Hormonal control of bone collagen synthesis in vitro: effects of parathyroid hormone and calcitonin. The effects of parathyroid hormone (PTH) on bone collagen synthesis were assessed in organ cultures of fetal rat calvaria by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and non-collagen protein (NCP) using purified bacterial collagenase. 1) PTH decreased the incorporation of labeled proline into CDP at concentrations similar to those which stimulate bone resorption in vitro. 2) This effect was observed in bones treated for 6 h, but not for 3 h; it was maximal at 24 h and was maintained for 96 h. Bones treated with PTH for 48 h and transferred to control media for 48 h showed recovery of CDP labeling to control values. 3) the effect was specific for bone collagen. There was little alteration in the incorporation of proline into NCP, and incorporation into collagen was not inhibited. 4) The effect could be ascribed to decreased collagen synthesis and not to changes in amino acid uptake, precursor pool size, or degradation of newly synthesized CDP. In 3 hour experiments, PTH did increase the labeling of CDP and NCP, but only at tracer concentration of proline in the medium, compatible with an early stimulation of amino acid uptake. 5) Similar inhibition was observed with purified bovine (1-84) PTH and synthetic bovine PTH (1-34) as well as with crude homologous PTH obtained from rat parathyroid gland culture fluid. Human (hCT) and salmon (sCT) calcitonin did not inhibit the effect of PTH on the labeling of CDP nor did they stimulate CDP labeling directly at concentrations which inhibited bone resorption. Dibutyryl cyclic-3',5'-adenosine monophosphate (D3cAMP) inhibited labeling of CDP at concentrations of .03 to .3 mM, thus mimicking the action of PTH. However, in this system DBcAMP inhibited 45Ca release, thus mimicking CT. We conclude that the direct effect of PTH on bone collagen synthesis is a slow reversible inhibition, not opposed by CT. This effect may be mediated by cAMP formation in bone cells.


Intracellular localization of calcium in the chromaffin cells of the rat adrenal medulla. The ulstrastructural localization of calcium in the chromaffin cells of the adrenal medulla was carried out by using potassium pyroantimonate during fixation. Calcium-containing deposits were either diffuse within the cytoplasm or associated with membrane-bounded organelles. Variable amounts of precipitates were found within the nucleus and in the Golgi complex. However, the major sites of calcium antimonate deposits were the secretory granules and the mitochondria. The morphological identification of calcium-storing organelles in the adreno-medullary cells may be useful in evaluating the involvement of such intracellular compartments during the secretory process.


Pulsatile release of LH and secretion of ovarian steroids in sheep during the luteal phase of the estrous cycle. The concentration of LH and progesterone in jugular venous plasma and the secretion of steroids by the ovary were measured every 10 minutes for 2 hours on days 12, 14, and 16 of the estrous cycle in 5 ewes with utero-ovarian autotransplants. A pulse of LH occurred about once every 2 hours, when the concentration rose from a basal level of 0.57 +/- 0.08 ng/ml to a peak of 2.97 +/- 0.57 ng/ml. Within 5 minutes of the pulse of LH, the secretion of estradiol (an exclusive product of the follicle) rose rapidly from a basal level of 0.75 +/- 0.12 ng/min to reach a peak value of 2.16 +/- 0.33 ng/min in about 30 minutes. In contrast, the secretion of progesterone from the corpus luteum, and the concentration of progesterone in the peripheral plasma changed very little following the pulse of LH. The secretion of androstenedione, which arises from the follicle and corpus luteum, increased from 3.03 +/-0.75 ng/min to 7.85 +/- 1.78 ng/min by 30 minutes after the pulse of LH. These findings indicate that the follicle, and possibly the stroma, respond rapidly to episodic fluctuations in the concentration of LH and are probably involved in the negative feedback loop between the ovary and the hypothalamic pituitary system. The fluctuations in the secretion of progesterone from the corpus luteum, on the other hand, are unrelated to pulses of LH.


Twenty-four-hour resting prolactin levels in male rats: the effect of septal lesions and order of sacrifice. Diurnal variation in resting prolactin levels was studied in male rats that were normal, sham-operated, or lesioned in the septal nuclei. Separate groups of rats were sampled by decapitation every 3 or 4 hours in two separate studies during a 24 h period consisting of 12 h light and 12 h dark. Male rats did not exhibit a significant diurnal variation in resting prolactin levels. A septal lesion had no effect on resting prolactin levels. Prolactin levels were found to be highly responsive to environmental disturbances and were subject to the effects of sequential sacrificing procedures. The possibility was raised that prolactin exhibits a diurnal variation in sensitivity to stress which varies as a function of circulating levels of corticosterone.


The diurnal rhythm of plasma aldosterone, plasma renin activity, plasma cortisol and serum growth hormone and subnormal responsiveness of aldosterone to angiotensin-II in the patients with normotensive acromegaly. The diurnal rhythm of plasma aldosterone concentration (PA), plasma renin activity (PRA), plasma cortisol (PC) and serum growth hormone (GH) were examined in 5 cases of normotensive acromegaly and the results were compared with the observations in normal subjects. Moreover, the response of PA to angiotensin-II infusion was studied in 6 cases of normotensive acromegaly. A normal diurnal rhythm with the lowest values in the evening or midnight and the highest values in the morning was observed in 3 of 5 cases in PA and 3 of 4 cases in PC. On the other hand, no apparent rhythm of GH was observed in any cases and that of PRA in 4 of 5 cases. Although there was a significant positive correlation between PA and PC, no significant correlation was demonstrated between PA and PRA. The response of PA to angiotensin-II fusion was significantly suppressed in normotensive acromegaly as compared to the normal subjects in spite of normal levels of PRA except for 1 case. The above observations were interpreted to suggest that the aldosterone regulation system is slightly altered in a certain number of patients with normotensive acromegaly in contrast to the normal subjects in which PRA is the main contributing factor. The low PA and suppressed response of PA toangiotensin-II infusion may suggest the defective action of angiotensin-II infusion on the adrenal gland.


Secretion of gastrin and calcitonin after ingestion of meat extract in patients with peptic ulcer. Serum gastrin and calcitonin were determined after administration of meat extract to patients with peptic ulcer in order to investigate what role gastrin and calcitionin are playing in peptic ulcer. Serum gastrin levels were elevated after ingestion of meat extract in 9 out of 11 patients with peptic ulcer. Serum calcitonin (CT) levels were elevated rather gradually in 7 subjects. It was suggested that increased endogenous gastrin levels may have stimulated the release of CT. When salmon calcitonin (SCT) was admininstered intravenously at a dose of 100 MRCu before administration of meat extract, the rise of gastrin and CT was suppressed in 67% of cases as to gastrin and in 43% as to CT.


Inhibition of transfer ribonucleic acid nucleotidyl transferase (EC 2.7.7.25) from Ehrlich tumor cells by proflavine sulfate and ethidium bromide. Transfer RNA nucleotidyl transferase from Ehrlich tumor cells was inhibited by proflavine sulfate and ethidium bromide but not by actinomycin D, chromomycin, rifamycin SV, rifampin, daunomycin, or alpha-amanitin. Complete inhibition of nucleotidyl transferase activity was attained at a final concentration of 1 mmol/1 proflavine sulfate, while 2 mmol/1 ethidium bromide was required to completely inhibit the enzyme. CMP incorporation into tRNA was more sensitive to both proflavine sulfate and ethidium bromide than was AMP incorporation.


Haptoglobin--haemoglobin interaction. Heterogeneity of the haptoglobin 1-1 molecule in its binding affinity for horse R/E-hemoglobin. The formation of two different complexes when haptoglobin (Hp) and haemoglobin (Hb) are mixed in a 1:1 molar ratio is demonstrated by isoelectrofocusing. In these two complexes, the affinity of Hp for Hb is shown to be different, since Hb can be displaced only from one of the complexes, by a further addition of Hp. This is confirmed by a quantitative study of the reaction stoichiometry, when [Hp]/[Hb] = 1 and [Hp]/[Hb] greater than 1, which allows an evaluation of the amount of each complex formed. All these data cannot be explained other than by the existence of two forms of Hp molecule and a reaction scheme which fits these experiments is proposed.


On the heterogeneity of native ribosomal subunits in Ehrlich-ascites-tumor cells cultured in vitro. Native small ribosomal subunits in cultured Ehrlich ascites tumor cells, analyzed by high-resolution CsCl isopycnic centrifugation, consist of at least five classes of particles. These particles with buoyant densities of 1.39, 1.42, 1.45, and 1.51 g/cm3 were designated as SI, SII, SIII, SIV and SV, respectively. They were different from the ribosome-derived 40-S subunits which have a density of 1.54 g/cm3. Native large ribosomal subunits consist of at least two classes of particles with densities of 1.57 g/cm3 (LI) and 1.59 g/cm3 (LII), respectively. The ribosome-derived 60-S subunits have the same buoyant density as LII particles. Labeling of Ehrlich ascites cells with radioactive uridine and a subsequent chase in the presence of RNA-synthesis inhibitors shows that radioactivity was incorporated first in precursor particles with a density of 1.48 g/cm3 and then subsequently appeared in SIII and SII particles. Met-tRNAf is found exclusively on native 40-S particles with densities of 1.42 and 1.49 g/cm3. This result has been observed in cells labeled with [35S] methionine in vivo as well as with tRNA charged in vitro. The possibility that SII particles contain 40-S initiation complexes is discussed.


