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D Modena

Publications and source records attributed to D Modena.

28 records · Page 2Linked to original sources
PubMed1989

Immunocytochemical characterization of mouse monoclonal ACTH antibodies with a note on staining conditions and control procedures.

Mouse monoclonal antibodies (MAbs) have been produced against porcine ACTH and tested for their immunocytochemical utility. Ten out of 12 MAbs reacted with formaldehyde-fixed human ACTH[1-39] and fragments thereof. Cytochemical fragment testing revealed that 6 of the 10 MAbs recognized epitopes in the vicinity of the region where porcine ACTH differs from mouse ACTH (amino acids 26, 29 and 31). Both tissue and cytochemical model data indicate that many of the MAbs detected porcine ACTH with somewhat higher potency than human and rat ACTH (rat ACTH[1-39] is identical to mouse ACTH[1-39]). MAbs Nos. 5, 8 and 12, in particular, revealed a highly satisfactory signal to noise ratio also in formalin-fixed, paraffin-embedded specimens. Most of the MAbs were potent in detecting both the high concentrations of ACTH congeners in corticotrophs and melanotrophs and the lower concentrations of such peptides in human antropyloric gastrin cells. Blocking of tissue endogenous peroxidase activity reduced reactivity towards the MAbs. This could be circumvented by use of biotinylated primary antibodies combined with avidin/streptavidin-alkaline phosphatase detection. Availability of MAbs and of the corresponding synthetic antigen also made some quantitative comparisons and analyses of appropriate control procedures possible.

Adrenocorticotropic Hormone↗
PubMed1986

Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus.

The Saccharomyces diastaticus structural gene STA2, encoding an exracellular glucoamylase (1,4-alpha-D-glucan glycohydrolase, EC 3.2.1.3.), has been cloned by complementation of a stao strain. A genomic library was initially constructed from a STA2 yeast strain in the yeast Escherichia coli shuttle cosmid vector pYCl. The Sta+ complementing function was further delimited to an 8.3 kb BglII fragment whose restriction map was found to be similar to related genomic regions of STA1 and STA3. Fusions of several DNA fragments derived from the 8.3 kb BglII fragment with a truncated E. coli beta-galactosidase gene resulted in two overlapping fragments that could direct the production of large fusion proteins in E. coli. These fusion proteins were immunoprecipitable by anti-glucoamylase II antibodies, confirming that the Sta+ complementing fusion was due to the expression of a gene that coded for a yeast glucoamylase. Measurements of the STA1, STA2 and STA3 RNA transcripts by RNA-DNA hybridization using an internal fragment of the cloned STA2 gene as the probe indicated that a common transcript of 2.5 kb is produced by each of the STA genes. Integrative disruption of the STA2 gene through homologous recombination was achieved by transforming a STA2 yeast strain to Sta- using an in vitro constructed donor DNA fragment that has the URA3 gene inserted within the coding region of the cloned glucoamylase gene. This was confirmed by tetrad analysis of crosses between strains carrying a disrupted STA2 and a functional STA2. Southern blot analysis using BamHI digested genomic DNA from 15 tetrads demonstrated consistent co-segregation and Mendelian inheritance of the Sta- phenotype with STA2::URA3. These data further confirm that the cloned DNA that showed Sta+ complementing activity carries a functional STA2 gene that encodes the yeast extracellular glucoamylase II.

Cloning, Molecular↗
PubMed1986

Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus.

In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.

Antibodies, Monoclonal↗
PubMed1986

Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Saccharomyces species.

Three unlinked, homologous genes, STA1, STA2, and STA3, encode the extracellular glycosylated glucoamylase isozymes I, II, and III, respectively, in Saccharomyces species. S. cerevisiae, which is sta0 (absence of functional STA genes in haploids), does carry a glucoamylase gene, delta sta, expressed only during sporulation (W. J. Colonna and P. T. Magee, J. Bacteriol. 134:844-853, 1978; I. Yamashita and S. Fukui, Mol. Cell. Biol. 5:3069-3073, 1985). In this study we examined some of the physiological and genetic factors that affect glucoamylase expression. It was found that STA2 strains grown in synthetic medium produce glucoamylase only in the presence of either Maltrin M365 (a mixture of maltooligosaccharides) or starch. Maximal levels of glucoamylase activity were found in cells grown in rich medium supplemented with glycerol plus ethanol, starch, or Maltrin. When various sugars served as carbon sources they all supported glucoamylase synthesis, although at reduced levels. In any given growth medium glucoamylase isozyme II synthesis was modulated by functionality of the mitochondria. Synthesis of glucoamylase is continuous throughout the growth phases, with maximal secretion taking place in the early stationary phase. In the various regimens, the differences in enzyme accumulation are accounted for by differences in the levels of glucoamylase mRNA. Both glucoamylase mRNA and enzyme activity were drastically and coordinately inhibited in MATa/MAT alpha diploids and by the presence of the regulatory gene STA10. Both effects were partially overcome when the STA2 gene was present on a multicopy plasmid. The STA2 mRNA and glucoamylase were coinduced in sporulating STA2/STA2 diploids. A smaller, coinduced RNA species was also detected by Northern blotting with a STA2 probe. The same mRNA species was detected in sporulating sta0 diploids and is likely to encode the sporulation-specific glucoamylase.

Culture Media↗
PubMed1983

Permanent decrease in activity of ornithine decarboxylase antizyme in rat liver during chemical hepatocarcinogenesis.

