Duodenal active transport of calcium and phosphate in vitamin D-deficient rats: effects of nephrectomy, Cestrum diurnum, and 1alpha,25-dihydroxyvitamin D3. Both the methanol:chloroform extractable material from the leaves of the Solanaceous plant, Cestrum diurnum (C.d.), and a 270 ng dose of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) increased the active absorption of calcium and phosphate across the proximal duodenum, studied in vitro, from sham-operated and nephrectomized (NPX) vitamin D-deficient rats. In these studies, conducted 24 h after surgery, the uremic state in the NPX animals markedly diminished the intestinal transport response to 1alpha,25-(OH)2D3 and also lowered baseline transport values across duodenum from the NPX vitamin D-deficient controls. Both C.d. and 1alpha, 25-(OH)2D3 elevated plasma Ca levels equally well in the sham-operated and NPX groups. The stimulation of intestinal Ca absorption in NPX animals indicates that, like the leaves of the South American plant, Solanum glaucophyllum, C.d. contains materials which can function in an analogous manner to compounds in the vitamin D group that have either a 1alpha hydroxyl group or its steric equivalent.


Prostaglandin-induced release of anterior pituitary hormones: structure-activity relationships. Prostaglandins (PG's) of the E, F, A, or B series were infused into a lateral ventricle of the brain of adult male rats, and the efficacy of each PG to stimulate the discharge of LH, prolactin, and TSH from the pituitary gland was determined. PGE2, PGF2alpha, and PGF2beta were found to be potent stimulators of LH release. The release was considerably less when PGE1, PGF1beta, PGA2, or PGB2 was infused. The basal release of LH was not altered by infusion of PGF1alpha, PGA1, PGB1, or the control solution. It is suggested that the cis double bond in the 5,6 position and the 11-hydroxyl group are essential for the LH releasing activity of PG's and that these functional groups may be of importance for activation of a receptor at the level of the brain. Prolactin secretion was stimulated 6 to 7-fold by the infusion of 5 mug of PGE2, but 20 mug had no stimulatory effect. PGF2beta caused a 3 to 4-fold increase in basal plasma prolactin concentration and was the only PG tested other than PGE2 that stimulated prolactin release. None of the PG's infused intraventricularly affected TSH secretion.


Testosterone metabolism in rabbits actively immunized with testosterone. Active immunization of male rabbits results in a marked increase in plasma testosterone (T) and in the fraction of T bound to circulating proteins. In order to identify the cause of the increased T levels, the half-life of T after castration, and the metabolic clearance rate (MCR) and production rate (PR) after a single injection of tritiated T were determined in normal and immunized rabbits. In the immunized animals the half-life of T was significantly longer, the MCR showed a 10-fold decrease (10.2 +/- 3.2 1/day compared to 108 +/- 43 1/day) and the PR was increased from 0.44 +/- 0.34 mg/day to 1.20 +/- 0.36 mg/day compared with the control group. The binding of T to circulating antibodies is considered to be the direct cause of the decreased MCR and, via the diminished negative feedback at the hypothalamic-pituitary levels, the indirect cause of the increased PR in the immunized animals.


The effect of diabetes, insulin, and the redox potential on leucine metabolism by isolated rat hemidiaphragm. The oxidation of leucine by hemidiaphragms of control and diabetic rats was studied in vitro. Rats were rendered diabetic with streptozotocin. Hemidiaphragms of diabetic rats produced approximately 50% more 14CO2 during incubation with 0.1 mM [1-14C]leucine than did control muscles. This was observed during incubation with or without glucose and in the presence or absence of a full complement of plasma amino acids. The concentration of leucine in the tissue water of hemidiaphragms from diabetic rats was greater than that in the control muscles before incubation. The specific activity of leucine at the end of 60 min incubation was not significantly different in diabetic and control muscles, indicating that the increased 14CO2 production represented stimulation of leucine oxidation. Hemidiaphragms of diabetic rats released more leucine into the medium during incubation than did control muscles. The stimulating effect of diabetes on leucine oxidation in vitro was reversible by insulin therapy prior to sacrifice. The addition of 5 mM pyruvate to a medium containing glucose inhibited 14CO2 production from [14C]leucine in control muscles, but stimulated leucine oxidation by hemidiaphragms of diabetic rats. Leucine oxidation by hemidiaphragms of diabetic rats was markedly stimulated by the addition of an electron acceptor, 0.02 mM methylene blue, suggesting that the NADH/NAD ratio may be rate-limiting for branched chain amino acid oxidation in muscles of diabetic rats, but not in muscles of controls. We suggest that the accelerated oxidation of branched chain amino acids by muscles may play a role in the acceleration of the muscle protein catabolism and gluconeogenesis which develop during insulin deficiency. The restraining effect of the cellular redox potential on branched chain amino acid oxidation may play a role in the eventual deceleration of protein catabolism during a prolonged fast.


The hypothalamic-neurohypophysial system of the rat: localization and quantitation of neurophysin by light microscopic immunocytochemistry in normal rats and in Brattleboro rats deficient in vasopressin and a neurophysin. The cellular distribution of neurophysin was examined in hypothalami and neural lobes of normal Long-Evans rats and Brattleboro rats deficient in vasopressin and a major neurophysin. Tissue sections were treated with antisera to bovine, human, and rat neurophysins, using immunoperoxidase bridge techniques. Antisera to oxytocin (OT) and vasopressin (VP) were applied to adjacent sections. Two distinct cell populations were discernible in both magnocellular nuclei on the basis of the intensity of cytoplasmic staining. About half of the magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei of homozygous Brattleboro rats with diabetes insipidus (DI) were devoid of immunoreactive neurophysin, OT, and VP. These cells were presumably the defective counterparts of those neurons that produce VP and its associated neurophysin in normal and heterozygous Brattleboro rats. The cells in homozygous DI rats which were stained with immunoreaction products to NP and OT were more concentrated in the dorsal part of the SON and in the periphery of the PVN. Spatial segregation of different neurons was also seen in the neural lobe, where clusters of stained axons were surrounded by bundles of nerve fibers lacking immunoreactive material. In normal rats and heterozygotes nearly all magnocellular neurons reacted immunologically with antiserum to neurophysin but with different intensities, so that "dark" and "light" cells could be distinguished. The darker cells in heterozygous Brattleboro rats had the same pattern of distribution as cells which contained OT. In homozygous DI rats, only some of those cells which contained neurophysin and OT exhibited a positive reaction with antiserum to VP due to slight reactivity with OT. The results obtained in the homozygous Brattleboro rat would suggest that OT and VP and their associated neurophysins are produced in different neurons in both the SON and PVN. However, in normal rats and in heterozygous Brattleboro rats, VP appeared to be present in both OT-positive and OT-negative neurons suggesting that some cells may have the capacity to synthesize two hormones.


Relation between individual differences in sexual behavior and plasma testosterone levels in the guinea pig. After 3 tests for male sex behavior, adult male guinea pigs were classified as either low-activity (LA, no ejaculations) or high-activity (HA, ejaculation during at least 2 behavior tests). In one experiment, resting levels of peripheral plasma testosterone (T), measured by radioimmunoassay, did not differ between the groups (LA=2.22 +/- 0.17 ng/ml, HA=2.09 +/- 0.11 ng/ml, X +/- SEM). However, plasma T levels were significantly higher in HA males 2 min after a sex test (2.27 +/- 0.24 ng/ml) than in LA males (1.40 +/- 0.20 ng/ml). A second experiment confirmed these results and also demonstrated that exposure of HA or LA males to an estrous female placed on the opposite side of a wire mesh barrier similarly led to higher T levels in HA than in LA males. These results indicate that a) behavioral differences between HA and LA guinea pigs are not attributable to differences in resting T levels, b) HA and LA males perceive the sex test situation differently, leading to slight increases in T in HA males and slight decreases in T in LA males, and c) these changes in T level are not dependent on copulation but can be induced by mere exposure to the sight smell and/or sound of an estrous female.


The effects of insulin and growth hormone on the release of somatomedin by the isolated rat liver. Livers from hypophysectomized (hypox) rats were perfused with oxygenated Waymouth's medium in a system which permitted continuous recirculation for separate 30 minute periods after which fresh medium was supplied. In most experiments 6 changes of medium were carried out over a 3 hour period. The somatomedin activity of each perfusate was determined by measuring its ability to stimulate sulfate uptake in hypox rat cartilage in vitro. For comparison between experiments the results are expressed as the per cent stimulation of sulfate uptake by the perfusate compared with the unperfused buffer. Without hormonal additions there was a progressive fall in the release of somatomedin activity during the 6 periods of study. When compared with the results without hormone, the addition of 1000 muU/ml of insulin per ml of medium during the 2nd to 6th period led to a significant increase in perfusate somatomedin activity at all periods. The addition of 100 muU/ml of insulin was without significant effect. The possible inter-relationship between insulin and growth hormone in the regulation of somatomedin release was studied with a dose of bGH of 250 ng/ml which had previously been shown to be insufficient by itsel to stimulate somatomedin release. When added to a medium containing 1000 muU/ml of insulin, this dose of bGH did not significantly stimulate somatomedin release beyond that obtained with insulin alone. However, when 250 ng/ml was added to a medium containing 100 muU/ml insulin, a significant stimulation of somatomedin release was observed while the addition of each hormone separately was without significant effect. These results support the hypothesis that insulin shares with GH the regulation of somatomedin release by the liver. Differences in insulin concentration may explain some clinical situations in which somatomedin concentrations cannot be correlated with GH levels.


The cell-free synthesis of the alpha subunit of human chorionic gonadotropin. The synthesis of the alpha subunit of human chorionic gonadotropin (hCG) was demonstrated in a cell-free system composed of polysomes derived from first trimester placenta and cell sap prepared from ascites tumor cells. The in vitro synthesized proteins labeled with [35S]methionine were shown to have at least 4 tryptic peptides that co-migrated with the same peptides from authentic hCG. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed the synthesis of a discrete protein, migrating with an apparent molecular weight of about 17,000, which contained methionine-labeled tryptic peptides found in the alpha subunit. The level of radioactivity in these tryptic peptides was five times greater with polysomes from first trimester placentae than with those from term placentae. The efficiency of total protein synthesis in both cases was about the same. These data strongly suggest that the decrease in blood levels of hCG after the first trimester is caused by a selective decrease in the rate of synthesis of the hormone.