Uptake and metabolism of fatty acids and analogues by cultured cardiac cells from chick embryo. Cultured heart cells from chick embryo, present as a confluent monolayer in a minimal medium, take up labelled fatty acids (from C6 to C22) bound to serum albumin with a rapid incorporation into neutral and complex lipids (visible at 15 s), while beta-oxidation is delayed (visible at 15 min). Fatty acids enter the cardiac cells through two mechanisms, one involving a readily saturable process, the other resembling passive diffusion. The saturable transport system recognizes fatty acids with a high affinity, Km from 4.5 to 16 muM. The V is from 0.03 to 0.3 nmol of fatty acid incorporated min-1 (10(6) cells)-1. Competitive inhibition occurs between fatty acids while glucose, aminoacids and lactate do not compete with palmitate for the entry into the cells. The analysis of unesterified fatty acids from the usual culture medium and from the cells show a gradient of concentration up to 54-fold; moreover experiments performed in minimal medium at 20 degrees C show that fatty acids accumulate up to 28-fold in the cells. Efflux rates can be measured after loading the cells at 20 degrees C; the curves are bimodal and the pseudo first-order rate constants thus determined are in the order of 0.5 min-1, as opposed to 2.5 to 5.0 min-1 for the passive-diffusion component calculated for the influx rates. Studies on the role of serum albumin at constant and at variable fatty acid-albumin molar ratios indicate that the rate of uptake is controlled by the total fatty acid concentration and not solely by the concentration of unbound fatty acids. No labelled fatty acid was found to be bound to the cytoplasmic fraction supposed to contain the fatty acids-binding protein; our results are in favour of the presence of a specific transport system for fatty acids either at the level of subcellular organelles or more likely at the surface of cardiac cells.


Histone-gene reiteration in the genome of mouse. Histone messenger RNA labelled to high specific activity has been isolated and purified from the mouse tissue-culture cell line L929. It had an electrophoretic mobility equivalent to a mean molecular weight of 1.4 X 10(5). The synthesis of this RNA was suppressed, but apparently not completely so, by inhibition of cellular DNA synthesis to more than 95% by addition of cytosine arabinoside. The synthesis of other RNA species remained unaffected by this treatment. Poly(A)-containing mRNA, representing a 20% contaminant of the electrophoretically purified histone mRNA, was removed by poly(U)-Sepharose chromatography. Histone mRNA thus purified was hybridized to DNA fractions enriched for histone genes of the sea urchin Psammechinus miliaris by actinomycin/CsCl gradient centrifugation. The mRNA was eluted from the hybrids and challenged with mouse DNA in vast DNA excess conditions. A (cot1/2) of 360 mol s 1(-1) was obtained from the RNA trace curve, suggesting a 10-20-fold reiteration of the histone genes in the haploid genome. Thus histone genes in mouse are much less highly reiterated than in sea urchins, but nevertheless are present in considerable excess over the number theoretically necessary for histone synthesis during the S phase of the cell cycle.


Binding affinities of retinol and related compounds to retinol binding proteins. Fluorimetric titrations were used to determine apparent dissociation constants of the all-trans isomers of retinol, retinoic acid, retinyl acetate and retinyl palmitate to human-retinol binding protein and chicken-retinol binding protein. Enhancement of the fluorescence of retinol and retinyl acetate when bound to the protein was utilized to establish the binding affinity of these compounds. With retinoic acid which is essentially a non-fluorescent compound, quenching of protein fluorescence due to energy transfer to the bound ligand from tryptophanyl residues served to determine the binding affinity. The various ligands display 1:1 molecular complexes with both types of retinol binding proteins. Retinol, retinoic acid and retinyl acetate were found to have similar binding affinities to both species of carrier proteins: For retinol K'd=1.9 X 10(-7) M with human-retinol binding protein and K'd=1.5 X 10(-7) M with chicken-retinol binding protein; for retinoic acid K'd-2.1 X 10(-7) M with human-retinol binding protein; for retinyl acetate and K'd=2.2 X 10(-7) M with chicken-retinol binding protein; for retinyl acetate K'd=2.2 X 10(-7) M with human-retinol binding protein and K'd=1.7 X 10(-7) M with chicken-retinol binding protein. Retinyl palmitate appeared to have weak association with either of the two retinol binding proteins, if at all. The above results suggest that both human and chicken retinol binding proteins behave similar with respect to the binding of the ligands. Non-polar interactions probably play a primary role in the binding and effects of functional groups and charges are of secondary importance.


The influence of hypertrophy on myocardial function. In patients with hypertrophy from long-standing left ventricle pressure (PL) and volume overload (VL) as well as in a group of controls basal left ventricular contractility was assessed by peak measured isovolumic velocity of shortening (Vpm), mean velocity of circumferential fiber shortening (mean VCF) and mean normalized systolic ejection rate (MNSER). The angiographically determined left ventricular muscle mass was elevated to a similar extent in PL and VL. As compared to the control group both hypertrophy had decreased contractile indexes. No significant differences of contractility existed between the groups with PL and VL. Hence it appears that in chronic mechanical overloading the extent of hypertrophy is probably more important for the decrease of contractility than the nature of the stimulis to hypertrophy. In 15 patients with aortic stenosis left ventricular muscle mass and isovolumic and ejection phase contractile indexes were determined preoperatively and 13.5 months after successful valve replacement by a tilting disc prosthesis. Postoperatively there was a significant (P less than 0.001) decrease of muscle mass from 182 to 114 g/m2. Mean VCF increased from 1.07 to 1.52 circ/sec (P less than 0.01); MNSER from 1.92 to 2.59 enddiastolic volumes/sec (P less than 0.01); Vpm from 1.17 to 1.60 ML/sec (P less than 0.001) and total pressure Vmax from 1.61 to 2.09 ML/sec (P less than 0.01). In 9 of 13 patients an abnormal or pathologic response to handgrip at the preoperative study became normal postoperatively. It is concluded that in aortic stenosis removal of the chronic pressure burden leads to regression of left ventricular hypertrophy associated with an improvement of resting and exercise contractile function. Pressure-deprived contractile indexes; ejection phase contractile indexes; chronic pressure load; chronic volume load; aortic valve replacement; postoperative hemodynamics.


Effects of acute and chronic methadone treatment of the uptake of 3H-5-hydroxytryptamine in rat hypothalamus slices. Effects of in vitro methadone and several other narcotics were investigated on 3H-5-HT uptake in rat hypothalamus slices. The results indicated that d, l-methadone and levorphanol had slightly greater inhibitory action on the uptake than the isomers, d-methadone and dextrorphan, respectively. Morphine, diacetylmorphine and the narcotic antagonist naloxone produced a considerably weaker inhibitory effect. After an acute injection of methadone, but not morphine, the uptake of 5-HT by hypothalamic slices of treated animals was inhibited. The chronic treatment of rats with methadone for 18 days had no significant effect on the uptake, but following the withdrawal of this treatment for 2 weeks the 5-HT uptake was significantly elevated. The inhibitory effects of in vitro methadone in the hypothalamus slices were not modified by the chronic drug treatment.


Direct dopaminergic action of lisuride hydrogen maleate, an ergot derivative, in mice. Lisuride hydrogen maleate induced stereotyped behaviour in normal as well as in reserpinized mice. It antagonized the motor depression and hypothermia induced by reserpine. On i.p. administration the compound was about as effective as apomorphine and D-amphetamine. As with apomorphine and in contrast to D-amphetamine the effects of lisuride hydrogen maleate in reserpinized mice were not impaired by additional treatment with alpha-methyl-p-tyrosine methylester. In untreated mice, the substance was very potent in lowering body temperature with significant hypothermia measured after dosages as low as 0.10 mg/kg i.p. Occurrence of stereotyped behaviour and hypothermia could be prevented by the dopaminergic antagonist haloperidol. From these data it is concluded that lisuride hydrogen maleate in addition to its interaction with serotoninergic systems is a potent dopaminergic agonist with a probably direct action on dopaminergic receptors. Further arguments in support of such an action of lisuride hydrogen maleate are, in addition to biochemical data, its serum prolactin lowering effect in rats, its strong emetic action in dogs and its effects on rat behaviour.


Behavioural effects of a new non-phenylethylamine anorexigenic agent: mazindol. Mazindol, a new anorexigenic agent which possesses a different chemical structure from phenylethylamine derivatives such as amphetamine, causes anorexia along with increases in locomotor activity and body temperature. Mazindol also induces stereotyped behaviour and, if injected into rats with unilateral nigro-striatal lesions, causes turning towards the lesioned side. Mazindol-induced anorexia is antagonized by pretreatment with alpha-methyl-p-tyrosine or pimozide. Pimozide pretreatment prevents the rotation induced by Mazindol in rats with unilateral nigro-striatal lesions. The involvement of dopamine in the mechanism whereby Mazindol elicits anorexia and turning behaviour is discussed.


Dissociation of stereotyped biting responses and oro-bucco-lingual dyskinesias. A comparison was made of the doses of neuroleptic and related agents required to inhibit the stereotyped biting/gnawing/licking response induced in the guinea pig by systemically administered apomorphine and d-amphetamine or by dopamine administered bilaterally into the striatum. Haloperidol. lenperone, fluphenazine (0.5-8 mg/kg i.p.) and fluspirilene (0.125-8 mg/kg i.p.) each inhibited the stereotyped behaviour induced by apomorphine and amphetamine but doses up to 16 mg/kg i.p. failed to modify the dyskinesias induced by intrastriatal dopamine. Pimozide similarly abolished the stereotypies (0.125-8 mg/kg i.p.) but a larger dose (16 mg/kg i.p.) also abolished the dopamine-induced dyskinesias. However, oxiperomide and spiroxatrine were both shown to possess marked anti-dyskinetic (1-2 mg/kg i.p.) as well as anti-stereotypic (0.25-5 mg/kg i.p.) properties. Thioridazine, clothiapine, clozapine, sulpiride and metoclopramide were generally inactive against the stereotypies and dyskinesias although 1.25-5 mg/kg s.c. morphine effectively abolished stereotyped behaviour and the dopamine-induced dyskinesias were inhibited in a small proportion of animals. It is suggested that the dopamine mechanisms involved with stereotypy induction differ from those activated by intrastriatally administered dopamine to induce abnormal oro-facial movements, and that, since only the effects of intrastriatal dopamine showed the same relative degree of resistance to neuroleptic inhibition as clinical dyskinesias, that this may be more applicable to the clinical situation than the stereotypy model.


Antagonism by sulpiride of three apomorphine-induced effects in rodents. Three effects of apomorphine (hypothermia and climbing behavior in mice, stereotyped behavior in rats) were studied. Sulpiride antagonized the two effects in mice but stereotyped behavior in rats remained unchanged. Pimozide and haloperidol antagonized the three effects. These results could be explained by the existence of two types of dopaminergic receptors or by the different accessibility to identical dopaminergic receptors located in different CNS areas.


