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Biomedical subjects

P Licht

Publications and source records attributed to P Licht.

At least 37 records · Page 2Linked to original sources

Purification and partial amino acid sequences of two distinct albumins from turtle plasma.

Two putative albumins, denoted Alb-1 (apparent molecular mass of 67 kDa) and Alb-2 (68 kDa), were purified from plasma of the emydid turtle (Trachemys scripta). Concentrations in serum or plasma were determined by radioimmunoassay using 125I-labeled Alb-1. In juvenile turtles (less than 2 years of age), serum concentrations of Alb-1 and Alb-2 were 2.72 +/- 0.23 mg/ml and 1.68 +/- 0.22 mg/ml, respectively, while concentrations in plasma pooled from adult turtles were 4.2 mg/ml and 2.6 mg/ml, respectively. The two albumins are immunologically distinct from one another as determined by both radioimmunoassay with 125I-labeled Alb-1 and Western blot analysis with antichicken albumin antiserum. Determination of the amino acid compositions of Alb-1 and Alb-2, and of albumin purified from plasma of the common snapping turtle (Chelydra serpentina), suggested that Alb-1 is more similar to albumins of other animals than is Alb-2. This was also indicated by Western blot analysis and by determining the N-terminal amino acid sequences of Alb-1 (40 residues) and Alb-2 (15 residues). Thus, it appears that two distinct forms of albumin are synthesized by T. scripta, possibly as a result of gene duplication and divergence.

Amino Acid Sequence↗

Abundant synthesis of transthyretin in the brain, but not in the liver, of turtles.

The binding of thyroxine to proteins in the blood plasma of the turtle, Trachemys scripta, was analyzed by incubation with radioactive thyroxine, electrophoresis and autoradiography. Albumin and an alpha-globulin were found to bind thyroxine; no thyroxine-binding transthyretin was detected in the prealbumin region. In contrast to blood plasma, a thyroxine-binding prealbumin was observed in medium from T. scripta choroid plexus incubated in vitro. RNA was extracted from brain tissue containing choroid plexus and from liver of T. scripta and Chelydra serpentina and analyzed by hybridization with transthyretin cDNA from the lizard Tiliqua rugosa. The brain RNAs contained substantial amounts of transthyretin mRNA, whereas only trace amounts of transthyretin mRNA were detected in RNA from liver. No transthyretin mRNA was observed in RNA from kidney. The results support the hypothesis that the expression of the transthyretin gene first evolved in the choroid plexus of the brain at the stage of the stem reptiles, whereas abundant transthyretin synthesis in liver evolved much later, and independently, in mammals and birds.

Animals↗

Pattern and source of secretion of relaxin in the reproductive cycle of the spotted hyena (Crocuta crocuta).

Female spotted hyenas are highly masculinized at birth and have no external vagina. Copulation with males and birth of young are accomplished through the central urogenital canal of the clitoris. This unusual adaptation requires remarkable changes in the elasticity of the connective tissues of the clitoris, without which neither copulation nor birth would be possible. We hypothesized that relaxin, a hormone that increases the extensibility of the connective tissues of the uterus and cervix of many other mammalian species, plays a role in the clitoral changes observed in hyenas. Serum relaxin was determined by specific RIA. Relaxin was not detected in serum of males, pubertal or nonpregnant adult females, or ovariectomized females. Immunoactive relaxin was detected in serum of juveniles at the time of initial growth of the urogenital meatus. High concentrations of immunoactive relaxin appeared in the serum of pregnant hyenas in the 2 wk preceding parturition. Immunoassays of extracts of hyena tissues and serum obtained from uterine and ovarian veins indicated that the placenta was the predominant source of relaxin, with possible ovarian contributions. Circulating relaxin decreased promptly following cesarean section near term. We conclude that relaxin secretion coincides with changes in extensibility of clitoral connective tissues 1) during growth of the clitoris in juveniles and 2) near the time of parturition in adults.

Animals↗

Purification and characterization of plasma vitamin D binding protein from the snapping turtle, Chelydra serpentina.

