The effects of castration and treatment with testosterone on the biliary excretion of (3H)aldosterone in rats. The rate of excretion of aldosterone radiometabolites into the bile duct cannulation, and the intravenous injection of (3H)aldosterone, was demonstrated to be markedly increased in male rats following castration. In 1 h, 72% of the injected 3H-radioactivity was excreted in the bile of castrated male rats compared with 26% in the intact male control rats. Castration of the males led to the increased biliary excretion of aldosterone metabolites and the elimination of the sex-dependence of this process in rats. The ovariectomy of female rats did not substantially increase the rate of excretion of aldosterone metabolites via the bile. Castrated male rats treated with testosterone excreted aldosterone metabolites into the bile at a slower rate. A similar treatment of ovariectomized female rats with testosterone also significantly slowed the rate of biliary excretion of the aldosterone metabolites. These findings suggest that the presence of androgens plays an important role in regulating the routes of hepatic metabolism of aldosterone and the rates of clearance of aldosterone and its metabolites from the plasma into the bile of rats.


Metabolism of single-component and high-molecular-weight radioactive insulin in rats. Single component (125I)iodoinsulin injected in rats was converted into a high-molecular-weight form. This form of radioactive insulin was shown to be metabolized significantly more slowly and to exhibit a more prolonged half-life than the single-component radioactive insulin. When injected in rats, it remained predominantly in the high-molecular-weight form without conversion into free, single-component insulin. A significant amount of radioactivity was shown to be in the kidneys of rats following the injection of single-component radioactive insulin. The radioactivity of kidney homogenates was shown to exhibiti molecular sizes similar to those of single-component insulin, gamma globulin, and insulin degradation products. The injection of the high-molecular-weight form of radioactive insulin did not produce a significant accumulation of radioactivity in the kidneys of the rats; the radioactivity remained primarily in the blood of the rats, a finding consistent with the observed prolonged half-life of this form of radioactive insulin. The radioactivity in the urine of the animals injected with the single component or the high-molecular-weight radioactive insulin was present only in the region corresponding to degradation products.


Effects of prostaglandins on RNA and casein synthesis in mammary gland explants of mice. Prostaglandins B2, E2, and F2alpha were found to stimulate RNA synthesis in mammary gland explants of mice in a prolactin-like manner. These effects were non-additive to maximally stimulatory concentrations of prolacin. Also, time-course studies showed that the effects of these prostaglandins only became apparent following at 2-4 hour lag period. The effects of these prostaglandins were not observed in explants which were not pretreated with insulin plus hydrocortisone, i.e., in tissues not responsive to prolactin Prostaglandins E1, A2, A1, B1, and F1alpha had no effects on the rate of RNA synthesis, but each of these prostaglandins abolished the responsivitiy of the explants to prolactin. Since DBcAMP, theophylline, and indomethacin were found previously to suppress the aciton of prolactin on RNA synthesis, the effects of these agents on the stimulatory effects of prostaglandins B2, E2, and F2alpha were tested. The incubation of explants with 5 mM DBcAMP or 5 mM thophylline abolished the effects of these prostaglandins on RNA synthesis. But incubation with indomethacin did not affect the responses to these prostaglandins. Prolactin-like effects of prostaglandins on casein synthesis were not observed. Prostaglandins E1, A2, A1, B2, F1alpha and indomethacin did, however, abolish the prolactin stimulation of casein synthesis. Furthermore, the preincubation of explants with prostaglandins B2, E2, F2alpha or arachidonic acid significantly shortened the time of onset of the prolactin stimulation of casein synthesis. The results are discussed with regard to a possible role of the prostaglandins as mediators for the actions of prolactin in the mammary gland.


Participation of serotonin in the phasic release of LH. I. Evidence from pharmacological experiments. Subcutaneous implantation of a silastic tubing containing crystalline estradiol in castrated female rats results in a circadian rhythm of LH release. Under such conditions, blockade of serotonin (5-HT) biosynthesis by p-chlorophenylalanine (PCPA) completely inhibits the afternoon elevation of plasma LH. Gonadotropin peaks remain inhibited as long as the concentration of the transmitter is effectively depleted. Intraperitoneal administration or intraventricular infusion of minute doses of the immediate precursor of the amine, 5-hydroxytryptophan (5-HTP), results in the reappearance of the next afternoon rise of plasma gonadotropin, whenever hypothalamic levels of 5-hydroxyindole acetic acid (5-HIAA), the main metabolite of 5-HT, are significantly increased over their value in PGPA-treated animals. The administration of methiothepin, a 5-HT receptor blocker, 9 hours of more prior to the next expected LH rise, similarly inhibits the cycle, whereas a dopamine receptor inhibitor has no effect under the same time conditions. We have concluded that serotoninergic neuron systems can have a positive, permissive effect on the transfer of neural information resulting in phasic gonadotropin release; this action of the amine is different from, but not contradictory to, the known inhibitory effect of 5-HT on the release of LHRH from the median eminence.


Metabolic clearance and secretion rates of porcine growth hormone in genetically lean and obese swine. The metabolic clearance rate (MCR) and the secretion rate (SR) of porcine growth hormone (pGH) have been examined in swine rendered genetically either lean or obese after 18 generations of selection for or against backfat thickness. At 15 weeks of age (when the muscle:fat ratio was greater than 1) the mean half-life (t1/2), MCR, and SR, for the obese, control, and lean swine were: t1/2 = 7.4, 8.9, and 9.8 min; MCR = 341, 279, and 158 ml/min; SR = 907, 802, and 520 ng/min, respectively. At 90 kg body weight (when muscle:fat ratio was less than 1, and the age was about 30 weeks) the data for obese, control, and lean swine were: t1/2 = 11.3, 12.0, and 11.7 min; MCR =305, 280, and 336 ml/min; SR= 535, 626, and 932 ng/min, respectively. The t1/2, MCR, and SR were not significantly different among the obese, control, and lean swine at either 15 weeks or 90 kg body weight. Comparing the two stages of development, the younger swine (15 weeks of age) had a shorter t1/2 (P less than .01), and secreted and cleared more pGH on a per kg body weight basis (P less than .05) than the older swine (90 kg bodyweight, about 30 weeks of age). However, the results suggest that the selection of swine for either leanness or fatness for 18 generations did not alter the MCR and SR of pGH. In addition, the differences observed between the younger and older swine suggest that GH is cleared at a more rapid rate and more GH is available per unit of mass in the younger animals.


Studies on a cell line from a functional rat thyroid tumor in continuous culture. A clonal cell line derived from a functional and transplantable rat thyroid tumor was established in continuous monolayer culture by the use of enzymatic dissociation followed by an alternate culture-animal passage procedure. After being implanted backin the animal and again plated in culture, epithelial-like cells aggregated and rearranged themselves over the bottoms of dishes in structures resembling cross sections of a normal thyroid gland. The same morphology and growth pattern were maintained after innumerable subcultures and freeze/thawing periods. Cells grew with a population-doubling time of about 24 h in serum-supplemented synthetic medium. Cell monolayers stained with periodic acid-Schiff (PAS) showed a uniformly epithelial-like morphology; their cytoplasm contained abundant PAS-positive material that was resistant to enzymatic digestion with amylase. Thin-layer chromatography of acid-butanol cell extracts in primary and clonal cultures, followed by a specific and sensitive staining method for iodine compounds, demonstrated the presence of monoiodotyrosine (MIT), diiodotyrosine, and triiodothyronine-thyroxine. In contrast, in similar extracts obtained from a cultured liver cell line, the only iodinated amino acid was MIT. Thus, with regard to the above criteria, this cell line preserved specialized thyroid cell morphology and function.


Circadian rhythms of serum renin activity and serum corticosterone, prolactin, and aldosterone concentrations in the male rat on normal and low-sodium diets. To investigate the control of aldosterone secretion, non-stress levels of serum aldosterone, corticosterone, and prolactin, and renin activity were determined at 4-h intervals during 24-h light-dark cycles in adult male rats on regular and low-sodium diets. Circadian rhythms of plasma aldosterone, prolactin, and corticosterone concentrations and of serum renin activity were demonstrated during a regular sodium diet. When the rats were on a low-sodium diet, a circadian rhythm of serum corticosterone and aldosterone concentration was observed, but there was no circadian variation in serum renin activity or in serum prolactin concentration. Serum aldosterone concentration correlated with serum corticosterone concentration (r = 0.48) and serum renin activity (r = 0.36) during a low-sodium diet. Serum prolactin concentration did not correlate with serum aldosterone concentration or serum osmolality. These data are compatible with a role for renin and ACTH, but not for prolactin, in the modulation of aldosterone secretion in the rat.


Synthesis of 17beta-estradiol by isolated ovarian tissues of the pregnant rat: aromatization in the corpus luteum. Corpora lutea from pregnant rats were incubated to determine their ability to produce 17beta-estradiol and to aromatize testosterone in vitro. Corpora lutea and non-luteal ovarian tissues were removed from rats on days 7, 15, and 22 of pregnancy, and these tissues were immediately frozen or incubated separately in medium 199 at 37 C in an atmosphere of 95% O2-5% CO2 for 4 h. 17Beta-Estradiol in tissue and medium were quantified by a highly specific radioimmunoassay. The estradiol content ivnariably increased in non-luteal tissues during incubation, while it decreased or remained the same in incubated corpora lutea. The synthesis in non-luteal tissues, which was 18 to 400-fold greter. The incubation of corpora lutea (5 to 25 mg of tissue) with testosterone (200 ng) on days 7, 15, and 22 of pregnancy resulted in a mean accumulation of 17beta-estradiol in medium of 2.5 x 103 pg/mg tissue, compared with a mean value of 6 pg/mg for luteal tissue removed from the same ovaries and incubated without testosterone. The incubation of corpora lutea from 15-day pregnant rats with (7alpha-3H)-testosterone resulted in 15% conversion to presumptive (7alpha-3H)17beta-estradiol, which was isolated identically to estradiol isolated for radioimmunoassay. Recrystallization to constant specific activity revealed a high degree of radiochemical purity (75%) of the isolated (3H)estradiol. Rat diaphragm muscle and rabbit corpora lutea did not aromatize testosterone to 17beta-estradiol in amounts detectable by radioirpora lutea in vitro is virtually diol by non-luteal ovarian tissues. However,the corpora lutea show a striking capacity to aromatize testosterone, which might explain the high estradiol content of the rat corpora lutea during pregnancy. The physiological significance of this aromatizing system and of 17beta-estradiol in the corpus luteum is unknown but may be related to the luteotropic action of estradiol in the pregnant rat.


