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ABSTRACT: Immunoreactive substance P was investigated in turtle lumbar spinal cord after sciatic nerve transection. In control animals immunoreactive fibers were densest in synaptic field Ia, where the longest axons invaded synaptic field III. Positive neuronal bodies were identified in the lateral column of the dorsal horn and substance P immunoreactive varicosities were observed in the ventral horn, in close relationship with presumed motoneurons. Other varicosities appeared in the lateral and anterior funiculi. After axotomy, substance P immunoreactive fibers were reduced slightly on the side of the lesion, which was located in long fibers that invaded synaptic field III and in the varicosities of the lateral and anterior funiculus. The changes were observed at 7 days after axonal injury and persisted at 15, 30, 60 and 90 days after the lesion. These findings show that turtles should be considered as a model to study the role of substance P in peripheral axonal injury, since the distribution and temporal changes of substance P were similar to those found in mammals.
Brazilian Journal of Medical and Biological Research 05/2003; 36(4):515-20. · 1.13 Impact Factor
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ABSTRACT: Glycogen phosphorylase (GP) and cytochrome oxidase (CO) activities were mapped histochemically in the brain of the turtle Trachemys dorbigni. In the telencephalon, both activities occurred in the olfactory bulb, in all cortical areas, in the dorsal ventricular ridge, striatum, primordium hippocampi and olfactory tubercle. In the diencephalon, they were identified in some areas of the hypothalamus, and in rotundus and geniculate nuclei. Both reactions were detected in the oculomotor, trochlear, mesencephalic trigeminal nuclei, the nucleus of the posterior commissure, torus semicircularis, substantia nigra and ruber and isthmic nuclei of the mesencephalon. In all layers of the optic tectum GP activity was found, but CO only labelled the stratum griseum centrale. In the medulla oblonga both enzymes appear in the reticular, raphe and vestibular nuclei, locus coeruleus and nuclei of cranial nerves. In the cerebellum, the granular and molecular layers, and the deep cerebellar nuclei were positive for both enzymes. The Purkinje cells were only reactive for CO. In the spinal cord, motor and commissural neurones exhibited a positive reaction for the two enzymes. However, CO also occurred in the marginal nucleus and in the lateral funiculus. These results may be useful as a basis for subsequent studies on turtle brain metabolism.
Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 11/1999; 124(2):113-22. · 2.23 Impact Factor
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ABSTRACT: Seven days after transection of the sciatic nerve NADPH-diaphorase activity increased in the small and medium neurons of the dorsal root ganglia of the turtle. However, this increase was observed only in medium neurons for up to 90 days. At this time a bilateral increase of NADPH-diaphorase staining was observed in all areas and neuronal types of the dorsal horn, and in positive motoneurons in the lumbar spinal cord, ipsilateral to the lesion. A similar increase was also demonstrable in spinal glial and endothelial cells. These findings are discussed in relation to the role of nitric oxide in hyperalgesia and neuronal regeneration or degeneration.
Brazilian Journal of Medical and Biological Research 05/1999; 32(4):489-93. · 1.13 Impact Factor
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ABSTRACT: Growth hormone binding proteins (GHBP) have been identified in the blood of many species. The aim of the present work is to study the physiological role of the GHBP in the turtle serum which we recently described. Binding studies were carried out using in vivo pharmacokinetic and chromatographic techniques as well as in vitro methods. When (125)I-GH was injected in physiological concentration into Chrysemys dorbigni turtles, the first step of pharmacokinetics was the binding of a significant fraction of the labeled GH by the GHBPs present in serum. The decay curve followed a three compartments model and gave the equation: Ae(-alphat) + Be(-betat) + Ce(-gammat). The fast compartment with t(1/2) of 14.4 min or 25.2 min, for hGH and bGH represents 30.3% and 18.9% of total radioactivity, respectively, at hypothetical time zero (not experi mental). Chromatographic studies reveal that this rapid compartment represents free GH. The second and third compartments represent complex forms between GH and GHBPs present in the turtle serum, and represent 70% and 80% of total radioactivity for hGH and bGH, respectively. In vitro chromatographic studies showed direct evidence of the presence of GHBPs in the turtle serum. The presence of these GHBPs changed the pharmacokinetics of labeled GH in plasma and the subsequent liver uptake of GH. The labeled hGH or bGH binds to turtle serum in similar proportion, but maximal liver uptake of these hormones are completely different (L/B ratio of 9.2 +/- 0.6 (n = 5) for ( 125)I-hGH and 4.8 +/- 0.3 (n = 7) for (125)I-bGH). The reasons for these differences could be that human GH binds to lactogenic and somatotropic receptors and bovine GH binds only to somatotropic receptors.