Vitamin D binding protein (DBP) was isolated from the plasma of the snapping turtle, Chelydra serpentina (mixed sex adult), and compared with the previously characterized dual function binding plasma protein that binds both thyroxine (T4) and vitamin D (tsTDBP) in an emydid turtle, Trachemys scripta. Purification of Chelydra serpentina DBP (csDBP) was accomplished by ion exchange chromatography, preparative polyacrylamide gel electrophoresis, and reverse-phase high-performance liquid chromatography. Estimates of increased purification (ca. 71-fold), recovery (ca. 1.5%), and corresponding plasma concentration (ca. 0.6 mg/ml) are confounded by interference with other proteins. A protein was identified that showed the same high affinity for 25-OH-cholecalciferol (vitamin D3) as the tsTDBP, and the two exhibited similar heat resistances in binding. The csDBP and tsTDBP had similar molecular weights by SDS-PAGE (ca. 58 kDa), showed immunological cross reactivity, and differed by only three residues, representing conservative substitutions, in the 30 NH2-terminal amino acid sequence. A slightly lesser homology (up to 89% similarity based on conservative substitutions) was seen with three mammalian DBPs. However, unlike tsTDBP, the DBP from the snapper did not bind T4. These data support the view that the DBP of emydid turtles secondarily evolved a second T4 binding site, and this is probably independent of the D3 binding domain.

Amino Acid Sequence↗

Cephalic veins in coronary artery bypass surgery.

Various alternative conduits for aortocoronary bypass grafting have been suggested when the saphenous vein quality is inadequate. During a 10-year period we have used the cephalic vein in 39 patients. Eighteen entered an angiographic follow-up study. A total of 31 arm vein grafts were used with 43 distal anastomoses. When calculating patency by number of patent distal anastomoses, this was 46% (median follow-up was 31 months), but calculating by the number of patent grafts we found 52% patency (median follow-up 31 months). In conclusion, we discourage others from using arm veins in aortocoronary bypass operations.

Aged↗

Substitution with testosterone as aromatizable substrate for induction of follicular maturation, estradiol production and ovulation in a patient with 17 alpha-hydroxylase deficiency.

17 alpha-Hydroxylase Deficiency (17 alpha-OHDS) is a rare defect of steroid biosynthesis, characterized by the inability to synthesize cortisol, androgens or estrogens, by the complete absence of follicular maturation, hypergonadotropic hypogonadism, primary amenorrhea and hypertension. Since the ovaries of such patients contain numerous primordial follicles, we hypothesized that the absence of spontaneous follicular maturation could be due to a lack of aromatizable substrate. To provide this substrate, testosterone was administered either by intra-ovarian injection or by vaginal administration. Ovarian stimulation was performed with human urinary gonadotropins. Follicular maturation and ovulation could be induced by this treatment, as determined from ultrasonography, the analysis of LH, estradiol and progesterone serum levels and the aspiration of oocytes from the mature follicles. Fertilization of these oocytes in vitro, however, did not succeed. We conclude that follicular maturation can be induced in 17 alpha-OHDS by gonadotropins when testosterone is provided as an aromatizable substrate and that estrogens are a necessary component of follicular maturation.

Adrenal Hyperplasia, Congenital↗

Glucoprivation counteracts effects of testosterone on daily torpor in Siberian hamsters.

We tested whether 1) glucose availability is a signal for initiation of torpor in male hamsters and 2) glucoprivation can override the inhibitory effects of androgens on daily torpor. Male hamsters maintained at ambient temperatures of 8-16 degrees C were injected with 2-deoxy-D-glucose (2DG), a glucose analogue that interferes with cellular glucose oxidation. 2DG (2,000-2,500 mg/kg body mass) induced torpor within 1 h of treatment in normal adult males in reproductive condition and in those bearing testosterone (T)-filled capsules that produced supraphysiological blood T concentrations; body temperatures were reduced from 37 to 25 degrees C for several hours. Latency to torpor was increased and duration of torpor was decreased in the T-treated hamsters. Food intake decreased substantially both on the day of torpor and on the succeeding day. Glucoprivation appears to counteract the potent inhibitory effect of androgens on torpor and induces a hypometabolic state that results in overall energy savings.

