The effect of UV-B radiation on Bufo arenarum embryos survival and superoxide dismutase activity.

The exposure of Bufo arenarum embryos to 300-310 nm UV-B at a dose of 4,104 Joule/m(2) resulted in 100% lethality within 24 hr while 820 Joule/m(2) was the NOEC value for short-term chronic (10 days) exposure. The dose response curves show that lethal effects are proportional with the dose and achieve its highest value within 48 hr post exposure. The superoxide dismutase (SOD) activity in amphibian embryos for sublethal UV-B exposures was evaluated by means of UV-B treatments with 273 (A), 820(B), 1368(C) and 1915(D) Joule/m(2) at 2 and 5 hours post irradiation. The SOD activity in units/mg protein in A, B, C and D at 2 hr after treatments were 80.72 +/- 14.29, 74.5 +/- 13.19, 39.5 +/- 6.99 and 10.7 +/- 1.89 respectively while for control embryos it was 10.88 +/- 1.31. At 5 hr after treatments the SOD values were similar to those found in control embryos. The results confirm the high susceptibility of amphibian embryos to UV-B and point out that the SOD activity is enhanced by low doses of UV-B irradiation achieving significantly higher values than in control embryos at 2 hr post exposure.

[Hyperhomocysteinemia: atherothrombosis and neurotoxicity]. / Hiperhomocist(e)inemia: aterotrombosis y neurotoxicidad.

The positive correlation existing between hyperhomocyst(e)inemia [HH(e)] and vascular disease has firmly been established through data derived from numerous epidemiological and experimental observations. Clinical data corroborate that homocysteine (Hcy) is an independent risk factor for coronary, cerebral and peripheral arterial occlusive disease or peripheral venous thrombosis. Hcy is a sulfhydryl-containing amino acid that is formed by the demethylation of methionine. It is normally catalyzed to cystathionine by cystathionine beta-synthase a pyridoxal phosphate-dependent enzyme. Hcy is also remethylated to methionine by 5-methyltetrahydrofolate-Hcy methyltransferase (methionine synthase), a vitamin B12 dependent enzyme and by betaine-Hcy methyltransferase. Nutritional status such as vitamin B12, or vitamin B6, or folate deficiencies and genetic defects such as cystathionine beta-synthase or methylene-tetrahydrofolate reductase may contribute to increasing plasma homocysteine levels. The pathogenesis of Hcy-induced vascular damage may be multifactorial, including direct Hcy damage to the endothelium, stimulation of proliferation of smooth muscle cells, enhanced low-density lipoprotein peroxidation, increase of platelet aggregation, and effects on the coagulation system. Besides adverse effects on the endothelium and vessel wall, Hcy exert a toxic action on neuronal cells trough the stimulation of N-methyl-D-aspartate (NMDA) receptors. Under these conditions, neuronal damage derives from excessive calcium influx and reactive oxygen generation. This mechanism may contribute to the cognitive changes and markedly increased risk of cerebrovascular disease in children and young adults with homocystunuria. Moreover, during stroke, in hiperhomocysteinemic patients, disruption of the blood-brain barrier results in exposure of the brain to near plasma levels of Hcy. The brain is exposed to 15-50 microM H(e). Thus, the neurotoxicity of Hcy acting through the overstimulation of NMDA receptors could contribute to neuronal damage in homocystinuria and HH(e). Since HH(e) is associated with certain neurodegeneratives diseases, in the present review, the molecular mechanisms involved in neurotoxicity due to Hcy are discussed.

Metallothionein induction and cadmium uptake in Bufo arenarum embryos following an acclimation protocol.

Bufo arenarum embryos at the end of their embryonic development were acclimated to cadmium (Cd) by means of a 10-day treatment protocol. Embryos were processed for metallothionein (Mt) isolation and Cd and zinc (Zn) contents were measured. The results showed that: (1) the uptake of Cd in the experimental embryos was 7 microg/g embryo (wet weight) representing a bioaccumulation of Cd 255 times higher than in the maintaining medium; (2) a major Mt-like fraction was Cd-induced 7.8 times that in control embryos; two other protein fractions also bound Cd and Zn but were induced by Cd only about 2 and 1.4 times; (3) the Zn concentration was about 44 microg Zn/g embryo (wet weight) and did not change significantly (p>0.01) in the experimental embryos with respect to controls, but in acclimated embryos the essential metal was released from the Mts. The enhanced Mt synthesis and release of Zn from the native Mts are discussed in relation to the acclimation phenomenon.