Archives of Physiology and Biochemistry 05/1999; 107(2):129-35.
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ABSTRACT: Seven days after transection of the sciatic nerve NADPH-diaphorase activity increased in the small and medium neurons of the dorsal root ganglia of the turtle. However, this increase was observed only in medium neurons for up to 90 days. At this time a bilateral increase of NADPH-diaphorase staining was observed in all areas and neuronal types of the dorsal horn, and in positive motoneurons in the lumbar spinal cord, ipsilateral to the lesion. A similar increase was also demonstrable in spinal glial and endothelial cells. These findings are discussed in relation to the role of nitric oxide in hyperalgesia and neuronal regeneration or degeneration.
Brazilian Journal of Medical and Biological Research. 01/1999;
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ABSTRACT: Bovine 125I-insulin was injected into the estuarine crab Chasmagnathus granulata in order to study its distribution and specific uptake by tissues. The highest radioactivity uptake occurred in both anterior and posterior gills, which reached maximum values at 30-60 min following labeled insulin administration. Heart and hepatopancreas concentrated a very low amount of radioactivity (only 9 and 3%, respectively, of that shown by gills). A significant reduction of the uptake was observed in the gills when an excess of unlabeled insulin was injected together with the labeled hormone. In vitro studies also showed specific uptake of 125I-insulin by the gills incubated at 25 degrees C, which reached a plateau after 120-min incubation, suggesting a saturable process. The inhibition of 125I-insulin uptake was dose dependent on unlabeled insulin. Glucagon did not compete with radioactivity uptake by gills in vivo and in vitro. Further characterization of insulin-binding sites was performed in gill membrane. The amount of unlabeled insulin that prevented 50% of the 125I-insulin uptake was 7.78 micrograms/ml, and the Scatchard plot analysis established the presence of binding site with Kd of 3.11 microM and Bmax of 0.14 microM (r = 0.99). Ovine prolactin was not able to prevent. 125I-insulin binding to gill membrane. These findings seem to indicate the presence of specific binding sites for insulin or insulin-like substance in crab gills, which deserves further studies.
Journal of Experimental Zoology 11/1997; 279(2):118-25.
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ABSTRACT: Proteins that bind growth hormone (GHBP) have been identified in the blood of many mammalian and avian species, but not in reptilian species. We carried out binding studies with the serum of turtles using chromatographic techniques as well as the dextran-charcoal separation method. As in other species, we found at least two different GHBPs: one with high MW and low affinity and the other with lower MW and higher affinity. The high affinity GHBP was partially purified using gel filtration and affinity chromatography, reaching a degree of purification of 11,000 times (0.17 nmol/g of serum protein in the serum vs 1900 nmol/g protein in the purified material). When the high affinity GHBP was characterized, it was found to have a dissociation constant (Kd: 2.6 +/- 0.7 nM) similar to those described for mouse or rat, but lower than those for chicken, rabbit or man. The binding capacity (Bmax) was 120 +/- 43 fmoles/mg of protein, which can be also expressed as 1.08 +/- 0.38 pmol/ml of serum. A preliminary MW estimation of 50-60 kDa was obtained for turtle higher affinity GHBP. The specificity of this high affinity GHBP is somatogenic, since bovine GH competes as well as human GH for 125I-hGH bound to binding protein, while ovine PRL competes only partially and with low affinity. Unrelated hormones, as insulin and glucagon, can not displace the 125I-hGH bound to turtle GHBP. A very important seasonal variation in turtle GHBP activity was observed: maximum binding was found in November (springtime), followed by a continuous decline over March and May.
Archives of Physiology and Biochemistry 05/1997; 105(2):167-74.