This study was undertaken to determine whether or not there is failure of cellular control of L-ornithine decarboxylase activity by its antizyme, the only known natural intracellular inhibitor protein for L-ornithine decarboxylase activity, in rat liver during hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene. The formation of hepatic ornithine decarboxylase antizyme was elicited by i.p. injections of putrescine into rats fed a basal diet and rats fed the carcinogenic diet. The activities of both hepatic ornithine decarboxylase and hepatic ornithine decarboxylase antizyme were measured every month for five months, i.e., until hepatoma was fully developed. During azo-dye hepatocarcinogenesis and in fully developed hepatoma the activity of hepatic ornithine decarboxylase antizyme was always significantly lower than in normal resting liver, with minima at the second and the third months. The hepatoma does not synthesize ornithine decarboxylase antizyme more slowly than normal liver, since the difference could be neither abolished nor lessened by lengthening the time available for antizyme formation. Our results strongly suggest that the high intracellular putrescine levels in the livers of rats during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis do not exert their normal control on hepatic ornithine decarboxylase activity because of a relative inability of these preneoplastic or neoplastic cells to make the ornithine decarboxylase antizyme.

Animals↗
PubMed1983

Restoration of normal ornithine decarboxylase antizyme activity in rat liver after acute carcinogen treatment.

This study was undertaken to see whether or not the decrease in ornithine decarboxylase antizyme activity caused in rat liver by a hepatocarcinogen could be reversed. Thioacetamide was administered only once, in a single i.p. injection and at a non-carcinogenic, non-necrogenic dose. The activities of both hepatic ornithine decarboxylase and hepatic ornithine decarboxylase antizyme were measured at intervals of hours after the injection of thioacetamide. The hepatic ornithine decarboxylase antizyme in thioacetamide-treated rats was minimal at 40 and 80 h after carcinogen administration. The reversal process requires a very long time, namely 450 h for normal levels of hepatic ornithine decarboxylase antizyme activity to be restored in treated rats. This time is much longer than that required to restore normal ornithine decarboxylase activity in liver of thioacetamide-treated rats. The results of this study, combined with those of the preceding paper, demonstrate that hepatocarcinogens cause a relative inability of rat liver cells to make the ornithine decarboxylase antizyme and that the irreversibility of this defect in cellular control of ornithine decarboxylase activity may be a constant feature in the neoplastic transformation of the rat liver.

Acetamides↗
PubMed1982

Degrees of malignancy in human primary central nervous system tumors: ornithine decarboxylase levels as better indicators than adenosylmethionine decarboxylase levels.

The levels of activity to ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase (AMD) were measured in various types of primary human tumors of the central nervous system (CNS) and whenever possible were related to the malignancy of the tumor graded according to histopathologic criteria. In astrocytomas ODC levels increased linearly and progressively from infratentorial pilocytic astrocytoma (grade I) to glioblastoma multiforme (grade IV) and corrected well with the degree of histologic malignancy of the tumor. AMD activity levels, however, correlated with tumor malignancy only up to grade III astrocytoma. Medulloblastomas exhibited an unusual dichotomy with regard to the levels of polyamine biosynthetic decarboxylases (PBD): Medulloblastomas had the highest ODC activities of all the CNS tumors tested but had low AMD activities. In tumors of neuroepithelial tissue ODC level increases and, when present, AMD level increases were not due to proliferation of new blood vessels, because CNS hemangioblastomas had very low levels of both PBD activities. No significant differences in either of the PBD levels were observed among the several variants of meningiomas tested, the meningotheliomatous, the transitional, and the fibrous meningiomas. However, atypical forms of meningioma, i.e., those with mitotic figures, whatever the histologic variants, had higher levels of ODC, but not of AMD, than the typical forms, i.e., those without mitotic figures.

Adenosylmethionine Decarboxylase↗
PubMed1981

Changes in th circadian rhythm of ornithine decarboxylase in rat liver during chemical hepatocarcinogenesis.

The chronobiology of ornithine decarboxylase (ODC) activity in livers was investigated in noninbred Sprague-Dawley rats fed for 5 months with a basal diet or diets with 3-methyl-4'-(dimethylamino)azobenzene (3-Me-DAB) that were oncogenic or caused bile duct hyperplasia (1-naphthylisothiocyanate) (NIT). After a transient disappearance of the ODC circadian rhythm during month 1 on the oncogenic diet, this rhythm in the livers of the rats was reestablished at 60 and 90 days and then disappeared for the next 2 months. When present, the ODC rhythm in 3-Me-DAB-treated rats had the same daily temporal pattern as that of the controls. In the livers of rats treated with NIT, the ODC circadian rhythm was never detectable, even after only 1 month of feeding. Generally, the 3-Me-DAB feeding induced higher levels of ODC activity than did the NIT feeding. The alternation of the appearance and the disappearance of ODC circadian rhythm might reflect changes in the cell population during neoplastic transformation. Te chronobiologic differences in ODC rhythm between the group fed 3-Me-DAB and the group fed NIT could be related to the different types of proliferating cells involved in the hepatic responses to the two drugs.

1-Naphthylisothiocyanate↗
PubMed1980

Levels of activity of the polyamine biosynthetic decarboxylases as indicators of degree of malignancy of human cutaneous epitheliomas.

The activities of ornithine decarboxylase and of S-adenosyl-L-methionine decarboxylase in human normal epidermis, in basal cell epitheliomas, and in squamous cell carcinomas of human skin have been compared. All 3 types of tissues have characteristic levels of each of these enzymes. The normal epidermis had the lowest levels of both ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase activities. The levels of the polyamine biosynthetic decarboxylases in basal cell epitheliomas were significantly higher than those of normal epidermis, but at the same time significantly lower than those present in squamous cell carcinomas. These results support the conclusion that in epithelial malignant tumors of human skin the extent of the increase in the activities of polyamine biosynthetic decarboxylases is well correlated with the neoplasm's growth rate, which is faster in the squamous cell carcinomas than in the basal cell epitheliomas.

Aged↗