Isolation, characterization and expression analysis of the ornithine decarboxylase gene (ODC1) of the entomopathogenic fungus, Metarhizium anisopliae

The gene ODC1, which codes for the ornithine decarboxylase enzyme, was isolated from the entomopathogenic fungus, Metarhizium anisopliae. The deduced amino acid sequence predicted a protein of 447 amino acids with a molecular weight of 49.3 kDa that contained the canonical motifs of ornithine decarboxylases. The ODC1 cDNA sequence was expressed in Escherichia coli cells; radiometric enzyme assays showed that the purified recombinant protein had ornithine decarboxylase activity. The optimum pH of the purified Odc1 protein was 8.0-8.5, and the optimum reaction temperature was 37 °C. The apparent K_m for ornithine at a pyridoxal phosphate concentration of 20 mM was 22 μM. The competitive inhibitor of ODC activity, 1,4-diamino-2-butanone (DAB), at 0.25 mM inhibited 95% of ODC activity. The ODC1 mRNA showed an increase at the beginning of appressorium formation in vitro. During the M. anisopliae invasion process into Plutella xylostella larvae, the ODC1 mRNA showed a discrete increase within the germinating spore and during appressorium formation. The second expression peak was higher and prolonged during the invasion and death of the insect. The ODC1 gene complements the polyamine auxotrophy of Yarrowia lipolytica odc null mutant.     

Human ornithine decarboxylase(ODC)-encoding gene: cloning and expression in ODC-deficient CHO cells

We have cloned a full-length human ornithine decarboxylase (ODC)-encoding gene from a genomic library of human myeloma cells which overproduce ODC due to a selective gene amplification. Correct expression of the cloned gene was assessed by transfecting it into a Chinese hamster ovary (CHO) cell mutant devoid of ODC activity. Transfection with a 10-kb BamHI DNA fragment of the genomic clone, conferred ODC activity to the recipient cells and relieved them of dependence on exogenous polyamines for growth. A set of 40 transformants was isolated, eight of which were further characterized. The transfected ODC gene appeared to be hypomethylated at the cytosine residues in the sequence CpG. The transfectants were all responsive to serum stimulation, but showed different levels of ODC expression depending on both copy number and integration site of the transfected ODC gene. ODC serum induction in the transfectants was sensitive to cycloheximide and polyamine additions, and the half-life of the enzyme was very short, like that in normal CHO cells. These results suggest that the human ODC gene we transfected contains all the elements needed for normal control of ODC expression.     

Ornithine decarboxylase (ODC) expression pattern in human prostate tissues and ODC transgenic mice.

Ornithine decarboxylase (ODC) is the key enzyme in the polyamine synthesis pathway and is overexpressed in a variety of cancers. We have performed a detailed immunostaining analysis of the expression of ODC in normal, benign prostatic hyperplasia (BPH), and cancerous prostate tissues. We conclude that ODC is overexpressed in both BPH and neoplastic tissues and that ODC overexpression appears to be an early event in prostate carcinogenesis. The extent of overexpression decreases as cancer progresses. Interestingly, ODC overexpression was also detected in patients who underwent androgen ablation therapy, suggesting ODC overexpression may contribute to the androgen-independent survival of prostate cancer cells. ODC is perinuclear localized in BPH samples but is diffusely cytoplasmic in cancer samples. Having shown ODC overexpression in human prostate cancer, we developed prostate-specific ODC transgenic mice to further investigate whether ODC overexpression alone is a causal factor in prostate carcinogenesis. RT-PCR and immunostaining confirmed that ODC was overexpressed in a subset of prostate epithelial cells. Although minor nucleoli enlargements in some tissues were detected, gross morphological changes were not observed in transgenic prostates. Therefore, overexpression of ODC alone in this subset of prostate epithelial cells is not sufficient to induce prostate carcinogenesis.     

The role of mitogens and lymphokines in the induction of ornithine decarboxylase (ODC) in T lymphocytes

Polyamine synthesis occurs early in lymphocyte activation after stimulation with antigen or mitogen. Ornithine decarboxylase (ODC) is the primary enzyme in the polyamine cascade. We have examined the induction of ODC by mitogens and/or lymphokines in human peripheral blood T lymphocytes. When isolated populations of monocytes and T lymphocytes were stimulated with phytohemagglutinin (PHA) there was little or no change in ODC activity. The combination of T lymphocytes and monocytes enhanced mitogen-induced ODC activity 10-fold. Several interleukin 1 (IL 1)-containing supernatants and fractionated human IL 1 were capable of substituting for monocytes in supporting PHA induction of ODC in T lymphocytes. Interleukin 2 (IL 2) and IL 2-containing supernatants were also capable of increasing ODC activity in T lymphocytes in the absence of monocytes. Lymphokines alone in the absence of PHA could not induce ODC. We conclude that both mitogens and monocytes are required for the induction of polyamine synthesis in T lymphocytes, and that supernatants containing IL 1 or IL 1 and IL 2 can substitute for monocytes in the induction of ODC in mitogen-stimulated T lymphocytes.     