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ABSTRACT: Since experimental diabetes in rats and mice is associated with impairment of several aspects of thyroid function, we determined glucose and amino acid uptake in vitro by isolated thyroid glands from normal and streptozotocin-diabetic rats. Adult male Wistar rats weighing 150-200 g were used. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg body weight) and after five days only rats with blood glucose levels higher than 250 mg/dl were used. The thyroid glands were preincubated in Krebs-Ringer bicarbonate buffer in the presence or absence of insulin (0.7 nM to 7 muM) for 90 min and then incubated with the same concentration of the hormone or its vehicle plus 0.2 microCi of [1-14C]-2-deoxy-D-glucose ([14C]DG) or [1-14C] methylaminoisobutyric acid ([14C]MeAIB) for 15 to 180 min. The uptake of [14C]DG or [14C]MeAIB by the thyroid glands of normal rats increased as a function of incubation time, and the presence of insulin (7 microM) induced a significant increase of labelled DG from 3.30 +/- 0.11 to 4.16 +/- 0.12 and of labelled meAIB from 1.79 +/- 0.06 to 3.10 +/- 0.17 tissue/medium ratio (T/M) and after 45 min of incubation. The lowest concentration of insulin that increased both [14C]DG and [14C]MeAIB transport was 7 nM. Thyroid glands from STZ rats exhibited lower basal values of [14C]DG (4.03 +/- 0.11 T/M) or [14C]MeAIB uptake (1.05 +/- 0.05 T/M) than glands from normal rats (4.62 +/- 0.13 and 1.70 +/- 0.08 T/M, respectively). Insulin produced a stimulatory effect on the transport of both substrates in STZ rats. However, the maximal stimulating concentration of the hormone did not restore [14C]DG and [14C]MeAIB uptake to control values (4.89 +/- 0.17 in STZ rats versus 5.44 +/- 0.17 T/M in controls for [14C]DG, and 1.51 +/- 0.11 in STZ rats versus 2.19 +/- 0.10 T/M in controls for [14C]MeAIB). These results indicate that insulin exerts a direct action on the thyroid gland, and its absence or reduction affects thyroid metabolism, contributing, at least in part, to the abnormality in thyroid function associated with diabetes mellitus.
Brazilian Journal of Medical and Biological Research 12/1996; 29(11):1549-55. · 1.13 Impact Factor
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Hormone and Metabolic Research 04/1994; 26(3):160-1. · 2.19 Impact Factor
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ABSTRACT: Insulin A and B chains from pancreas of the turtle Chrysemys dorbigni have been purified to homogeneity, and their primary structures have been determined. The sequence of the A chain is G-I-V-E-Q-C-C-H-N-T-C-S-L-Y-Q-L-E-N-Y-C-N, and that of the B chain is A-A-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-S-P-K-A. The amino acid sequence of Chrysemys insulin is identical to that of another turtle (Pseudemys scripta), the chicken, and turkey. When compared with alligator insulin, it has three conservative substitutions in the B chain. However, there are seven substitutions when compared with the insulin of the rattlesnake.
General and Comparative Endocrinology 01/1992; 84(3):355-9. · 3.27 Impact Factor
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ABSTRACT: The presence of specific insulin binding sites in the thyroid gland of the turtle Chrysemys dorbigni has been previously reported. The purpose of the present work was to investigate the probable action of insulin on the uptake of [14C]deoxy-D-glucose ([14C]DG) and [14C]alpha-aminoisobutyric acid ([14C]AIB) in turtle (C. dorbigni) thyroid glands in vitro. Thyroid fragments (+/- 40 mg) were incubated at 25 degrees in Krebs-Ringer-bicarbonate buffer containing 0.2 microCi of [14C]DG or [14C]AIB without or with bovine insulin at different periods of time. The uptake of [14C]DG and [14C]AIB increased with incubation time. The presence of insulin (7 x 10(-6) M) in the incubation medium during 240 min did not modify the [14C]DG uptake. However if the thyroid fragments were previously incubated with insulin (60 min) and then incubated (240 min) with the same concentration of the hormone, the [14C]DG uptake was markedly increased. This stimulatory effect of insulin was dose-dependent. In similar experimental conditions, insulin also produced a significant increase in the uptake of [14C]AIB. Therefore, these findings strongly support the hypothesis that insulin might exert a direct action on the thyroid function.
General and Comparative Endocrinology 05/1991; 82(1):8-13. · 3.27 Impact Factor
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ABSTRACT: Immunoreactive insulin was demonstrated immunohistochemically with antibodies to human and porcine insulin by the avidin-biotin-peroxidase complex method in open-type gastrointestinal cells from sections of the antral stomach and of the upper, middle and lower intestine of the turtles Chrysemys dorbigni and Phrynops hilarii. In both species the concentration of cells positive for insulin-like material was higher in the gastric antrum than in the gut. The localization of insulin-like material in gastrointestinal mucosal cells of turtles is an unusual finding among vertebrates, because the insulin-containing cells migrate from the mucosal epithelium of the intestine early in vertebrate evolution to the acinar pancreas. The chemical nature of the gastrointestinal insulin-like material and its physiological role remain to be determined.