Effect of hysterectomy on serum luteinizing hormone concentrations and on corpus luteum function in the rat. Serum progesterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were estimated in intact and hysterectomized pseudopregnant rats. Progesterone concentrations increased significantly from day 2 of pseudopregnancy (PSP) onwards, and a plateau was reached on days 4 and 5. No significant differences in progesterone concentrations between intact and long-term hysterectomized animals were observed until day 10 of PSP. In the intact rats, progesterone concentrations decreased after day 8, but in the hysterectomized rats they remained elevated until days 18-20 of PSP. No differences between FSH concentrations in intact and hysterectomized rats were found. In hysterectomized rats, however, LH concentrations were significantly lower than those in the intact rats on days 2-7 of PSP. When hysterectomy was performed on day 2 of PSP (acute hysterectomy) lower LH concentrations were also found in the hysterectomized animals compared with sham-operated animals. No differences in progesterone concentrations were found between sham-operated controls and acutely hysterectomized animals on days 3-8 of PSP. It was found that LH concentrations in ovariectomized long-term hysterectomized rats were lower than those in ovariectomized, but otherwise intact, animals. In contrast to its effect on tonic LH secretion, long-term hysterectomy had no influence on the surge of LH during the afternoon of proestrus. In both intact and long-term hysterectomized rats the duration of PSP was not significantly altered after transplantation of an ovary under the kidney capsule and the removal of the ovaries in situ. It is concluded that hysterectomy leads to a decreased LH secretion by the pituitary gland, but has no influence on the maximal progesterone concentrations during PSP. The significance of these findings, particularly with regard to the prolongation of PSP after hysterectomy, is discussed.


Thyroid hormone metabolism during liver regeneration in rats. The metabolism of thyroid hormones was studied during the prereplicative period of liver regeneration. After partial hepatectomy, serum thyroxine (T4) and triiodothyronine (T3) levles progressively fell, and reached a nadir at 12 h proportional to the quantity of liver tissue exised. The diminution (60-80%) in serum iodothyronines was related specifically to partial hepatectomy because laparotomy, ether anesthesia, and other stressful surgical procedures did not induce similar changes. At least 3 phenomena appear to be involved: 1) increased utilization and turnover of thyroid hormone by the regenerating liver remmant. 2) diminished hormone secretion by the thyroid gland between 6-12 h after surgery, and 3) a slightly reduced concentration of serum iodothyronine carrier proteins. The results support the concept that the liver participates in the metabolic regulation of T2 and T4 which in turn, control hepatocellular growth. It is suggested, however, that additional unknown factors control increased hepatic thyroid hormone turnover after partial hepatectomy.


Electron microscope immunohistochemical localization of thyroglobulin in the rat thyroid gland. In order to make a precise identification of the organelles containing thyroglobulin (TG) in the thyroid follicular cells, an electron microscope immunohistochemical localization of TG was conducted in the rat thyroid gland. In both control and TSH-treated rats, TG was detected in the follicular lumen, in large vesicles which correspond to the classical colloid droplets, in apical vesicles, and also in the apical cytoplasm. When the formation of colloid droplets was prevented by the previous administration of L-thyroxine, a positive reaction for TG was observed only in the colloid and apical vesicles, and diffusely in the cytoplasm. These results clearly establish for the first time the presence of immunoreactive TG in apical vesicles, luminal colloid and colloid droplets, and cytoplasm (possibly cell sap). They confirm previous indirect evidence suggesting that apical vesicles are involved in the transport of TG and that colloid droplets whose formation is induced by TSH, do contain TG.


Control of cell growth. IV. Growth properties of a new cell line established from an estrogen-dependent kidney tumor of the Syrian hamster. A permanent cell line has been established in culture from an estrogen-dependent kidney tumor of the Syrian hamster. The cells were selected from mixed populations by three methods: 1) animal passage enrichment, 2) growth on petri dishes, and 3) serum-type dependence. The inoculation of these cells, designated the H-301 line, into host animals showed tumor formation in estrogen-treated males or estrogen-treated females, but no tumor formation in normal or castrated hamsters of either sex. The estrogen-dependent tumor formation in male hamsters by H-301 cells was inhibited by progesterone, hydrocortisone, and testosterone. When H-301 cell growth was examined in culture, the addition of estrogens or other steroid hormones to the medium over the range of 0.1 to 10 ng/ml showed no growth-stimulating or inhibiting effects. Medium prepared with charcoal-extracted serum (to reduce by 90% the endogenous levels of steroid hormones) supported growth as well as medium prepared with normal serum. Furthermore, the addition of either estrogens, testosterone, or antiestrogens to medium prepared with charcoal-extracted serum showed no significant effects upon growth. We have shown also that culture medium prepared with serum from young calves did not support the growth of the cells, and the addition of estrogens to this medium did not change this growth pattern. However, the addition of fetal calf serum to medium already containing calf serum promoted maximal growth. Evidence is presented that the growth-promoting agent in fetal calf serum was inactivated by a 90 C heat treatment. Furthermore, the ether-soluble fractions from fetal calf serum and horse serum did not significantly improve growth when added to calf serum containing medium. We conclude that estrogens or other steroid hormones alone are not sufficient to promote the growth of the H-301 cells, but that heat-labile serum factors are required.


Ontogeny of sensitivity to growth hormone in rat diaphragm muscle. In order to delineate the ages of onset and decline in sensitivity to growth hormone (GH) in rats, the ability of ovine GH in vitro to stimulate amino acid uptake and protein synthesis was studied in diaphragm muscle. GH (25 mug/ml) produced a significant, but small (12-17%), stimulation of alpha-aminoisobutyric acid (AIB, 1 mM) uptake in diaphragms from fed intact rats 7-24 days old, but not from 21-day fetuses or from fed rats 4, 30, 50, 100, or 130 days old. When intact rats were fasted for 24 h, both the magnitude and consistency of the responses to GH increased considerably. In fasted rats, GH significantly stimulated AIB transport by 10,29, 77,111, and 58% at ages 4, 7, 11, 15, and 23 days, respectively; GH was not effective at age 3 or 30 days. At age 15 days, 0.5 mug/ml GH stimulated AIB transport by 96%. GH (25 or 0.5 mug/ml) significantly increased leucine incorporation into protein at age 15 days in fasted intact rats. When rats were hypophysectomized at age 28, 47, or 98 days and tested 2-3 days later, the basal rate of protein synthesis fell, but the basal rate of AIB transport did not; however, GH now stimulated both AIB uptake and amino acid incorporation into protein. These results suggest that rat diaphragm muscle first becomes sensitive to GH at about age 4-7 days (AIB transport), but that further development may be necessary for the anabolic action of the hormone (protein synthesis) to be seen. Stimulation of AIB uptake is maximal about 15 days after birth. Thereafter, diaphragm muscles from normal rats becomes progressively more refractory to the acute stimulatory effects of GH on transport processes. Hypphysectomy of rats 30 days old or more leads to the re-establishment of GH-responsiveness by the AIB transport system. This cannot be attributed merely to a lowering of the basal transport rate; rather hypophysectomy appears to result in the loss of an inhibitor of growth hormone's acute stimulatory actions on transport processes. Previous work suggests that this refractory state is produced in normal rats by occasional surges of endogenous growth hormone. The dominance of the refractory state appears about the time of weaning. This may protect the weaned rat, whose diet is more variable than it is during the suckling period, from periodic depletion of amino acids and glucose from the plasma in response to intermittent surges of GH, thereby allowing GH to produce a sustained stimulation of protein synthesis and growth.


Calcitonin secretion in vitro. II. Regulatory effects of enteric mammalian polypeptide hormones on trout C-cell cultures. We have studied the effects of several porcine enteric polypeptide hormones on trout calcitonin (tCT) secretion in long-lived monolayer cultures of calcitonin-secreting (C-) cells derived from trout ultimobranchial glands. Gastrin, pancreozymin (CCK-PZ), glucagon, and secretin have dose-related stimulatory effects on tCT secretion; the distinct effects of secretin on tCT secretion are in contrast to its lack of CT secretagogue activity in some mammals. Synthetic peptide hormones and/or their structural analogs have variable secretory effects which correspond in general to the potency of the analogs for the well-recognized biological actions of these various peptide hormones in mammals. Although enteric peptide hormones are present in fish, their physiological role in the regulation of CT secretion has not been studied. These in vitro studies indicate a possible role for these hormones in the control of tCT secretion and support the concept that there are differences in the regulators of C-cell function in higher and lower vertebrates. In vitro studies of fish CT secretion are needed to establish the physiological significance of these in vitro studies of fish C-cell function.


The effect of growth hormone on parathyroid function in rats. The effect of excessive growth hormone (GH) on parathyroid function in rats was evaluated in order to determine whether the GH-induced increase in serum calcium (Ca) may be mediated via stimulation of parathyroid hormone (PTH) secretion. Rats receiving injections of bovine GH 2 mg daily for 4 weeks showed a progressive significant increase in both serum Ca and immunoreactive PTH (iPTH) after the second week. Rats receiving daily injections of rat GH, 0.25 mg daily for 3 weeks, followed by 0.5 mg daily for 3 additional weeks, showed a significant increase in serum iPTH during administration of the higher dose, and also a significantly increased parathyroid gland weight and PTH content. The concomitant increase in serum Ca and iPTH suggest that GH stimulates the parathyroid glands to increase PTH secretion, which in turn causes an increase in serum Ca. The parathyroid response increases with the increase in dose and duration of GH stimulus. The data suggest that the hypercalcemia often observed in acromegaly may be due, at least in part, to GH-induced excessive secretory activity of the parathyroid glands.


Comparative effectiveness of preoptic and tuberal stimulation for luteinizing hormone release and ovulation in two strains of rats. Proestrous rats under pentobarbital anesthesia during the 'critical period' were electrically stimulated in either the medial preoptic area (mPOA) or the anterior basal tuber. In Osborne-Mendel (O-M) and Charles River CD rats, comparisons were made of the ovulatory responses to trains of matched biphasic pulse pairs of different microamperage, 30 sec on and 30 sec off for 45 min. In CD rats, comparisons were made of the relative efficiencies of stimulation through a coaxial platinum electrode near the midline and a pair of platinum electrodes spaced bilaterally 2 mm apart across either the mPOA or the tuber. Other comparisons, with either type of electrode in the basal tuber, were made between 4-day and 5-day cyclic CD rats with respect both to the ovulatory responses and to the serum concentrations of LH 90 min after the start of the 45-min stimulus. In both O-M and CD rats, tuberal stimulation was more effective for ovulation than mPOA stimulation. CD rats were much less responsive to mPOA stimulation (spaced electrodes) than O-M rats, but the responses of both strains to tuberal stimulation were essentially alike. In 4-day cyclic CD rats, the ovulatory responses to mPOA stimulation with the coaxial electrode were much superior to those from spaced-electrode stimulation. In contrast, tuberal stimulation with either type of electrode gave equivalent ovulation frequencies and equivalent tubal ovum numbers in both 4-day and 5-day cyclic rats. However, the serum LH concentrations disclosed somewhat better responses to stimulation with the coaxial electrode (coaxial/spaced=1.57). The advantage of stimulation with the coaxial electrode may be due to high current density or, at least in part, to its more medial location. The results are compatible with the concept of preoptic-tuberal neuronal system, diffuse rostrally and convergent upon the medial basal tuber, controlling the ovulatory surge of LH.