Cartilage sulfation and serum somatomedin in rats during and after cortisone-induced growth arrest. The uptake of sulfate by rib cartilage in vitro and in vivo and the serum somatomedin activity by bioassay were determined in male rats during and after cortisone-induced growth arrest. Experimental treatment consisted of subcutaneous injections of cortisone acetate in a dose of 2.5 mg/rat/day for 4 days, beginning at 29 to 30 days of age in Buffalo rats, or 5 mg/rat/day for 4 or 5 days, beginning at 39 to 41 days of age in Long-Evans rats. Groups of hypophysectomized rats were studied in parallel in one experiment. The sulfate uptake in the controls declined linearly with increasing age in both the in vitro and the in vivo studies. Hypophysectomy resulted in a constant low level of sulfate uptake in vitro. At the end of cortisone treatment, the in vitro sulfate uptake was approximately midway between that of the hypophysectomized rats and that of controls; at 7 days recovery, it was at the control level; at 14 days it showed an additional rise above the control value; from 14 days to 35 days it declined parallel with but above the sulfate uptake of controls. The in vivo sulfate uptake was depressed by cortisone treatment. During recovery it approximately control values at recovery day 21. In succeeding recovery periods in vivo sulfate uptake remained at control levels. Serum somatomedin activity was significantly reduced during cortisone treatment; it returned to the control level by 21 days of recovery. The incubation of the cartilage of controls and cortisone-treated rats at 14 days of recovery with and without the presence of normal rat serum, cortisone recovery serum, or hypophysectomy serum resulted in significantly higher sulfate uptake in the cortisone-treated rat cartilage in each medium. These sera did not differ significantly in their stimulation of sulfate uptake in either cortisone recovery cartilage or control cartilage. Both treated and control cartilage had greater sulfate uptake with larger doses of serum added to the medium. The dose-response curves were parallel during treatment and early recovery; but the slopes of the dose-response curves of the cartilage of cortisone-treated rats were greater than those of the controls during late recovery. It is concluded that the increased in vitro sulfation after 14 days of recovery in cortisone-treated rats signifies a persistent alteration in cartilage metabolism. Normal in vivo sulfate uptake during that time may be the result of humoral controls. A mechanism other than the impairment of somatomedin production is probably involved in the failure of catch-up growth after glucocorticoid treatment in the rat.


The metabolic clearance rates and interconversion of cortisol and cortisone in pregnant and nonpregnant baboons. Pregnancy alters the pattern of maternal cortisol (F) metabolism and increases the maternal serum cortisol-binding capacity (CBC) of baboons. To determine whether these changes are associated with alterations in F clearance,the metabolic clearance rate (MCR) and interconversion (p) of F and cortisone (E) were measured by continuous infusion of (3H)F and (14C)E in 9 regularly menstruating and 7 pregnant baboons (Papio papio). In nonpregnant animals, the values (X +/- SE) for MCR-E (488 +/- 48 1/day) were greater (P less than 0.001) than those for MCR-F (214 +/- 22 1/day). The p value for the conversion of E leads to F (62.8 +/- 4.7%) was greater (P less than 0.001) than that for the reaction F leads to E (41.6 +/- 3.7%), indicating that F formation is favored. Consistent with MCR-E greater than MCR-F, the per cent of F bound to proteins other than albumin (75 +/- 2) was greater (P less than 0.001) than the per cent of E bound (52 +/-3). The production rate  (MCR x peripheral concentration; mug/min) of F (55.1 +/- 7.9) was greater (P less than 0.001) than that of E (28.5 +/-3.9) with essentially all of the F being secreted directly (secretion rate 51.2 +/- 7.9 mug/min). Essentially all of the E produced was derived from circulating F, vitually none being secreted directly (secretion rate 4.6 +/- 3.9 mug/min). Pregnancy did not alter the MCR-F (190 +/- 23 1/day), MCR-E 525+/- 51 1/day), per cent of F (79 +/- 3), or per cent of E (49 +/-3) bound,or F (57.2 +/- 9.2 mug/min) or E (35.5 +/- 4.9 mug/min) production rates. CBC (mug F/100 ml) was significantly (P less than 0.01) elevated (25.3 +/- 2.3, nonpregnant vs 35.1 +/- u.6, pregnant). In addition, p E leads to F was increased (75.5 +/- 1.8%) as was p F leads to E (54.3 +/- 3.7%; P less than 0.01). We have concluded that the MCR-F during pregnancy is more dependent on alterations in maternal metabolism than on the increased serum CBC characteristic of gestation. We suggest that the latter factor may be important in regulating the physiologic levels of the other steroids which bind to it.


The formation of 5alpha-androstane-3alpha, 17beta-diol by dog prostate. The 3-keto reduction of (1.2-3H)dihydrotestosterone to 3alpha and 3beta-androstanediol has been assessed in homogenates of 31 dog prostates that varied in size from less than 1 to greater than 15 g. The formation of both androstanediols was demonstrable in microsomes and cytosol, and both NADH and NADPH were effective cofactors for the two reactions. The formation of 3beta-androstanediol per unit weight of prostate or per mg protein did not show a clear-cut relation with the size fo the gland, but 3alpha-androstanediol formation per mg microsomal protein increased as a linear function with size so that the rates were on an average 10-fold greater in prostates greater than 15 g than in those less than 5 g in weight.


Acute changes in norepinephrine content in the median eminence induced by orchidectomy or testosterone replacement. Median eminence catecholamines were measured in individual samples from intact, castrated, or sham-operated male rats, in order to correlate changes in catecholamine content with the early post-castration rise in serum LH levels. Both norepinephrine (NE) and dopamine (DA) were measured by an enzymatic-isotopic assay and advantage was also taken of a method recently developed that makes it possible to obtain the median eminence free of the surrounding hypothalamus. Determinations were made at 4, 8, or 24 h after surgery. All animals were killed between 1400 and 1600 h. It was observed that castration increases the ME content of NE significantly at all the times studied, with peak values 8 h after orchidectomy. Sham operation induced a significant decrease in NE content in the ME 4 h after surgery. By 8 h the NE values had returned to intact control levels. Both castration and sham operation induced a significant decrease in DA levels 4 h after surgery. By 8 h, DA values had returned to intact control levels in both groups. When the NE levels in castrated rats had reached the highest value, serum LH values had not departed from control levels, indicating that the change in NE content in the ME takes place prior to the expected rise in LH. Testosterone replacement therapy (40 mug/100 gBW) of castrated rats blocked the increase in NE content observed 8 h after castration. Dopamine levels were not affected by this treatment. These results support the concept that the NE contained in median eminence catecholaminergic terminals participates in the feed-back regulation of LH secretion in the male rat, while DA is apparently not involved under the present conditions.


Studies on the extra-hepatic effects of glucagon in the eviscerated rat. The in vivo effects of glucagon on the metabolism of extra-hepatic tissues have been investigated in eviscerated, functionally hepatectomized rats with intact kidneys. In these animals, even pharmacological amounts of exogenous glucagon did not significantly alter plasma glucose, FFA, or amino acids, compared with saline treatment. The possible secondary release of adrenal catecholamines following such doses of glucagon appeared to be similarly ineffective in increasing the peripheral tissue mobilization of substrates. It was only when the eviscerated animals were pretreated with insulin that the subsequent administration of glucagon or epinephrine elicited significant elevations in plasma FFA. The concomitant evisceration and adrenalectomy did not produce results which were significantly different from evisceration alone. Both kinds of animals required insulin pretreatment before a lipolytic response to glucagon or epinephrine could be demonstrated. This suggests that severe insulin insufficiency itself elicits almost maximum catabolism in these animals and that the further addition of other catabolic hormones such as glucagon or epinephrine cannot increase these catabolic effects, as manifest in plasma concentrations of FFA. These data show an extra-hepatic lipolytic effect of glucagon in vivo, but do not illuminate the significance of this effect in the intact animal.


Presence of melatonin in plasma and urine or pinealectomized rats. Melatonin was shown to occur in rat plasma and urine after pinealectomy by bioassay, radioimmunoassay, and thin-layer chromatography. Total daily excretion of melatonin in pinealectomized rats was about 20% of control. Light-dark rhythms of plasma melatonin levels and urinary excretion rates disappeared after pinealectomy.


Catecholamine involvement in episodic luteinizing hormone release in adult ovariectomized rats. This study was intended to examine the role of hypothalamic norepinephrine (HNE) and dopamine (HDA) in episodic luteinizing hormone (LH) release in adult ovariectomized rats. Unrestrained, unanesthetized rats with indwelling right atrial cannulae were bled continuously (30, 50, or 100 mul of whole blood/4-6 min for 3-4 hours), and the blood samples were analyzed for LH by radio-immunoassay. In other individual rats, changes in the hypothalamic levels of norepinephrine and dopamine after drug administration were determined by a radioisotopic-enzymatic catechol-O-methyl transferase assay. alpha-Methyl-p-tyrosine significantly decreased HNE and HDA concentrations but failed to alter episodic LH release. Two dopamine-beta-hydroxylase inhibitors (U-14,624 and FLA-63) caused marked reductions in HNE, small but not statistically significant increases in HDA, and an inhibition of episodic LH secretion. Apomorphine, a dopamine receptor stimulator, caused a transient (50-60 min) but marked inhibition of episodic LH release. Saline injection had no effect. Pimozide, a blocker of dopamine receptors, prevented the inhibitory effects seen following apomorphine. Although not studied in detail, pimozide alone did not appear to alter episodic LH secretion. These data suggest that in adult ovariectomized rats norepinephrine may be an excitatory neurotransmitter in the modulation of episodic LH release. The activation of dopamine receptors may be capable of inhibiting this release process. However, the apparent inability of pimozide alone to alter episodic LH discharge suggests that under physiological conditions dopamine may not play a role in the modulation of episodic LH secretion.


Electrical stimulation of the arcuate nucleus in ovariectomized rats inhibits episodic luteinizing hormone (LH) release but excites LH release after estrogen priming. The effect of electrical stimulation of the arcuate nucleus of the hypothalamus (ARH) on the blood levels of luteinizing hormone (LH) was studied in ovariectomized (ovx) rats. Unanesthetized, unrestrained rats were bled continuously (30 or 50 mul whole blood/4-6 min) through indwelling right atrial cannulae for 1 1/2-2h prior to the onset of stimulation. The bleeding was continued for the following three hours, during which electrical stimulation was applied for two 60 min periods separated by a 60 min non-stimulation period. Whole blood was analyzed for LH by radioimmunoassay. Electrical stimulation of the ARH in ovx rats inhibited the episodic LH release characteristic of these animals. The inhibition was rapid in onset, beginning within minutes, and lasted for most if not all of the stimulation period or longer. The blood LH levels returned to control values prior to the second stimulation period during which the inhibitory response was again seen. Stimulation in other hypothalamic areas outside the ARH was generally ineffective, although repeatable inhibition of episodic LH release did occur in 2 of 3 rats stimulated in the periventricular nucleus. In a second study, ovx rats were primed with 5 mug estradiol benzoate/100 g body weight/day for 2 days prior to stimulation. In these rats, stimulation of the ARH or median eminence produced increases in LH release, whereas stimulation in other hypothalamic areas outside the ARH was ineffective. The parameters of electrical stimulation that produced increases in LH release were the same as those that caused the inhibition of episodic LH release. The results demonstrate that electrical stimulation of the ARH inhibits LH release in ovx rats, but increases LH release if the animals have been primed with estrogen.