Brazilian Journal of Medical and Biological Research 02/1991; 24(3):327-31. · 1.13 Impact Factor
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ABSTRACT: The diabetogenic action of streptozotocin (SZ) was investigated in the turtle Chrysemys dorbigni after a 1- or 14-day fast. SZ (130 or 250 mg/kg) was injected intravenously, and blood glucose and plasma insulin were measured. Pancreatic endocrine cells were stained immunohistochemically by the immunoperoxidase avidin-biotin-peroxidase complex method. Only 14% of the SZ-treated turtles showed hyperglycemia. Prolonged fasting did not increase the percentage of hyperglycemic animals. In control turtles, insulin (beta)-, glucagon (alpha)- and somatostatin (delta)-immunoreactive cells were detected in increasing order of frequency. The qualitative changes seen in cells from the hyperglycemic SZ-treated turtles were more evident in beta and delta cells.
Brazilian Journal of Medical and Biological Research 02/1989; 22(8):1033-7. · 1.13 Impact Factor
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ABSTRACT: The difficulty in obtaining cerebrospinal fluid (CFS) in an efficient and simple manner in rats prompts us to introduce a new technique that makes use of a cannula placed in the lateral ventricle. The cannula is implanted with a stereotaxic apparatus and the CSF is collected with a glass capillary tube. The technique has proved to work well for experiments in which the CSF must be free of blood. It also permits the collection of volumes of CSF sufficient for radioimmunoassays, and may be used in chronic experiments.
Physiology & Behavior 02/1987; 41(5):523-4. · 2.87 Impact Factor
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ABSTRACT: The characteristics of the specific binding of labelled insulin to turtle thyroid microsomes were investigated. Binding experiments were performed in Krebs-Ringer bicarbonate buffer (pH 7.4) at 25 or 4 degrees C for different periods of time. Dissociation of the labelled insulin from the binding sites was also evaluated. It was found that the binding is dependent on time, temperature and microsomal protein concentration, with an optimum pH of 8.0. Unlabelled insulin and pro-insulin competed with the labelled insulin, binding in direct proportion to their biological activities, while glucagon and growth hormones did not compete for the binding sites. Scatchard plot analysis established the presence of binding sites of high and low affinities, and the rate of dissociation of bound insulin was considerably increased by the addition of unlabelled insulin. Both results are compatible with a negative co-operativity site-site interaction model. Trypsin abolished the insulin binding. These findings indicate that the microsomes from the turtle thyroid gland contain specific binding sites for insulin. However, pre-incubation of microsomes with phospholipase C or S-adenosyl-L-methionine (SAM), or incubation in the presence of 2 mol NaCl/l did not increase the specific insulin binding. Therefore, the binding properties are similar to those observed in mammalian insulin-responsive tissues except for the absence of the effects of 2 mol NaCl/l, phospholipase C or SAM, which suggests the absence of masked insulin-binding sites.
Journal of Endocrinology 02/1986; 108(1):157-62. · 3.55 Impact Factor
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ABSTRACT: Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with 125I-insulin in the presence or absence of unlabeled insulin, in order to study its specific uptake. At 24 degrees, the specific uptake reached a plateau at 180 min of incubation. The dose of bovine insulin that inhibited 50% of the 125I-insulin uptake was 2 micrograms/ml of incubation medium. Most of the radioactive material (71%) extracted from the gland, after 30 min incubation with 125I-insulin, eluted in the same position as labeled insulin on Sephadex G-50. Only 24% eluted in the salt position. After 240 min incubation, increased amount of radioactivity appeared in the Na125I position. When bovine insulin was added together with the labeled hormone, a substantial reduction of radioactivity was observed in the insulin and Na125I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with 125I-insulin either at 24 or 6 degrees. The percentage of trichloroacetic acid (TCA)-soluble radioactive material in the dissociation medium increased with incubation time at both temperatures. However, the degradation activity was lower at 6 than at 24 degrees. The addition of bovine insulin to the incubation buffer containing 125I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with 125I-hGH or 125I-BSA showed values of uptake, dissociation, and degradation similar to those experiments in which an excess of bovine insulin was added together with the labeled hormone. Thus, by multiple criteria, such as specific uptake, dissociation, and degradation, the presence of insulin-binding sites in the turtle thyroid gland may be suggested.