Animals↗

Primary structure of solitary form of gonadotropin-releasing hormone (GnRH) in cichlid pituitary; three forms of GnRH in brain of cichlid and pumpkinseed fish.

GnRH is a decapeptide family with at least nine distinct structures. Vertebrates, except for most placental mammals, have more than one of these GnRH forms within the brain. We report chromatographical and immunological evidence that three forms of GnRH are in the brains of both cichlid (Haplochromis burtoni) and pumpkinseed (Lepomis gibbosus) fishes. We argue that the three forms correspond to those previously described as sea bream GnRH (sbGnRH), chicken GnRH-II and salmon GnRH. In contrast, only one GnRH form was present in the pituitary of the cichlid and is identified as sbGnRH by amino acid sequence. This is the first report in which the primary structure of GnRH is determined from pituitary tissue. The N-terminus was identified by monitoring the digestion of the peptide by pyroglutamate aminopeptidase with matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The amidation of the C-terminus was established using an esterification procedure for monitoring with MALDI-MS. This report supports the idea that three forms of GnRH within one species is widespread in the order Perciformes. The present study establishes sbGnRH as the third GnRH form in H. burtoni and predicts that sbGnRH is synthesized in preoptic neurons, then transported to the pituitary in the preoptic-hypophyseal axons for the release of one or both gonadotropins.

Amino Acid Sequence↗

Factors influencing testosterone metabolism by anuran larvae.

The effects of experimental conditions--steroid concentration, temperature, treatment duration, tadpole density, and delivery mode--on testosterone (T) metabolic rates were examined in larval anurans. T uptake into tadpoles was measured by scintillation counting of [3H]T and metabolism of T was studied by thin layer chromatography in two anuran species, the African clawed frog (Xenopus laevis) and the western toad (Bufo boreas). T was extensively metabolized by both species, but the effects of each experimental condition on T metabolism varied between species. In the study of delivery mode, tadpoles injected with T dissolved in saline metabolized T faster than animals in groups where the steroid was added directly to the water. Animals injected with T dissolved in oil metabolized T the slowest, but T was short-lived in all cases. The current study demonstrates that concentration, temperature, treatment duration, tadpole density, and delivery mode may all affect steroid metabolic rates, but these effects vary between species in unpredictable ways. Since steroid metabolism may either activate or deactivate steroids, these factors are important in understanding the potency of steroids in tadpoles. A review of the literature dealing with steroid effects in anuran larvae illustrates these problems.

Analysis of Variance↗

Structure of a repilian plasma thyroxine binding protein indicates homology to vitamin D-binding protein.

Biochemical analyses revealed that a thyroxine (T4)-binding protein (TBP) in the blood of some turtles exhibits a high degree of structural homology to mammalian vitamin D-binding protein (DBP), instead of mammalian T4-binding protein (TBG). Like DBP, TBP is rich in Cys (ca. 28/molecule). The 31 NH2-terminal amino acids of TBP show 68% identity to the corresponding region of mammalian (rat, mouse, and human) DBP and one peptide from Lys-C proteolysis shows similar homology to residues 35-49 of DBP (the predicted sterol binding domain). Another peptide shows a 75% identity to a sequence corresponding to residues 134-154 of DBP, but seven others showed no homology to any proteins in GenBank. Digestion with N-glycosidase F reduced the M(r) of TBP by only 4.5K compared with 14K for TBG; T4-binding activity of TBP was unaffected. TBP (and DBP) also behaves as a much smaller molecule (approximately 57 kDa) than TBG (> 70 kDa) by size-exclusion chromatography, despite similarities in their estimated sizes from sodium dodecyl sulfate electrophoresis (approximately 60 kDa). Binding studies confirm that the turtle T4-binding protein likely also represents the major blood DPB. Thus, in the turtle, a single binding protein resembling DBP performs two major transport functions which are normally served by proteins representing two different multigene families in mammals.