Hiperhomocist(e)inemia: aterotrombosis y neurotoxicidad. / [Hyperhomocysteinemia: atherothrombosis and neurotoxicity]

The positive correlation existing between hyperhomocyst(e)inemia [HH(e)] and vascular disease has firmly been established through data derived from numerous epidemiological and experimental observations. Clinical data corroborate that homocysteine (Hcy) is an independent risk factor for coronary, cerebral and peripheral arterial occlusive disease or peripheral venous thrombosis. Hcy is a sulfhydryl-containing amino acid that is formed by the demethylation of methionine. It is normally catalyzed to cystathionine by cystathionine beta-synthase a pyridoxal phosphate-dependent enzyme. Hcy is also remethylated to methionine by 5-methyltetrahydrofolate-Hcy methyltransferase (methionine synthase), a vitamin B12 dependent enzyme and by betaine-Hcy methyltransferase. Nutritional status such as vitamin B12, or vitamin B6, or folate deficiencies and genetic defects such as cystathionine beta-synthase or methylene-tetrahydrofolate reductase may contribute to increasing plasma homocysteine levels. The pathogenesis of Hcy-induced vascular damage may be multifactorial, including direct Hcy damage to the endothelium, stimulation of proliferation of smooth muscle cells, enhanced low-density lipoprotein peroxidation, increase of platelet aggregation, and effects on the coagulation system. Besides adverse effects on the endothelium and vessel wall, Hcy exert a toxic action on neuronal cells trough the stimulation of N-methyl-D-aspartate (NMDA) receptors. Under these conditions, neuronal damage derives from excessive calcium influx and reactive oxygen generation. This mechanism may contribute to the cognitive changes and markedly increased risk of cerebrovascular disease in children and young adults with homocystunuria. Moreover, during stroke, in hiperhomocysteinemic patients, disruption of the blood-brain barrier results in exposure of the brain to near plasma levels of Hcy. The brain is exposed to 15-50 microM H(e). Thus, the neurotoxicity of Hcy acting through the overstimulation of NMDA receptors could contribute to neuronal damage in homocystinuria and HH(e). Since HH(e) is associated with certain neurodegeneratives diseases, in the present review, the molecular mechanisms involved in neurotoxicity due to Hcy are discussed.

Total homocysteine levels in plasma: high-performance liquid chromatographic determination with electrochemical detection and glassy carbon electrode.

An isocratic high-performance liquid chromatography with electrochemical detection (HPLC-ED) method for the determination of total plasma homocysteine [H(e)] has been developed. The electrochemical detection is performed using a glassy-carbon electrode that is not specific for thiol groups. We have tried to solve the problem of specificity focusing our work on chromatographic resolution and have obtained good results without coelution of other thiol compounds or any substances mentioned as common interferences for carbon electrode methods: uric acid, ascorbic acid and salicylates. Thirty samples a day can be assayed for total homocysteine with a lower limit of detection of 2 pmol, and a limit of quantification of 1.0 micromol/l, with a coefficient of variation (C.V.) <20%. For a concentration of total plasma homocysteine of 9.36 micromol/l, the intra- and inter-assay C.V.s were of 3.86% and 5.55% respectively. The analytical recovery achieved in the preparation of the samples ranged from 85.0% to 98.3% and the electrochemical response was linear up to 100 micromol/l.

Metallothioneins and cadmium uptake by the liver in Bufo arenarum.

Bufo arenarum females were treated daily with 0.5 mg Cd kg(-1) during 10 days to evaluate the uptake of this heavy metal and the induction of metallothionein synthesis in the liver. The liver incorporated 26% of the Cd administered, about 6.5 times higher than the average uptake of the other tissues of B. arenarum. Three protein fractions from the B. arenarum liver bound Cd, and were induced by this xenobiotic up to approx. 24 times above the basal level of these proteins.

GnRH molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes). Immunological and chromatographic evidence for the presence of a novel molecular variant.

Gonadotropin-releasing hormone (GnRH) molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes), were characterized by gradient reverse phase high performance liquid chromatography (RP-HPLC). Eluted fractions were tested in radioimmunoassays with different antisera. The results show that the brain extract contains three forms of GnRH: one is immunologically and chromatographically similar to cIIGnRH (chicken II), and another is similar to sGnRH (salmon). A third GnRH appears to be chromatographic and immunologically different from the nine other known forms of the vertebrate hormone. This is the only variant present in the pituitary gland.

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column.

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (cIGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.

Immunoreactive GnRH suggesting a third form of GnRH in addition to cIIGnRH and sGnRH in the brain and pituitary gland of Prochilodus lineatus (Characiformes).

Molecular variants of GnRH (gonadotropin-releasing hormone) in brain and pituitary extracts of the South American characiforme Prochilodus lineatus were studied using a combination of reverse-phase high-performance liquid chromatography and radioimmunoassay with different antisera. In brain extracts our study revealed that this fish has at least two different types of GnRH: cIIGnRH (chicken II) and sGnRH (salmon), and possibly a third variant of this molecule. In pituitary extracts we could find only two immunoreactive peaks corresponding to sGnRH and the possible third form.