Alpha-flupenthixol-induced hyperactivity by chronic dosing in rats. Socially reared and isolation-reared rats treated chronically since weaning with alpha-flupenthixol showed elevated levels of spontaneous locomotor activity compared with control treated rats. However, chronic apomorphine treatment had no effect on spontaneous locomotor activity. Chronic alpha-flupenthixol treatment enhanced stereotyped behaviour after 1.5 mg/kg d-amphetamine or 0.5 mg/kg apomorphine in the socially reared condition. 'Spontaneous stereotypies' were also observed in the chronic alpha-flupenthixol-treated animals. Chronic apomorphine treatment did not affect stereotyped responding. The results are discussed in terms of 'behavioural supersensitivity'.


[Fatal poisoning by water dropwort (Oenanthe crocata)]. The authors relate clinical and toxicological data concerning a recent deadly intoxication by OEnanthe crocata absorption and stress upon awkwardness of clinical diagnosis. Most frequent cases were observed by veterinarians in bovine and porcine intoxications. This plant grows in wet meadows of western France and south-west of England. In spite of its rarity, this intoxication is mostly dangerous and oftenest lethal.


The effects of ventricular fluid osmolality on bulk flow of nascent fluid into the cerebral ventricles of cats. The effects of ventricular fluid osmolality on the bulk flow of nascent fluid into the cerebral ventricles of anesthetized cats was measured during ventriculocisternal perfusion. This nascent fluid consists of both cerebrospinal fluid (CSF) and fluid which results from an osmotic gradient between ventricular fluid and the blood and/or brain. Perfusions were carried out with both mock CSF and with solutions containing either sucrose, urea, or NaCl. Differences between the normal bulk flow rate of nascent CSF and bulk flow rate measured during perfusion with anisotonic solutions were linearly related to corresponding differences in osmolality of the effluent fluid from the ventricles. The coefficients of somotic flow using sucrose (0.231 mul/min per mOsm) and NaCl (0.224) were similar, and greater than that using urea (0.156). During perfusion with sucrose when effluent osmolality increased by 200 mOsm (63% of normal), bulk flow rate of nascent fluid increased by 50 mul/min (200% of normal). Flow was undetectable when the effluent osmolality was 190 mOsm (decrease of 135 mOsm), although osmotically active particles continued to enter the ventricular system. Intravenous injection of acetazolamide reduced these coefficients to similar values of 0.0963 for NaCl, and 0.0955 for urea. In all experimental conditions no changes were found in cerebral water content. These results suggest that the increased bulk flow which occurs during perfusion with hypertonic solutions originates from the choroid plexus.


Effect of alcohol ingestion on the epithelial cell population in rat small intestine. Chronic administration of alcohol to well-nourished rats led to striking changes in the small intestinal cell population. The present experiments corroborate the view that alcohol is directly toxic to the small intestine.


Copper utilization during embryogenesis of Palaemon lamarrei. The pattern of copper utilization during the embryogenesis of the freshwater prawn, Palaemon lamarrei has been described. Throughout the incubation period, lasting for 14 to 16 days, the egg of P. lamarrei is permeable to salts and against a concentration gradient, it absorbs 11 mug of salt from the freshwater medium. Out of this total salt uptake, as much as 0.143 mug is due to the absorption of copper. Intake of copper, as a function of incubation time, exhibit a more or less similar trend to that of total salt intake and this can be attributed to the increased synthesis of haemocyanin.


Is feminine differentiation of the brain hormonally determined? The androgen insensitive, genetically male rat pseudohermaphrodite displays neither masculine or feminine sexual behavior when primed with the appropriate sex hormones. Although in the absence of androgen imprinting the animal develops anatomically as female, our results suggest that feminine differentiation of the brain requires active imprinting by perinatal hormone(s), possibly adrenal progesterone.


Man's strategy in domestication - a synthesis of new research trends. The minimum brain size possible in the relevant wild species and certain colour types which, because of alterations in the neurotransmitter system caused by the respective colour genes, are related to behavioural traits diverging from the wild animals norm appear to be first-rate bases for domestication either separately or in combination.


[4-aminobutyrophenones with hypotensive activity]. A series of new butyrophenones was synthesized, the aim being to reduce neuroleptic activity and enhance the hypotensive effects of this class of drugs. The compounds were screened for toxicity according to the Irwin scheme and tested in anaesthetized cats for their effects on systemic arterial pressure. The most interesting compound, i.e. 1-(4'-fluorobenzoyl)-3-pyrrolidinyl-propane, was tested also in anaesthetized rabbits, cats, and dogs and in concious dogs. Moreover its central nervous system effects were tested in rats and mice. The compound proved practically devoid of neurological effects and showed interesting hypotensive activity.


[Immunodepressive activity of phenobarbital chemically bound to the protein carrier]. Experiments conducted on mice immunized with sheep's erythrocytes proved that phenobarbital, even in toxic doses does not produce any immunodepressive effect. But when it is covalently bound by the diazo-method with a protein-carrier phenobarbital becomes a strong immunodepressant. Then, however, it does not affect the central nervous system, nor influence the immunodepressive activity of cyclophosphamide--an agent metabolically activated in the liver. Free phenobarbital mitigates the effect of cyclophosphamide on the immunological response in mice.


Effect of anti-luteinizing hormone antibodies on the gonadotropic cells of the pituitary in immature male rats: an electron microscopic study. Treatment of immature rats with antigonadotropic serum results in atrophy of gonads and castration-like changes of gonadotroph cells in the pituitary gland. Newborn male albino Wistar rats were daily and subcutaneously inoculated with 0.1 to 0.8 ml of anti-luteinizing hormone (LH) sera from the first 24 hours of age, through the first 5 weeks. For controls, a similar schedule with normal rabbit serum was used. Rats were killed at weekly intervals and pituitaries were fixed for electron microscopy. The anti-LH titer, determined by microhemagglutination, was between 1:2, 560 and 1:10,240; the greatest contaminant was follicle-stimulating hormone, with the antiserum ranging from 1:160 to 1:320. Absorbed antisera, checked by double agar diffusion against unabsorbed antisera, showed no precipitin lines, indicating no antigen excess. There was no significant cytologic difference between control and experimental pituitaries during the 1st week of treatment. At the end of the 2nd week, gonadotroph cells contained a highly developed Golgi complex; by the 3rd week these cells contained pronounced, dilated, saclike ergastoplasmic cisternae, with few secretory granules in the cytoplasm of one type; by the 4th week, ergastoplasmic cisternae had broken down to form the first large vacuoles of "castration" LH gonadotrophs, some of which contained typical granules. Numerous mitotic and chromophobe cells were also observed. At the end of the 5th week castration LH cells persisted, in addition to mitotic cells, chromophobes, and numerous follicle-stimulating hormone gonadotrophs with degenerated features. The effect of anti-LH antisera on pituitary gonadotrophs could be explained as a neutralization of endogenous LH, probably not only inhibiting the long feedback mechanism, but also the short feedback loop at the level of the same pituitary cells.


Studies on sperm antigenicity, 5. In vivo and in vitro cellular reactivity in guinea pigs sensitized with fractions of human spermatozoa. In vitro delayed type hypersensitivity was demonstrated with peritoneal exudate cells from guinea pigs immunized with different preparations of human and guinea pig seminal components emulsified with complete Freund's adjuvant-H37Ra. The seminal components were intact human spermatozoa (HuSp); intact guinea pig spermatozoa (GPSp); human seminal plasma (HuSePlFr); and fractions of human spermatozoa obtained by centrifugation of the homogenate at 5,000 X g (5S30 and 5p30), at 20,000 X g (20S30 and 20p30), and at 144,000 X g (144p120). Cellular sensitivity was demonstrated in vivo by skin testing and in vitro by the macrophage inhibition technique. Peritoneal exudate cells from guinea pigs sensitized with fractions 5p30 and 20p30 elicited a delayed hypersensitivity reaction which could be detected only with intact human spermatozoa. Other human spermatozoal fractions (5S30, 20S30, and 144p120) were weak immunogens. Sensitization of guinea pigs with fractions of human spermatozoa, in addition to causing delayed hypersensitivity reactions, elicited low titers of spermatoxic antibodies. Antibodies to human spermatozoal fractions 5S30, 5p30, 20S30, and 20p30 cross-reacted with intact human spermatozoa and intact guinea pig spermatozoa. It is postulated that the existence of "spermatozoa-specific" coating antigen(s) derived from other components of the reproductive tract might be responsible for human spermatozoal antigenicity.


Testicular response to exogenous gonadotropins in the syndrome of feminizing testes. The steroid response of testes to exogenous gonadotropin administration was studied before castration in a patient with the complete form of the testicular feminization syndrome. Urinary steroid determination were made along with measurements of plasma testosterone during adrenal suppression with dexamethasone and after stimulation with human menopausal gonadotropin (HMG) and HMG plus human chorionic gonadotropin (HCG). Our data provide evidence that the gonads in the testicular feminization syndrome synthesize steroids normally. Estrogens and testosterone are secreted by the testes, and the Leydig cells of the testes are capable of responding to exogenous HMG and HCG stimulation. These findins are consistent with the hypothesis that there is no abnormal testicular steroidogenesis in these genotypic males. Additional evidence is provided that these gonads secrete testosterone in amounts comparable to those of normally functioning testes. Failure of virilization in the presence of androgen secretion as indicated by previous authors is probably the result of a deficiency of androgenic action at the end organs. The presence of sparse pubic and axillary hair in the mother and the finding of the same type of karyotype (46,XY) in a sibling suggest that hereditary factors play a role in this disorder.


[The importance of an additional retropubic urethroply by the pereyra or the Marshall-Marchetti-Krantz method in the primary operative treatment of stress incontinence involving rotational descent of the urethra (author's transl)]. Between 1971 and 1974, 270 patients underwent a hysterectomy and anterior colporrhaphy for severe stress incontinence of Type Green II with loss of the posterior urethrovesical angle and rotational descent of the urethra. All these patients had an additional retropubic urethropoly either of the Pereyra of Marshall-Marchetti type or an additional sling operation. 176 patients were followed up. The total series showed a cure rate of 57%, an improvement rate of 28% and persistent or recurrent incontinence in 14.3% of the cases. The importance of pre-operative diagnosis by cystometry and sphincterometry is stressed. An irritable bladder is emphasized as a contraindication to an operation for stress incontinence. The conclusion is reached that the primary stress incontinence operation is the most decisive and the most likely to succeed. Operations for recurrent stress incontinence show a much lower cure rate. Since the anterior colporrhaphy according to Stoeckel or Kelly is not capable of curing severe forms of stress incontinence with rotational descent of the urethra, our results show that an additional retropubic urethropoly is desirable and justified in these cases.


