Minor contribution of an internal ribosome entry site in the 5'-UTR of ornithine decarboxylase mRNA on its translation

The mechanism of synthesis of ornithine decarboxylase (ODC) at the level of translation was studied using cell culture and cell-free systems. Synthesis of firefly luciferase (Fluc) from the second open reading frame (ORF) in a bicistronic construct transfected into FM3A and HeLa cells was enhanced by the presence of the 5′-untranslated region (5′-UTR) of ODC mRNA between the two ORFs. However, cotransfection of the gene encoding 2A protease inhibited the synthesis of Fluc. Synthesis of Fluc from the second cistron in the bicistronic mRNA in a cell-free system was not affected significantly by the 5′-UTR of ODC mRNA. Synthesis of ODC from ODC mRNA in a cell-free system was inhibited by 2A protease and cap analogue (m⁷GpppG). Rapamycin inhibited ODC synthesis by 40-50% at both the G₁/S boundary and the G₂/M phase. These results indicate that an IRES in the 5′-UTR of ODC mRNA does not function effectively.     

Multiple species of ornithine decarboxylase in rat tissues: Effects of dexamethasone

Several species of ornithine decarboxylase were separated by chromatography of rat thymus and kidney extracts on DEAE-Sepharose CL-6B. One major and one minor species were absent from thymus of rats two hours after hormone treatment but otherwise, the elution profile was identical to thymus from control animals. The elution patterns of ODC activity in kidneys of rats treated 2.5 or 5 hours before sacrifice with dexamethasone differ from that of control kidney and from each other. Enzyme from kidneys early after hormone treatment is eluted earlier than enzyme from control tissue, while at 5 hours, the enzyme is eluted much later than in the control. This suggests that the hormone-induced activity is subsequently modified.     

Effect of diabetes on the induction of ornithine decarboxylase by refeeding.

Refeeding of starved rats that had previously been schedule-fed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours. In diabetic rats, refeeding caused a smaller increase in enzyme activity in liver and none at all in muscle. When insulin was administered together with food to the diabetic rats, ornithine decarboxylase in muscle increased to levels greater than those observed in refed controls. The activity of the enzyme in liver also increased; however, the increase was still less than that observed in refed control rats. The data indicate that the induction of ornithine decarboxylase in liver and muscle following food ingestion is altered in diabetes. In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboxylase in skeletal muscle.     

Transneuronal induction of adrenal ornithine decarboxylase through descending spinal pathways

Induction of adrenal ornithine decarboxylase (ODC) was studied in rats that had previously undergone hemisection of the spinal cord at the C5–C6 level. Control rats were sham-operated. In the latter, the stress of immobilization produced symmetrical increases in ODC of the adrenal medulla. In rats with cord hemisection a quantitatively similar increase was observed only on the intact side, the response of adrenomedullary ODC of the lesioned side being significantly smaller. Induction of adrenomedullary ODC in hemisected rats by administration of apomorphine or of 2-deoxyglucose resulted in the same pattern of response as after immobilization. Induction of adrenocortical ODC by immobilization or apomorphine was not affected by cord hemisection. The results demonstrate the existence of both ipsilateral and contralateral components of descending spinal pathways for the induction of adrenomedullary ODC, the ipsilateral fibres exerting a preponderant effect.     

Absence of inactivation or phosphorylation of ornithine decarboxylase by nuclear protein kinase NII and of immunological cross-reactivity between RNA polymerase I and ornithine decarboxylase

Incubation with protein kinase NII did not result in phosphorylation or inactivation of mouse kidney ornithine decarboxylase. Partially purified ornithine decarboxylase preparations contained a protein kinase activity and stimulated the activity of RNA polymerase I. However, these properties were due to contaminating protein(s) since further purification reduced the kinase activity and removal of the ornithine decarboxylase with a specific antiserum did not abolish the ability to stimulate RNA polymerase I. Antibodies to RNA polymerase I did not interact with ornithine decarboxylase and antibodies to ornithine decarboxylase did not interact with RNA polymerase I. These results indicate that: a) mammalian ornithine decarboxylase activity is not regulated by phosphorylation by protein kinase NII or the contaminating kinase, and b) the ability of impure preparations of ornithine decarboxylase to stimulate RNA polymerase I is due to a contaminating unrelated protein.     