Direct stimulation by growth hormone of glucagon and insulin release from isolated rat pancreas. The acute effects of bovine growth hormone (bGH) upon the release of glucagon and insulin from the isolated perfused rat pancreas were investigated in the presence of 5.6 mM glucose. Glucagon and insulin release occurred in response to 10(-9), 10(-8) and 10(-7)M bGH within 1-2 min and dissipated within 4 min; a significant increase was seen at 24 sec for glucagon and at 48 sec for insulin. In this concentration range, a dose-response relationship was established for both islet hormones. Upon the addition of 5 mM fumarate, glutamate, and pyruvate, glucagon and insulin release occurred also in response to 10(-11) and 10(-10)M bGH. In the absence of glucose, in response to 10(-7)M bGH, the magnitude of glucagon release was similar to that seen with 5.6 mM glucose; but the release of insulin failed to occur. In the presence of 16.7 mM glucose, the release of glucagon was blunted and that of insulin augmented. The data indicate that growth hormone can stimulate acutely the release of glucagon and insulin at physiological as well as pharmacological concentrations. These rapid and short-lived effects are likely to be direct and not mediated through the generation of somatomedin. The stimulatory effect of growth hormone appears to favor the release of glucagon under conditions of glucose lack, and of insulin under conditions of glucose abundance. These results indicate that growth hormone may have a tonic effect on islet hormone release.


Influence of synthetic somatostatin upon growth hormone release from perifused rat pituitaries. Basal release of pre-labeled, stored tritiated rat growth hormone ([3H]rGH) from perfused rat pituitary explants is a constant fraction of pituitary [LH]rGH content. Synthetic somatostatin (SRIF) inhibits the release of pre-labeled, stored [3H]rGH in a dose-dependent fashion. Prolonged exposure to SRIF results in an immediate and continuous inhibition of [3H]rGH release. In the in vitro perifusion system, the maximal inhibition, which is achieved with 25 nM SRIF, results in a rate of [3H]rGH release which is 30 to 40 per cent of basal release. Pulses of SRIF produce inhibition of [3H]rGH release followed by rebound release after withdrawal of SRIF. When the time of exposure to SRIF is held constant, both the SRIF-induced inhibition and the rebound release of [3H]rGH are dose-dependent. A similar progressive response is seen when a constant SRIF concentration is pulsed for variable periods of time. There is no net inhibition of [3H]rGH release by pulses of SRIF.


Ultradian growth hormone rhythm in the rat: effects of feeding, hyperglycemia, and insulin-induced hypoglycemia. Temporal patterns of plasma GH, immunoreactive insulin (IRI), and glucose were defined by obtaining serial blood samples from freely-moving male rats bearing chronic intracardiac venous cannulae.  Blood was withdrawn every 15 min for periods of 6 h.  Plasma GH and IRI were determined by radioimmunoassay.  The typical ultradian rhythm of GH secretion was evident in each undisturbed animal (peaks greater than 200 ng/ml; troughs less than 1 ng/ml; mean period:  3.40 +/-0.08 h).  Basal plasma IRI and glucose levels fluctuated minimally.  There was no significant correlation between plasma GH and IRI, GH and glucose, or IRI and glucose levels in unfed rats.  The rhythmic GH secretory patterns of feeding animals (mean period: 3.12 +/-0.16 h; peaks greater than 200 ng/ml; troughs less than 1 ng/ml) were similar to those of non-feeding animals (mean period:  3.34 +/-0.15 h; peaks greater than 200 ng/ml; troughs less than 1 ng/ml) despite large fluctuations in plasma IRI levels and a wide variation in the number and size of the meals taken.  No consistent relation was observed between the ingestion of meals and the bursts of GH secretion.  The mean period of the GH rhythm was not significantly altered by hyperglycemia (mean period; 3.25 +/- 0.08 h), although the amplitude of the pulses of half of the hyperglycemic rats was markedly depressed. Insulin-induced hypoglycemia caused a significant depression in the amplitude of the GH pulses; however, the pattern of this response was not consistent.  Despite wide variability in the GH response, the magnitude and time course of recovery of the plasma glucose levels was similar in all animals. These results suggest that GH secretion in the rat is regulated primarily by an endogenous ultradian rhythm which is not dependent on changes in plasma glucose or IRI levels, and continues to function independently of feeding behavior.  It is unlikely that GH is an important physiologic regulator of glucose homeostasis in this species.


Negative feedback control of LH by testosterone: a quantitative study in male rats. Adult male rats were castrated and implanted sc with testosterone (T)-filled Silastic capsules of different sizes which produce stable plasma T levels proportional to capsule length.  T and LH levels were determined in blood samples obtained at 3-day intervals for 21 days.  Plasma T values greater than 1.8 ng/ml suppressed plasma LH to levels below the intact range, while a plasma T of less than 0.5 ng/ml yeilded no significant inhibition of the post-castration LH rise.  Animals with a plasma T of about 0.7 ng/ml, however, showed intact, castrate, or immediate LH levels.  The feedback-effective range for all animals was between 0.5 ngT/mg (castrate-like LH response) and 1.8 ng/ml (LH at or below intact levels), but individual rats seemed to have a more restricted feedback range than that indicated by these group data.  To test this hypothesis further, rats were implanted at the time of castration with five 5-mm T-filled capsules.  One capsule was removed each week, producing step function drops in plasma T, and allowing examination of the LH responses of individual animals.  Plasma t levels greater than 1.6 ng/ml maintained LH values at or below intact levels.  However, when plasma T dropped to 1.1 ng/ml, some rats showed castration-like LH increase and others did not respond until after a further drop to 0.6 ng/ml.  It was concluded that the feedback-effective range of plasma T is very restricted and differs in individual rats.


Seasonal profiles of plasma luteinizing hormone, testosterone and estradiol in the ram. Plasma profiles of luteinizing hormone (LH), testosterone (T), and estradiol (E2) have been determined in five mature rams during the primary breeding season (September) and again when breeding activity was low (May). Blood samples collected every half h for 24 h during September or May showed that LH was released into the general circulation as distinct peaks. Although this secretory pattern was not the same for all rams and seasonal differences were noted in certain individuals, it is suggested that an inherent rhythm persists within each ram. With the exception of one ram in September, obvious T peaks were preceded by LH peaks greater than 2 ng/ml (LH-T interval less than 60 min). Season had no effect on the number and magnitude of LH peaks or on the mean concentrations; however, basal levels of LH were higher (P less than 0.01) in September than in May. Seasonal differences in peripheral T levels were more dramatic with mean, baseline, and peak concentrations elevated (p less than 0.01) during the fall. The number of T peaks, like those of LH, was not affected by season. Large variations in circulating levels of E2 made any possible relationship between this hormone and LH or T difficult to identify. Furthermore, a diurnal or seasonal rhythm could not be established for E2 in this study. It is concluded that seasonal differences occur in secretory patterns of LH and T and that a cause and effect relationship between these two hormones exists. Under conditions of this study, neither a consistent relationship between E2 and LH or T, nor a seasonal difference in mean E2 levels, was apparent.


Evidence for adrenergic mediation of cholinergic inhibition of prolactin release. Pilocarpine, a cholinergic agonist, significantly reduced the high levels of serum prolactin in estrogen-primed male rats and in female rats on the late afternoon of proestrus. In male rats treated with reserpine, chlorpromazine, haloperidol, or pimozide, serum prolactin levels were greatly elevated. Subsequent treatment with pilocarpine failed to reduce serum prolactin concentrations in these rats. When atropine, a cholinergic antagonist, was injected ip in doses of 3 to 250 mg/kg into male rats, prolactin release was not altered. However, when atropine was injected prior to pilocarpine, it prevented the reduction in serum prolactin by the latter drug. Methyl-atropine, which does not enter the CNS, did not prevent pilocarpine from inhibiting prolactin release. These results suggest that cholinergic inhibition of prolactin release is mediated via adrenergic neurons, and thus support a role for a cholinergic link in hypothalamic regulation of prolactin release.


The insulinotropic action of gastric inhibitory polypeptide in the perfused isolated rat pancreas. Gastric inhibitory polypeptide (GIP) produced a dose-related increase in immunoreactive insulin (IRI) from the perfused isolated rat pancreas. The doses employed were within physiological limits. This effect was glucose-concentration-dependent in that there existed a threshold concentration of glucose above which GIP exerted the insulinotropic action, and that, at a fixed concentration of GIP, increased glucose concentrations stimulated IRI release in more than an additive manner. A biologically active fragment of the GIP molecule was isolated and purified. All criteria have been satisfied that GIP is an insulinotropic hormone.


Gonadotrophin stimulation of progesterone synthesis by midpregnancy mouse ovarian cells in vitro. The ability of cultured midpregnancy mouse ovarian cells to synthesize progesterone de novo and from oxogenous pregnenolone has been assessed. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of delta5,3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity, but is unaffected by aminoglutethimide, which inhibits the cholesterol side-chain cleavage enzyme complex (desmolase). Since there is little metabolism of the formed progesterone, the ability of ovarian cells to convert exogenous pregnenolone to progesterone in vitro reflects the activity of 3beta-HSD in these cells. Cultured ovarian cells are also capable of endogenous progesterone production in the absence of added pregnenolone, although the absolute amount of progesterone produced is considerably less than that produced from exogenous pregnenolone. Since endogenous progesterone accumulation is almost completely blocked by the addition of aminoglutethimide to the culture medium, it is concluded that this response does represent de novo progesterone synthesis. Neither bovine luteinizing hormone (LH) nor human chorionic gonadotrophin (hCG) affects 3beta-HSD activity in cultured ovarian cells. The ability of the cells to secrete or to further metabolize the progesterone formed is also unaffected. However, both LH and hCG stimulate endogenous progesterone production within one hour of their addition to the culture medium. The stimulation, 2-10 fold in several experiments, can be maintained for at least six days of culture, and is not a result of an increase in the growth rate of the ovarian cells. As would be expected, the stimulation is blocked by the addition of aminoglutethimide to the culture medium. Taken together, these facts suggest that gonadotrophic hormones stimulate progesterone production by ovarian cells specifically by their action at steps prior to the conversion of pregnenolone to progesterone.


