enzyme modifications (12, 13) also may be involved. A major reason for difficulties in studying the regulation of this enzyme is that it represents only 0.01-0.05% of the total cellular protein, even when maximally induced. Nonetheless, purification of the enzyme to apparent homogeneity (5,(14)(15)(16)(17) and development of radioimmunoassays (5, 6) have permitted studies on OrnDCase turnover.We reasoned that further understanding of the factors that regulate OrnDCase would be facilitated if a probe were available that would allow direct measurement of OrnDCase mRNA sequences. Kidneys of testosterone-treated mice were chosen as a source for isolation of OrnDCase mRNA because androgen administration increases enzyme protein in this organ to a higher concentration than is known in most tissues (2, 5, 6). In the presen...

A cDNA encoding for the glucoamylase P enzyme (GAMP) of the fungus Hormoconis resinae was introduced into the cellulolytic filamentous fungus Trichoderma reesei under the control of the promoter of the major cellulase gene (cbh1) of Trichoderma. The transforming vector plasmid used was found to be integrated into the genome of T. reesei at various locations and in multiple copies. The size of the GAMP secreted by Trichoderma varied because of different glycosylation patterns. The best transformant strains secreted about 700 mg/l of active GAMP, which is 20-fold more than obtained with H. resinae.

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Mesothelioma represents an aggressive tumor type with high resistance to all treatment modalities. Its pathogenesis is strongly associated with exposure to asbestos fibers and probably with free radicals. One of the most important free radical scavenging enzymes, mitochondrial manganese superoxide dismutase (MnSOD), has been shown to be elevated in mesothelioma (K. Kahlos et al., 1998, Am. J. Respir. Cell Mol. Biol. 18:570-580). In the present study, we could detect intense ultrastructural accumulation of MnSOD in the mitochondrial compartment of malignant mesothelioma cells. There was no association between the immunohistochemical reactivity and the most common and functional polymorphic variant of MnSOD, the Ala to Val amino acid change at 9 position (16th amino acid from the beginning of the signal sequence), in the 31 mesothelioma cases investigated. Comparative genomic hybridization and fluorescence in situ hybridization did not reveal any changes in chromosome 6, where the MnSOD gene is located. Sequencing of the MnSOD promoter region in four mesothelioma cell lines showed similar nucleotide variables in the malignant and nonmalignant cells. Therefore, the intense expression of MnSOD in the mitochondria of mesothelioma cells does not appear be associated with any major chromosomal alterations or the polymorphism of MnSOD gene. Association with oxidative/nitrosative stress in mesothelioma using nitrotyrosine immunostaining pointed to a tendency for more intense reactivity in those mesotheliomas with higher MnSOD expression (P = 0.069).

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