[Induction of second trimester abortion by infusion of intraamniotic hypertonic and extraamniotic physiological saline solution (author's transl)]. In a group of 84 women in the second trimester of pregnancy abortion was induced by intramniotic transabdominal instillation of 20 per cent NaCl. In a second group of 91 women the abortion was induced by means of extraamniotic physiological infusion of saline solution. The only complication observed in the first group was an increasing fever. In the second group there were better results. The fetus abortion was complete and in a shorter time. We assume that the new method is the method of choice because it gives no complications and may be easily performed. It may be used also in cases of missed abortion or intrauterine fetal death.


Conversion of adiasporic and mycelial stages of Emmonsia crescens examined in the scanning electron microscope. Conversion of adiasporic and mycelial stages of the fungus Emmonsia crescens, the causative agent of adiaspiromycosis, was observed by a scanning electron microscope. Multilocular germination of adiaspores and their surface structures are described.


[Studies on medico-legal diagnosis in cold district. 1. On the production of ethylalcohol in the outdoor cadavers in winter (author's transl)]. Some diagnostic problems in legal medicine were studied in dog cadavers left outdoors in the snow during winter in Sapporo. It was found that the cadaveric blood did not deteriorate till the end of 7 weeks and ethylalcohol or other Widmark positive substances were not produced in the cadavers during this period.


[Some evaluations of the Widmark method (author's transl)]. Some evaluations were made on the Widmark method is designed for determination of blood ethanol concentration. A reagent of potassium dichromate-sulfuric acid mixture was found potent within 3 weeks after preparation when kept air-tight in hard glass ware without exposure to light, and 0.01 N sodium thiosulphate reagent was potent within 2 weeks under similar conditions. No change was observed in the potassium dichromate-sulfuric acid mixture reagent even after heating at 50-60 degrees C for 2 hours. Heating for distillation and oxidation of ethanol was satisfactory with air bath in an electric thermostat.


[Acyl-CoA synthetase activity of long-chain mono and dicarboxylic acid in beef liver preparations (author's transl)]. Higher omega-oxidation activities in the diabetic mammal and the starved one suggest that omega-oxidation mechanism plays an important role under these conditions. Dicarboxylic acid that is the final product of omega-oxidation can be metabolized further by beta-oxidation, subsequently, formation of succinyl-CoA and short-chain dicarboxylic acid might be increased in the liver. The physiological significance of omega-oxidation might consist in supplying the substrate of TCA cycle for utilization of acetyl-CoA and excreting the short-chain dicarboxylate in urine resulting in the decrease of ketone bodies in the blood, especially in diabetes and starvation. On the bases of these information, it is important to investigate the metabolism of dicarboxylic acids. Generally, fatty acids must be activated before they enter the metabolic pathway. By in vitro studies with rat liver homogenate, we have recently demonstrated that octadecaned-ioic acid must be activated by ATP-Mg2+ and CoA as monocarboxylic acid is. However, it has not been studied to compare the activity of acyl-CoA synthetase on mono and dicarboxylic acid. So, in this report, we assayed the activity of acyl-CoA synthetase in beef liver preparations using palmitic or hexadecanedioic acid (C1;16) as substrate. The results are as follows 1) Activation capacity of the supernatant of sonicated mitochondria was less than that of sonicated microsome for either palmitate or hexadecanedioate. 2) Activation capacity for hexadecanedioate was less than that for palmitate in both supernatant of sonicated mitochondria and that of sonicated microsome. 3) In our experiment, it might be suggested that the subcellular distribution of hexadecanedioate activation is almost identical with that of palmitate activation.


High HL-A5 frequency in Behçet's disease. HL-A antigens of 35 patients with Behçet's disease and 36 normal Japanese were determined by a standard microlymphocytotoxicity test. The frequency of HL-A5 among patients was significantly higher than the controls. No significant differences were observed in the distribution of other HL-A antigens.


[On-line, high-speeded data analysis of cardiovascular function using hybrid systems (author's transl)]. In recent years application of computer techniques to various problems in biology has increased. In the field of anesthesia, where rapid changes appearing in a short time must be followed, the conventional, mannual methods are not applicable. So we have developed on-line computer analytical methods of cardiovascular function. Hybrid systems are the preferred methods in many biological problems, either because of efficiency and lower cost, or because of real time, on-line, closed-loop capabilities for direct use during experiments. As a computer output we used a graphic display computer and cathod ray tube devices. We applied these systems to analize and calculate cardiac work with other circulatory parameters. We got a ventricular function curve in visible form on CRT within 30 seconds after introducing the signals of left ventricular work and ventricular end-diastolic pressure into the analog computer. It was also useful to calculate vascular input impedence and myocardial maximal velocity of shortening. Finally causes of input errors in these analytical methods were discussed.


Induction and suppression of reagins in the neonatal mouse. Neonatal SWR mice are capable of synthesizing reagins when immunized with a mixture of concanavalin and ovalbumin or a mixture of Bordetella pertussis, alum and ovalbumin. Reaginic antibody-forming cells can be found in the spleen, lymph nodes, bone marrow and Peyer's patches. Tolerance with respect to IgE can be induced by the injection of deaggregated ovalbumin into neonatal mice.


Plasma prolactin and its relationship to risk factors in human breast cancer. The prolactin concentration has been determined in plasma from ostensibly healthy women living on the Island of Guernsey. There were 102, 42, and 41 women who had a mother, sister or maternal aunt, respectively, with breast cancer. The remaining 184 women in this study claimed to have no known family history of breast cancer and were used as a control group. The increased risk of breast cancer due to family history was not associated with a raised mean prolactin level compared to the control group. However, in the luteal phase of the menstrual cycle, daughters of breast cancer patients had significantly raised levels of prolactin at 19.00 h. There were no abnormalities in the mean plasma prolactin levels for the above groups associated with differences in age at first child, age at menarche, interval between age at menarche and first child, and body weight of post-menopausal women. (All these factors have been reported to influence breast cancer risk). It is concluded that prolactin has no obvious function in the aetiology of breast cancer. If it is involved, the mechanism by which it acts must be subtle and concerned with the homeostatic control governing nycthemeral prolactin rhythms.


Human tumor cells grown in fetal calf serum and human serum: influences on the tests for lymphocyte cytotoxicity, serum blocking and serum arming effects. Peripheral blood lymphoid cells (PBL) from cancer patients and normal donors were tested against three melanoma cell lines grown in either 10% fetal calf serum (FCS) or 2.5-5% human AB serum in order to determine if the heterologous membrane (HM) antigen or other FCS antigens acquired from the bovine serum supplement could influence lymphoid cell-mediated cytotoxicity in vitro. FCS-grown melanoma cells were more susceptible than the AB serum-grown subline to lymphocyte cytotoxic effects. Arming effects by autologous sera on normal donor lymphocytes and to a lesser extent on lymphocytes of cancer patients were more pronounced on the FCS-grown M12 melanoma cells. This effect was abrogated when the cells were grown in human AB serum for at least 8 weeks. The non-HM tumor-associated antigen remained at the same original low level. Blocking effects were more evident on the AB-grown M14 melanoma line. These data suggest that the FCS antigens on the cell surface may have been responsible for the augmented PBL cytotoxicity. The anti-FCS antibody present in normal and cancer patients' blood induced an antibody-dependent cellular cytotoxicity (ADCC). Elimination of arming activity against HM or other FCS antigens from AB-grown cells may have made the serum blocking factors more apparent. However, cytotoxicity against tumor cells by PBL from normal donors was still apparent even on the human serum-grown cells, suggesting that a different antigen-antibody system was also responsible for this "non-specific" activity.


Defective leukotaxis in patients with lepromatous leprosy. Sera from patients with lepromatous leprosy contain a leukotactic (chemotactic) inhibitor that irreversibly inhibits a variety of chemotactic factors. The presence of this inhibitor correlates with lack of skin reactivity to a variety of antigens. The inhibitor appears to be similar to a serum factor recently termed the chemotactic factor inactivator. The presence in leprosy sera of this inhibitor may be responsible for some of the defects of cellular inflammatory responses found in patients with lepromatous leprosy.


Comparative study of the 48-hour response to soluble antigens obtained from human and armadillo leprosy material in lepromatous leprosy patients and normal persons, contacts of leprosy patients. We prepared antigens by precipitating with 80% ammonium sulfate supernatants of human and armadillo antigen at a concentration of 160 X 10(6) bacteria per ml. The precipitate was resuspended, dialyzed and filtered. The antigen obtained was inactivated with trypsin during 30 minutes. The tests made with these antigens were negative for the 48-hour test in lepromatous patients and highly positive in normal persons who were contacts of leprosy patients.


Search for low-energy conformations of a neurotoxic protein by means of predictive rules, tests for hard-sphere overlaps, and energy minimization. A method to obtain models for the three-dimensional structure of the neurotoxin alpha from Naja nigricollis from its amino acid sequence is explored here. Empirical predictive rules were used to estimate the positions of helices, extended structures and bends; advantage was taken of the availability of 14 homologous sequences for the neurotoxins in an attempt to increase the reliability of these predictions. Unassigned residues were allowed to take up several possible conformations determined from the frequencies of occurrence of each type of conformation of that residue in x-ray structures of many proteins. The conformational space of the molecule was explored initially by testing for hard-sphere overlaps and approximate closure of disulfide loops with the aid of a computer; this procedure yielded a limited number of conformations, whose conformational energies were then determined and minimized by optimizing the backbone and side-chain dihedral angles of each residue. Five compact conformations with low energy were found for this neurotoxin. The procedure used here provides an illustration as to how empirical protein algorithms may be used to limit the conformational space, in which energy minimization has to be carried out.