Exaggeration of experimental hypertension in rats by contraceptive steroids (Enovid). The possibly additive pressor effects of contraceptive steroids were studied by treating hypertensive rats chronically with Enovid. Renal hypertensive rats were unaffected by drug treatment during the first 5 weeks, but, from the 6th to the 8th week, Enovid-treated rats had much higher systolic pressures than those given corn oil alone. However, these differences dwindled and became insignificant from the 9th to the 16th week despite continued treatment. Subsequently, other rats were pretreated with Enovid or corn oil for 5 weeks before hypertension was induced by implanting deoxycorticosterone acetate (Doca). In contrast to the equivocal results obtained previously, Doca hypertension was consistently more pronounced in rats treated with Enovid than in those given corn oil. This pressure difference was later verified by direct measurement of phasic aortic pressures from indwelling catheters and by the postmortem finding that Enovid-treated rats had larger hearts than corn oil-treated ones. The exact mechanism by which Enovid enhances Doca hypertension is still undetermined, but sympathetic hyperactivity was considered an unlikely explanation, since responses to posterior hypothalamic stimulation, norepinephrine, or ganglion blockade with pentolinium were unaltered.


Thyroidal iodine metabolism during the development of the chick embryo. Stable iodine was measured in the thyroid gland of the chick embryo from day 9 to day 20 of incubation in order to evaluate quantitatively the functional development of the gland. Total iodine content increased progressively during incubation. From day 9 to day 17 of incubation, this increase resulted from the increases of pellet-bound iodine and of soluble iodine. Afterwards, it essentially paralleled the increase of the soluble thyroglobulin-bound iodine which reflected the increase in both thyroglobulin content and the degree of iodination of the thyroglobulin. The total iodine, thyroglobulin-bound iodine and thyroglobulin (TG) content, increased as power functions of time during incubation, with critical times on days 11 and 15. Their concentrations also increased during the whole incubation period, while the iodide concentration remained roughly constant (25 ng/mg) from day 13 to day 19. Only one iodoprotein, 19.5 S TG, was found, and its heterogeneity of iodination was demonstrated during the whole period of incubation studied (from day 11 to day 20). The degree of dissociation with sodium dodecyl sulfate (SDS) of the TG into 12 S subunits decreased as the degree of iodination of the TG increased. Throughout embryonic development, iodine was bound more and more to TG molecules, which were resistant to dissociation with SDS. While the average iodine content of the TG increased, no appreciable changes were found in iodotyrosine and iodothyronine percentages of TG-bound iodine: monoiodotyrosine, 26%; diiodotyrosine, 43%; thyroxine 12%; 3,5,3'-triiodothyronine, 2.5%. As a consequence, a linear relationship existed for each iodoamino acid between the number of its residues per mole of TG and the iodine content of TG (127I atoms per mole)-- about 30 atoms of iodine was required to form 1 mole of T4. The low efficiency of the TG of the chick embryo as a thyroidal hormone-forming protein was compensated for by its high degree of iodination.


Characterization of acid phosphatase isoenzymes in human skin fibroblasts. The multiple acid phosphatase isoenzymes of cultivated skin fibroblasts were investigated in an effort to further clarify the basis of the observed molecular heterogeneity. Treatment with neuraminidase did not alter the migration of isoenzymes present prior to treatment with neuraminidase did not alter the migration of isoenzymes present prior to treatment, but additional isoenzymes were detectable after treatment. Variation of enzymes, permitting prediction of net charge and molecular size differences. Isoelectric focusing between pH 5.0 and pH 8.0 demonstrated three isoenzymes of acid phosphatase in the total cell homogenate and in the lysosomal fraction, two of which appeared to have similar isoelectric points.


Response of plasma and red cell glutathione reductase of several mammalian species and domestic chickens to flavin adenine dinucleotide. Glutathione reductase (GR) activity was determined in the plasma and red cells from a number of species (human, horse, cattle, sheep, chicken, rabbit, rat). The species with the highest plasma activity (horse and cattle) had the lowest red cell activity. Unlike red cell GR, plasma GR was not significantly stimulated by the addition of flavin adenine dinucleotide (FAD). In species with high plasma GR activity the determination of whole blood FAD activity coefficients may not be a good index of riboflavin status.


Ultrasonic measurement of arterial blood pressure in conditioned thoroughbreds. Indirect systolic and diastolic arterial pressure measurements of 456 Thoroughbred horses of various ages and sex stabled at 9 different race tracks were measured with an ultrasonic-Doppler device interfaced with a sphygmomanometer cuff. The mean systolic pressure was 111.8 +/- 13.3 mmHg and the mean diastolic pressure was 69.6 +/- 13.8 mmHg in this population.


The current position of A.I. in horse breeding. This short review article describes the various techniques currently available for artificial insemination in the horse. The collection and use of raw and extended semen is discussed together with the more recent developments in freezing semen. The expected conception rates with both fresh and frozen semen are quoted. The possible benefits in disease control and stud management are discussed, as well as the difficulties in controlling the use of A.I. from the Breed Registration Authorities point of view.


A case of peritoneal mesothelioma in a thoroughbred mare. The report describes a case of abdominal neoplasia in a mare diagnosed ante-mortem by the technique of paracentesis abdominis. Histopathological findings suggest that the lesion was a mesothelioma, primarily involving the greater omentum.


Cytology of diffuse mesothelioma in the thorax of a horse. Examination of effusions from body cavities must include a search for neoplastic as well as inflammatory cells. Neoplastic cells found in the thorax are generally derived from adenocarcinomas or mesotheliomas. Mesotheliomas are relatively uncommon and the neoplastic cells found in effusions are difficult to differentiate from activated cells in inflammatory effusions. An ante-mortem diagnosis of mesothelioma was made in a mare on the basis of the large volume of fluid produced, the pleomorphic mesothelial cells, the polyp formation and the absence of PAS staining material after digestion of the cell impression with maltase diastase.


A preliminary study of the measurement of plasma osmotic pressure in equine medicine. The normal plasma osmotic pressure was established in 205 Thoroughbred horses. Samples taken from horses suffering from a variety of diseases showed that measurement of this parameter might prove a valuable non specific test for assessing the severity of some diseases in which there are alterations in metabolism.


Therapy of a horse with diarrhoea of unknown aetiology. A 5 year old Thoroughbred stallion with diarrhoea of unknown aetiology was referred to Davis. Treatment was aimed at terminating diarrhoea and restoring normal fluid status. Laboratory aids were utilised to establish where inbalance and deficits were present. Antibiotics and corticosteroids were used as an adjunct to fluid therapy. The case history and rationale of treatment of fluid disorders resulting from diarrhoea are discussed.


Studies of new intracellular proteases in various organs of rats. Participation of proteases in degradation of ornithine aminotransferase in vitro and in vivo. Homogenates of the muscle layer of rat small intestine irreversibly inactivated endogenous ornithine aminotransferase at 37 degrees C. Addition to the homogenate of coenzymes and the various keto-acids which act as substrate inhibited conversion of the holoenzyme to the apoenzyme and its subsequent degradation. Addition of protease inhibitors including soybean trypsin inhibitor, chymostatin and phenylmethylsulfonyl fluoride almost completely prevented inactivation of he enzyme. Immunological activity decreased during inactivation of the enzyme, but its rate of decrease was much slower than that of loss of enzyme activity. Antigen-antibody precipitates from homogenates containing inactivated enzyme, were separated by electrophoresis on sodium dodecylsulfate-polyacrylamide gels. In this way breakdown products of the enzyme were found, indicating that the enzyme in homogenates was inactivated by limited proteolysis. These results obtained in vitro support our previous suggestion (1975) of a stepwise mechanism for degradation of pyridoxal enzymes.


Characterization and replication control of plasmids from Enterobacter cloacae DF 13. It has been shown previously that Enterobacter clocacae DF13 harbours at least five different size classes of plasmids. A 45 x 10(6)-Mr self-transmissible R factor determining resistance against tetracyclin, sulfanilamide, streptomycin and chloramphenicol, a 6.0 x 10(6)-Mr bacteriocinogenic factor without sex factors activity and cryptic plasmids in the size classes of Mr 1.3 x10(6), 2.8 x 10(6) and 8.0 x 10(6) respectively. The present work deals with the determination of the homogeneity and molecular relationship of 1.3 x 10(6)-Mr (mini) and 2.8 x 10(6)-Mr (midi) cryptic plasmids and the 6.0 x 10(6)-Mr (maxi) bacteriocinogenic factor, their kinetics of replication and their replication control in response to inhibition of protein synthesis.


The involvement of RNA in the initiation of DNA synthesis in mammalian cells. Artifacts arising during caesium-salt density-gradient centrifugation which simulate a covalent attachment of RNA to newly synthesized DNA. Artifacts were encountered during caesium salt density gradient centrifugation which simulated results expected if newly synthesized DNA is covalently attached to RNA. Newly synthesized DNA (in baby hamster kidney cells, BHK-21/C13), pulse-labelled with [3H]thymidine for 10 min at temperatures below 25 degrees C, banded at a greater buoyant density than mature [14C]DNA (heat-denatured nucleic acids) when centrifuged to equilibrium in caesium chloride gradients. Some of the newly synthesized RNA, labelled with [3H]uridine, banded at a buoyant density slightly greater than DNA in caesium sulphate gradients. These results were not obtained when nucleic acids were pulse-labelled at 37 degrees C, nor when samples were heat-denatured in the presence of formaldehyde. This suggests that nucleic acids can aggregate during centrifugation; this is discussed in relation to the molecular size of the DNA.


The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L). Identification of the trypsin-reactive site, partial-amino-acid sequence and further physico-chemical properties of the major inhibitor. The two principal isoinhibitors P-5 and P-6 isolated earlier from the seeds of chick peas (Cicer arietinum L.) by a procedure involving biospecific affinity chromatography on active, matrix-bound trypsin are shown to be the virgin and trypsin-modified forms of the same inhibitor. The virgin inhibitor P-5 consists of a single peptide chain of 66 amino acid residues cross-linked by seven disulfide bridges. Upon interaction with the matrix-bound trypsin under the conditions used the virgin inhibitor P-5 is about 50% converted to P-6 by cleavage of a single Lys-Ser linkage at position 14-15 in the molecule. The resulting tetradecapeptide remains bound to the rest of the molecule by two or four disulfide bridges, but the two fragments representing residues 1-14 and 15-66 separate readily by molecular-sieve chromatography following reductive or oxidative cleavage of the disulfide linkages. The sequence 1-25 was established by Edman degradation.