Pressor action of centrally perfused angiotensin II in rats with hereditary hypothalamic diabetes insipidus. A method was devised for the perfusion of the cerebral ventricles in conscious rats. Using this method a basal secretion rate of 15 +/- 3 pg of immunoreactive angiotensin II per min was calculated. This material was suggested to be of extrarenal origin. In comparison to findings in normal Long-Evans rats, pressor responses to intraventricular perfusions of angiotensin II were reduced in rats heterozygous for hypothalamic diabetes insipidus and virtually absent in rats homozygous for the hypothalamic deficiency whether they were treated with vasopressin or not. The pressor response to intraventricular angiotensin II is suggested to be related to the release of vasopressin.


Maturation and survival of spermatozoa in the epididymis of pregnenolone treated hypophysectomized rats. Adult hypophysectomized male rats were injected daily with 2 mg pregnenolone for 30 days. In these rats the testosterone concentrations in peripheral blood (0.5 ng/ml) and testes (46 ng/g) were appreciably lower than in intact rats (4.1 ng/ml and 167 ng/g, respectively). In hypophysectomized animals treated with pregnenolone the testes and epididymides were much better maintained than the prostates and seminal vesicles. High concentrations of dihydrotestosterone were found to be present in the head (12 ng/g) and body (8 ng/g) of the epididymis of these rats. Although the number of spermatozoa in the distal part of the epididymis of pregnenolone-treated hypophysectomized rats was only 23% of the number found in intact control animals, the spermatozoa were fertile.


Sympathetic nerve endings in the pineal gland protect against acute stress-induced increase in N-acetyltransferase (EC 2.3.1.5.) activity. Injection of the antidepressant desmyethylimipramine (DMI, 10 mg/kg) into intact rats or into rats in which the superior cervical ganglia had been decentralized caused a marked enhancement of the swimming stress-induced increase in pineal gland acetyl-CoA:serotonin N-acetyltransferase (N-acetyltransferase, EC 2.3.1.5) activity. DMI is known to block uptake, the transport of catecholamines by nerve endings. It was found that DMI had no effect on enzyme activity in superior cervical ganglionectomized (SCGX) rats which were swimming stressed. The pineal glands of these animals are devoid of nerve endings. In unstressed intact or unstressed surgically altered rats, injection of DMI caused only a minor increase in N-acetyltransferase activity, which was much smaller than that seen after stress. After 5 h in organ culture sympathetic nerve endings within the pineal gland are still intact. At this time DMI treatment of pineal glands taken from intact rats shifted the dose-response curve for epinephrine (EPI) stimulation of N-acetyltransferase activity by two orders of magnitude, but caused only a slight change in the dose-response curve for isoproterenol, which is not taken up into nerve endings. In contrast, DMI treatment in organ culture had no effect on the dose-response curve for EPI in denervated pineal glands. These results support the hypothesis that the response of pineal N-acetyltransferase activity to stimulation by stress is influenced by uptake. It would appear that in addition to terminating neuronal adrenergic transmission, this transport process physiologically protects the pineal gland against nontranssynaptic adrenergic stimulation.


Parathyroid hormone as a possible causal factor in osteopetrosis of the tl rat. The possibility of parathyroid dysfunction as a causal factor in the osteopetrosis of the tl rats was explored by evaluating serum calcium (Ca) and immunoreactive parathyroid hormone (iPTH) concentrations in this strain of rats, as compared with those of phenotypically normal littermate (LM) and non-littermate (NLM) control groups. The mean serum Ca concentration in the tl rat was not significantly different from that of the NLM group, although it was less than that of the LM group. However, all Ca values were within the normal range. The mean serum iPTH concentration in the tl rats was not significantly different from those of either control group. The data indicate that the adult tl rat has adequate but not excessive PTH secretion. Therefore, osteopetrosis and its manifestations in the adult tl are not caused by an abnormality of parathyroid function.


Anesthetics and thyrotropin secretion in the rat. The effects of seven anesthetics (thiopentone, 50 mg/kg ip; pentobarbitone, 50 mg/kg ip; chloral hydrate, 300 mg/kg ip; urethane, 1,5 g/kg, 1/2ip, 1/2sc; ether; methoxyflurane, 1,5%; halothane, 2%) on basal serum TSH concentrations and on the cold-induced as well as the TRH-induced TSH responses were studied in Sprague-Dawley rats. The basal TSH level in female rats were decreased by ether and halothane at 30 min and somewhat increased by pentobarbitone and chloral hydrate. The cold-induced (4C, 30-60 min) TSH response of the warm-adapted male rats (30 C, 7 days) was decreased by all of the anesthetics studied, but the effect of pentobarbitone was not significant. The TRH-induced (50 ng iv) TSH response in female rats was totally abolished only by deep ether anesthesia but augmented by bariturates and chloral hydrate. It is concluded that all of the anesthetics studied can modify the secretion of TSH by their central effects. Ether in high concentration seems to be effective also at the pituitary level. The use of anesthetics may be a source of error when studying the neurotransmitter control of TSH-TRH secretion in the rat.


Influence of superoxide dismutase and catalase on the stimulation by phagocytosis of L-thyroxine and L-triiodothyronine deiodination in the human leukocyte. Neither superoxide dismutase, which destroys the superoxide radical by catalyzing its conversion to hydrogen peroxide, nor catalase prevented the stimulation by phagocytosis of the deiodination of L-thyroxine or L-triiodothyronine in human leukocytes. On the other hand, superoxide dismutase led to an increase in the proportion of iodinated material, which remained at the origin during chromatography in a butanol-acetic acid-water solvent system, whereas catalase had the opposite effect. The data suggest that neither the superoxide radical nor hydrogen peroxide plays a major role in the stimulation of deiodination of hormone, but that once deiodination has occurred, hydrogen peroxide is more important than that superoxide radical in subsequent iodination reactions within the leukocyte.


Supersensitivity of anterior pituitary dopamine receptors involved in the inhibition of prolactin secretion following destruction of the medial basal hypothalamus. The medial basal hypothalamus of ovariectomized rats was destroyed using a modified Halász knife. Large increases in prolactin secretion were observed 1 and 14 days following the lesions. Long- and short-term lesioned animals were anesthetized with chloral hydrate and treated with various doses of apomorphine (0.05, 0.2, 2, 5 mg/kg). Blood samples were obtained before and 10, 30 and 60 minutes after the injection. Both the 0.05 and 0.2 mg/kg doses caused significantly greater and longer-lasting inhibition of prolactin in long-term than in short-term lesioned animals. Since the MBH was totally destroyed this study suggests that anterior pituitary dopamine receptors involved in the inhibition of prolactin secretion become supersensitive in long-term lesioned rats.


Secretion of an FSH-inhibiting factor by cultured Sertoli cells. Levels of LH and FSH released in vitro by rat anterior pituitary cells which were either co-cultured with isolated rat Sertoli cells or were grown with spent media recovered from the cultured Sertoli cells were measured by radioimmunoassay. The amounts of FSH released by pituitary cells grown for three days with Sertoli cells isolated from 31-36 day old rats or spent media from the cultured Sertoli cells, were significantly (P less than 0.01) lower compared to control pituitary cultures grown with fresh chemically defined medium. In contrast, the levels of LH were similar to the controls. The selective inhibition of FSH release was not observed when pituitary cells were co-cultured with rat spleen or kidney cells or with ruptured Sertoli cells. The FSH-inhibiting Sertoli-cell factor (SCF) was found to be a heat-labile macromolecule. It is suggested that the SCF may be secreted by the Sertoli cells in vivo, and regulate FSH secretion via negative feedback mechanism at the pituitary level.


Dual effect of clomiphene citrate on pituitary gonadotropin secretion in postmenopausal women. Fifteen postmenopusal women were given 25 mg, 50 mg of clomiphene citrate daily by mouth for 3 consecutive days. Significant (p less than 0.01)decrease of serum LH (73%, 65%) and FSH (76%, 76%) resulted after administration of 25 mg and 50 mg clomiphene citrate respectively. The suppressive effect was sustained for at least 4 days after cessation of this drug. On other hand decreased LH and FSH levels after administration of ethinyl estradiol increased again by simultaneous administration of clomiphene citrate. These results suggest deficient subjects but anti-estrogenic effects are evident when serum estrogen levels are restored in subjects with estrogen deficiency.


Studies on in vitro synthesis and secretion of human chorionic gonadtropin and its subunits. Human chorionic tissues were cultivated in vitro in they presence of 3H-proline in order to study the synthesis of the hCG and its subunits by the placenta. After terminating the culture, tissue extracts and media were individually gel-filtrated on Sephadex G-100. The eluted fractions were radioimmunoassayed for hCG, hCGalpha and hCGbeta and were measured for 3H-radioactivity. Label incorporation was determined by immunoprecipitation. Elution profiles of tissue extracts showed the existence of large immunologic forms of hHCG, hCGalpha and hCGbeta emerging near the void volume. The amounts of these large immunologic species in chorionic tissue gradually decreased during the course of cultivation. 3H-proline was almost exclusively incorporated into the large immunologic forms of hCG, hCGalpha and hCGbeta within the chrionic tissue during the 5-hour exposure. A great quantity of hCGalpha was found in the media after the 3-day culture, while the amount of hCGbeta found in the media was minute. After a 15-minute pulse, the 3H-radioactivity peak within the chorionic tissue appeared in the void volume, conicidental with hCG immunoreactivity. During the chase period, there was shift of the 3H-radioactivity peak associated with hCG immunoreactivity from the void volume to the more retarded area. In the media until after the 60-minute chase, no labeled hCG and its subunits appeared. Within the media after 3-hour chase, no labeled hCG peak associated with 3H-radioactivity was more retarded on Sephadex G-100 than that within the tissue extract after the 15-MINUTE pulse. These results suggest that the large immunologic forms of hCG, HCGalpha and hCGbeta are synthesized as the earliest detectable biosynthetic forms and that the may then be converted to small molecule species. In the culture of molar trophoblastic tissues after a 15-minute pulse, considerable amounts of hCG and its subunits accompanied by high 3H-radioactively had already been secreted into the media. These observations suggest that protein synthesis by molar trophoblastic tissue is markedly enhanced as compared with that by normal chorionic tissue and that immunoreactive materials synthesized in molar trophoblastic tissue may be secreted more radily than those synthesized a in normal chorionic tssue.