Activity of placental enzymes of carbohydrate and lipid metabolism in normal, toxemic and small-for-date pregnancies. The specific activity of placental enzymes with a regulatory function in the pathways of glycolysis, gluconeogenesis, NADPH generation and fatty acid synthesis decrease during gestation in the rat. Similar decreases occur in the activity of enzymes of the human placenta when compared between early gestation and term, with the exception of the activity of enzymes related to gluconeogenesis which tend to increase as gestation advances. In term placentas from pregnancies complicated by preeclamptic toxemia, enzyme activities were significantly lower than those in placentas from normal pregnancies, irrespective of whether the baby was of normal weight or small-for-date. This indicates an accelerated decline in placental metabolic function in toxemia. In contrast, in placentas from nontoxemic pregnancies with small-for-date babies the enzyme activities were significantly higher than those in normal term placentas. It is suggested that the decrease in placental enzyme activity, which is associated with placental growth during the course of normal gestation, is retarded in nontoxemic pregnancies with small-for-date babies, apparently due to the arrested aging of the placenta.


Colonization of soil by Histoplasma capsulatum: II. Effect of bat guano added to an area of reduced colonization. Application of bat guano to an area with reduced but detectable colonization by Histoplasma capsulatum had no quantitative effect on the fungus. After seeding the guano treated area and an untreated area with a soil culture of the fungus, a quantitative effect was noted in the treated area but the period of survival did not appear to be extended. Twenty-four months after seeding, the fungus was no longer detectable in the study areas and they have remained negative for more than a year.


Acquired retinal folds in the cat.

Acquired retinal folds in the cat. Retinal folds were found in 5 cats. The apparent cause of the folding was varied: in 1 cat the folds appeared after a localized retinal detachment; in 2 cats the condition accompanied other intraocular abnormalities associated with feline infectious peritonitis; 1 cat had active keratitis, and the retinal changes were thought to have been injury related; and 1 cat, bilaterally affected, had chronic glomerulonephritis.


Narcolepsy in seven dogs.

Narcolepsy in seven dogs. Narcolepsy in 7 dogs was tentatively diagnosed on the onset of cataplexy, before or during young adulthood. Confirmatory polygraphic sleep recordings were done in 3 of the dogs. In 2 dogs, treatment with neostigmine did not cause the signs to disappear, thus ruling out myasthenia gravis; trials with imipramine reduced catapletic attacks. Data from case histories, polygraphic recordings, drug trials, and clinical tests were used to compare and contrast the disease in man and in the dog.


Villonodular synovitis of the equine metacarpophalangeal joint. Villonodular synovitis was diagnosed as the cause of lameness in 14 horses. The diagnosis was based on the history, clinical signs, and arthrographic findings. Treatment consisted of surgical excision and radiation therapy. The response to treatment was favorable in 13 horses.


Castration of adult male dogs: effects on roaming, aggression, urine marking, and mounting. In a retrospective study involving 42 dogs, it was concluded that the objectionable male behavioral patterns of roaming, fighting with other males, urine marking in the house, and mounting of other dogs or people are amenable to alteration by other castration in adulthood. Roaming was reduced in 90% of the dogs.


The effect of neonatal treatment of male mice with antiandrogens and of females with androgens on the development of the os penis and os clitoridis. The os penis in mice and rats is composed of a proximal intramembranous and endochondral osseous element and a distal cartilaginous, ossifying element. Female mice, but not rats, have a small os clitoridis which corresponds to the intramembranous part of the proximal element of the os penis. In mice of either sex a dense mesenchymatous formation ventral to the urethra is the anlage for the bones of the external genitalia. In the early postnatal period the proximal part of the os penis develops as bone at the outer and as cartilage at the basal end of the anlage, while in females a minute focus of ossification differentiates into the small os clitoridis without passing through a cartilaginous phase. The distal element of the os penis is formed later than the proximal rod and grows at a slower rate. Neonatal treatment with an antiandrogen inhibits the increase in size and calcification of the os penis. Neonatal castration is an even more effective inhibitor. Neonatal treatment with testosterone or dihydrotestosterone, but not with oestradiol, stimulates the growth of the bony proximal os clitoridis, but induces only a rudimentary collagenous distal element. The differences between mice and rats in the response of the tissues of the clitoris to androgenic treatment are discussed, particularly as regards the differentiation of proximal and distal elements.


Pleuromutilins. Fermentation, structure and biosynthesis. Derivatives of pleuromutilin, formed during the fermentation of pleuromutilin, were isolated and their structure determined. 14-Acetyl-mutilin and mutilin as well as different unsaturated fatty acid esters of pleuromutilin were identified. The proportion of each derivative formed depends to a considerable degree on the conditions of the fermentation process. The possible biosynthetic pathways are shown.


An intraluminal transducer/telemetry system for oviductal motility studies. A method for measurement of changes in lumen diameter of the oviduct has been developed. Miniature intraluminal "doughnut-shaped" transducers, 1.0-1.2 mm in diameter, constructed from conductive silicone rubber are implanted in the rabbit isthmus and respond to localized changes--increases or decreases--in lumen diameter. Multiple transducer implants in the same tube permit rate as well as direction of contractions to be determined. A custom-designed, micropower dual-channel telemetry system was fabricated to record motility from unrestrained animals. Typical data collected using this system to determine the effects of induced ovulation on instantaneous changes in lumen diameter are presented.


Utilization of exogenous pyrimidines as a source of nitrogen by cells of the yeast Rhodotorula glutinis. Uptake and intracellular transformation of pyrimidines supplying cells of the yeast Rhodotorula glutinis with nitrogen have been studied. The amine nitrogen of cytosine was found to be the easiest to utilize. The presence in the medium of inorganic ammonia along with cytosine had a slight effect on cytosine deaminase (EC 3.5.4.1) activity. The uracil produced entered into the nutrient medium with no fission break of the pyridmidine ring. In the absence of any other source of nitrogen, the cells of the yeast R. glutinis utilized nitrogen of the pyrimidine ring of oxypyrimidines. Catabolism of uracil followed the reductive pattern, with release of carbon dioxide; this was accompanied by synthesis of the key enzyme of pyrimidine catabolism, dihydrouracil dehydrogenase (EC 1.3.1.1), whose activity rose 10-fold. With thymidne as the sole source of nitrogen, the lag-phase growth of the yeast cells was maximum. Catabolism of the pyrimidine ring of thymine was possibly preceded by its transformation into uracil. With no source of nitrogen easily utilized, the uridine 5'-monophosphate content in the generally acid-soluble pool rose. Our discussion of the regulation of catabolism of exogenous pyrimidine bases by the yeast R. glutinis takes into account the fact that transformations of pyrimidine bases are determined by how easily the cells can use a particular base as a source of nitrogen.


Repair of deoxyribonucleic acid in ultraviolet light-sensitive and -resistant Dictyostelium discoideum strains. Some responses of the cellular slime mold Dictyostelium discoideum to ultraviolet light (UV) irradiation were investigated by analyzing two aspects of deoxyribonucleic acid (DNA) excision repair in the vegetative cells: (i) the fate of thymine-containing dimers and (ii) the production and rejoining of single-strand breaks. Experiments were done with the parental, radiation-resistant NC-4 strain and with the radiation-sensitive gammas-13 strain. The majority (greater than 85%) of the thymine-containing dimers produced in both strains by an energy fluence of 100/Jm2 were removed from the acid-insoluble DNA fraction within the first 3 to 4 h of reincubation in the dark. Moreover, as measured by alkaline sucrose gradients, single-strand breaks appeared in the DNA of both NC-4 and gammas-13 irradiated cells very rapidly and at low temperatures. This was presumed to be a result of the incision (nicking) step of excision repair as performed by UV-specific endonuclease(s). In NC-4 the time required for dimer excision correlated with the sealing of breaks as well as with the UV-induced division delays. In gammas-13 the single-strand breaks were closed at a slower rate than in NC-4. However, this was not accompanied by more extensive division delays.


Beta-actinin-like protein from plasmodium. A beta-actinin-like protein was isolated from plasmodia of the slime mold. The chain weight was the same as that of actin (43,000), but the amino acid composition was significantly different. The action of plasmodium beta-actinin on actin was the same as that of beta-actinin from rabbit skeletal muscle: inhibition of the recombination of F-actin fragments; formation of Mg polymer; inhibition of interfilamental interaction of F-actin and retardation of depolymerization of F-actin. The only difference observed was its sensitivity to trypsin: plasmodium actinin was less quickly digested by trypsin than rabbit beta-actinin.


Affinity chromatography of trypsin and related enzymes. II. An affinity adsorbent containing glycylglycyl-L-arginine. An affinity adsorbent for trypsin [EC 3.4.21.4] (GGA Sepharose) was prepared. Glycylglycyl-L-arginine (GGA) was synthesized by a simple procedure and was immobilized on agarose gel. This adsorbent proved to have essentially the same characteristics as AP Sepharose, which is an affinity adsorbent containing tryptic peptides of protamine (1). GGS Sepharose was specific for native trypsin and had a stronger affinity at lower pH's (6-5) than at the optimum pH of trypsin action (8.2). It also proved to be suitable for analytical experiments because of its relatively weak affinity. By comparison of the elution profiles of trypsin from GGA Sepharose under various conditions, the nature of the interaction of trypsin with the adsorbent could be studied. It was found that alpha- and beta-trypsin could be distinguished. In the presence of arginine and N-substitute arginines, the elution of trypsin was accelerated. From the extents of the accelerating effects, the affinities of these compouunds could be compared.