Molecular modifications associated with Aging of globin messenger RNA in vitro. Using polyacrylamide gel elution-electrophoresis in aqueous medium, highly purified rabbit globin mRNA can be fractionated into several populations of molecules differing by their mean poly(A) content. Both alpha and beta globin mRNA are heterogenous with respect to their electrophoretic mobilities. With the conditions used no separation of alpha and beta globin mRNA occurs during electrophoresis. From the specific radioactivity distribution in the different mRNA fractions one can conclude that the polyadenylate sequence at the 3' end of globin mRNA molecules becomes shorter with aging. This shortening occurs on alpha as well as beta, globin mRNAs and the extent of heterogeneity in poly(A) content is similar for both globin mRNAs. Furthermore, using two different methods of mRNA fractionation (polyacrylamide gel elution-electrophoresis and elution of poly (U)-Sepharose-bound mRNA at increasing temperatures) it is shown that old mRNA molecules differ from relatively young messages in their ability to direct cell-free globin synthesis. Modifications reducing template activity in vitro thus seem to take place during globin mRNA aging.


Decreased rate of metabolism induced by a shift of the double bond in prostaglandin F 2alpha from the delta5 to the delta4 position. The synthesis of an isomer of prostaglandin F 2alpha,9alpha,11alpha,15(S)-trihydroxyprosta-4-cis,13-transdienoic acid is described. The metabolism of this compound in the rat has been investigated. The rate of degradation by beta-oxidation was slowed down considerably. Thus 10-20% of the injected isomer was excreted in the urine unchanged indicating a longer half-life in the circulation than for prostaglandin F 2alpha. More over 2% was excreted as C20 metabolites, 11-18% as C18 metabolites and 8-15% as C16 metabolites. This relative resistance to degradation by beta-oxidation is of considerable biochemical and pharmacological interest.


Regulation of phenylalanine ammonia-lyase activity in cell-suspension cultures of Petroselinum hortense. Apparent rates of enzyme synthesis and degradation. The time courses for induced changes in the phenylalanine ammonia-lyase activity at five different stages during the growth cycle of cell-suspension cultures from parsley (Petroselinum hortense Hoffm.) were investigated. Large increases in the enzyme activity, induced either by irradiation or by dilution of a cell culture into fresh medium, were followed by an expotential decline to the initial low level. The maximum inducible level of specific enzyme activity varied within a range of about six-fold, depending on the mode of induction and the growth stage of the cell culture.


Nuclease S1 cleavage and the primary structure of mitochondrial DNA. The single-strand-specific nuclease S1 from Aspergillus oryzae rapidly converts superhelical mitochondrial DNA (African Green Monkey cells, Vero ATCC; CCL 81) into nicked circular DNA. These nicked mitochondrial DNA molecules contain two nicks, one in each strand. The phosphodiester backbones are cleaved during this reaction at or near sites that are alkali-labile. In a second slow reaction the circular mitochondrial DNA is converted into a linear duplex DNA. Permutation tests indicate that this linear DNA represents a nonpermutated collection of DNA molecules. These results suggest that two of the alkai-labile sites in the phosphodiester backbones of the mitochondrial chromosome are closely spaced on opposite strands and at specific positions.


The interdependence of magnesium with spermidine and phosphoenolpyruvate in an enzyme-synthesizing system in vitro. The DNA-dependent syntheses of different enzymes of the bacteriophages T3 and T7 were studied in an Escherichia coli system in vitro with respect to the optimal Mg2+ concentration and its interdependence with substituting (e.g. spermidine) and complexing agents (e.g. phosphoenolpyruvate). The following results were obtained. 1. The optimal conditions for the syntheses of the different enzymes were not identical. The optima for RNA polymerase synthesis were 8 mM Mg2+, 10 mM P-pyruvate and 3 mM spermidine; for S-adenosyl-L-methionine cleaving enzyme synthesis, 6 mM Mg2+, 6 mM P-pyruvate and 3 mM spermidine; and for lysozyme synthesis, 13-18 mM Mg2+, 28 mM P-pyruvate and 3-0 mM spermidine. 2. The optimal conditions for the synthesis of analog enzymes (RNA polymerases and lysozymes) from the two templates were identical with experimental error. 3. Mg2+ and spermidine substituted for each other in relation to the number of their charges. 4. The apparent complexing of one Mg2+ molecule required the addition of 3-5 P pyruvate molecules. 5. Under the optimal conditions the enzyme-synthesizing activity was higher by more than a factor of 10 compared to previously described systems.


L-alpha-Hydroxyacid oxidase isozymes. Purification and molecular properties. L-alpha-Hydroxyacid oxidase isozymes from rat liver (A isozyme) and kidney (B isozyme) have been isolated in a high state of purity with specific activities of 61 and 14.7 microkatals per gram protein respectively. The subunit molecular weights determined by sodium dodecylsulphate polyacrylamide gel electrophoresis were 40000 +/- 3000; the mouse A and B isozymes were also partially purified and their subunit molecular weights shown to be 37000.


The purification and properties of cyclohexanone oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871. 1. Cyclohexanone oxygenases from Norcardia globerula CL1 and Acinetobacter NCIB 9871 have been purified 12-fold and 35-fold respectively and each gives a single symmetrical sedimentation peak in the ultracentrifuge and a single protein band on 2.25 nm average pore radius polyacrylamide gels. 2. The enzyme from N. globerula has a molecular weight of 53000 while that from Acinetobacter has a molecular weight of about 59000. Each is a single polypeptide chain with one mole of bound FAD per mole of protein that does not dissociate during purification. Acidification of the Acinetobacter enzyme in the presence of (NH4)2SO4 releases the bound FAD and yields native apoenzyme from which the active holoenzyme can be reconstituted. The apparent dissociation constant for the FAD is 40 nM.


Structure of flavin adducts with acetylenic substrates. Chemistry of monoamine oxidase and lactate oxidase inhibition. The photoreaction of flavoquinones (lumiflavin, riboflavin, FMN etc. and their 3-alkylated derivatives) with propargylamine-type acetylenic substrates, R4 -Calpha identical to Cbeta -CgammaHR3 -NR2R1, yields a mixture of two adducts,which result from covalent Calpha fixation of the Cgamma-deprotonated substrate to either position C(4a) or N(5) in the flavin nucleus. The N(5) adduct is a dihydroflavin-5-trimethine-cyanine with very intense (xi greater than 20000 M-1 cm-1) absorption maxima in the region 380-450 nm depending on the R1,R2. This absorption allows recognition of minute amounts of this species of flavocyanine even in complex mixtures. Flavocyanines can be reconverted to starting flavin by base. It spectral properties are identical with those obtained for the pargyline-flavin inhibitor complex from bovine kidney or pig liver monoamine oxidase.


Synthesis of albumin via a precursor protein in cell suspensions from rat liver. The mechanism of the biosynthesis of albumin was studied in cell suspensions from rat liver. The cells were prepared by continuous perfusion of the liver in situ with 0.05% collagenase and 0.10% hyaluronidase and incubated under conditions optimized for the incorporation of amino acids into protein. Seven minutes after starting the incubation L-[1-14C]leucine was added, followed after 25 min by a 15  or 30-min chase with an 830-fold excess of non-radioactive L-leucine. Total protein, an albumin-like protein, and albumin were isolated from samples withdrawn immediately of total protein was found to remain constant after addition of the non-radioactive L-leucine, whereas that of the albumin-like protein decreased and that of albumin increased with incubation time. The increase in albumin radioactivity accounted for the decrease in radioactivity of the albumin-like protein, suggesting that the latter is a precursor of albumin. The precursor protein differed from albumin by an oligopeptide extension at the N-terminal end.


Influence of chain length and unsaturation on the effects of fatty acids on phosphoglyceride biosynthesis in isolated rat and pig hepatocytes. Hepatocytes isolated from rat or pig by collagenase perfusion were incubated with [3H]glcyerol and different albumin-bount fatty acids. Among C22 fatty acids docosahexaenoic acid stimulated phosphatidylethanolamine synthesis in rat hepatocytes most effectively. Addition of docosahexaenoic acid plus either palmitic or stearic acid resulted almost in the same stimulation whereas combinations of this acid with lauric or myristic acid had no effect. Lauric acid and myristic acid alone inhibited phosphatidylethanolamine synthesis. The chain length specificity for monoenoic fatty acids was similar, the hexadecenoic and octadecenoic acids (both cis and trans) being most stimulatory. The addition of 0.2 mM ethanolamine markedly stimulated phosphatidylethanolamine synthesis, but most effects of fatty acids were similar in its presence or absence.


An enzyme complex for the dehydrogenation of phytoene in Phycomyces. Phycomyces strain C5, carrying mutation carB10, accumulates phytoene instead of beta-carotene. Heterokaryons containing C5 nuclei and different other nuclei carrying the wild type carB allele accumulate significant amounts of phytofluene, zota-carotene and neurosporene. From quantitative analyses of carotenes and nuclear proportions in the heterokaryons we conclude that four copies of the carB gene product, assembled in an enzyme complex, act sequentially in the conversion of phytoene to lycopene.


Synthesis of a cleavable protein-crosslinking reagent for the investigation of ribosome structure. This communication describes a simple method for synthesizing cleavable bifunctional imido esters of different chain lengths. These reagents, which form covalent crosslinks between lysine residues of proteins, contain a disulfide bond which is cleaved under mild conditions by reducing agents such as 2-mercaptoethanol. The reagents are synthesized via the dithiobisnitrile which is prepared in high yield by reacting the appropriate omega-activated nitrile with sodium polysulfide and is then converted quantitatively to the diimidate. Three such reagents were prepared: dimethyl 3.3'-dithiobispropionimidate, dimethyl 4,4'-dithiobisbutyrimidate, and dimethyl 6-6'-dithiobiscaproimidate. The first was synthesized from acrylonitrile, and the others from the appropriate omega-bromonitriles. Experiments with the bispropionimidate and a test protein, pancreatic ribonuclease, have shown the reagent to be effective in producing multimeric crosslinked complexes, from which monomeric proteins can recovered after treatment with 2-mercaptoethanol. The reagents are suitable for studies of ribosomal structure.


Purification of the mRNAs for Ewe alphaS-casein and beta-casein by immunoprecipitation of polysomes. Specific polysomes synthesizing alphas-casein and beta-casein were immunoprecipitated from total polysomes of the lactating ewe mammary gland. The polysome - anti-casein complex was immunoprecipitated by anti-immunoglobulins. 22%, 32% and 10% of polysomes were immunoprecipitated with saturating amounts of anti-alphas-casein, anti-beta-casein and anti-chi-casein respectively. Poly(U)-Sepharose chromatography of the immunoprecipitated RNAs permitted the isolation of the corresponding poly(A)-containing RNA which migrated as one major band in polyacrylamide gel electrophoresis. As judged by the contamination with the messenger activity for one of the other caseins, the purity of the mRNA for alphas-casein as well as for beta-casein was estimated to be between 75% and 80%.