Intratesticular tissue fluid (ITF) for the micro-bioassay of LH. Intratesticular tissue fluid (ITF) was obtained by centrifugation of the decapsulated testis of the adult rat. Testosterone level in ITF was increased by direct injection of LH into the testis and was lowered by anti-LH serum. Based on the result of previous reports with testosterone in testicular vein blood, a simplification of the LH bioassay without imparing sensitivity and accuracy was undertaken using ITF as the material for testosterone measurement. Dose-response relationship between injected LH and testosterone concentration in ITF was observed. The sensitivity of the assay was 5 ng for LH (NIH-LH-S15). FSH (NIH-FSH-S10) and prolactin (NIH-P-S11) had no detectable activity on testosterone production. This assay method depending on biological activities of LH will be helpful for unbiased evaluation of the result obtained by radioimmunoassay.


Free amino acids in the caput and the cauda epididymis of adult rats. To examine the content and the composition of free amino acids in the intraluminal fluid of rat epididymis, the fluids were obtained by light pressure on the dissected tissues. The amount of the total free amino acids in the pressed fluid from the caput epididymis was significantly higher than those of the cauda epididymis and the testis. Glu and Gln were predominant amino acids in the caput, and their amounts occupied more than half of the total ninhydrin reactive compounds. Such a high concentration of Glu and Gln was not observed either in the cauda or in the testis. Castration decreased Glu and increased Gln in amount. Testosterone treatment to castrated animals did not restore Glu and Gln contents in the pressed fluid from the caput epididymis to the level observed in intact rats completely. Therefore, it was assumed that a large amount of Glu in the caput was due to many factors; secretion and metabolism of epithelial cells of the gland which might be regulated by androgen, inflow of rete testis fluid, and sperm metabolism of amino acids in the epididymis. The results obtained from the caput epididymis to which the efferent duct of the testis was ligated also supported this interpretation.


Heterotypical sexual behavior in male rats: individual difference in lordosis respones. Castrated Wistar male rats were primed with varying amounts of estradiol benzoate (EB) for successive 2 days and progesterone (P) on the third day 6-8 hr prior to the  behavioral test. The tests were performed 4 times at 2-3 weeks intervals. As a priming procedure for the first behavioral test, 50 mug EB and 0.5 mg P were given. The quantity of P was kept constant therafter. For the second test, the dose of EB was increased to 100 mug, but decreased again to 50 mug and further to 10 mug for the third and fourth tests, respectively. The inciedence of animals showing lordosis was quite low, and was not significantly changed by increasing or decreasing the dosage of EB during the series of the behavioral tests. Forty-three out of 51 animals never showed lordosis at all 4 behavioral tests. In contrast, 5 out of 8 rats which responded to mountings by the males continued to display lordosis behavior throughout the series of the successive 4 tests. This consistence of individual responeses during the series of the behavioral tests may indicate the possible existence of individual difference in lordosis responde in male rats.


Immunological studies on LATS-immunogloblin by the reaction with staphylococcal protein A. The reaction of LATS activity with Staphylococcal Protein A, a specific binding protein with the Fc part of human IgG(1), IgG(2) and Ig(4), was examined. When IgG(1), IgG(2) and Ig(4) subclasses were removed from LATS positive sera or LATS-IgG fractions by affinity chromatography on Protein A-Sepharose, LATS activity decreased. Almost all LATS activity was found in the fraction that reacted with Protein A. It is suggested that LATS has an expression of a very distinct immunoglobulin G structure, and that LATS activity is distributed mainly in the fraction containing IgG(1), Ig(2) and Ig(4) in LATS positive serum;


Effects of synthetic progestational steroids on liver and serum lipids in rats. Effects of norethisterone (NT) purified norethisterone (pure NT), norethynodrel (NE), medroxyprogesterone acetate (MAP), chlormadinone acetate (CMA) and desoxycorticosterone acetate (DOCA) on serum and liver lipid levels and serum lipoproteins were examined in both intact and estradiol-treated male rats. NT and NE caused a decrease in serum cholesterol and phospholipid levels, an increase in liver cholesterol level, no significant change in triglyceride levels of both serum and liver, with a significant change in serum lipoprotein patterns; a decreased in alpha- and beta-lipoproteins and a marked increase in pre beta-lipoprotein. Pure NT decreased serum cholesterol without causing any change in lipoprotein pattern. MAP, CMA and DOCA causee almost no effect on lipid levels in serum and liver, but CMA and DOCA increased alpha-lipoprotein and decreased beta- and pre beta-lipoproteins. An acute treatment with estradiol caused a decrease in alpha- and beta-lipoproteins and an increase in pre beta-lipoprotein with a decrease in serum lipid levels and an increase in liver lipids. By contrary, a chronic treatment with a marked hypercholesterolemia. This increase of alpha-lipoprotein in estradiol-treated rats was prevented by NT and NE, not affected or rather decreased by MAP but further increased with CMA and DOCA. These data suggest that the effects of synthetic progestational steroids on lipids are classified into two groups, 19-nortestosterone derivatives and 17alpha-hydroxyprogesterone derivatives including DOCA. The former caused a decrease in serum lipid levels with an increase of pre beta-lipoprotein and adecrease of alpha-lipoprotein. The latter caused almost no change or a slight increase in serum lipid levels with a decrease in pre beta-lipoprotein and an increase in alpha-lipoprotein, though it was not found in MAP.


Long-term effects of vasectomy on the pituitary-gonadal system of rat and mouse. A long-term vasectomy operation in rat and mouse did not reveal any consistent change in the weights of testis and androgen dependent organs. Histological structure of the testis and caput epididymis remains normal after vasectomy. The androgenic activity of the testis remains normal as reflected by fructose content of seminal vesicles. Pituitary cytology remains unaltered. Yellowish cysts were observed frequently in the cauda epididymis of experimental animals, associated with increased epididymal weights. The protein and RNA content of the testis did not show appreciable change. A significant increase was observed in the protein and RNA content of epididymis after bilateral vasectomy. There was no effect of bilateral vasectomy on sialic acid contents of testis and epididymis of rats and seminal vesicular fructose contents of mouse.


Competitive protein binding assay of oestradiol-17 beta and oestrone using uterine cytosol prepared from HCG-treated rabbits. Uterine cytosol was prepared from rabbits after treatment with human chorionic gonadotrophin (HCG) and used for the determination of oestradiol-17 beta and oestrone by competitive protein binding (CPB) assay. The so obtained uterine cytosol gave higher percentage of binding and proved to be more stable when stored at --20 degrees C than cytosol obtained from the pregnant uterus.


Effect of human chorionic gonadotropin on the testicular delta 5-3 beta-hydroxysteriod dehydrogenase and spermatogenesis in hibernating toad (Bufo melanostictus). A histochemical study of testicular delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-OHD) and a quantitative study of seminiferous tubules in hibernating toad following the administration of HCG revealed stimulation in the activity of the enzyme delta 5-3 beta-OHD and the spermatogenesis by increasing spermiation, meiotic division and the maturation of secondary spermatogonia.


Comparative investigations on thyroglobulin biosynthesis in rabbit and guinea-pig thyroid in vitro. The dynamics of biosynthesis of thyroglobulin (TG) subunits and their polymerization were studied comparatively in rabbit and guinea-pig thyroid in vitro. The incorporation of 14C-leucine into soluble, microsome-bound proteins (total and per mg) and into various fractions of soluble proteins was followed during incubation for 5 to 300 minutes. Some differences in TG synthesis between rabbits and guinea-pigs were observed, namely in the dynamics of 14C-leucine incorporation, the rate of subunit aggregation, the relationship between soluble and particulate proteins and the sedimentation characteristics of newly synthesized and pre-formed TG. The incorporation of 14C-leucine into soluble and microsome-bound proteins increased with time of incubation, but in rabbits the level of incorporation was much higher than in guinea-pigs. In rabbits, the ratio between radioactivity in soluble and particulate proteins decreased with time. In guinea-pig, this ratio increased with time of incubation and was much higher than in rabbits. Sucrose density gradient analysis of the newly formed soluble proteins showed that 12S fraction was synthesized in relatively large amounts, during early incubation of rabbit thyroid, although it was no found as a native protein in the thyroid extract of this species. After prolonged incubation the quantity of 12S protein decreased with a simultaneous increase in the amount of newly formed TG. The quick transformation of 12S subunit into TG is probably the reason why native 12S protein is absent in rabbit thyroid gland. However, in guinea-pigs the transformation into TG is much slower so that a considerable amount of radioactivity remained in the 12S fraction even after 300 min incubation. The results from rabbit experiments suggest that the 12S protein, probably represents a precursor of TG. In guinea-pig, the origin of 12S protein is twofold, as subunit of TG and as a product of its dissociation.


[Diabetes mellitus and Addison's disease)]. The authors diagnosed diabetes mellitus together with Addison's disease in a 38 year-old male patient. They are discussing therapeutic difficulties arising from the biglandular hypofunctional condition and the problems of the pathogenesis.


Lengthening of the duration of action of DDAVP by "ineffective" doses of clofibrate in patients with pituitary diabetes insipidus. Duration of action of single intravenous and intranasal doses of 1-Deamino-8D-Arginine Vasopressin (DDAVP) was lengthened by "ineffective" doses of clofibrate (not affecting water metabolism when administered alone) in patients with pituitary diabetes insipidus. This interaction was observed when as low doses as 0.02 mug DDAVP were administered intravenously to one patient and as high doses as 1 and 2 mug DDAVP were given intravenously to another patient. Administration of less or more DDAVP than "optimal" for the interaction resulted in decreased or absent potentiation by clofibrate. The exact cause of the interaction is unknown.


Treatment of diabetic coma with low-dose injections of insulin. Twenty-one patients in severe diabetic coma were treated with small doses of insulin at a rate of 4.1 units per hour (total dose about 100 units per 24 hours). Using single doses of 4 to 10 units by the intravenous or intramuscular routes the fall of blood glucose was steady in all cases. In the treatment of diabetic coma this regimen of insulin administration has proved simple, safe and effective since 1946. Main dangers during recompensation of diabetic coma are: hypovolaemia with oliguria -- anuria, dysequilibrium syndrome with cerebral edema and hypokalaemia. Therefore early intensive and adequate intravenous fluid and electrolyte replacement is the most important part of treatment. Most of the cases in this study were undiagnosed diabetics (14) and elderly patients (9). Three patients older than 65 years and a 56-year old diabetic died. In this context the most important aspects of treatment to avoid death are: prevention of diabetic coma and adequate fluid and electrolyte replacement especially in geriatric patients.