Chondroitin sulfate glycosyltransferases in cultured chondrocytes. Turnover, oscillatory change during growth, and suppression by 5-bromodeoxyuridine. Activity levels of three glycosyltransferases (UDP-D-xylose:core protein xylosyltransferase, UDP-D-galactose:D-xylose galactosyltransferase, and UDP-N-acetyl-D-galactosamine: (GluUA-GalNAc-4-sulfate)3 N-acetylgalactosaminyltransferase), which are involved in the biosynthesis of chondroitin sulfate, were measured during the development of chrondrocytes in monolayer culture. Maxima in enzyme specific activities occurred on Day 8 of culture and preceded the maximum rate of synthesis of chondroitin sulfate proteoglycan (Day 10) by 2 days. The activity of xylosyltransferase was lowered by treatment of cells with inhibitors of RNA and protein synthesis, whereas the activities of the other two glycosyltransferases were relatively stable to treatments with these drugs. In the presence of puromycin, xylosyltransferase decayed with a half-life of 2 to 3 hours in rapidly growing cells, while galactosyltransferase decayed with a half-life or approximately 12 hours, indicating that the chrondroitin sulfate glycosyltransferases are not coordinately synthesized and degraded. 5-Bromo-2'-deoxyuridine also caused a greater decrease in xylosyltransferase activity than in the activities of the other two glycosyltransferases. The differential effect of 5-bromo-2'-deoxyuridine on glycosyltransferase activity was also showm by correlating the residual enzyme activity with the potential for synthesis of chrondroitin sulfate chains as measured in the presence of beta-xylosides.


Time of occurrence of disulfide linking between procollagen chains. The disulfide bonds that link together single collagen pro-alpha chains into trimers, prior to their secretion, were found to be synthesized after the pro-alpha chains were completed and released from polysomes and not while the pro-alpha chains were still nascent. After a 4- or 8-min incubation of cell cultures derived from embryonic chick sternum with [3H]proline, the nascent chains isolated from polysome preparations contained negligible amounts of disulfide-linked chains. After a 20-min incubation, some very large disulfide-linked material was found associated with the polysome preparation, but this material proved to be noncollagenous and also appeared to represent completed, and not nascent, chains. The presence of this proline-labeled species was confirmed in analyses of whole cell lysates, in which it is present as a mixture of monomers of approximately 290,000 molecular weight and disulfide-linked dimers. Pulse-chase experiments also gave results that were consistent with the conclusion that released single pro-alpha chains are the precursors of released disulfide-linked pro-alpha chains. Such experiments also showed that pro-alpha chains could continue to form disulfide-linked species in the presence of inhibitors of peptide chain elongation.


Folate metabolism in mammalian cells in culture. I Partial characterization of the folate derivatives present in L1210 mouse leukemia cells. Chromatographic procedures were developed which permit the separation of the monoglutamylfolic acid derivatives from each other and from their polyglutamyl congeners. Using these methods, the cellular folate derivatives in logarithmically growing L1210 cells were found to consist entirely of polyglutamyl derivatives. When ascorbate extracts of these cells were digested with hog kidney gamma-glutamylcarboxypeptidase and then chromatographed on DEAE-cellulose columns, the cellular folates eluted coincident with tetrahydrofolate, 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, and 5-methyltetrahydrofolate, with minimal amounts associated with folate (approximately 0.8%) or dihydrofolate (approximately 0.4%). When these experiments were repeated on cells previously depleted of folates which were then grown on 0.4 muM pteroylglutamic acid (a concentration insufficient to maintain an optimal growth rate) more than 95% of the folates were again found in polyglutamyl form. These results imply that the polyglutamyl derivatives found are not storage forms of enzymatically active cofactor, but rather are the actual cofactor forms used by these mammalian cells.


Rate-limiting steps for the esterase and dehydrogenase reaction catalyzed by horse liver aldehyde dehydrogenase. Horse liver aldehyde dehydrogenase, like other aldehyde dehydrogenases, is capable of hydrolyzing esters such as nitrophenyl acetate. Pre-steady state and burst kinetics were performed using substrate levels of enzyme to determine whether the rate-limiting step occurred prior to or after the formation of an acyl enzyme intermediate. A burst was found by both techniques for the dehydrogenase reaction; but no burst was found for the esterase reaction. These data show that the rate-limiting step for the two reactions catalyzed by the enzyme differs. For dehydrogenase it occurs after the formation of the acyl intermediate, but for esterase it occurs prior to its formation.


Comparison of the physical properties of chemically prepared and tissue-endogenous equine apoferritins. To support the case for a biological applicability of previous experiments performed on chemically prepared apoferritin, its hydrodynamic and circular dichroic properties were compared to those of tissue-endogenous apoferritin. The molecular weights and sedimentation coefficients of the two differently prepared apoferritins were identical. Similarly, their circular dichroic spectra between 310 and 200 nm were identical. These data support the hypothesis that chemically prepared apoferritin is identical with tissue-endogenous apoferritin. Ferritins exhibited low ultraviolet CD spectra quite similar to apoferritin except amplitudes were often as much as 8% less. Apoferritin subunits, prepared by acid dissociation, did not possess the positive 292 and 286 nm bands exhibited by the native molecule, although hydrodynamically the subunit behaves as a globular protein. This suggests the presence of tryptophanyl and tyrosyl residues at the subunit contact sites. Two characteristics of the apoferritin CD spectrum were proposed as means to evaluate the quality of apoferritin preparations. These are a ratio, [theta]286 [theta]222 = -4.5 X 10(-3) +/- 0.2 X 10(-3), and the wavelength region, 258 to 262 nm, at which the near ultraviolet CD spectrum goes from a positive to a negative sense.


Fluorescent excitation transfer immunoassay. A general method for determination of antigens. A general immunochemical method for the assay of haptens and proteins has been devised and applied to morphine, a morphine-albumin conjugate, and human immunoglobulin G. A fluorescein-labeled antigen and a quencher-labeled antibody are employed. By use of fluorescein and rhodamine as the fluorescer and quencher, respectively, dipole-dipole-coupled excitation energy transfer can occur within the antigen-antibody complex. The resulting quenching of fluorescence can be inhibited by competitive binding with unlabeled antigen, Alternatively, separate antibody samples can be labeled with fluorescein and rhodamine, respectively. Unlabeled antigen causes aggregation of the separately labeled components with resultant quenching. Using the latter method, experiments suggest that up to about 20 anti-morphine antibody binding sites will associate with morphine-albumin conjugates. When an excess of the conjugate is present the antibodies appear to assemble in clumps on the protein surface. Mathematical analysis of the quenching of fluorescein-labeled morphine by rhodamine-labeled anti-morphine gives an approximate fit to the quenching data, but the calculations are very dependent on the assumptions used.


Reiteration frequency of delta-crystallin DNA in lens and non-lens tissues of chick embryos. delta-Crystallin gene is not amplified during lens cell differentiation. The differentiation of embryonic chick lens fibers is characterized by an increased rate of synthesis of the lens protein, delta-crystallin, and accumulation of delta-crystallin mRNA. In the present study, the number of delta-crystallin genes in lens and non-lens tissues of embryonic chicks has been determined to test whether the accumulation of delta-crystallin mRNA during lens fiber differentiation is associated with an amplification of delta-crystallin genes. DNA from embryonic chick lens fibers, embryonic chick lens epithelia, or whole chick embryos with the eyes removed, was annealed with [3H]DNA complementary to delta-crystallin mRNA. Analysis of the annealing reactions indicated that the sequences for delta-crystallin are in the unique fraction of the chick genome, and are not amplified in the lens during embryonic development.


Subunit composition of delta-crystallin from embryonic chick lens. Analysis of methionine-containing tryptic peptides and cyanogen bromide peptides. The predominant protein in the embryonic chick lens, delta-crystallin, is composed of four subunits with molecular weights near 50,000. The degree to which these 4 polypeptides are the same or dissimilar was explored in delta crystallin purified from 15-day-old embryonic chick lenses by relating the numbers of methionine-containing tryptic peptides and cyanogen bromide (CNBr) peptides derived from the native protein to the average number of methionine residues per subunit. Amino acid analyses indicated that 1 mol of native delta-crystallin contains approximately 32 methionine residues, leading to an average of 8 methionine residues per subunit. Approximately equal amounts of 8 methionine-containing tryptic peptides were resolved by two-dimensional thin layer separation on cellulose sheets and by isoelectric focusing in polyacrylamide gels. Nine CNBr peptides were resolved by a combination of electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gels and chromatography on SDS-hydroxylapatite columns. The additive molecular weight of the 9 CNBr peptides was very close to the delta-crystallin subunit molecular weight of 50,000. Thus, the subunits of 15-day-old embryonic chick delta-crystallin have similar sequence of encoded amino acids.


Echography: a new approach to the diagnosis of adrenal hemorrhage of the newborn. A case of adrenal hemorrhage of the newborn, diagnosed by ultrasound, is presented. All other diagnostic modalities were found to be non-specific. Ultrasonic evaluation is recommended in the evaluation of pediatric abdominal masses. This is a safe, non-invasive, rapid and easily performed study. It is felt that the use of diagnostic ultrasound in evaluating neonates for adrenal hemorrhage wil obviate the necessity of more invasive procedures, such as exploratory laparotomy.


A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Subcellular localization of the protease in porcine skeletal muscle. A study was done to determine whether the Ca2+-activated muscle protease (CAF) that removes Z disks from myofibrils in the presence of Ca2+ is located in a sedimentable subcellular organelle. Porcine skeletal muscle cells were diced finely with a scalpel and were suspended in 0.25 M sucrose, 4 mM EDTA with a VIRTIS homogenizer. Filtration of the suspended muscle through four layers of cheesecloth removed most of the myofibrils and stromal protein. Nuclear (1,000 gavg for 15 min), mitochondrial-microsomal (50,000 gavg for 60 min), and supernatant fractions were assayed for succinic dehydrogenase, acid ribonuclease, cathepsin D, and CAF activities. Approximately 96% of total succinic dehydrogenase activity, 81% of cathepsin D activity, and 45% of acid ribonuclease activity, but only 14% of total CAF activity, were found in the nuclear and mitochondrial-microsomal fractions. Cathepsin D activity in the nuclear and mitochondrial-microsomal fractions was decreased if assays were done without prior treatment to rupture membranous structures; hence, our cell rupture and homogenization procedures preserved some intact lysosomal organelles. The results indicate that the small amount of CAF activity in the nuclear and mitochondrial-microsomal fractions was due to contamination by supernate and that CAF is not located in a membrane-bounded subcellular particle. Because CAF is active at the intracellular pH and temperature of living skeletal muscle cells and is in direct contact with the cytoplasm of muscle cells, its activity must be regulated by intracellular cellular Ca2+ concentration to prevent continuous and indiscriminate degradation of myofibrils.