Amino Acid Sequence↗

Thyroxine-binding protein represents the major vitamin D-binding protein in the plasma of the turtle, Trachemys scripta.

Structural homology between the high-affinity thyroxine (T4)-binding protein (TBP) in the plasma of the turtle, Trachemys scripta, and vitamin D-binding proteins (DBP) of mammals prompted an investigation of plasma vitamin D binding in the turtle. Several lines of evidence indicate that the TBP represents the primary binding protein for 25-OH-cholecalciferol (D3) in the turtle plasma. D3-binding protein in whole plasma migrates in the same position as TBP by size-exclusion chromatography and polyacrylamide gel electrophoresis; it is electrophoretically distinct from sex hormone-binding proteins. D3 binding to purified TBP alone is enhanced (up to sevenfold) in the presence of plasma proteins, including albumin; with this correction, the D3-binding activity of plasma corresponds to expected TBP titers. Plasma selectively stripped of TBP by affinity chromatography and purified turtle albumin have only trace D3-binding activity. Variations in physiological state (thyroidal status, age, and sex) previously associated with variable T4 binding (and TBP levels) in T. scripta show correlated variability in plasma D3 binding. D3 binding is also highly correlated with T4 binding (r = 0.81; P < 0.001) for plasma samples taken from 30 adults representing 10 different species of Trachemys. D3 binding in plasma exhibits a high-affinity site (Ka = 2.3 x 10(8) M-1) and a second lower-affinity (Ka = 2 x 10(6) M-1), higher-capacity site; capacities are highly variable. Purified TBP has a comparable high affinity (Ka = 2.8 x 10(8) M-1), with a capacity close to 1 mol/mol. Binding of T4 and D3 are not competitive, indicating separate binding sites for the two ligands; in fact, T4 tends to enhance the affinity and the capacity for D3. A single protein in turtle plasma ("TBP/DBP") functions in the transport of two different hormones normally served by two distinct binding proteins (representing different multigene families) in mammals. These results have implications for the mediation of T4 effects on growth.

Animals↗

Influence of androgen on the GnRH-stimulated secretion and biosynthesis of gonadotropins in the pituitary of juvenile female bullfrogs, Rana catesbeiana.

The influence of androgen on pituitary sensitivity to gonadotropin-releasing hormone (GnRH) was investigated in juvenile female bullfrogs. Newly metamorphosed bullfrogs were treated in vivo for 7 days or their pituitaries were treated in vitro for 24 hr with 5 alpha-dihydrotestosterone (DHT). Pituitary sensitivity to GnRH was assessed by incubating glands with 100 ng/ml GnRH. The secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was quantified by separate radioimmunoassays. Gth biosynthesis was quantified using immunoprecipitation to measure the incorporation of [35S]methionine. Prior exposure to DHT, in vitro or in vivo, significantly elevated the GnRH-induced secretion of LH and FSH. However, DHT had a differential influence on the two gonadotropins; both the basal and the GnRH-induced secretion of LH was enhanced, whereas only the GnRH-induced secretion of FSH was elevated. DHT did not significantly alter Gth biosynthesis. Treating older frogs (5 months postmetamorphic) in vivo with DHT (for 7 days) combined with a GnRH agonist (GnRHa) for 3 days enhanced the GnRH-stimulated Gth biosynthesis compared to treatment with either DHT or GnRHa alone. Therefore, while DHT may act on the pituitary to enhance gonadotropin secretion in response to GnRH, this action does not result in a concomitant increase in Gth biosynthesis. Juvenile female bullfrogs may require an increase in both GnRH and DHT in order to stimulate Gth biosynthesis.

Androgens↗

Dopaminergic inhibition of gonadotropic release in hibernating frogs, Rana temporaria.