Methylation analysis of glucosaminitol and glucosaminyl-glucosaminitol disaccharides. Formation of 2-deoxy-2-(N-acetylacetamido)-glucitol derivatives. N-Acetylglucosaminitol, N-acetylglucosaminyl-beta 1',4-N-acetylglucosaminitol (chitobiitol), the corresponding trimer (chitotriitol) and N-acetylgucosaminyl-beta 1'6-N-acetylglucosaminitol were subjected to methylation analysis using combined gas-liquid chromatography/mass spectrometry. The preparation were subjected to permethylation according to Hakomori, followed by acid hydrolysis, reduction and peracetylation. From N-acetylglucosaminitol and its 4-0 and 6-0 substituted derivatives, methylated N-acetylacetamido derivatives were obtained. The possible conditions for the formation of these products are discussed.


[alpha-L-Rhamnosidase from Fagopyrum esculentum: purification and some properties (author's transl)]. An alpha-L-rhamnosidase from the seeds of Fagopyrum esculentum (saracen corn) has previously been identified, and the effect of the enzyme on rhamnoisic bonds has been studied with various flavonoid glycosides. This alpha-L-rhamnosidase can be useful in structural studies, and a preliminary report of this study has appeared. The present paper describes the extensive purification of the enzyme and the determination of its properties. The purification involved extraction, ammonium sulfate fractionation and chromatography on Sephadex G 75, DEAE-Sephadex and Ultrogel AcA-44. The alpha-L-rhamnosidase was purified about 9600 fold and the final enzyme preparation was practically pure according to the criteria of disc electrophoresis. The molecular weight of this alpha-L-rhamnosidase, calculated from data obtained by disc gel electrophoresis and gel filtration, was about 70 000. Isoelectric focusing established the isoelectric point to be 3.7. The behaviour of the enzyme on a concanavalin-A-Sepharose column suggests the presence of residues resembling alpha-D-mannose or alpha-D-glucose in the protein. The various kinetic parameters, Kcat, Km and the Kcat/Km ratio have been determined at pH 5 on the following substrates: p-nitrophenyl-alpha-L-rhamnoside and rutinose (6-O-alpha-L-rhamnosyl-D-glucopyranose). All kinetics exhibit a Michaelian behaviour and the Km for the former substrate was 0.33 mM and for the latter, 2.2 mM. The Kcat/Km ratio corroborates the greater specificity of the enzyme for p-nitrophenyl-alpha-L-rhamnoside. L-Rhamnose, L-lyxose, 6-deoxy-D-glucose and methyl-alpha-D-mannoside were shown to behave strictly as competitive inhibitors of alpha-L-rhamnosidase activity; it seems that the methyl group of L-rhamnose is important for substrate binding to the enzyme.


Effect of specific antibodies on D-glyceraldehyde-3-phosphate dehydrogenase. The inhibition of rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase by specific antibodies produced in rabbits has been studied. The results suggest that no influence on the enzyme active site is caused by the interaction with antibody, the inhibition being due entirely to the restricted accessibility for substrates of a part of dehydrogenase molecules included in the immune precipitate. Soluble complexes of the enzyme with monovalent Fab antibody fragments retain full catalytic activity. Modification of 8 -SH groups per mole of glyceraldehyde-3-phosphate dehydrogenase with p-chloromercuribenzoate results in no alterations in the quantitative precipitin curve, thus supporting the conclusion about the different localization of species-specific antigenic determinants of the enzyme and its active center. Interaction with monovalent Fab fragments of antibody stabilizes the structure of the dehydrogenase. Eight molar equivalents of Fab fragments almost completely protect the enzyme from cold inactivation in the presence of 0.15 M NaCl. Complex formation with Fab fragments does not prevent, however, the ADP-induced inactivation of the enzyme.


Carbohydrate in pancreatic ribonucleases. A survey of the presence and compositions of carbohydrate chains attached to pancreatic ribonucleases is given. Carbohydrate chains may occur at asparagine residues in Asn-X-Ser/Thr sequences at four exposed sites of the molecule (positions 21, 34, 62 and 76). These sites form part of highly variant sequences in pancreatic ribonucleases with the consequence that the enzymes from very closely related species may differ in the presence or absence of carbohydrate. In a number of ribonucleases Asn-X-Ser/Thr sequences occur which carry carbohydrate in only a part of the molecules. The occurrence of Asn-X-Ser/Thr sequences entirely without any carbohydrate also has been demonstrated. Compositions of the carbohydrate moieties of ribonucleases from cow, sheep, pig, whales, giraffe, okapi, moose, horse, coypu, chinchilla, and guinea-pig are presented. Striking differences in complexity have been found, both between chains attached to the same site in different species (cow and giraffe), between chains attached to different sites of the same enzyme in one-species (pig) and even between chains attached to the same site in a single species (chinchilla).


Mechanism of the stimulatory effect of fructose on ethanol oxidation in perfused rat liver. The stimulatory effect of fructose on ethanol oxidation was studied in livers from fasted rats perfused with Krebs-Henseleit-bicarbonate buffer in a non-recirculating system. Two series of experiments were performed: (A) ethanol was infused with stepwise increasing concentrations (0.1-20 mM) in the presence of 4 mM fructose; (B) fructose was infused with stepwise increasing concentrations (0.5-10 mM) in the presence of 2 mM ethanol. From measured metabolic rates the following parameters were calculated: energy-rich phosphates consumed for fructose metabolism which were provided from oxidative phosphorylation (delta approximately P); reducing equivalents derived from stimulated ethanol utilization which were disposed by mitochondrial oxidation (delta2H). Under the various conditions studied a linear relationship between these parameters was observed. The ratio delta approximately P/delta2H was about 2.0. It is suggested that fructose stimulates ethanol oxidation indirectly by increasing the energy consumption of the liver due to the production of glucose from fructose. Consequetnly, the rate of oxidative phosphorylation is increased and, therefore, the capacity of the respiratory chain for oxidizing reducing equivalents derived from ethanol is enhanced. The data support the more general hypothesis that the rate of ethanol oxidation depend upon the rate of hepatic energy consumption in a given metabolic state.


Isolation and characterization of the 'photosynthetic' phosphoglycerate kinase from Beta vulgaris. 1. Phosphoglycerate kinase has been isolated from a photosynthetic plant tissue, Beta vulgaris leaves. The purification procedure is described. 2. The best preparation had no detectable impurity on electrophoresis, and had a specific activity comparable with the same enzyme from other sources. 3. The molecular weight was not distinguishably different from that of the yeast or muscle enzyme, as measured by polyacrylamide-dodecylsulphate electrophoresis. Measurement of aromatic and sulphydryl residues indicated a close similarity with the yeast enzyme. The enzyme appears to have substantially lower isoelectric point than phosphoglycerate kinases from other sources. 4. Kinetic studies indicated that the affinities for the substrates MgATP2- and 3-phosphoglycerate were not significantly different from those of the 'glycolytic' yeast enzyme. There was no evidence that the B. vulgaris enzyme had specific properties making it more suitable for its gluconeogenic rather than glycolytic role.


Catabolism of cholesterol by bovine adrenal-cortex enzymes: in vitro formation of oxygenated sterols and side-chain cleavage products. The identification of sterols and steroids formed by incubation of [7alph-3H, 26-14C]cholesterol with mitochondrial enzymes from bovine adrenal cortex is reported. Only 17% of the radioactivity associated with cholesterol metabolites was found in pregnenolone, whereas 15% was reliable to oxygenated sterols and 6% to steroid compounds. The significance of the formation of these compounds is discussed particularly as regards oxygenated cholesterol derivatives.


[Study of the biosynthesis of phleic acids, polyunsaturated fatty acids synthesised by Mycobacterium phlei (author's transl)]. Because of their structures, phleic acids (general formula: CH3-(CH2)m-(CH=CH-CH2-CH2)n-CO2H; main component: m = 14, n = 5) cannot be synthesized by the same kinds of enzymatic systems as other natural polyunsaturated fatty acids. By using specifically labelled 14C compounds, we have tested the ability of different molecules to be incorporated in the phleate skeletons by Mycobacterium phlei. The localisation of radioactive carbon atoms has been studied by chemical degradation of labelled phleates, isolation and purification of the degradation products, and determination of their specific radioactivity. When M. phlei cells are incubated with labelled acetate, the unsaturated and saturated parts of the molecules of phleic acids are unequally labelled. The radioactivity of succinate monoester on the one hand and fatty acids (mixture of myristic and palmitic acids) on the other hand, measured after oxidative degradation of phleate esters, shows a constant ratio under definite conditions. Whether [1-14C]acetate or [2-14C]acetate is used for incubation, the same ratio is observed. Therefore acetate is the precursor of the unsaturated part as well as of the saturated part of the phleate molecules. By using labelled fatty acid esters, it has been found that palmitic acid is the precursor of phleates with m = 14, while myristic acid is the precursor of phleates with m = 12. Stearic and eicosanoic acids are not incorporated without degradation. The hypothesis of a condensation of a saturated fatty acid with a preformed polyunsaturated molecule was examined. Search for such a molecule in the lipids of M. phlei gives negative results. Pentaunsaturated phleate arising from palmitate is more abundant than pentaunsaturated phleate arising from myristate, while the reverse is true for hexaunsaturated phleates. These observations make very unlikely such an hypothesis. An elongation process fits well with the observed facts provided that this process involves elongation by two acetate units simultaneously, making elongation by four carbon atoms at a time. Such a requirement would be easily satisfied if two molecules of acetate are condensed together before their utilization in the elongation process. In such a hypothetical process, crotonate would be the most probable substrate of the elongation reaction.


Acetylcholinesterase and its association with lipid. Human erythrocyte acetylcholinesterase preparations which vary in lipid content, from lipid-rich to lipid-poor, have been successfully prepared using deoxycholate. It was found that the lipid content of the enzyme decreased gradually as the deoxycholate concentration used in its preparation was increased. The binding of lipid to lipid-poor preparations of the enzyme has been investigated. It was found that the activity of such preparations was highly dependent on their phospholipid contents. Maximum specific activity was associated with a fixed phospholipid content. The lipid-poor enzyme was highly activated by addition of either endogenous(membrane) or exogenous lipid. Based on the data presented, it was concluded that acetylcholinesterase is a phospholipoprotein, and its activity is highly dependent on its phospholipid component.