Biological membranes as bilayer couples. III. Compensatory shape changes induced in membranes. We have previously proposed the hypothesis that asymmetric membranes behave like bilayer couples: the two layers of the bilayer membrane can respond differently to a particular perturbation. Such a perturbation, for example, can result in the expansion of one layer relative to the other, thereby producing a curvature of that membrane. In experiments with erythrocytes and lymphocytes, we now demonstrate that different membrane perturbations which have opposite effects on membrane curvature can compensate and neutralize one another, as expected from the bilayer couple hypothesis. This provides a rational basis, for example, for understanding the effects of amphipathic drugs on a variety of cellular phenomena which involve shape changes of membranes.


Sulfhydryls and the in vitro polymerization of tubulin. The free sulfhydryls of brain tubulin prepared by cyclic polymerization procedures both with and without glycerol have been examined. The average free sulfhydryl titer of tubulin prepared with glycerol (7.0 sulfhydryls/55,000 mol wt) is greater than that of tubulin prepared without glycerol (4.0 sulfhydryls/55,000 mol wt). Diamide, a sulfhydryl-oxidizing agent, inhibits the polymerization of tubulin. Diamide also disperses the 20S and 30S oligomers of tubulin seen in analytical ultracentrifuge patterns of tubulin solutions and, depending on the temperature at which diamide is added, converts all or part of the oligomeric material to 6S dimers. Electron microscopy demonstrates that diamide also destroys the 450-A ring structures characteristic of tubulin solutions. All diamide effects are reversible by the addition of 10 mM dithioerythreitol, a sulfhydryl-reducing agent. That diamide interacts with sulfhydryls on tubulin is directly demonstrated by a 50% decrease in the free sulfhydryl titer of tubulin measured after diamide treatment. Concentrations of CaCl2 which inhibit polymerization also decrease the free sulfhydryl titer of tubulin.


Direct association of messenger RNA labeled in the presence of fluoroorotate with membranes of the endoplasmic reticulum in rat liver. Liver rough endoplasmic reticulum (RER) membranes were isolated from rats given [3H]orotic acid for 48 h (ribosomal RNA [rRNA] label) or for 3 h along with 5-fluoroorotate; this latter procedure permits the labeling of cytoplasmic messenger RNAs (mRNAs) in the absence of rRNA labeling. More than 50% of the labeled mRNA remained attached to membranes of the RER after complete removal of ribosomes with a buffer of high ionic strength in the presence of puromycin. Under similar conditions, membranes retained 40% of their polyadenylate as determined by a [3H]-polyuridylate hybridization assay. Treatment of mRNA-labeled endoplasmic reticulum membranes with pancreatic RNase indicates that the polyadenylate and possibly nonpolyadenylate-pyrimidine portions of the messenger are involved in the binding of mRNA to the membranes. The implication of these results in furthering our understanding of the mechanisms of the translational regulation of genetic expression is discussed.


Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs. Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.


A highly infectious 'mycoplasma' that inhibits meiosis in the fungus Coprinus. We have discovered a cytoplasmically inherited infectious agent that inhibits meiosis in a species of Coprinus, a basidiomycetous fungus. From infectivity, filtration, centrifugation and ultrastructural studies we believe the agent to be a mycoplasma. The agent is highly infectious to several strains of the host species and is capable of spreading rapidly through infected hosts. No pathological effect has been seen on any aspect of growth or differentiation of the fungus except for the inability of infected strains or undergo meiosis. The failure of meiosis results in mushrooms that do not produce the normal black spores and are therefore pale in colour. The paleness represents a simple assay for the presence and activity of the infectious agent. Infected hosts do not display any ultrastructural abnormalities in the vegetative stages, only in the cells in which meiosis should occur. In the meiotic cells, at the time when normal cells are undergoing synapsis and synaptinemal complexes are forming, the vacuoles of the infected cells become occupied with vesicular, membrane-bound bodies resembling in shape and form mycoplasmas. Extracts from infected clones may be filtered through 0.2-mum filters and retain full infectivity. The infectious material may be pelleted from such extracts at only 10 000g. Migration experiments, as well as the filtration studies, rule out involvement directly of nuclei. The high rate of infection and spread of the mycoplasma through the host, combined with the anatomical simplicity of the host, make this an ideal system in which to study the basis of infection. The singularity of the pathological effect make this host-parasite association useful in studying both the underlying mechanisms of mycoplasma pathogenicity and to investigate the regulation of meiosis. This is only the second report of mycoplasmas in fungi.


The relationship of luteinizing hormone secretion to sleep in women during the early follicular phase: effects of sleep reversal and a prolonged three-hour sleep-wake schedule. The relationship of luteinizing hormone (LH) secretion to sleep in adult women was investigated in two ways: an acute 180 degrees sleep-wake cycle reversal in a group of six women and a schedule in which a young woman engaged in a three hour sleep-wake cycle (two hours awake, one hour allowed for sleep continuously for ten days--the study was carried out on the eighth day). Each subject in the reversal study had a baseline period during which plasma samples were collected every twenty minutes for twenty-four hours and nocturnal sleep was monitored electrophysiologically during the early follicular phase of the menstrual cycle. During a succeeding cycle, the study was repeated after sleep-wake reversal. LH secretory patterns were analyzed by comparing the 24-hour mean plasma LH concentration with the hourly averages in percentage terms, using Stage 2 sleep onset as the zero point. LH secretion was depressed to approximately the same degree in both the baseline and reversal studies. The average hourly percentage difference from the 24-hour mean for the four-hour period following sleep onset was -13.4% and -13.1% for the baseline and reversal, respectively. These percentage deviations represented practically the entire negative deviation for the 24-hour period in both studies. The difference between the first four-hour period after sleep onset and the second was significant. The subject on a three-hour cycle had a baseline in which a large decrease in LH secretion occurred after sleep onset (-52.2% during the third hour). Her LH secretory pattern during the three-hour sleep-wake schedule was characterized by a fall during sleep periods, particularly when slow wave sleep (SWS) predominated. However, no correlation was found between specific sleep stages and LH secretion in the six women of the reversal study. These results confirm a relationship of LH secretion to sleep in adult women, one which is different from that described during puberty.


Treatment of women with the galactorrhea-amenorrhea syndrome with pyridoxine (vitamin B6). Three women with the galactorrhea-amenorrhea syndrome and elevated prolactin concentrations experienced a return of regular ovulatory menses within 37-94 days after starting pyridoxine treatment (200-600 mg/day). In each the galactorrhea ceased and serum prolactin levels were maintained in the normal range while taking pyridoxine. In two other women with prolonged secondary amenorrhea but without hyperprolactinemia or galactorrhea, pyridoxine at dosages up to 600 mg/day did not restore ovulatory menses. Pyridoxine treatment was also ineffective in decreasing profuse galactorrhea in one woman with normal prolactin levels and regular ovulatory menses. In the three women effectively treated with pyridoxine, the galactorrhea returned, serum prolactin levels increased, and the menses ceased after discontinuing pyridoxine. These results imply that pyridoxine, by decreasing the excessive secretion of prolactin, may be useful in the long-term medical management of women with hyperprolactinemia and the galactorrhea-amenorrhea syndrome.


The regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the isolated perfused rat liver. The effect of perfusion of an isolated rat liver on hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was studied. In liver removed during the basal period of the diurnal cycle of enzyme activity, a 227 +/- 41% increase in enzyme activity occurred after 3 h of a plasma-free perfusion. This could be prevented by the addition of cycloheximide or pure cholesterol (dispersed with lecithin) to the perfusate. In contrast, the continuous addition of taurocholate or taurochenodeoxycholate, alone or in combination, at a variety of rates did not prevent the increase in enzyme activity. The added bile salts were efficiently extracted from the perfusate and excreted in the bile. The addition of these bile salts to a cholesterol-enriched perfusate did not alter the effect obtained with cholesterol alone. If the perfusate contained whole serum, the increase induced by perfusion in the basal period was smaller (88 +/- 27%) than with plasma-free perfusate. Again, the major bile salts of the rat failed to prevent the increase in enzyme activity induced by liver perfusion. If livers were removed and perfused at the height of the diurnal cycle of enzyme activity, the enzyme activity remained high (2 +/- 10% increase) rather than decreasing, as occurs in vivo. If cholesterol was added to these perfusions, a 52 +/- 4% decrease was induced. Bile salt addition induced no decrease. From the results it is concluded that the major bile salts are not direct regulators of hepatic cholesterol synthesis, but pure cholesterol, in the absence of bile salt or lipoprotein, is able to initiate the mechanism that represses hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.


Detection of antibodies of the IgM class in sera of patients recently infected with influenza viruses. Sucrose density gradient ultracentrifugation can be used to detect specific 19S antibodies of the IgM class in the sera of patients recently infected with influenza A virus, provided steps are taken to remove non-specific inhibitors of haemagglutination. The usefulness of the procedure for the diagnosis of influenza requires further evaluation.


Induction of parental behavior in Japanese quail (Coturnix coturnix japonica). The Japanese quail is a precocial species, and because of its relatively rapid development, sexual maturation in about 40 days after hatching, and prolific breeding capacity, it promises to become an organism well suited for avian research. One stumbling block has been the inability to induce, with any consistency, parental behavior in laboratory stocks. The study reported herein demonstrates a method for establishing a self-perpetuating population under seminaturalistic conditions. In addition, given the limitions of finite space, chick mortality appears to have been mostly density dependent, thus indicating that the increase in the size of the population is circumscribed in part by population density.


Attenuation of gastric lesions by psychological aspects of aggression in rats. Rats that fought with each other in response to electric shock showed reduced gastric lesions in comparison with animals that received the same shocks alone so that fighting behavior did not occur. Also, gastric lesions were similarly reduced in animals that fought even though they could not physically contact one another because of a barrier between them, so that the "protective" effect of fighting derived from release of, or display of, fighting behavior and did not require physical combat. A second experiment showed that animals that received shock together but did not engage in fighting behavior showed no reduction of gastric lesions, so that the protective effect of fighting was not an artifact of animals receiving shock together. Possible explanations for why fighting behavior reduces gastric lesions are discussed.