[Stimulation of ovarian function in infantile rats with testosterone alone or in combination with chorionic gonadotropin or clomiphene citrate]. Infantile female rats were subcutaneously injected with 2.5 mg testosterone within two days. The majority of the rats responded to this treatment with opening of the vagina, and about 50% developed an estrus. However, corpora lutea were not formed. Equally treated animals received additionally 0.2 i.u. HCG -- a dose which is inffective when given alone -- distributed on the second and third day of experiment. After this treatment an increased number of rats reacted with estrus and the development of corpora lutea. In infantile female rats, follicles stimulated by testosterone were also transformed into corpora lutea by an injection of 0.025 mg clomiphene citrate.


Histochemical observations on the adrenal gland of bat Vesperugo pipistrellus (Dobson). The presence of delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSDH), 11beta-hydroxysteroid dehydrogenase 11beta-HSDH), 17beta-hydroxysteroid dehydrogenase (17beta-HSDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) has been histochemically demonstrated in the zona glomerulosa, fasciculata and reticularis and also in the cortical cells that are found interspersed in the medulla of the adrenal gland of the bat, Vesperugo pipistrellus. The present study indicates that the adrenal cortex of the bat is capable of synthesizing corticosteroids and also sex steroids.


Regulation of nitrogen catabolic enzymes in chick liver -- effects of amino acids. It is well-known that in the chick, dietary protein increases the levels of several hepatic enzymes that are involved in nitrogen metabolism and excretion. However, the biochemical mechanism of this response is essentially unknown. The experiments presented in this paper show that the chick is responding to alpha-amino nitrogen and not to any specific amino acid. Furthermore, it is shown that this system responds to endogenous sources of nitrogen as well as dietary protein and that the xanthine dehydrogenase response involves regulation of enzyme synthesis without changing the rate of degradation.


Changes in hepatic fatty acid degradation and blood lipid and ketone body content during development of the rat. The following have been measured during the development of the laboratory rat: the rate of oxidation of palmitoylcarnitine, decanoylcarnitine, and 14C-palmitate by liver mitochondria; the concentrations of ketone bodies in the blood; the plasma concentrations of non-esterified fatty acids, glycerol, and triglyceride. In each case, a rise after birth and a fall at weaning were observed. These changes can be correlated with the dietary changes which occur at these times. However, during the suckling period, when a constant high fat content diet is consumed, further marked changes in the parameters measured were observed which cannot be related to nutritional factors.


A comparative study of glytaminase isozymes in rat tissues. The three kinds of glutamineses in rat kidney and liver were defined in comparative terms by their properties and were separately purified. Substantial purification was obtained by polymerization and depolymerization of the kidney isozyme that is activated by phosphate, These isozymes differ most strikingly in the activators of their reactions:maleate and a high concentration of phosphate, respectively, for the two kidney isozymes, and a low concentration of phosphate for the liver isozyme. The kidney isozyme that is activated by phosphate was also activated by a much lower concentration of ATP and by other complex phosphates. They also differ in physical properties: the maleate-activated isozyme was heat resistant (50 degrees C) and associated with insoluble submitochondrial particles; both phosphate-activated isozymes were heat sensitive and could be solubilized from their respective mitochondria; the phosphate-activated isozyme of kidney polymerized in phosphate-borate solution while that of liver did not. The characteristics of the kidney isozyme that was activated by high phosphate were shared by the glutaminases in adult brain, transplanted tumors, and in fetal liver and kidney. The similarity to the kidney enzyme was confirmed by the use of polymerization in phosphate borate of the isozyme from a mammary carcinoma to effect its purification.


The development of creatine kinase in rat skeletal muscle. Changes in isoenzyme ratio, protein, RNA and DNA during development. The activity of cytosolic creatine kinase in rat skeletal muscle rises stepwise during development. The increases occur simultnaeously with transient increases in DNA content. The second increase is accompanied by a rise in total protein, soluble sarcoplasmic protein and RNA/DNA ratio. Such changes are not observed at 20 days after birth, when creatine kinase finally accumulates to the adult level. Transient higher amounts of the MB and BB isoenzymes are observed after the first and second stepwise increase. The increase in creatine kinase activity observed after birth is predominantly due to an activation of the M gene. The BB isoenzyme is still present in adult skeletal muscle, but contributes little to the total activity.


Nature of the multiple forms of alanine aminopeptidase. Alanine aminopeptidase (particle-bound aminopeptidase, EC 3.4.11.2) isolated in a highly purified state from human liver, kidney, and pancreas were investigated with regard to their different electrophoretic mobilities. While the molecular weights are identical, different isoelectric points were found. The results of carbohydrate analysis point to N-acetylneuraminic acid as the main factor that causes the differences in electrophoretic behaviour of the alanine aminopeptidases investigated.


Percutaneous tendon splitting--method and results. The paper briefly describes the percutaneous tendon splitting operation as performed in general practice and evaluates its effectiveness on the basis of races run during a 2 year follow-up period. The results are compared with similar figures for normal Standardbred trotters. A higher percentage of horses subjected to splitting of the suspensory ligament failed to remain sound long enough to run more than 10 races during the follow-up period. The percentage of horses returning to racing whose superficial flexor was operated upon was somewhat lower than the control group.


Fractures of the pedal bone in the horse. A series of 79 fractures of the pedal bone in various types of horses are reported and the clinical features described. The fractures were of 3 types--intra-articular or extra-articular, involving the wing of the pedal bone, and fractures of the extensor process. Forty-five horses with a fractured wing of the pedal bone were treated conservatively and 11 by immobilisation of the bony fragment with a compression screw. Conservative treatment was usually successful in the extra-articular fractures. Intra-articular fractures treated conservatively in animals under 3 years had a good prognosis. The results were not so satisfactory in older horses and surgical treatment proved to be the method of choice. All, but one, of the 23 fractures of the extensor process were treated conservatively. The prognosis was not always good, as secondary osteoarthritis of the interphalangeal joint sometimes developed.


Epidermoid cyst of the brain in the horse. An aged Connemara mare had a neurological illness lasting 3 weeks and characterised by episodes of dullness, head-pressing, staggering gait and hyperexcitability. The signs recurred at intervals of 3 to 4 days, with intervening periods of apparent normality. Post-mortem examination revealed an epidermoid cyst on the dorsal surface of the occipital lobe of the left cerebral hemisphere. The cyst was lined by stratified squamous epithelium and contained desquamated keratinised material and blood. The lesion is considered to be a congenital malformation which enlarges and produces space-occupying signs.


Four cases of Tyzzer's disease in foals in England. A rodent pathogen, Bacillus piliformis, has been recognised as causing a rapidly fatal hepatitis in 4 foals in England. The disease in foals has been recognised in America since 1973. A clinico-pathological account of the 4 cases is given and the differential diagnosis discussed. The 4 foals' ages fell within a very narrow range (24-34 days). Some of the properties of this unusual intracellular pathogen are reviewed. For the first time in the equine the bacillus was seen in association with myocardial lesions. There are marked differences in the epidemiology of the disease in the mouse and in the foal, which may result from the very different management practices in the 2 species. It is suggested that the dams of affected foals may be carriers of the disease.


A practical health programme for prevention of parasitic and infectious diseases in horses and ponies. A scheme of combined anthelmintic treatment and vaccination is described. The seasonal veterinary activities are as follows: March (all horses); parasitological examination of faeces, anthelmintic treatment, immunization against influenza and tetanus (booster dose). June: parasitological faeces examination of foals, anthelmintic treatment of all horses, immunization of foals against tetanus (1st dose). August: anthelmintic treatment of all horses and immunization of foals against influenza (1st dose) and tetanus (2nd dose). October: parasitological faeces examination and anthelmintic treatment of all horses, and immunization against influenza of foals (2nd dose) and yearlings (2nd booster dose). December (for all horses): anthelmintic treatment.


The importance of blood gas measurement in the diagnosis of an intraventricular septal defect in a horse: a case report. An 18 month Thoroughbred gelding was diagnosed on cardiac catheterization as having an interventricular septal defect. Right side cardiac blood pressures were within the normal range and confirmation of the diagnosis came from changes in the blood pO2 and pCO2 between the right atrium and right ventricle. The significance of these values is discussed.


Growth rate in thoroughbred yearlings and two year olds. One hundred and four animals aged 13-18 months were measured at monthly or two monthly intervals, as were comparable numbers of animals 19-36 months old. Height, girth and circumference of the cannon bone below the knee were recorded, measurements for colts and fillies are shown separately. The average height at 18 months was 148 cm (15.01/2 h.h.) and at 36 months 156 cm (15.31/2 h.h.).


The adipokinetic effect of hyperthermic stress in man. The purpose of this investigation was to assess the alteration in serum free fatty acid concentrations during heat stress and dehydration. Each subject was exposed to heat stress in an environment chamber on 2 separate occasions. During the first exposure the subjects remained seated until the core temperature was elevated 1.4degreesC resulting in a mean weight loss of 1.66 kg due to dehydration. The second condition involved water replacement equal to the weight loss of the initial dehydration condition. Blood samples were obtained prior to heat exposure, when the core temperature was elevated 0.7degreesC and 1.4degreesC. They were subsequently analyzed for free fatty acids (FFA), glucose and lactin acid. Heart rates and core temperatures were monitored at 4 min intervals. During the dehydration condition the mean change in serum FFA was 0.9 muEq/ml in contrast to 0.2 muEq/ml for the rehydration condition. Serum levels of glucose increased moderately throughout the exposure (8 mg-%).


Structure and function of cardiac mitochondria in exhausted guinea pigs. The ultrastructure and functional capacity of mitochondria from hearts of exhausted guinea pigs were compared to those from rested animals. Electron micrographs of heart ventricles were examined for ultrastructural alterations, and functional capacity was studied by oxygen polarography. General swelling of mitochondria from exhausted animals was indicated by a 41% reduction in mitochondrial surface/volume ratio and a 22% increase in volume density of mitochondria from exercised animals when compared to those from controls. The yield of isolated mitochondria from exhausted guinea pigs was similar to that for control animals. In the presence of all substrates tested the rate of oxygen consumption was lower in the exhausted animals than in the controls. The ADP/O ratios and respiratory control indices for pyruvate, succinate and ascorbate were not systematically affected by exercise. With glutamate as substrate respiratory control was reduced in exercised guinea pigs. These experiments suggest that the structure of myocardial mitochondria from exhausted animals is sufficient to maintain normal ADP/O ratios but is not adequate to maintain normal rates of respiration.


Effects of physical training on ventilatory equivalent and respiratory exchange ratio during weight supported, steady-state exercise. Thirty-three college men participated in a 9-week endurance training program. An equal number of subjects served as controls. Pre- and post-test metabolic measurements were made during 10 min of submaximal exercise (1080 kpm/min at 60 rpm) and 15 min of recovery. Measurements included oxygen consumption, CO2 production, ventilatory equivalent (Ve/VO2 ratio) and respiratory exchange ratio (R). A three factor design variance analysis was used to analyze the effects of training on min-by-min exercise and recovery Ve/VO2 ratio and R. For the experimental group training resulted in a significant improvement in ventilatory efficiency during exercise, as well as a significant decrease in R. During recovery, Ve/VO2 and R decreased significantly for both groups although the magnitude of change was greater for the group that trained. Apparently, there was a significant habituation effect due to test procedures for the control group. The results are discussed in terms of lactate production and substrate utilization during exercise.