The influence of a dopaminergic antagonist, metoclopramide (MET), and an agonist, bromocriptine (BROMO), on reproductive status was examined in female frogs, Rana temporaria. MET induced advanced ovulation during hibernation, suggesting dopaminergic inhibition of gonadotropin (LH) release during this period. BROMO did not decrease plasma LH in intact females in comparison with vehicle (VEH)-treated controls (VEH: 11 +/- 6 vs BROMO: 5 +/- 4 ng/ml) or in sham-lesioned (SL) females (SL; 12 +/- 5 vs SL + BROMO: 9 +/- 8 ng/ml). However, BROMO significantly depressed the rise in plasma LH following lesioning (L) which disconnected the hypothalamus from the medium eminence-pituitary complex (L + BROMO: 29 +/- 10 vs L: 74 +/- 30 ng/ml; P < 0.002). Taken together with previous results of lesion studies, these data point to an important role of dopaminergic inhibition in the regulation of seasonal reproduction in this frog.

Animals↗

The nature and distribution of gonadotropin-releasing hormones in brains and plasma of ranid frogs.

Highly specific antisera and reversed-phase HPLC were used to examine the nature and distribution of gonadotropin-releasing hormone (GnRH) in the brains and plasma of three species of Rana frog previously reported to vary in the types and brain distributions of GnRH. The three species examined, Rana pipiens, Rana esculenta, and Rana ridibunda, exhibit differences in total concentration and relative concentrations of GnRH in brain areas, but all three contain the mammalian and chicken-II forms of GnRH (mGnRH and cGnRH-II). Both forms were found in the telencephalon and diencephalon, but mGnRH is consistently the most abundant form in the preoptic-hypothalamic area (e.g., ratios of mGnRH:cGnRH-II > 3:1 in hypothalamus-median eminence), whereas, cGnRH-II is the most abundant in the telencephalon and the only form measured in the cerebellum and medulla. The total content of cGnRH-II in the whole brain is about 1.5-2 times higher than that of mGnRH, due to the larger size of the areas outside the preoptic-hypothalamic area. These general patterns were the same for adults and juveniles. We found no evidence of a third form of GnRH corresponding to salmon GnRH in hypothalamus or other brain areas of R. esculenta as previously reported. These analyses also revealed the presence of both forms of GnRH in plasma draining the hypothalamic area, but the concentration of cGnRH-II is relatively higher than that in the corresponding hypothalamic tissue, suggesting differentially greater release or slower degradation of this form. These results do not support the conclusion that the ranid frogs are highly variable in the nature and distribution of GnRH in the brain, although they suggest that both forms of GnRH are potentially involved in the direct regulation of pituitary gonadotropes.

Amino Acid Sequence↗

The relation of the dual thyroxine/vitamin D-binding protein (TBP/DBP) of emydid turtles to vitamin D-binding proteins of other vertebrates.

The relation of a dual binding protein, involved in the transport of both thyroxine (T4) and vitamin D3, in the blood of emydid turtles to blood proteins in other vertebrates was examined immunologically. Binding studies with 25-OH-[3H]cholecalciferol (D3) confirmed the presence of a D3-binding protein (DBP) in the plasma of a wide variety of chelonian species representing both major suborders, as well as other species of reptiles, amphibians, birds, and mammals. Analysis by polyacrylamide gel electrophoresis (PAGE) showed that these binding proteins exhibited variable electrophoretic mobilities and in some species multiple DBPs were observed. Western blot analysis of these gels and additional tests with SDS-PAGE showed that an antiserum against the dual T4/D3-binding protein (TBP/DBP) from the turtle Trachemys scripta cross-reacted with the DBPs of diverse turtles, one crocodilian (Alligator), and birds; however, there was little or no cross-reaction with plasma from squamate reptiles (three snakes, four lizards), one crocodilian (Osteolaemus), amphibians (two anurans, one urodele), or mammals (six eutherians, one metatherian). The cross-reacting proteins (DBPs) all exhibit a similar M(r) (approximately 60 K). Adsorption of plasma by TBP/DBP-affinity chromatography confirmed this phylogenetic pattern of cross-reactions between the anti-TBP/DBP serum and functional DBPs in chicken, turtle, and alligator blood (affinity adsorption effectively eliminated D3 binding); there was only weak cross-reaction with DBP in a lizard and the second crocodilian (50% reduction in D3 binding) and none with DBP of snakes or mammals (D3 binding was unaffected). Results suggest that the plasma protein (TBP/DBP) that exhibits dual, high-affinity binding of T4 and D3 in one turtle family evolved from the more "primitive" vitamin D-binding protein of stem reptiles; thus, the high-affinity T4-binding site on this molecule is probably a derived characteristic of DBP in the Emydidae.

