Different turnover of rat fetal and placental ornithine decarboxylases

The half-lives of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) have been studied in fetuses and placentas from 18-day-pregnant rats. While the turnover of fetal and placental SAMDC were slightly different (t1/2 = 38 and 75 min, respectively) the half-lives of fetal and placental ODC differed markedly. T1/2 of fetal ODC was 15 min, similar to other mammalian ODCs, but placental ODC showed a relatively high half-life, about 160 min. According to that, placental ODC was more resistant than the fetal enzyme to in vivo hyperthermic treatment (40 degrees C, 1 h). Our results suggest that the degradative mechanisms for ODC in rat placenta could be regulated differently to those in other mammalian tissues.     

Gender-related differences in carnosine,anserine and lysine content of murine skeletal muscle

Summary. The aminoacyl-imidazole dipeptides carnosine (-alanyl-L-histidine) and anserine (-alanyl-1-methyl-histidine) are present in relatively high concentrations in excitable tissues, such as muscle and nervous tissue. In the present study we describe the existence of a marked sexual dimorphism of carnosine and anserine in skeletal muscles of CD1 mice. In adult animals the concentrations of anserine were higher than those of carnosine in all skeletal muscles studied, and the content of aminoacyl-imidazole dipeptides was remarkably higher in males than in females. Postnatal ontogenic studies and hormonal manipulations indicated that carnosine synthesis was up-regulated by testosterone whereas anserine synthesis increased with age. Regional variations in the concentrations of the dipeptides were observed in both sexes, skeletal muscles from hind legs having higher amounts of carnosine and anserine than those present in fore legs or in the pectoral region. The concentration of L-lysine in skeletal muscles also showed regional variations and a sexual dimorphic pattern with females having higher levels than males in all muscles studied. The results suggest that these differences may be related with the anabolic action of androgens on skeletal muscle.     

The preovulatory rise of ovarian ornithine decarboxylase is required for progesterone secretion by the corpus luteum

Ovarian progesterone secretion during the diestrus stage of the estrous cycle is produced by luteal cells derived from granulosa and thecal cells after the differentiation process that follows ovulation. Our results show that blockade of the preovulatory rise of ovarian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by treatment with the specific inhibitor alpha-difluoromethylornithine (DFMO) leads to a significant decrease in the ovarian progesterone content and a dramatic fall in the plasma levels of this hormone during the following diestrus. The same inhibition was produced in spite of the fact that both luteinizing and follicle stimulating hormones were given concomitantly with DFMO. On the other hand, the acute rise in the plasma progesterone levels observed after administration of human chorionic gonadotropin to mice at different periods of the estrous cycle was not affected by DFMO administration. Our results indicate that although elevated levels of ODC are not required for acute ovarian steroidogenesis, the preovulatory peak of ovarian ODC activity observed in the evening of proestrus may be critical for the establishment of a constitutive steroidogenic pathway and progesterone secretion by the corpus luteum during the diestrus stage of the murine estrous cycle.     

The flavodoxin from Helicobacter pylori: structural determinants of thermostability and FMN cofactor binding

Flavodoxin has been recently recognized as an essential protein for a number of pathogenic bacteria including Helicobacter pylori, where it has been proposed to constitute a target for antibacterial drug development. One way we are exploring to screen for novel inhibitory compounds is to perform thermal upshift assays, for which a detailed knowledge of protein thermostability and cofactor binding properties is of great help. However, very little is known on the stability and ligand binding properties of H. pylori flavodoxin, and its peculiar FMN binding site together with the variety of behaviors observed within the flavodoxin family preclude extrapolations. We have thus performed a detailed experimental and computational analysis of the thermostability and cofactor binding energetics of H. pylori flavodoxin, and we have found that the thermal unfolding equilibrium is more complex that any other previously described for flavodoxins as it involves the accumulation of two distinct equilibrium intermediates. Fortunately the entire stability and binding data can be satisfactorily fitted to a model, summarized in a simple phase diagram, where the cofactor only binds to the native state. On the other hand, we show how variability of thermal unfolding behavior within the flavodoxin family can be predicted using structure-energetics relationships implemented in the COREX algorithm. The different distribution and ranges of local stabilities of the Anabaena and H. pylori apoflavodoxins explain the essential experimental differences observed: much lower Tm1, greater resistance to global unfolding, and more pronounced cold denaturation in H. pylori. Finally, a new strategy is proposed to identify using COREX structural characteristics of equilibrium intermediate states populated during protein unfolding.     

Effects of central administration of lead, cadmium and other divalent cations on body temperature in mice

The intracerebroventricular administration of lead or cadmium to mice produced a marked decrease in body temperature. The maximum decreases in body temperature produced by the central administration of the heavy metals (1.97±0.3°C for lead and 2.87±0.6°C for cadmium) were similar to those obtained after acute peripheral administration of these compounds. The decreases in body temperature obtained after central administration of calcium, magnesium or zinc were significantly smaller than those produced by the heavy metals. The results indicate that the central nervous system is implicated in the hypothermia induced by these divalent cations and suggest that the alteration of membrane ionic permeability by these substances at hypothalamic sites may be related to the control of body temperature.     

Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).     

Hyperthermia and the neurotoxicity of exogenous glutamate in infant rats

A substantial elevation of the excitatory neurotransmitter glutamate can be produced in the brain of 3-day old rats, either after subcutaneous injection of monosodium glutamate (4 mg/g), or by hyperthermic treatment (40°C, 3 h). In the glutamate-treated animals a large increase in the GABA levels has also been observed while the elevation of this amino acid in the hyperthermic animals is insignificant. Although the magnitude of the increase of glutamate in both cases is rather similar, in the hyperthermic animals no cerebral lesions such as those produced in the glutamate-treated animals could be observed. Therefore, high extracellular levels of glutamate seem to be required to produce the variety of neurotoxic effects related to this excitatory amino acid.     

Differential expression of ornithine decarboxylase antizyme inhibitors and antizymes in rodent tissues and human cell lines

Ornithine decarboxylase antizyme inhibitors, AZIN1 and AZIN2, are regulators and homologous proteins of ornithine decarboxylase (ODC), the rate limiting enzyme in the biosynthesis of polyamines. In this study, we have examined by means of real-time RT-PCR the relative abundance of mRNA of the three ODC paralogs in different rodent tissues, as well as in several cell lines derived from human tumors. With the exception of mouse and rat testes, ODC mRNA was the most expressed gene in all tissues examined (values higher than 60%). AZIN2 was more expressed than AZIN1 in testis, epididymis, brain, adrenal gland and lung, whereas the opposite was found in liver, kidney, heart, intestine and pancreas, as well as in all the cell lines examined. mRNA abundance of the three antizymes (AZ1, AZ2 and AZ3) that interact with ODC and antizyme inhibitors was also analyzed. AZ1 and AZ2 mRNA were ubiquitously expressed, AZ1 mRNA being more abundant than that of AZ2, although the ratio was dependent on the mouse tissue. In carcinoma-derived cells AZ1 was more expressed than AZ2, whereas in neuroblastoma-derived cells AZ2 mRNA was much more abundant than that of AZ1. AZ3 was expressed exclusively in rodent testes, where it was the most abundant of the three antizymes (~80%). This study is the first comparative-quantitative analysis on the expression of antizymes and antizyme inhibitors in different types of mammalian cells.