[Modified acetylcellulose etrols for molded frames of corrective eyeglasses]. / Modifitsirovannye atsetiltselliuloznye étroly dlia lit'evykh oprav korrigiruiushchikh ochkov.

The mode and cyclic pattern of tensions capable to disturb continuity of plastic frames and to change their shapes in long-term use are defined. The relations for their amplitudes are derived and limit values found. The process of filling a mould is studied as well as macrostructures and internal defects that develop in a casting. Creepage of the plastic frame is investigated when relaxation loading is applied. To assure prolonged strength and stability of the shape some criteria for optimizing the compositions of AC-etrols are proposed. Replacement of the AC and plasticization with low-molecular plasticizers are shown to affect the chosen criteria for which appropriate levels are ascertained. The optimum modifications of the AC-etrols used for mould frames are specified.

Relationship between type I and type II estrogen binding sites in the rat uterus.

Binding parameters of type I and type II sites for (3H)-estradiol were studied in cytosol of the rat uterus using the Scatchard method and a computer program based on the analysis of Rodbard et al. The data showed that type II sites present low binding capacity, in addition to low affinity, in contrast to the previously reported high capacity for this binding molecule. These results suggest that type II sites may not be crucial in the action of estradiol at the uterine level.

Estradiol binding and phosphatase activity in the uterus after castration and chronic diabetes: effect of molybdate and estrogen replacement.

We have studied the cytosolic estrogen receptor in uterus of rats after castration and diabetes induction with Streptozotocin, and the relationship of estradiol (E2) binding with phosphatase activities. Ovariectomy (OVX) and diabetes produced a significant reduction in Type I and II binding sites, but did not affect the equilibrium dissociation constants. The reduction of receptor levels was partially reversed by homogenization and incubation with 20 mM molybdate (MoO4) and also by chronic treatment with E2. Considering the possibility that the increase in E2 binding in the presence of MoO=4 was due to phosphatase inhibition, the activities of these enzymes hypothetically involved in receptor dephosphorylation and inactivation were determined in uterine homogenate and cytosol from intact, OVX, and diabetic rats with and without E2 treatment. Chronic OVX and diabetes induced stimulation of alkaline, acid and neutral phosphatase activities. On the contrary, the increment of estrogenic receptors due to E2 treatment was not correlated with changes in phosphatase activity. It is possible that this effect was due to the protection of the receptor or to the induction of receptor synthesis by E2. Molybdate inhibited acid and neutral phosphatases and increased alkaline phosphatase, which suggest that neutral and acid phosphatases are identical and that they were responsible for the receptor inactivation. However, it is unclear at present the relationship between the increment of alkaline phosphatase and the reduction of receptors.

Estradiol binding and phosphatase activity in the uterus after castration and chronic diabetes: effect of molybdate and estrogen replacement.

We have studied the cytosolic estrogen receptor in uterus of rats after castration and diabetes induction with Streptozotocin, and the relationship of estradiol (E2) binding with phosphatase activities. Ovariectomy (OVX) and diabetes produced a significant reduction in Type I and II binding sites, but did not affect the equilibrium dissociation constants. The reduction of receptor levels was partially reversed by homogenization and incubation with 20 mM molybdate (MoO4) and also by chronic treatment with E2. Considering the possibility that the increase in E2 binding in the presence of MoO=4 was due to phosphatase inhibition, the activities of these enzymes hypothetically involved in receptor dephosphorylation and inactivation were determined in uterine homogenate and cytosol from intact, OVX, and diabetic rats with and without E2 treatment. Chronic OVX and diabetes induced stimulation of alkaline, acid and neutral phosphatase activities. On the contrary, the increment of estrogenic receptors due to E2 treatment was not correlated with changes in phosphatase activity. It is possible that this effect was due to the protection of the receptor or to the induction of receptor synthesis by E2. Molybdate inhibited acid and neutral phosphatases and increased alkaline phosphatase, which suggest that neutral and acid phosphatases are identical and that they were responsible for the receptor inactivation. However, it is unclear at present the relationship between the increment of alkaline phosphatase and the reduction of receptors.

Relationship between type I and type II estrogen binding sites in the rat uterus.

Binding parameters of type I and type II sites for (3H)-estradiol were studied in cytosol of the rat uterus using the Scatchard method and a computer program based on the analysis of Rodbard et al. The data showed that type II sites present low binding capacity, in addition to low affinity, in contrast to the previously reported high capacity for this binding molecule. These results suggest that type II sites may not be crucial in the action of estradiol at the uterine level.

Changes in nuclear translocation of estradiol-receptor complex in anterior pituitary and uterus of rats with streptozotocin diabetes.