[Separation of structural studies of the molecular species of monomycolates and dimycolates of alpha-D-trehalose present in Mycobacterium phlei (author's transl)]. Mixtures of dimycolates of alpha-D-trehalose (cord factor) and monomycolates have been isolated from Mycobacterium phlei and separated as trimethylsilyl derivatives according to the polarity of the fatty acid residues. The free glycolipids can be recovered by mild hydrolysis. Silylation and disilylation reactions did not induce any isomerisation. The structure of these trehalose esters has been determined by using a series of reactions elaborated on synthetic acyl-sugar models. Free hydroxyl groups are transformed into tetrahydropyran ethers, deacylated by dimsyl sodium, methylated and the sugar derivatives are hydrolyzed. The O-methyl-sugars obtained contain a methyl ether group located at the position where the acyl group was present initially. Identification by gas chromatography of the O-methyl-sugars thus allows the location of the fatty acid residues in the glycolipid. It has been demonstrated that no migration occurs. Two types of 6-monomycoloyl-alpha-D-trehalose have been isolated, differing by the nature of the mycolic acid residues. One of them, called MA, contains "alpha-phlei mycolic acid". The other one, called MB, contains "gamma-phlei mycolic acid" which is the ester of 2-eicosanol with the omega-carboxyl function of a dicarboxylic mycolic acid. Three types of 6,6'-dimycoloyl-alpha-D-trehalose (cord factor) have been obtained. Two of them are symmetrical diesters, containing either two residues of alpha-mycolic acid, or two residues of gamma-mycolic acid. The last one is an unsymmetrical diester, the hydrolysis of which gives one mole of alpha-mycolic acid and one mole of gamma-mycolic acid. The ratio of the different diesters in the cord factor fraction might be explained by some equilibrium between the different 6-ester groups or by a transformation in situ of one species into another one by biochemical modification of the mycolic acid residues.


Incorporation of inorganic sulfate in rat-liver Golgi. Partial subcellular distribution and specifically the incorporation of 35SO4 in the Golgi apparatus of rat liver was studied. The isolated Golgi fraction was placed on a further sucrose gradient for subfraction and in all samples the total incorporated label, the trichloroacetic-acid-soluble and insoluble, and chloroform/methanol-soluble and insoluble amounts of label were measured. Approximately 2.3% of the total injected 35S was found in the liver, of which 2% was localized in the isolated Golgi fraction. Further subfractionation of the Golgi has shown that the majority of the 35S is localized at densities 1.12-1.13 g/cm3 in the gradient. In the isolated Golgi fraction 85% of the label was present in the trichloroacetic-acid-soluble and 15% in the insoluble part. Approximately 1% of the label in the Golgi fraction was soluble in chloroform/methanol. On the bases of radioactivity per weight of protein and total radioactivity, the Golgi fractions had the highest activity, except the first supernatant which had higher radioactivity. Briefly, a significant amount of the 35SO4 can be found in the isolated rat liver Golgi 15 min after the injection of the label, and the majority of this label is localized in the material sedimenting at density 1.12-1.13 g/cm3 after subfractionation of the Golgi apparatus.


Thermal stability of immobilized enzymes circular dichroism, fluorescence and kinetic measurements of alpha-chymotrypsin attached to soluble carriers. The temperature-induced unfolding of alpha-chymotrypsin and of chymotrypsin covalently bound to two soluble transparent carriers, dextran and a copolymer of maleic acid anhydride and acrylic acid, has been studied by tryptophan fluorescence emission, circular dichroic and kinetic measurements. It has been shown that the structural and functional properties of the enzyme when bound to the anionic copolymer are strongly influenced by electrostatic interactions. A number of reference experiments with anionic polyelectrolytes and the hydrogenated monomers of the copolymer suggest that these changes are brought about by the cooperative ion pair formation between protein and polyanionic matrix.


Subcellular distribution and properties of poly(A)-containing RNA from cultured plant cells. Cultured sycamore cells rapidly incorporate [3H]uridine or [32P]orthophosphate into rRNA precursors and polydisperse RNA. Mature rRNA accumulates only after a lag period of approximately 40 min. Fractionation of pulse-labelled cells and analysis of the RNA shows that after 30 min the rRNA precursors, together with some polydisperse RNA, are confined to the nucleus. In consequence radioactive polydisperse RNA can be isolated from polyribosomes in the complete absence of labelled rRNA. Approximately 40% of this RNA is retained by an oligo(dT)-cellulose column and by this criterion is judged to contain poly(A) sequences. A smaller proportion of nuclear polydisperse RNA also contains poly(A). The tendency for poly(A)-containing RNA to aggregate complicates molecular weight determinations. Denaturation of poly(A)-containing RNA in 8 M urea prior to gel electrophoresis produces a broad peak of RNA with an average Mr = 10(6). Analysis of the nucleotide composition of total cell poly(A)-containing RNA shows that it contains 41% AMP. Roughly 6% of this RNA is resistant to digestion by ribonuclease A and T1. AMP is the only nucleotide detectable in these fragments. From their mobility during electrophoresis in 8 M urea at 60 degrees C with 5.8-S, 5-S and tRNA as molecular weight markers it is concluded that the poly(A) regions contain an average of 160 nucleotides.


Studies of malformation syndromes of man XXXXIA: anatomical studies in the Hanhart syndrome--a pathogenetic hypothesis. Two infants with the Hanhart syndrome, i.e. micrognathia, microglossia, terminal deficiency of all limbs and imperforate anus in one, were dissected and studied in detail. The interrelationships of the muscular and skeletal defects suggested that they were the result of incomplete rather than abnormal morphogenesis. We speculate that the oral and limb abnormalities resulted from deficient mesodermal proliferation caused by disturbances in the ectodermal-mesodermal interactions beginning about the 4th week of development. The imperforate anus may also relate to the proposed defect.


Studies of malformation syndromes of man XXXXI B: nosologic studies in the Hanhart and the Möbius syndrome. We reviewed etiologic and phenotypic aspects of those orofacial and limb anomalies usually diagnosed as Hanhart syndrome and Mobius syndrome but also those described, among others, under names such as aglossia-adactylia syndrome, gloss-palatine ankylosis, ankyloglossia superior, peromelia and micrognathia, cleft palate/lateral synechiae syndrome, and the Charlie M. syndrome. By coding the degree of severity of the limb defects it was possible to compare these cases quantitatively and to determine the nosologic significance of associated cranial nerve palsies and chest abnormalities. We analyzed 7 personal and 62 previously reported cases and found: 1. that the severity in the upper limbs and, particularly, malformations of the feet, but not the presence or absence of cranial nerve palsies, is a significant feature in the differentiation of cases, and 2. that the group of patients with cranial nerve palsies includes some with limb defects similar to those in the Hanhart syndrome and others with features which overlap the manifestations of the Poland syndrome. Still other cases had cranial nerve palsy as an isolated trait or as a component manifestation of several different syndromes. These findings permit re-definition and nosologic delimitation of the various syndromes as follows: 1. The Hanhart-syndrome: usually severe limb defect of at least one hand or foot, frequently associated with severe oral abnormalities and sometimes also with cranial nerve palsy. Most cases reported as aglossia-adactylia syndrome, aglossia-hypomelia syndrome, and some cases reported as glossopalatine ankylosis, ankyloglossia superior and Mobius syndrome describe instances of the Hanhart syndrome. 2. The Poland-Mobius syndrome: we suggest this term to refer to those cases of "Mobius syndrome" which have a chest defect and/or symbrachydactyly of the type seen in the Poland syndrome. We suspect that these cases of the "Mobius syndrome," and most of the cases which are usually diagnosed as Poland syndrome represent a different spectrum of the same condition, hence the term Poland-Mobius syndrome. 3. The autosomal dominant cleft palate/lateral synechiae syndrome delineated by Fuhrmann et al. and other apparently less frequent conditions are mentioned in the discussion. Cranial nerve palsy obviously occurs in several etiologically distinct conditions. An analogous situation is present, although less obvious, in the Hanhart and the Poland-Mobius syndrome. Both of these conditions are formal genesis malformation syndromes which implies that they are etiologically non-specific developmental field complexes. In the Hanhart syndrome Bersu et al. postulate a common pathogenetic disturbance for oral and limb defects, thus suggesting that the manifestations represent a single anomaly rather than a "syndrome." This anomaly, for which we suggest the term Kettner anomaly, may occur not only in the Hanhart syndrome but also in other conditions. Similarly, the Poland anomaly, i.e...


[Lipic content and fatty acid pattern in human milk and cow's milk (author's transl)]. The lipid content and the fatty acid pattern in human colostrum, transitional and mature molk and in pasteurised cow's milk and in "Vorzugsmilch" (special untreated milk) were investigated. The analyses were done in pooled daily milk of 15-20 women to archieve representative mean values for human milk. The lipid content amounted to 1.87 g/100 ml in human colostrum, 2.82 g/100 ml in transitional mild and 3.45 g/100 ml in mature human milk. The increase of lipid content during lactation is attributed to maturation of the milk and is not influenced by maternal diet. During maturation of human milk decreases the portion of the unsaturated fatty acids from 57 to 53%, the portion of saturated fatty acids increases accordingly from 43 to 47%. The decrease of the unsaturated fatty acids is mainly due to the decrease of oleic acid while the portion of linoleic acid increases from 10.8 to 12.0%. In the same duration the linoleic acid portion of the energy content of human milk increases there with from 3.3 keal % in colostrum to 5.6 kcal % in mature human milk. Lipid content and fatty acid pattern of pasteurised cow's milk and "Vorzugsmilch" were compared with the corresponding values in human milk. The importance of the differences in lipid content and fatty acid pattern of human milk and cow's milk for the nutrition of healthy young infants is discussed.


Acid-base parameters and alveolar-arterial oxygen tension gradients in healthy neonatal and postneonatal infants. In a randomly selected group of asymptomatic neonatal and postneonatal infants, acid-base parameters and oxygen tension measurements in arterial blood (radial artery) were carried out during air and oxygen breathing. Acid-base results showed a mild, partially compensated metabolic acidosis, indicating adequate pulmonary function. Arterial oxygen tension was in the normal range for this age group. Calculated alveolar oxygen tension levels demonstrated alveolar-arterial gradients of about 30 mm Hg during air breathing and 320 mm Hg during O2 breathing. Volumes of R-L shunts calculated from these gradients, were in the order of magnitude commonly found at this age, i.e., about 20% of right cardiac output. No differences were discernible between infants of appropriate weight for gestational age and small-for-date infants.


Repeated circadian growth hormone, luteinizing hormone, and follicle-stimulating hormone in children: influence of 9-alpha-fluorohydrocortisone. Growth hormone (GH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in plasma of 5 children at different pubertal stages and suffering from moderate orthostatic complaints, were measured by radioimmunoassay (RIA). Parameters for secretory capacity were arginine loading and circadian hormonal patterns in hourly intervals from 8 a.m. to 8 p.m. and in half-hourly intervals from 8 p.m. to 8 a.m. before and after 6 weeks treatment with 9-alpha-fluorohydrocortisone (9-alpha-F, Astonin-H, Fa. Merck, Darmstadt, 0.1--0.2 mg/day. Plasma GH during arginine tests and in circadian levels remained unchanged, but circadian LH showed a consistent slight rise in all children. With pubertal development, magnitude and timing of GH peaks increased in boys, and rather decreased in the two girls toward late puberty. Episodic fluctuation of LH and FSH were more marked during sleep, and increased in the three bosy as puberty advanced. Similar intraindividual patterns for GH and LH, but not for FSH were noted in 4 children. Timing of GH and LH peaks appeared to be correlated. An intrinsic hereditary long-term regulatory principle is discussed.