Role of ornithine decarboxylase in granulosa-cell replication and steroidogenesis in vitro

We investigated the role of ornithine decarboxylase in ovarian steroidogenesis and granulosa-cell replication under basal and hormonestimulated conditions $\text{in}\text{vitro}$. Enzyme activity was markedly (>95 or >99%) reduced by DL-difluoromethyl-ornithine or 1,3-diaminopropane, which significantly impaired granulosa-cell replication in log-phase cultures. However, inhibition of ornithine decarboxylase activity augmented basal and hormonestimulated steroid production per cell, an effect abolished by cyanoketone, a specific inhibitor of steroid synthesis. Both the anti-proliferative and the steroidogenic effects of enzyme inhibition were substantially reversed by putrescine, the end-product of the reaction. Thus, ornithine decarboxylase, or polyamines, may be required for granulosa-cell replication, while deprivation of these compounds facilitates the expression of more differentiated cell function, such as steroid synthesis.     

皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶活性及其基因表达的影响

采用高效液相色谱和原位杂交技术研究了皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶 (ODC)活性及ODCmRNA表达的影响。结果显示 ,大鼠完整肝脏中ODC水平较低 ,2 / 3肝切除 (PH)后 3h ,不同处理组ODC活性开始升高 ,6h达到最高值 ,其中 ,去肾上腺 NaCl组和糖皮质激素受体拮抗剂RU4 86处理组的酶活性高于对照组 (去肾上腺假手术组 ) ,而去肾上腺 皮质酮处理组的酶活性低于对照组 ,36h恢复到肝切除前水平 ;完整肝脏的ODCmRNA水平极低 ,PH后表达量迅速增加 ,5h达到最大值 ,不同处理组mRNA水平的高低顺序与酶活性一致 ,12h降至肝切除前水平 ;在PH前 12h给大鼠注射RU4 86 (10mg/kg体重 ) ,取得了与去肾上腺 NaCl处理鼠相似的结果。以上结果表明 ,在PH诱导的再生肝细胞中 ,ODCmRNA表达量的增加和 /或减少是造成ODC活性改变的原因之一 ,皮质酮对ODC活性及其mRNA的表达具有抑制作用 ,主要表现在肝再生的早期 ,该作用可能是通过受体实现的     

Expression and post-transcriptional regulation of ornithine decarboxylase during early Xenopus development.

In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.     

Epidermal ornithine decarboxylase and polyamines in mice exposed to 50 Hz magnetic fields and UV radiation

We studied the influence of magnetic fields (MFs) and simulated solar radiation (SSR) on ornithine decarboxylase (ODC) and polyamines in mouse epidermis. Chronic exposure to combined MF and SSR did not cause persistent effects on ODC activity or polyamines compared to the animals exposed only to UV, although the same MF treatment was previously found to accelerate skin tumor development. In an acute 24-h experiment, an elevation of putrescine and down-regulation of ODC activity was observed in the animals exposed to a 100-μT MF. No effect was seen 24 h after a single 2-MED (minimal erythemal dose) exposure to SSR. The results indicate that acute exposure to 50 Hz MF does exert distinctive biological effects on epidermal polyamine synthesis. Bioelectromagnetics 19:388–391, 1998. © 1998 Wiley-Liss, Inc.     

NaCl induced ornithine decarboxylase and DNA synthesis in rat stomach mucosa

A sensitive method is described that detects an alteration in the structure of tRNA that is caused by cadmium but not by magnesium or zinc ions. The chromatographic system, RPC-5, separates Drosophila tyrosyl-tRNA into two fractions. These two isoacceptors differ by a single position in the anticodon where either a guanosine or queuine resides. Cadmium ions apparently interact with the tRNA and prevent the chromatographic separation. This is the first instance where cadmium is shown to cause a selective change in nucleic acid structure. The RPC-5 system seems to be uniquely useful in detecting such a change.     

Endogenous praecaecal and total tract losses of nitrogen in pancreatic duct-ligated minipigs

The pancreatic duct-ligated minipig (PL) is an established model of pancreatic exocrine insufficiency (PEI) with a significant decrease of nutrient digestibility. This study aimed to quantify and compare endogenous losses of nitrogen (N) (ileal and faecal) in minipigs receiving an almost N-free diet. Altogether, 12 Göttingen minipigs (7 PL and 5 control animals) fitted with an re-entrant ileo-caecal fistula were used. In Study 1, ileal digesta was collected over a period of 12 h on seven consecutive days, including one 24 h collection, when animals were fed a diet containing 0.49 g N/kg dry matter (DM). In Study 2, faeces were collected for 10 consecutive days. In Group PL, the amount and DM content of ileal digesta were higher (p < 0.05), while N concentration was lower than in the Control. The ileo-caecal N flux [g/kg DM intake] was about 2.5 times higher in Group PL (5.47 ± 1.15) than in the Control (1.91 ± 0.59) (p < 0.05). The amount of faeces did not differ, but faecal N losses were higher in Group PL (p < 0.05). Endogenous faecal N losses [g N/kg DM intake] of the Control group (1.17 ± 0.72) were comparable with earlier studies, while those of Group PL were 2.6 times higher (3.09 ± 1.34). In contrast, urinary excretion of N did not differ between the Control and Group PL. In conclusion, PEI caused markedly increased endogenous N losses. Therefore, the impact of reduced digestibility of nutrients on endogenous N losses might be relevant for apparent protein digestibility rates and should be taken into account.     