We have studied estradiol (E2) receptor nuclear translocation in anterior pituitary and uterus of ovariectomized control and diabetic rats one month after diabetes induction with Streptozotocin. Animals were pretreated with a low (0.5 microgram/100 g) or a high (25 micrograms/100 g) E2 dose 60 min before killing for the pituitary nuclear translocation. We observed that with the low E2 dose, nuclear translocation was reduced in pituitary from diabetic rats; the low dose given for 4 days also resulted in reduced induction of cytosolic progestin receptors in the pituitary and lower serum prolactin response in the diabetic group. With the 25 micrograms/100 g E2 dose, E2-receptor translocation, and the biological activity of E2 (induction of progestin receptors and serum prolactin response) were in the normal range. Serum E2 in controls and diabetics treated with E2 were not different. In the uterus, both low and high E2 doses given for 4 days resulted in significantly reduced nuclear translocation and uterine weight increment in the diabetic animals. These results suggest a brain-pituitary disturbance in addition to a peripheral (uterine) relative insensitivity to sex hormones as contributing factors to the reproductive failure of diabetic animals.

Variability of levels of specific estrogen binding in a human endometrial adenocarcinoma cell line.

Previous reports from this laboratory have described marked changes in the levels of specific estradiol (E2) binders in cultured endometrial adenocarcinoma cells (HEC-1B) that occur during the first 30 h after replating. In the present study, binding levels were measured daily during a period of 8 days. HEC-1B cells were incubated with 100 nM [3H]-estradiol at 4 and 30 degrees C, in the presence or absence of 10 microM diethylstilbestrol and concentrations of bound ligand were determined in the nuclear and cytoplasmic fractions. It was found that concentrations of specific estradiol binders increased the first day after plating and declined thereafter. Saturation analysis of estrogen binding sites in cytosol of HEC-1B cells labeled with [3H]-E2 or [3H]-estriol (e3), at concentrations ranging from 0.1-100 mM, showed two plateaus for E2 binding (at about 20 and 60 nM) but only one (at about 40 nM) for E3, when dextran-coated charcoal was used to separate free and bound ligand. The constants of dissociation of E2 and E3 for the high affinity binder were about 4 and 20 nM, respectively.

Uptake and binding of [3H]aldosterone by the anterior pituitary and brain regions in adrenalectomized rats.

The uptake of [3H]-aldosterone by the brain and anterior pituitary (AP) was studied after i.v. injection of the isotope into adrenalectomized rats. The AP showed the higher uptake ratio (i.e., radioactivity in tissue/radioactivity in blood), while the brain regions examined (hippocampus, hypothalamus, amygdala, cerebellum and cortex) contained lower levels of radioactivity, although they concentrated the hormone from blood. Neither [3H]-corticosterone nor [3H]-18-hydroxydeoxycorticosterone accumulated in the AP as much as [3H]-aldosterone, while [3H]-corticosterone's uptake was greatest in the hippocampus. Competition experiments demonstrated that [3H]-aldosterone uptake in the AP was inhibited by pretreatment of animals with excess aldosterone, corticosterone and dexamethasone, whereas aldosterone and corticosterone but not dexamethasone competed in the brain regions. Binding sites were demonstrated in vitro in cytosol fractions from AP and several brain regions. Scatchard plot analysis demonstrated high affinity, low capacity binding sites in cytosol from AP and hippocampus. These results suggest the AP and brain areas may be considered as targets for aldosterone, although the functions of the mineralocorticoid in these tissues are a matter of speculation.

Receptors, enzymes, and hormonal responses of endometrial cells.

Studies of hormone-related biochemical parameters of human endometrium are facilitated by the availability of a variety of types of endometrial tissue resulting from changes in hormonal environment during the menstrual cycle or from decidual, hyperplastic, and neoplastic transformations. Estrogen and progesterone receptors levels, rates of metabolism of ovarian hormones, enzymatic activities regulated by hormones or affecting intracellular concentrations of steroids, rates of production of prolactin and prostaglandins, and endogenous levels of hormones have been measured in samples of endometrial tissue and in separated glands and stroma. These studies have also included an evaluation of effects of estrogens and progestins administered to patients or added to the medium during in vito incubations of endometrium under organ culture conditions. Epithelial cells derived from isolated glands and stromal cells have been grown in primary cultures and also subcultured. These studies have revealed two mechanisms by which progestins exert an antiestrogenic effect on the endometrial tissue: viz, they were found to provoke a decline in estrogen receptor levels and to increase the metabolism of E2 through stimulation of estradiol 17 beta-dehydrogenase activity. In addition, studies of cells in culture have led to the unexpected finding of wide and rapid fluctuations in the levels of estrogen receptors, thus providing an opportunity to investigate factors involved in the regulation of concentrations of specific E2-binding sites.