Oxygen deficit and repayment in submaximal exercise. Oxygen deficit and repayment ratios were investigated at various work loads, intensities and durations. An active baseline was used (walking at 60 m/min) from which deficit and repayment values were calculated. Oxygen uptake (VO2) and core temperatures were measured in 30 males at baseline and during treadmill running (140 m/min) for randomly assigned durations (0.5 ...20 min). Measurements were also made during a 30-min recovery period at baseline work. Results indicated: 1) No difference in O2-repayment between steady-state work and work prior to steady state (P greater than 0.10). 2) O2-repayment was independent of work duration (P greater than 0.10). 3) When workload and intensity were controlled, O2-deficit was not significant factor in O2-repayment (P greater than 0.10). 4) Work intensity (work VO2/VO2 max) was the most significant factor in O2-repayment accounting for 69% of the variance (r equals 0.83, P less than 0.001). Small increments in core temperature and ventilation were not significant factors in O2-repayment. When a working baseline is used, the magnitude of O2-repayment after exercise is independent of the work duration or the attainment of steady state. The extent of O2-repayment after exercise is mainly dependent upon the physiological intensity of the work and the absolute workload (R=0.89, P less than 0.001).


[Examination of blinking-rate in a simulated four hour driving task]. In a driving simulation task performance and physiological measurement of strain was registered on 3 subjects in 9 experiments. The time-variant behaviour of blinking-rate was examined together with performance, self-rating and physiological strain. The correlating results make blinking-rate a possible indicator for activation of willingness to good performance. A multidimensional concept for assessment of the degree of individual fatigue is presented and validated in respect of working-time.


Motor unit differentiation and integrated surface EMG in voluntary isometric contraction. The role of the differentiation of motor units (MU's) in the relation between integrated surface electromydgram and force was studied here for the biceps brachii, in the course of static contraction in man. The global EMG of the biceps brachii was recorded by two silver electrodes fixed to the skin. The MU's activity was simultaneously lead off by three bipolar wire electrodes inserted in three points of the muscle. MU firing frequency and recruitment were estimated by counting the action potentials on the three elementary records. The comparison between the increase of the integrated surface EMG and MU recruitment as a function of force shows that MU recruitment in itself cannot account for the increase in the integrated global EMG, particularly for high values of force. The difference between MU recruitmentand integrated global EMG, which can neither come from a possible MU's synchronisation as was shown in the discussion, is being suggested to be connected to the progressive firing of "phasic" MU's. This type of recruitment may also play an important role in the gradation of isometric force in normal man.


Hemodynamic responses to acute hematocrit and blood volume alterations in rats. Previous investigations of polycythemia in rodents failed to delineate the effects of the increased hematocrits from the effects of possible changes in blood volume. The hemodynamic responses of anesthetized rats to acutely produced increases in hematocrit and blood volume were therefore studied in this experiment. Acute isovolumetric polycythemia decreased O2 transport from a control value of 44.9 to 35.1 ml/min/kg 60 min after the exchange-transfusion. However, the high venous O2 content (over 12 Vol.-%) following the production of isovolumetric polycythemia suggested that there was still sufficient oxygen available to the tissues. Hypervolemia significantly modified the response to acute polycythemia. When hematocrit was maintained at a constant level, acute hypervolemia increased O2 transport (to 54.6 ml/min/kg after 60 min), and when acute hypervolemia and polycythemia were induced simultaneously, O2 transport was transiently increased (to 57.5 ml/min/kg after 15 min) and then returned to a value similar to the control level.


Step increment versus constant load tests for determination of maximal oxygen uptake. Ten male subjects performed five maximal treadmill running tests at 7 mph. Tests included two (test-retest) progressive, step increment (2 1/2% grade elevation), discontinuous tests (DCT); a progressive, step increment, continuous test (ct) and two constant load tests (CL and CL +2 1/2%). A DCT test was performed first for establishment of peak elevation levels as constant load tests were performed at the peak elevation level attained (CL) and at a level 2 1/2% higher (CL + 2 1/2%). The second DCT test and the remaining three tests were administered randomly. Peak preformance capability (operationally defined as duration at highest grade elevation) was markedly reduced during progressive tests as compared with constant load tests. There was a similar reduction in peak performance capability during the CT test as compared with DCT test. Maximal oxygen uptake (VO2max) was quite similar among the various tests. It was concluded that VO2max attained during progressive, step increment, tests is unaffected by cumulative submaximal work. Discontinuous and continuous progressive tests provide similar VO2max results.


The effect of various levels of strenuous to exhaustive exercise on reaction time. Reaction times and heart rates of 10 subjects walking and running on a motor-driven treadmill were recorded. The exercise consisted of four 3-min stages: Stage 1, 2.5 mph at 12% grade; Stage 2, 3.4 mpg at 14% grade; Stage 3, 4.2 mph at 16% grade; Stage 4 5.0 mph at 18% grade. Subjects were given three separate tests at 1-week intervals. There was an increase in reaction time with increased exercise, but this effect diminished significantly over days. Also, when heart rate was 80% of maximum during the post-test, reaction time was not significantly different from the pre-test. Optimum reaction time was obtained at approximately 40% of maximum heart rate.


The aerobic power of several groups of laborers in Colombia and the United States. Physiological responses to exercise and anthropometry were measured in several groups of physical laborers. The groups included Colombian sugar cane cutters, sugar cane loaders and general farm laborers. In addition, migrant workers and civil service laborers from the United States were studied. The Colombian populations were generally shorter, weighed less and had less body fat than the United States group. The nutritional state of the Colombian groups, as measured by the hematocrit, hemoglobin, serum albumin and total blood protein, appeared normal. The aerobic power of the Colombia populations was significantly larger than the United States groups. The larger aerobic power of the Colombian groups is probably related to greater physical fitness associated with the physical demands of their occupations.


Functional significance of collateral blood flow in working skeletal muscle of the dog. The semitendinosus muscle of the dog is supplied by two separate arteries and drained by two corresponding veins. In the muscles used in this study no blood entering via the distal artery was found to leave via the proximal vein during perfusion through both arteries (orthograde perfusion). Therefore, collateral flow (CF) could be determined as proximal venous outflow during occlusion of the proximal artery. During orthograde perfusion total blood flow averaged 12 ml x min-1 x 100 g-1 at rest and 58.4 ml x min-1 x 100 g-1 during exercise. CF was found to average 6.2 ml x min-1 x 100 g-1 at rest and increased to 9.2 ml x min-1 x 100 g-1 during exercise. CF was sufficient to cover the metabolic demand of resting muscle. During exercise the O2-uptake (VO2) of the distal muscle portion was increased 13.4 fold in comparison to a 3.1 fold increase in the proximal muscle portion. The average contractile power decreased by 46%. Additional infusion of adenosine into the distal artery resulted in an increase of CF to 11.4 ml x min-1 x 100 g-1 and of orthograde flow to 71 ml x min-1 x 100 g-1. The average contractile power of the muscle increased by 13%. Both orthograde flow and CF were found to decrease with increasing muscle load. But this decrease was significantly more pronounced in the case of CF especially at a lower range of loads. It is concluded that after acute occlusion of orthograde flow, CF is limited by the number, the size and the dilatory capacity of precapillary network vessels. Furthermore, CF is influenced considerably by changes of extravascular support.


A comparison of maximum oxygen uptake determination by bicycle ergometry at various pedaling frequencies and by treadmill running at various speeds. Tests of performance on a bicycle ergometer have routinely been standardized with pedaling frequencies of up to 60 rpm. Only rarely have higher speeds been used. It may be hypothesized than a higher VO2, more closely approximating the maximum attainable by treadmill running, may be achieved in bicycle ergometry if higher pedal frequencies are used so that premature fatigue of the leg musculature does not obscure the comparison. Even in treadmill running it remains equivocal which combination of speed and grade of running will produce a maximum VO2. Five male subjects performed maximally at pedal frequencies of 60, 80, 100 and 120 rpm and running speeds of 6.0, 6.5, 7.0, and 7.5 mph, on a bicycle and treadmill respectively. Power output on the bicycle and increasing grade sequence on the treadmill were maintained constant for each speed investigated. The highest VO2 attained in each test was measured and compared both within and between the modes of testing. It was confirmed that peak VO2 during bicycle ergometry is significantly less than VO2 max attained in treadmill running. Pedal frequencies of 80 and 100 rpm produced optimal VO2's in ergometry. No differences were noted between VO2's determined at any speed of treadmill running. Subjects preferred 60 or 80 rpm in ergometry and 6.0 and 7.0 mph in treadmill running; 120 rpm and 7.5 mph in bicycling and treadmill running, respectively, were generally disliked.


Muscle-pO2 in trained and untrained non-anesthetized guinea pigs and in men. pO2-measurements were performed by micro-Pt-electrodes a) in the gastrocnemius muscles of physically trained and untrained non-anesthetized guinea pigs, and b) in the tibialis anterior muscles of physically trained and untrained men, and of patients after a 2-3 weeks bedrest. Physical training raises the mean pO2-values in the resulting muscles of guinea pigs and men from 30-40 Torr to 50-55 Torr whereas physical inactivity (2-3 weeks bedrest) reduces the values to 17-18 Torr.


A new formula for estimating metabolic rate. A formula for estimating metaoblic rate using inlet volume flow and oxygen fractionation of outlet air is derived. The formula is simplified and the error induced is evaluated and compared with that of Weir's formula.


Reaction of N-(3-pyrene)maleimide with thiol groups of reticulocyte ribosomes. The reaction of N-(3-pyrene)maleimide with thiol groups of rabbit reticulocyte ribosomes offers a possible fluorescent probe for studying ribosomal structure and conformation. At relatively low concentrations of N-(3-pyrene)maleimide a group of 30-40 readily reactive sulfhydryl residues is derivatized. The major ribosomal proteins containing these thiol groups are identified as S2 + S3, S5, S7, S8, S29, S31, S32, L1, L5, L6, L10 + L14, L15, L18 + L19, and L36. Ribosomal activity, as measured by the nonenzymic binding of phenylalanyl-tRNA and polyphenylalanine synthesis, is inhibited by this degree of reaction with N-(3-pyrene)maleimide. The inhibition is relieved by the prior binding of polyuridylic acid to the ribosomes while the extent of derivatization by N-(3-pyrene)-maleimide is diminished only slightly. The average relative polarization of the fluorescence of the ribosomal bound N-(3-pyrene)maleimide changes significantly with the degree of derivatization of ribosomal thiol groups or with the binding of polyuridylic acid, indicating the value of such a fluorescent thiol-derivatizing agent as a probe of ribosomal structure.