Phosphorylation of rat heart ornithine decarboxylase by type-2 casein kinase

Highly purified preparations of rat heart ornithine decarboxylase are readily phosphorylated by rat liver type-2 casein kinase-TS at the same 54 KDa protein band which is also radiolabeled by 3H-DFMO. The reaction, which is stimulated by polylysine leads to the incorporation of up to 0.8 mol P/mol ornithine decarboxylase at seryl residue(s) included in a single 8.6 KDa CNBr fragment. Partially purified preparations of ornithine decarboxylase contain a type-2 casein kinase which promotes the phosphorylation of ornithine decarboxylase at the same CNBr fragment affected by rat liver casein kinase-TS.     

Effect of experimental diabetes on ornithine decarboxylase activity of rat tissues

Measurements have been made of the activity of ornithine decarboxylase of liver, heart, kidney and brain in alloxan-diabetic and control rats. In all these tissues this enzyme had decreased markedly at four weeks after induction of diabetes. These results are discussed in relation to the hormonal control and cyclic nucleotide regulation of ornithine decarboxylase.     

Post-transcriptional inhibition of ornithine decarboxylase induction by zinc in a difluoromethylornithine resistant cell line

Addition of Zn^2+_ to cell medium inhibited the induction of ornithine decarboxylase (ODC) activity in ODC overproducing L1210-DFMO^r cells. A significant effect was observed at a concentration as low as 0.01 mM, however a more marked inhibition was caused by the addition of 0.1 mM Zn²⁺. The inhibition of the induction of ODC activity was accompanied by a proportional decrease in the content of immunoreactive ODC protein, whereas the level of ODC mRNA, detemined by a solution hybridization RNase protection assay, was not affected signigicantly. Instead, some acceleration of ODC turnover was observed. the addition of 0.1 mM Co²⁺ or Mn²⁺, but not of other divalent metal ions, also inhibited ODC induction; differently from Zn²⁺ however, these metals affected cell viability and/or cell growth. Removal of endogenous Zn²⁺ by a chelator also provoked a strong decrease of ODC induction, which was reversed by Zn²⁺. However, addition of Zn²⁺ in excess of the chelator proved to be markedly inhibitory. These results indicate that both a restricted Zn²⁺ availability and an enhanced presence of the metal can inhibit the induction of ODC in L1210-DFMO^r cells.     

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-α)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCδ) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKδ pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.     

Cancer therapy and prevention by green tea: role of ornithine decarboxylase

Summary. Green tea which is widely consumed in China, Japan and India, contains polyphenolic compounds, which account for 30% of the dry weight of the leaves. Most of the polyphenols are flavanols, of which (−)-epigallocatechin-3-gallate (EGCG) is most abundant. Epidemiological studies revealed that the incidences of stomach and prostate cancers are the lowest in the world among a population that consumes green tea on a regular basis. It has also been reported that the quantity of green tea consumed, plays an important role in reducing cancer risk and in delaying cancer outbreak and recurrence. Various systems were used to confirm anti-cancer activities of green tea and/or EGCG. These included experimental animals in which cancer was induced chemically. Cultured cells transformed chemically or by oncogenes were also used. These studies clearly demonstrated that green tea or EGCG have anticancer and cancer preventive properties. The mechanisms of these activities have also been studied in details. It has been shown that green tea and its active components interfere with signal transduction pathways. Thus the activities of various protein kinases are inhibited, the expression of nuclear proto-oncogenes declines and the activity of ornithine decarboxylase (ODC) is reduced. ODC, which catalyzes the rate-limiting step in the biosynthesis of polyamines is closely linked with cellular proliferation and carcinogenesis. Inhibitors of ODC, like α-difluoromethylornithine (DFMO) have long been used for cancer prevention and therapy. It has been suggested that polyamine depletion by green tea could offer one explanation for its anti-cancer activities. Received July 27, 2001 Accepted September 8, 2001     

Histochemical localization of ornithine decarboxylase with a labelled suicidal enzyme inhibitor

We describe a new technique for cytochemical localization of ornithine decarboxylase by the use of a synthesized conjugate of rhodamine bound to α-difluoromethylornithine a suicidal inhibitor of the enzyme. The labelled inhibitor retained its specificity and irreversibility towards ornithine decarboxylase inhibition. Using this technique we have localized the enzyme in specific regions of the developing rat cerebellum. This novel technique may be generally applicable to other enzymes.