General and Comparative Endocrinology 12/1985; 60(2):306-14. · 3.27 Impact Factor
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ABSTRACT: Insulin binding to rat adrenal glands was studied in vivo by i.v. injection of 125I-insulin either alone or together with an excess of unlabelled hormones (insulin, glucagon, prolactin, or growth hormone). In addition, isolated glands from normal or streptozotocin diabetic rats (STZ) were incubated in vitro with 125I-insulin and varying concentrations of unlabelled insulin. Both experiments showed specific binding sites in the adrenal glands. Furthermore the glands from diabetic rats bound more insulin than the glands from controls. The insulin stimulatory effect on the deoxyglucose (14C-DG) uptake was examined in isolated glands from normal and STZ rats. Adrenal glands from control rats exhibited higher basal values of 14C-DG uptake than glands from STZ rats. Insulin induced a stimulatory effect on the 14C-DG transport in glands from both control and diabetic rats. Adrenal glands from STZ rats responded much earlier to exogenous insulin, however the maximal stimulating concentration of the hormone did not restore the 14C-DG uptake to control values. The lowest concentration of insulin that increased the 14C-DG transport was 3 X 10(-8) M. The adrenal gland must be considered a target organ for insulin by both criteria, insulin specific binding and stimulatory action on the deoxyglucose transport.
Hormone and Metabolic Research 01/1985; 16 Suppl 1:77-81. · 2.19 Impact Factor
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ABSTRACT: Insulin labeled with 125I was injected into turtles to study its specific uptake by the thyroid gland. The gel filtration behavior of labeled material in blood and thyroid gland was determined in order to ascertain if the uptake is specific. Most animals were pretreated with KI to saturate the gland with iodide. Maximum specific uptake of radioactivity by the thyroid gland was only detected in animals pretreated with KI. A significant dose-related reduction (ED50 = 0.5 micrograms/kg) was observed when unlabeled insulin was administered simultaneously with 125I-insulin. Prolactin, glucagon and growth hormone (2 mg/kg) did not affect 125I-insulin uptake. Most of the radioactive material extracted from the turtle thyroid 15 min after 125I-insulin injection coeluted with 125I-insulin on Sephadex G-50. This peak decreased as a function of time after 125I-insulin administration. Similar elution patterns were found for thyroid extracts from turtles previously treated with KI. The labeled hormone in the gland was rapidly degraded or processed to both higher and lower molecular weight compounds. Prior administration of KI suppressed the former, whereas when unlabeled insulin was injected simultaneously with 125I-insulin the amount of degradation products was reduced. The demonstration of radioactive degradation products is consistent with the intracellular receptor-mediated degradation hypothesis. These findings indicate the presence of specific insulin-binding sites in the thyroid gland.
Brazilian Journal of Medical and Biological Research 02/1984; 17(1):65-74. · 1.13 Impact Factor
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ABSTRACT: Insulin labeled with 125I was injected into turtles (Chrysemys dorbigni) to study its specific uptake by tissues. The maximum specific uptake of radioactivity by turtle tissues was obtained 1 hr after administration of [125I]iodoinsulin. Besides liver and adipose tissue, specific uptake of labeled insulin was detected in some endocrine glands, such as pituitary and adrenals. Both glands were as active in concentrating labeled insulin as liver and adipose tissue. A significant reduction of the uptake was observed when unlabeled insulin was injected together with the labeled hormone. This reduction was dose dependent, and the concentration of unlabeled insulin that prevented 50% of the tissue uptake of [125I]iodoinsulin was of 1 to 10 μg/kg body weight. These doses were able to induce blood glucose decrease in the turtle. Prolactin, growth hormone, or glucagon were unable to displace labeled insulin uptake. The major proportion of the radioactive material extracted from liver and pituitary 1 hr after [125I]iodoinsulin injection into turtle coeluted with [125I]iodoinsulin in Sephadex G-50 column. The presence of radioactive degradation products are consistent with the intracellular receptor mediated degradation hypothesis. These findings suggest the presence of specific insulin binding sites in liver, adipose tissue, pituitary, and adrenal glands from turtles.
General and Comparative Endocrinology 10/1982; 48(1):89-97. · 3.27 Impact Factor
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General and Comparative Endocrinology 07/1981; 44(2):171-6. · 3.27 Impact Factor