[Circular dichroism of heterodetic cyclic cystinyl tripeptides (author's transl)]. The circular dichroism of two series of heterodetic cyclic tripeptides, t-butoxycarbonyl-L-cysteinyl-X-cysteine disulfide methyl esters [X=Ala, Val, Leu, Phe, Glu(OBut), Lys(Z)] and L-cysteinyl-X-cysteine disulfide methyl ester formates, and a corresponding series of t-butoxycarbonyl-L-cysteinyl-(glycyl)n-cysteine disulfide methyl esters (n=1-5) was investigated. The interaction between the disulfide group and the amide bonds results in a long-wavelength Cotton effect at 300 nm, which exhibits a strong solvent dependence. In contrast to the long-wavelength disulfide band and to the n-pi amide Cotton effect a bathochromic shift is observed from less polar to strongly polar solvents. Within the series of N-terminal protected tripeptide esters the influence of the central chiral amino acid X is small, but it increases in the non-protected peptide esters. The circular dichroism is dominated by the influence of the conformation of the disulfide ring, which is essentially the same in both series. Due to interactions of the disulfide group with the amide chromophores, the sign of the long-wavelength disulfide Cotton effect cannot be used for the determination of the helicity of the CSSC group. Infrared spectra of the protected compounds indicate trans-peptide bonds in the solid state, which may also be true for the solutions. A pronounced time-dependence is observed for the circular dichroism of t-butoxycarbonyl-L-cysteinyl-glycyl-cysteine disulfide methyl ester after dissolving in ethanol. This is due to a uniform conformational change of the ring structure as shown by spectroscopic analysis using extinction and ellipticity diagrams. In the series of the cyclic cystinyl peptides containing glycyl residues a strong electronic disulfide-amide interaction is observed is observed for the tripeptide, being still observable for the tetra-peptide. The comparison of the glycyl tripeptide with the tripeptides containing chiral central amino acids reveals a negative contribution to the circular dichroism at 260 nm due to these L-residues.


The enthalpy of interaction of urea with some globular proteins. The enthalpies of interaction of urea with five globular proteins, ribonuclease A, trypsin, beta-lacto-globulin, ovalbumin and bovine serum albumin have been measured in aqueous solution at pH 7.0, I=0.005 M and 25 degrees C over a range of urea molality m from 0-15 mmol g-1 (where a 1 molal solution contains 1 mmol g-1). For all the proteins the interaction is exothermic, and there is an appreciable heat evolution at low urea concentrations, m less than 5 mmol g-1, which increases sharply at higher urea concentrations when the proteins undergo unfolding. If account is taken of the endothermic enthalpies of unfolding of the native proteins, the enthalpies of interactions of urea per unit mass denatured protein lie in the range -45 to -75 J g-1, corresponding to an average binding enthalpy of -23 kJ mol-1 bound urea.


Investigation of the covalent bond structure of peptide-matrix systems by Edman degradation of support-fixed peptides. Two model peptides Ala-Ser-Asp-Tyr-Leu and Ala-Ser-Glu-Tyr-Leu have been synthesized and coupled with an without protecting groups to the amino groups of polystyrene resins. The yield in the binding operation was 93-97% by using water-soluble carbodiimides as condensing reagents. Edman degradation of the products and quantitative estimation of the amino acid phenylthio-hydantoins, as well as amino acid analysis of the partially and completely degraded resins, showed about 7-10% of the fixed peptides not to be involved in the degradation procedure. That part of the material which was not involved in the Edman degradation during the first step also remained inactive in the following degradation operations. The analytical data presented in the paper explain not only the course of the Edman degradation of peptides linked via the side-chain carboxylic groups to the resin, but they also describe the chemical reactivity of the various carboxylic groups in the binding operation. 22% of the peptides are bound to the resin by side-chain carboxylic groups in the case of the aspartate-containing peptide, 26-27% in the case of the glutamate-containing peptide. 75-77% and 72-75% respectively of the peptides are fixed to the resin via the C-terminal carboxylic group. No more than 3% of the fixed material may be bound bifunctionally to the support.


Light chains from slow-twitch muscle myosin. Myosin light chains have been isolated from slow-twitch soleus muscles of rabbit and cat. Two chemically related light chains of molecular weight about 22 000 have been identified from their thiol sequences in each species, and these have been further characterized by amino acid analysis and peptide mapping studies. These light chains are related to the alkali light chains of rabbit fast-twitch muscles and to the larger cardiac light chain from bovine heart muscle. The presence of two chemically related but phenotypically distinct light chains within single muscles suggests the presence of myosin isoenzyme or that the myosin molecule has a different light chain associated with each subfragment-1 head. Although the stoichiometry of these two light chains had not been determined, the former of these two conclusions is favoured by analogy with experiments on fast-twitch myosins. In addition to these related light chains, soleus muscle myosin, like fast-twitch myosins, contains a third light chain of about 19 000 molecular weight. Unlike the corresponding light chain of rabbit fast-twitch myosin, this 19 000-Mr light chain contrains no cysteine residues. The distribution of thiol peptides together with the characteristic mobilities of these light chains on polyacrylamide gels in the presence of sodium dodecyl sulphate provides a 'fingerprint' of myosins from different muscle types. This has been used to look for the presence of fast-twitch myosin in soleus muscle, the results showing both rabbit and cat soleus muscles are homogeneous in their myosin type within the sensitivity of detection of the methods. These techniques have also been used to confirm the reciprocal transformation of light chains in myosins isolated from cross-reinnervated muscles. Finally the relationships between these criteria for different myosin types and histochemical procedures for muscle fibre typing are discussed.


Enhancement of hydroxyindole-O-methyltransferase and DNA-dependent RNA polymerase activities induced by oestradiol in rat pineals in culture. 1. Hydroxyindole-O-methyltransferase and DNA-dependent RNA polymerase activities were determined in the pineal gland removed from the ovariectomised rat and cultured under various experimental conditions. 2. The transferase activity declined very slowly during 24 h of incubation. 17beta-Oestradiol significantly increased the transferase activity within 2h after the addition, and the extent of increase was dose-dependent within the concentration range from 0.1 to 15 nM, being increased by 80% at 15 nM. Enhancement of the transferase activity by oestradiol was abolished not only by inhibitors of protein synthesis (cycloheximide and puromycin), but also by those of RNA synthesis (actinomycin D and alpha-amanitin). It was also blocked by clomiphene citrate, an agent which is known to inhibit the binding of steroid hormones to their respective receptors. 3. RNA polymerase activity (forms A and B) declined rapidly during the initial period of pineal culture. Oestradiol (15 nM) increased the RNA polymerase B activity by 50% within 2 h after the addition. The increase was dose-dependent within the concentration range from 0.1 to 15 nM, and was abolished by clomiphene citrate. 4. The possibility is suggested that the pineal is a target organ of oestradiol, and that the steroid-induced reaction sequence in the pineal conforms to that is known in other target organs.


Study of the interaction of 4-bromomercuriocinnamic acid with alpha-chymotrypsin by 79 Br and 81Br pulsed nuclear-magnetic resonance. Br- has been used as a nuclear magnetic resonance (NMR) probe to study the reversible association of alpha-chymotrypsin and an Hg-labelled substrate (4-bromomercuriocinnamic acid, BrHgCin) which rapidly exchanges Br-. T1 was measured for 79Br and 81Br, using a pulse spectrometer. Values of the parameters that determine T1, Obs in aqueous solutions of KBr (pH=5.5) containing alpha-chymotrypsin and BrHgCin are reported. It is found that the rate of Br exchange is diffusion-limited and faster than the rate of reorientation of the BrHgCin-alpha-chymotrypsin complex. The rate constant for the formation of the covalent BrHgCin-alpha-chymotrypsin complex determined by this technique agrees well with previously published data. The rapid rate of Br exchange with the complex, however, is incompatible with the side chain of BrHgCin being entirely buried in a nonpolar pocket on the enzyme but compatible with the side chain being exposed to the solution. The contribution to the NMR signal from the non-covalent complex is negligible.


Studies on the structure and function of chick-oviduct chromatin. 1. Fractionation by ECTHAM-cellulose chromatography and physico-chemical characterization. Chick oviduct chromatin was separated into a ribonucleoprotein fraction and two chromatin fractions (early and late eluting). We utilized a gentle procedure in which moderately hydrated chromatin was subjected to chromatography on a weak ionic-exchange resin (ECTHAM-cellulose) eluted with a combined pH-salt gradient. Chemical analysis of the early (fraction I) and late (fraction II) eluting fractions revealed that their histones were identical and their nonhistone proteins were markedly different. Control experiments showed that these differences were not due to protein rearrangements during chromatin preparation and/or fractionation. The physical properties of fraction I and II differed in certain aspects. The aggregation response of fraction I to increasing concentrations of monovalent cations was five times lower than that of fraction II but the aggregation response to divalent cations was identical. Thermal denaturation assays of DNAs isolated from fractions I and II revealed identical derivative profiles of hyperchromicity vs temperature, thereby indicating similar base composition in the two fractions. Circular dichroism, spectra of the purified DNAs isolated from both fractions showed identical B-type conformations. However, DNA renaturation kinetics analyzed by computer technique indicated that fraction I DNA contained less than half the amount of highly repetitive sequences as compared to either unfractionated chromatin or fraction II. Circular dichroism spectra of fraction I and II chromatins (at room temperature) showed significant differences in a wavelength region were only DNA is optically active (i.e. 255-320 nm). These results indicated that the DNA complexed to proteins in fraction II assumed a more C-type conformation than the DNA in fraction I. The differences in the circular dichroism spectra could not be accounted for by differences in the RNAs or protein chromophores contained in fraction I and fraction II. When the circular dichroism spectra of fraction I and II were recorded at 55 degrees C, the differences between the two fractions were abolished. These results were interpreted to mean that the differences in the DNA conformations found in fractions I and II were due to the differences in their nonhistone proteins. These proteins were effective in maintaining DNA conformation differences only when they were in their native form but not when heated to 55 degree C. Comparison of the sedimentation coefficients of fractions I and II with their calculated molecular weights suggested a more extended structure in fraction I as compared to a more compact structure in fraction II. Only small differences were observed between fraction I and fraction II with respect to either buoyant density analysis in a metrizamide gradient or in the number of phosphate charges accessible to polylysine.