Characterization,heterologous expression and functional analysis of mevalonate diphosphate decarboxylase gene (MVD) of Candida albicans

Mevalonate diphosphate decarboxylase (MVD) catalyzes the conversion of mevalonate diphosphate to isopentenyl diphosphate, a key building block for a large family of functionally important biomolecules. We have cloned the gene encoding MVD from Candida albicans, and report the first characterization of such a gene from an opportunistic fungal pathogen. Sequence analysis revealed that the MVD comprises 362 amino acid residues with a predicted molecular weight of 39.5 kDa, sharing minimal identity with the human analogue. Analysis of the genomic sequence indicated that the coding frame is interrupted by a small intron of 51 bp. Southern analysis of genomic DNA demonstrated that MVD is a single-copy gene. Furthermore, Southern analysis of electrophoretic karyotypes of C. albicans obtained by pulsed-field gel electrophoresis showed that MVD is located on chromosome 1. Northern analysis revealed that the level of MVD expression is affected by (1) the carbon source in the growth medium, (2) the growth phase, and (3) the growth form of the fungus (yeast-like or hyphal). To demonstrate the biological function of C. albicans MVD, complementation experiments were carried out with an S. cerevisiae strain (erg19(ts)) that is temperature-sensitive for MVD activity. A single copy of the C. albicans MVD gene, under the control of the NOP1 promoter, was able fully to complement the erg19(ts) phenotype, and expression of the epitope-tagged C. albicans MVD was detectable by Western analysis. Furthermore, the low degree of sequence identity between C. albicans MVD and its human analogue raises the possibility that fungal-specific inhibitors can be developed for the enzyme. Thus, C. albicans MVD appears to be an interesting candidate that could be targeted for the development of anti-fungal agents.     

Structure and expression of the ornithine decarboxylase gene of Chlamydomonas reinhardtii

A cDNA was cloned encoding ornithine decarboxylase (ODC) of the unicellular green alga Chlamydomonas reinhardtii. The polypeptide consists of 396 amino acid residues with 35–37% sequence identity to other eukaryotic ODCs. As indicated by the phylogenetic tree calculated by neighbour joining analysis, the Chlamydomonas ODC has the same evolutionary distances to the ODCs of higher plants and mammalians. The Chlamydomonas ODC gene contains three introns of 222, 133, and 129 bp, respectively. As revealed by Northern-blot analyses, expression of the Chlamydomonas ODC gene is neither altered throughout the vegetative cell cycle nor modulated by exogenous polyamines.     

Solid-phase immunoenzyme analysis of Coccidioides immitis antigens

A highly effective and specific solid-phase enzyme immunoassay system has been developed. The enzyme immunoassay is a highly sensitive technique for the detection and identification of C. immitis cellular and metabolic antigens. This technique is suitable for the study of strain differences in the antigenic composition of C. immitis, rendered harmless by different methods. The expediency of the preliminary sonification of cell suspensions of C. immitis, the causative agent of coccidioidomycosis, has been experimentally confirmed.     

Developmental regulation of ornithine decarboxylase (ODC) in rat testis: comparison of changes in ODC activity with changes in ODC mRNA levels during testicular maturation.

These studies were undertaken to analyze the changes in testicular ornithine decarboxylase (ODC) mRNA levels and ODC activity in rats from birth to maturity. Levels of ODC mRNA were initially low in animals aged 10-17 days. Beginning at 21 days, ODC mRNA levels began to rise, reaching maximal levels by 40 days (p less than 0.01). The size of the 2.2- and 2.6-kb ODC mRNAs did not appear to change with age, as determined by Northern blot analysis. The increase in ODC mRNA that began at 21 days paralleled the increase in testis weight. This increase in ODC mRNA preceded the appearance of rat protamine-1 mRNA, a germ cell-specific mRNA found in round spermatids, which was first detected on Day 40. In contrast, levels of sulfated glycoprotein-2 mRNA, which, in the testis, is found exclusively in Sertoli cells, were highest at Day 17 and thereafter declined gradually with age. Unlike the increase in ODC mRNA levels, ODC activity was highest in 10-day-old animals and thereafter declined steadily with age, reaching minimal levels by 40 days (p less than 0.01). Thus, the increase in testicular ODC mRNA levels was in marked contrast to the decrease in testicular ODC activity. Incubation of cytosolic extract from 40-day-old animals with that from 10- or 17-day-old animals inhibited ODC activity approximately 50%, when compared to cytosols from 10- or 17-day-old animals. Dialysis of cytosol from 40-day-old animals prior to incubation with cytosol from 10-day-old animals relieved this inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bursera fagaroides, effect of an ethanolic extract on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica

The effect of an ethanolic extract from the stem bark of Bursera fagaroides on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica was evaluated. For this purpose, increasing concentrations of the extract, up to 8.0 mg/mL, were added to amoeba cultures or ODC reaction mixtures, which were incubated at 37 °C. Metronidazole and G418 were added as controls. After 1.5 and 72 h, the ODC activity in vitro and growth, respectively, were determined. Results revealed a strong inhibition of growth with IC₅₀ values on the order of 0.05 mg/mL. ODC activity, on the other hand, was inhibited by 12% and 50% at concentrations of 4.0 and 8.0 mg/mL, respectively.     

Cell-cycle-dependent expression of human ornithine decarboxylase

A human ornithine decarboxylase (ODC) gene probe has been isolated from a Jurkat T-cell cDNA expression library, sequenced, and used to analyze ODC mRNA levels in untransformed human lymphocytes and fibroblasts stimulated to proliferate by various mitogens. The partial cDNA sequence is 86% homologous to the mouse ODC cDNA, and Northern blots indicate that the human and mouse mRNA species are similar in size. ODC mRNA is barely detectable in quiescent human T lymphocytes and undetectable in density-arrested W138 fibroblasts. Following stimulation of T-lymphocyte proliferation with phytohemagglutinin, the ODC mRNA level rises to a peak around mid G1 phase and decreases as the cells enter S phase. Serum stimulation of density-arrested fibroblasts results in an elevation of the ODC mRNA level which persists throughout the cell cycle. Epidermal growth factor (20 ng/ml) but not insulin (10 mg/ml) or dexamethasone (55 ng/ml) stimulates ODC expression in quiescent W138 fibroblasts. Southern blots suggest that human cells have a single copy of the ODC gene.