Amphibians↗

Role of aromatization in testosterone-induced inhibition of luteinizing hormone secretion in female turtles, Trachemys scripta.

Both 17 beta-estradiol (E2) and testosterone (T) were shown to inhibit in vitro pituitary LH secretion in the turtle Trachemys scripta. Since T was approximately 500 times less potent than E2, and 5 alpha-dihydrotestosterone was even less active than T, the inhibitory action of T may result from its aromatization to estrogen. We utilized both in vivo and in vitro approaches to elucidate the roles of T and estrogen in the negative feedback of pituitary LH secretion. Gonadectomy of adult (vitellogenic) females significantly elevated plasma LH. Adult females treated with fadrozole (an aromatase inhibitor) with or without daily injections of keoxifene (an antiestrogen) also showed an increase in plasma LH to a level comparable to that observed in gonadectomized females, whereas plasma LH levels of juvenile females treated with fadrozole remained undetectable. In vitro LH secretion in response to GnRH in juvenile females was significantly inhibited by 48-h exposure to 50 ng/ml T or 100 pg/ml E2. Both fadrozole (200 microM) and keoxifene (200 nM) significantly blocked this T-induced inhibition of LH secretion, demonstrating that T lacks intrinsic inhibitory activity. Confirmation of the inhibition of aromatase activity by fadrozole comes from metabolic studies of 1 beta-[3H]androstenedione using turtle brain, ovary, and pituitary. In vitro, fadrozole altered the metabolism of 1 beta-[3H]androstenedione and inhibited aromatase activities in these tissues. These results indicate that the inhibitory effect of T is largely mediated through its aromatization to estrogen, and that estrogen is primarily responsible for the suppressed LH secretion in vitellogenic adult turtles.

Androstenedione↗

Lack of self-regulation of human chorionic gonadotropin biosynthesis in human choriocarcinoma cells.

Human gestational trophoblastic neoplasms overexpress hCG/LH receptors. Whether this overexpression is a reflection of a loss of self-regulation of hCG biosynthesis was investigated using JAR human choriocarcinoma cells. The results show that exogenous hCG did not affect steady state hCG alpha and hCG beta mRNA or dimer hCG protein levels in JAR cells. The JAR cells, however, responded to 8-bromo-cAMP with an increase in hCG alpha mRNA levels, suggesting that cAMP-mediated regulation of the hCG subunit genes was intact in the cells. Disruption of receptor function by a receptor antibody, which resulted in an increase in hCG alpha mRNA levels and hCG secretion in normal trophoblasts, had no effect on JAR cells. Unlike normal trophoblasts, which contain a predominant receptor transcript of 1.8 kilobases (kb), with minor higher molecular size (7.5 and 5.4 kb) transcripts occasionally seen, JAR cells contain a higher abundance of multiple transcripts (7.5, 5.4, 3.5, and 1.8 kb), with the predominant transcript being 5.4 kb. In addition, although normal trophoblasts contain an 80-kilodalton receptor protein, JAR cells contain only a 50-kilodalton hCG/LH receptor isoform. In contrast to the effects of exogenous hCG on normal placental tissue in vitro, it was unable to down-regulate receptor transcripts or receptor protein in JAR cells. In summary, JAR cells lack the ability to self-regulate hCG biosynthesis. This loss could explain how hCG can reach very high levels in gestational trophoblastic disease compared to those in normal pregnancy.

Choriocarcinoma↗