Aorto-pulmonary relationship, arterio-ventricular alignment, and ventricular septal defects in complete transposition of the great arteries. A study of the conal anatomy of 36 specimens of complete transposition of the great arteries (TGA) is reported. The aim of the investigation was to verify if the two main anatomic features of the malformation, i.e. abnormal aorto-pulmonary and arterioventricular relationships, were strictly related to the maldevelopment of the conal structures. The findings indicate that in the cases with mitro-pulmonary fibrous continuity, there is a wide variation in subaortic conal development. and no correlation exists between the anterior position of the aorta and the length of the subaortic parietal conus. Therefore, differential conal absorption does not seem to play a role in the presumptive lack of rotation in TGA. TGA presenting with bilateral conus and biventricular pulmonary trunk origin are relatively frequent in this survey, but no correlation was observed between overriding of the pulmonary artery and development of the subpulmonary parietal conus. Consequently, in TGA the morphogenetic process of transfer of the pulmonary artery above the left ventricle does not seem to depend only on the absorption of the subpulomonary parietal conus. Ventricular septal defects (VSDs) in TGA are related frequently to conal pathology, either to some failure in conal septal growth or to malalignment of the normally developed conal and sinusal components of the ventricular septum.


Selective aortic root angiography in the hypoplastic left heart syndrome. Selective aortic root angiography was performed in 18 patients with the clinical diagnosis of hypoplastic left heart syndrome in order to allow more precise correlation with echocardiography. Aortic valve atresia and severe hypoplasia of the left heart chambers subsequently were substaniated at necropsy in each instance. Wide variation in the size and shape of the aortic root and ascending aorta was demonstrated angiographically in these patients. In some patients, despite aortic valve atresia, the dimensions of the aortic root and sinuses of Valsalva at angiography were almost normal. In addition, this technique allowed the demonstration of primitive vascular communications between the coronary artery and left ventricular cavity in 1 patient (coronary-cameral fistula). The realization that the aortic root may approach nearly normal size in some patients with the hypoplastic left heart syndrome is essential to the echocardiographic diagnosis of this disease and to the differential diagnosis from other cardiac and noncardiac causes of severe neonatal congestive heart failure.


The pulse in patients with atrial fibrillation: its irregularity and inequality. In 32 patients with atrial fibrillation the distributions of the R-R intervals and the carotid pulse records were analyzed in an attempt to define and quantify 'irregularity' and 'inequality' of the pulse and its relationship to the mean ventricular rate. Using our definition or irregularity and inequality of the pulse (the percentage scatter around the mean) we arrived at the conclusion that: (1) the same degree of irregularity is maintained with different mean ventricular rates in the range studied by us (digitalis administration had no significant effect on this irregularity (2) the 'inequality' between beats decreases at slower ventricular rates: (3) a relationship with the postextrasystolic potentiation is suggested.


An analysis of pulsus alternans in aortic stenosis. Pulsus alternans was studied hemodynamically in 5 patients with calcific aortic stenosis. Left ventricular function was assessed by angiographic volume analysis and by force-velocity analysis. All cases showed alternation of max and min dP/dt and kVmax. Inconsistent factors were an alternating left ventricular enddiastolic pressure, alternating left ventricular enddiastolic volume, alternating left ventricular endsystolic volume, ejection fraction, enddiastolic wall stress and wall stiffness. All cases had a high ventricular mass, but the critical value of ventricular mass or the ratio of mass to left ventricular enddiastolic volume were also inconsistent. Pulses alternans is primarily due to alternating contractility.


The effect of atropine on calculated sinoatrial conduction time in man. This study investigates the effect of atropine on the length of the return cycles after premature atrial stimulation in man. On the assumption that sinus node automaticity is not changed by premature depolarization of the sinus node, sinoatrial conduction time (SACT) was calculated from the differences between the length of the return cycles and the spontaneous cycle length. 11 patients were studied before and after the injection of atropine. In all cases atropine caused an increase in heart rate. In 8 patients the return cycles exhibited a typical behavior. In 6 patients without signs of sinus node dysfunction, the mean calculated SACT was 109 msec, whereas 2 patients with a sick sinus syndrome (SSS) had a mean calculated SACT of 190 and 225 msec, respectively. 3 patients with SSS demonstrated an atypical pattern of the postextrasystolic pauses. In 6 patients without sinus node dysfunction, atropine caused a reduction of calculated SACT about 35%. In 2 patients with SSS the reduction was 38 and 49% of the control value, respectively, whereas in the remaining 3 patients with SSS who had an atypical return cycle pattern, a normalization occurred after the administration of atropine. We conclude that, besides its effects on sinus node automaticity, atropine has also a marked effect on sinoatrial conduction, even in patients with the sick sinus syndrome.


Clinical setting and prognostic significance of atrial fibrillation complicating acute myocardial infarction. Clinical setting, presence and degree of heart failure (HF) of 80 consecutive patients who developed atrial fibrillation (AF) following acute myocardial infarction were compared to that of 620 patients with acute myocardial infarction and sinus rhythm admitted during the same period. 33 of 163 (20%) with initially mild HF developed AF compared to 5 of 74 (7%) with initially severe HF, and 42 of 463 (9%) without HF (P less than 0.01). Patients with mild HF who developed AF had a higher incidence of deterioration of clinical condition (39 vs 14%, P less than 0.005) and a higher mortality rate (52 vs 22%, P less than 0.001) than patients with mild HF and sinus rhythm. The presence of AF influenced neither the development of HF and mortality rate in patients without HF nor the degree of HF and mortality in patients with initially severe HF. Pericarditis was present in 12 of 42 (28%) with AF occurring in the absence of HF, compared to 3 of 38 (8%) with AF and initially mild HF and to 56 of 620 (9%) with sinus rhythm (P less than 0.05). (1) in the absence of HF, AF may be the consequence of pericarditis; (2) in patients with mild HF, AF may be consequence of HF; (3) the contribution of AF to the subsequent clinical course is only of prognostic significance in patients with mild HF.


Ventricular arrhythmias prior to discharge after acute myocardial infarction. One hundred consecutive patients in sinus rhythm and without routinely prescribed antiarrhythmic therapy were investigated for ventricular ectopic beats (VEB) prior to discharge after an acute myocardial infarction. The registration was performed for three hours during the day, including light exercise, and three hours at night. The overall VEB frequency was 70%. 34% had uniform VEBs, 22% multiform VEB'S, ANd 11% paired VEB'S 2% HAD THE R-on-T phenomenon and 1% ventricular tachycardia (VT). Ventricular irritability was more common in patients discharged after reinfarction, an estimated large infarct, if they had shown VT or ventricular fibrillation in the acute phase or when treated with diuretics only because of heart failure. There was no lowering of the VEB incidence at night, 17 patients having had ventricular arrhythmias at night only. In 5 cases the VEB'S were of a higher degree during exercise than at rest.


Initial vectors of ventricular premature beats and anterior fascicular conduction defects. Vectorcardiographic studies on ventricular premature beats (VPB'S), occurring in records wherein the sinus beats were associated with an intraventricular conduction defect attributed to anterior fascicular delay or block, did not identify the exact site of a presumed reentry mechanism. The hypothesis that the orientation of the initial vectors of the VPB might reveal either anterograde or retrograde emergence from the anterior fasciulus iwth a reentry PVB was not strongly supported, although one or the other mechanism was compatible with the early vectors of certain VPBs. The VPBs revealed greater angular change on the spatial orientation of sequential early vectors (5--10--15 milliseconds) than occurred in sinus beats. Recurring VPBs, which appeared to casual visual inspection steriotyped in form, showed considerable variation in the orientation of the early vectors, indicating, in the cases studied, that VPBs, in the strictest sense, may be associated with differing initial entrance pathways to the myocardium and thus may be 'multiform'.


Hemodynamic effects of verapamil and practolol in man. The hemodynamic effects of verapamil and practolol were investigated in a group of 10 selected patients (7 with coronary artery disease) undergoing cardiac catherization. The drugs were given i.v. in a dose of 0.1 mg/kg, alone or in combination, before and after controling the heart rate by atrial pacing. Measured hemodynamic parameters included aortic and left ventricular pressure and its first derivative, cardiac output, and echocardiographically derived left ventricular dimensions. Each drug given individually caused minor hemodynamic changes, but the combination of practolol and verapamil when the heart rate was fixed by atrial pacing invaribly caused a reduction in LV dp/dt max which was independent of preload and afterload. It is concluded that both drugs have a mildly negative inotropic effect when administered at this dose intravenously to patients with normal or mildly impaired left ventricular function. The myocardial depression was more apparent when practolol preceded verpamil. Caution must be exercised when using these drugs in combination in patients with impaired myocardial function.


Comparison of verapamil and practolol in paroxysmal supraventricular tachycardia. 40 patients with paroxysmal supraventricular tachycardia were allocated to two different treatment groups according to their year of birth. Those patients born on odd years were given 5 mg of practolol intravenously, those born on even years receiving 5 mg of verapamil, and if the tachycardia continued, the same injection was repeated after 5 minutes. If practolol treatment failed, the patient was given 5 mg of verapamil half an hour later, and vice versa. Verapamil restored sinus rhythm in 19 patients out of 20. Practolol proved effective in 8 out of 20. In patients who were switched over to the other treatment verapamil was effective in 9 out of 11 and practolol in 1 patient. Side effects were few. Marked hypotension occurred in 2 patients, in 1 patient after 5 mg of verapamil, and after 5 mg of practolol in the other. It is concluded that verapamil is a very effective agent in the treatment of paroxysmal supraventricular tachycardia. Selection of patients is emphasized to increase the safety of its use.


Triglyceride metabolism in acute starvation: the role of secretin and glucagon. Plasma lipid and hormone levels have been measured during 72 hours total starvation in nine healthy subjects, to assess the relative importance of hormones and substrates in human triglyceride metabolism. Plasma free fatty acid and glycerol concentrations rose steadily on each day of starvation. Plasma triglyceride concentrations rose on the second and third days, from a control level of 649 +/- 67 mg/1 to a maximum of 1001 +/- 66 mg/1. Plasma cholesterol concentrations remained unchanged while glucose concentrations fell and insulin did not change. Plasma glucagon (C-GLI) levels doubled while secretin levels, reported previously, rose threefold. It is suggested that during acute starvation the rise in triglyceride concentration results from the increased availability of free fatty acids, and that elevated secretin and glucagon levels enhance lipolysis and hence provide substrates for triglyceride synthesis.


Influence of ureteral catheter size on urinary flow rate and glomerular filtration rate in the rat. The influence of ureteral catheter diameter on renal function was studied in rats during different diuretic states. Urinary flow rate and glomerular filtration rate were measured on each rat using ureteral catheters of two different internal diameters. In non-diuretic rats, urinary flow rate and glomerular filtration rate did not change according to the catheter diameter. In diuretic rats, urinary flow rate was from 40 to 70% lower with the smaller catheter. Differences became more marked as urinary flow rate increased. Glomerular filtration rates were about 20% lower when measured with small catheters. These results clearly demonstrate that the use of small diameter ureteral catheters during diuretic states leads to a decrease in glomerular filtration rate and urinary flow rate. The results also emphasize the importance of using large diameter catheters during such experimental conditions.


Renin-aldosterone axis in ethanol intoxication and hangover. The renin-aldosterone system was studied in human volunteers during ethanol intoxication and hangover. Plasma renin activity increased more than 100%, when 1.5 - 2.3 g ethanol per kg body weight was ingested over a three hour period. During hangover the increase even exceeded 200%. Plasma aldosterone concentration decreased during ethanol intoxication, but increased greatly during hangover. It is suggested that the stimulation of the renin-aldosterone axis during ethanol intoxication and hangover is due to dehydration and increased activity of the sympathetic nervous system.


On the mechanism of the coronary dilator effect of serotonin in the dog. In experiments designed to determine the nature of the coronary dilator effect of serotonin the influence of intracoronary administration of the amine on coronary perfusion pressure, heart rate and ventricular contractile force was assessed in anesthetized open-chest dogs in which the left coronary artery was perfused with blood at a constant rate. Serotonin elicited dose-related decreases in coronary perfusion pressure and increases in contractile force, and lowered heart rate slightly. The dilator response was antagonized by methysergide, slightly potentiated by practolol and unaffected by reserpine. The inotropic effect was partially antagonized by methysergide and completely blocked by practolol and reserpine. It is concluded that serotonin induces coronary dilatation by direct stimulation of specific receptors that this effect is independent of the cardiac stimulation produced by the amine, and the latter response is mediated through beta1-adrenoceptors activated by released norepinephrine.


Bronchopulmonary pharmacology of some prostaglandin endoperoxide analogs in the dog. This investigation examined quantitatively the bronchopulmonary effects of two prostaglandin endoperoxide analogs (cyclic ethers) in the spontaneously breathing, anesthetized dog. Responses to these agents were measured by computer analyses and compared to those produced by prostaglandin F2alpha (PGF2alpha). In a dose range of 0.03-1.0 mug/kg i.v., both cyclic ethers produced dose-related alterations in pulmonary airway resistance dynamic lung compliance, expiratory airflow rate, tidal volume and respiratory frequency. These pulmonary changes were significantly greater than those observed for PGF2alpha (0.3-3.0 mug/kg). The onset of activity was prompt (less than 1 min) and recovery was complete within 5-10 min. Atropine pretreatment greatly reduced the airway resistance effects of all three test agents, thus suggesting some cholinergic component to their potent bronchoconstrictor activities. These cyclic ethers are stable forms of prostaglandin endoperoxides and may represent invaluable pharmacologic tools for the further study of intermediates in prostaglandin synthesis.


Comparative effects of four migraine prophylactic drugs on an isolated extracranial artery. Clonidine and methysergide constrict the rabbit auricular artery by activating smooth muscle alpha-adrenoceptors. Clonidine inhibits and methysergide enhances responses to stimulation of the sympathetic nerves. Both drugs sensitise the artery to a variety of vasoconstrictor stimuli, although not to potassium chloride. This weak generalised sensitisation may depend on clonidine and methysergide themselves being vasoconstrictor since sensitisation after clonidine did not occur when vasoconstriction was abolished with phentolamine. The marked potentiation of 5-HT and tryptamine observed during clonidine perfusion may reflect a property of the tryptamines since it was observed during perfusion with histamine but not when histamine vasoconstriction was abolished with mepyramine. Cyproheptadine and pizotifen neither constrict the artery nor sensitise it to vasoconstrictor agents. They inhibit responses to nerve stimulation, alpha-adrenoceptor agonists, potassium chloride and particularly histamine. Sensitisation of blood vessels reinforced by direct vasoconstriction may contribute to the mechanism of action of clonidine and methysergide in migraine. Conversely, alterations in vascular function may be less important to the antimigraine actions of either cyproheptadine or pizotifen.


Antiarrhythmic and hemodynamic effects of an aminosteroid (Org 3001) in the digitalized dog. Org 6001 (3alpha-amino-5alpha-androstan-2beta-ol-17-one-hydrochloride) is an orally non-hormonal aminosteroid possessing antiarrhythmic activity. In 13 dogs the efficacy of the drug against ouabain-induced ventricular tachycardia (VT) was studied. VT was produced by a mean dose of 67.5 +/- 18.7 mug/kg ouabain, administered by continuous infusion of 25 mug/min. 20 min after the onset of VT an 0.125 mg/kg/min infusion of Org 6001 was initiated, doubling the dose every 10 min. In all dogs VT was reverted into normal sinus rhythm (NSR) by a dose of 9.72 +/- 7.07 mg/kg (0.87-20.75 mg/kg) Org 6001. The duration of VT ranged from 27-61 min (mean 47.1 +/- 11.4 min), including the 20 min waiting period. NSR persisted in 8 dogs until the experiment was terminated (90 min after onset of VT), while in 5 dogs VT returned after 3-23 min sufficient to revert VT into NSR. A bolus injection of Org 6001 (10 mg/kg) gave an immediate return to NSR in 3 dogs, in which VT was provoked again by administration of a second dose of ouabain after the 90 min period had elapsed. Though the interaction of ouabain makes a quantitative analysis of the negative inotropic effects difficult, it appeared that there uas no major hemodynamic deterioration during and after treatment with Org 6001. During digitalization there was a significant increase in the first derivative of left ventricular systolic pressure (peak LVdP/dt) from 2340 +/- 600 to 3650 +/- 1070 mm Hg/sec and in peripheral resistance, while heart rate decreased. During VT, left ventricular systolic pressure (LVSP) and mean aortic pressure (MAP) dropped by approximately 20 mm Hg, while heart rate increased significantly. After treatment with Org 6001, LVSP and MAP further decreased to 128 +/- 30 mm Hg (p less than 0.05) areased to 128 +/- 30 mm Hg (p less than 0.05) and 112 +/- 20 mm Hg respectively. Peak LVdP/dt fell from 3650 +/- 1390 to 2780 +/- 970 mm Hg/sec (p less than 0.05). Heart rate had dropped to 126 +/- 22 beats/min (p less than 0.05). During the first 30 min after Org 6001 infusion was stopped none of the parameters showed significant changes, although peak LVdP/dt rose slightly. It is shown in the present investigation that Org 6001 has effective antiarrhythmic properties in controlling ouabain-induced VT with acceptable cardiodepressant actions.


Time-course effects of oral guanethidine administration on cardiovascular and autonomic effects on dogs. The time-course effects of guanethidine (2.5 mb/kg/day, p.o.) administered for 2 days, 7 days and 7-8 months were investigated in dogs. Guanethidine treatment for 2 days failed to produce any significant alterations in sympathetic neuronal activity to the myocardium, resting neurogenic tone in the perfused hind limb vasculature and lumbar sympathetic neuronal transmission. Following guanethidine administration for a peroid of 7 days both cardiac and lumbar sympathetic neuronal activity was markedly and significantly depressed with concomitant reduction in heart rate and resting neurogenic tone to the hind limb. However, continued treatment with guanethidine for a period of 7-8 months resulted in complete restoration of cardiac as well as lumbar sympathetic neuronal activity. Both heart rate and resting neurogenic tone were also returned to placebo levels. The development of tolerance of these sympathetic nerves to guanethidine treatment was not associated with any alterations in the activity of adrenergic receptors. Further, there was a significant and time-dependent increase in cholinergic vasocilator activity in the lumbar sympathetic chain which could account for the exertional hypotension noted in the patients receiving guanethidine treatment.


Pharmacological comparison of R(+), S(-) and racemic thiopentone in mice. The i.p. LD50's of the enantiomorphs of thiopentone have the following increasing order of lethality R(+), racemate and S(-). Whereas the racemate and the S(-) compound have similar therapeutic indices, the R(+) compound has the highest value. The S(-) isomer is the most potent anesthetic agent, followed by the R(+) and the racemate. In the ability to block pentylenetetrazol- and strychnine-induced seizures, the compounds are ranked in the following order of decreased potency: S(-), racemate and R(+). The differences in potency between antipodes have been related to absolute steric configuration of the molecules. Differences in potency of the stereoisomers have been discussed in relationship to known metabolic conversions of thiopentone and its rapid penetration into body fat depots.


Frequency dependence of the alpha-adrenoceptor-mediated positive inotropic effect in guinea pig heart. Evidence for the role of alpha-adrenoceptors in the production of a positive inotropic effect has been obtained chiefly in heart preparations stimulated at a low rate (1 or 1.4 Hz). However, our previous results suggested that the alpha-receptor-mediated positive inotropic effect could be frequency dependent. In the present study cumulative concentration-inotropic effect curves were obtained in guinea pig ventricle strips treated with adrenaline and stimulated at 1 and at 2.5 Hz. Phentolamine (3 X 10(-6) M) and practolol (10(-6) M) were employed as antagonists. Phentolamine antagonized the effect of low concentrations of adrenaline (10(-9)-10(-8) M) in preparations driven at 1 Hz, but did not modify the curve of the agonist at the higher stimulation rate. In preparations driven at 1 Hz, practolol antagonized the effect of adrenaline at concentration above 3 X 10(-8) M without affecting the curve at lower concentrations, the beta-blocker competitively anatagonized the effect of the agonist at 2.5 Hz. It was concluded that a shortening of the interval between beats abolishes the alpha-mediated positive inotropic effect.


Study of the laxative properties of the disodium salt of the sulfuric diester of 3,3-bis-(4-hydroxyphenyl)-7-methyl-2-indolinone (DAN-603 in the rat. The influence of DAN-603 (disodium salt of sulphuric diester of 3,3-bis-(4-hydroxyphenyl)-7-methyl-2-indolinone) on the propulsive motility of the rat digestive tract was studied by means of indicators (charcoal and pyrvinium pamoate) and radioactive tracers (133BaSO4). The results showed that DAN-603 increases selectively the colon motility without modifying the speed of gastric, intestinal (small intestine) and caecal emptying.


Butyrophenone influences on the opiate receptor. Interaction of neuroleptic drugs with the opiate receptors was investigated by inhibition of the stereospecific binding of 3H-naloxone. Benperidol and pimozide, with IC50's of 0.3-0.5 muM, were more potent than the classical opiates meperidine and propoxyphene. A systematic structure-activity relationship was evident with the basic opiate structure of a benzene and a piperidine ring preserved in active compounds. No correlation between neuroleptic activity and binding to the opiate receptor was demonstrated.