Topical pharmacology of imidazole antifungals. Four imidazole derivatives have now undergone extensive open and comparative trials as topical agents in dermatomycoses and vaginal candidosis. They are chlormidazole (Chemie Grünenthal), clotrimazole (Bayer), miconazole (Janssen) and econazole (Janssen, Cilag-Chemie); all also have some antibacterial activity. Many other imidazoles have been marketed, usually as antiprotozoal or anthelminthic agents, and some of these have some antimycotic activity as well as other miscellaneous therapeutic properties. The mode of action of imidazole antimycotic agents is discussed; after prolonged topical application to animals and human subjects, systemic absorption is negligible. All four agents which are available as cream, powder, lotion or vaginal tablets have many sucessful studies to their credit, often with clinical and mycological cure rates of over 80% in a variety of dermatomycoses and in vaginal candidosis. The relative value of these topical agents is discussed, and it is suggested that in severe and extensive dermatomycoses consideration should be given to the systemic use of miconazole in support of topical therapy.


Effect of urea form and method of feeding on rumen ammonia concentration. Rumen ammonia was compared in six rumen fistulated, lactating Holstein cows fed urea twice daily in a concentrate mixture, urea fed once per day in a complete ration, and a gelatinized starch-urea product fed twice daily in the concentrate mixture. Samples were taken during 24-h intervals at the end of each 3 wk. Sharp and similar peaks in rumen ammonia followed feeding of urea and gelatinized urea-starch product twice daily in the concentrate mixture. A much lower ammonia peak followed the once daily feeding of the complete ration. Use of complete rations offered ad libitum eliminates the need for products with sustained ammonia release.


Comprehensive evaluation of fatty acids in foods. VII. Pork products. To derive food table values of fatty acid composition, a comprehensive review was made of the literature available since 1960 on the lipid composition of pork products. Factors affecting pork lipid composition are discussed. Diet of the animal was found to have a major influence. Few significant differences were found in the grams fatty acid per 100 gm. fat of meat from the areas of the loin, belly, leg, and shoulder. Mean fatty acid distributions were derived for the separable lean and separable fat of pork. Methods of converting literature data into units suitable for food table purposes are briefly described. Tables of fatty acids per 100 gm. fat are given for brains, heart, kidney and liver, and for the lean and fat portions of pork cuts. The tables show that on a grams-per-100-gm. -fat basis, these four organ meats contain less total fatty acids and are richer sources of polyunsaturated fatty acids than are skeletal muscle or adipose tissue. Tabulations are also given for fatty acids in 100-gm. portions of raw and cooked pork cuts, sausages, luncheon meats, variety meats, and three popular sausages made with beef and pork meat.


Evaluation of four methods for separation of lymphocytes from normal individuals and patients with cancer and tuberculosis. An optimal technique was sought for lymphocyte recovery from normal and chronic diseased individuals. Lymphocytes were separated by four techniques: Plasmagel, Ficoll--Hypaque, a commercial semiautomatic method, and simple centrifugation using blood drawn from ten normal individuals, ten cancer patients, and ten tuberculosis patients. The lymphocyte mixture obtained after using each method was analyzed for percent recovery, amount if contamination by erythrocytes and neutrophils, and percent viability. The results show that the semiautomatic method yielded the best percent recovery of lymphocytes for normal individuals, while the simple centrifugation method contributed the highest percent recovery for cancer and tuberculosis patients. The Ficoll-Hypaque method gave the lowest erythrocyte contamination for all three types of individuals tested, while the Plasmagel method gave the lowest neutrophil contamination for all three types of individuals. The simple centrifugation method yielded all viable lymphocytes and thus gave the highest percent viability.


Serine biosynthesis in human hair follicles by the phosphorylated pathway: follicular 3-phosphoglycerate dehydrogenase. The phosphorylated pathway of serine biosynthesis was demonstrated in human hair bulbs and sheaths by the formation of phosphoserine and serine from (14C)3-phosphoglyceric acid. The initial and rate limiting enzyme of the pathway, 3-phosphoglycerate dehydrogenase (3-PGDH) was demonstrated by enzyme determinations in human and rat hair follicles, human epidermis, and chicken epidermis. Follicular 3-PGDH was characterized using a sensitive fluorometric assay with NADH as a co-substrate. Monovalent cations (Na+, K+, Li+, or NH4+) were necessary for full enzyme activity. p-Hydroxymercuribenzoate inhibited activity, and activity was 3 times higher with NADH as a co-substrate than with NADPH. The apparent Km for the substrate hydroxyphosphopyruvic acid was 32.8 muM, and the apparent Km for NADH 4.8 muM similar to the Kms for other mammalian 3-PGDHs. Enzyme activity was not altered by parenteral corticosteroids, a high carbohydrate diet, low protein diet, or starvation. Enzyme activity decreased over the first 12 days of life in newborn rats. The phosphorylated pathway of serine synthesis provides a potential nondietary and nonhepatic source of serine, glycine, and their products in keratinizing tissues.


Mechanochemical coupling in flagella. IV. Absence of coupling between metabolism and motility in Chaetopterus spermatozoa. The metabolic rate of suspensions of spermatozoa from the annelid worm, Chaetopterus, does not change significantly when the viscosity of the suspension is increased by the addition of methyl cellulose. At high viscosities, where the spermatozoa swim with about 1/3 the normal beat frequency and 1.5-1.8 times the normal bend angle, the calculated power output against external viscous resistances increases to 4-5 times its normal value. This large increase in power output can be accommodated without an increase in metabolic rate only if internal viscous resistances to flagellar bending are relatively low.


Locomotion of flagellates with mastigonemes. Theoretical hydrodynamic analyses of the locomotion of flagellates with mastigonemes are presented and particular comparison is made within experimental data on Ochromonas malhamensis. The first part of the paper analyses locomotion assuming the mastigonemes are rigid and maintain a fixed and normal position relative to the flagellum. The predicted propulsive velocity of 60 mum/sec for Ochromonas agrees well with the observed values of 55-60 mum/sec. It is shown that the propulsive system of Ochromonas represents a compromise between the need for efficient rectilinear propulsion and the need to manoeuvre and accelerate. The effect of rigid mastigonemes which are maintained at non-zero angles to the flagellar normal is also calculated and demonstrates a significant degradation of performance when this angle is greater than about 10 degrees. The latter part of the paper investigates the more complex but more realistic situation in which the mastigonemes flex during the motion according to the instantaneous hydrodynamic forces imposed upon them. The cyclical flexing history of a mastigoneme with passage of a flagellar wave and the consequent velocity of propulsion are obtained for a variety of geometric configurations and structural mastigoneme stiffnesses. It is demonstrated that there exists a relatively small transition range in the values of mastigoneme flexibility below which the mastigonemes are essentially rigid and above which they become totally ineffective hydrodynamically so that the flagellum can be regarded as essentially smooth. Since the transition value of the modulus of elasticity is about 5 dynes/mum2 (or stiffness of 3.5 X 10(-16) dyne cm2) for the mastigonemes of Ochromonas it would appear that the actual value must be in excess of this. Comparison is made with the structural properties of the micro-tubules in eukaryote cilia and flagella and with prokaryote flagella. The latter comparison suggests that the mastigonemes of Ochromonas are just rigid enough to produce the observed propulsive effect.


Effect of intra-uterine foreign bodies and of prostaglandin administration on progesterone secretion during the oestrous cycle of the guinea-pig. The response of the guinea-pig corpus luteum to the luteolytic influence of glass beads placed in the uterus, or to prostaglandin administration, was followed by assay of the progesterone content of blood samples collected daily. Following the introduction of glass beads into the uterus early in the cycle, the secretion of progesterone was curtailed. Treatment with prostaglandin F2alpha over days 4-6 or 6-8 of the cycle temporarily depressed progesterone release without shortening the life of the corpora lutea. When the drug was administered over days 8-10, 10-12 or 12-14 the depression in progesterone was not followed by any recovery. These observations indicate that the response of the corpora lutea to a luteolytic influence changes during the oestrous cycle.


Dioestrous progesterone and pro-oestrous luteinizing hormone in 4- and 5-day cycles of female rats. Female rats with showed 5-day ovarian cycles spontaneously, but 4-day cycles for periods of variable length after induction of pseudopregnancy, were examined. While the time of onset of the ovulatory LH surge at pro-oestrus was the same in both 4- and 5-day cycles, dioestrous progesterone concentrations were dissimilar. The most prominent feature was a sharp increase during the night of the first day of dioestrus of the 4-day cycle. The results are interpreted as suggesting that dioestrous steroid concentrations rather than pro-oestrous LH concentrations are important in establishing cycle duration.


Development and application of a heterologous radioimmunoassay for ovine follicle-stimulating hormone. A highly specific and sensitive heterologous double antibody radioimmunoassay for ovine follicle-stimulating hormone (oFSH) is described in detail. The assay using a rabbit antiserum to human FSH and either 125I-labelled rat FSH or 125I-labelled oFSH as tracer is specific for FSH. A maximum cross-reaction (B/Bo = 50%) of 0-1% was observed with other ovine, rat or human pituitary hormones or human chorionic gonadotrophin. Serum levels of oFSH in samples collected daily throughout the oestrous cycle showed large individual variations. In five out of nine animals a peak of FSH was observed on the day of oestrus.


Cardiovascular dynamics of the chelonia during apnoea and lung ventilation. 1. In both Pseudemys and Testudo, blood pressures are identical in all chambers of the ventricle. Systolic pressures are not measurably different in the ventricle and systemic arteries but are 0-5-2-0 cm H2O lower in the pulmonary artery due to the resistance of the pulmonary outflow tract. Diastolic pressures are the same in all systemic arteries but are substantially lower in the pulmonary artery. It is concluded that the systemic and pulmonary circulations are perfused by a single pump during both apnoea and lung ventilation. 2. Flow profiles in pulmonary and systemic arteries are characteristicallly different. Substantial changes in cardiac output may occur during intermittent breathing and apnoea, especially if there are large fluctuations in heart rate. Pulmonary flow increases during lung ventilation due to vasodilation of the lung vasculature. Systemic is also affected though the increase is usually smaller. 3. Any separation in the blood pumped by the single ventricle must be maintained by laminar flow patterns and the composition of the blood in each of the major arteries should reflect their relationship to these patterns.


Heat exchange in relation to blood flow between thorax and abdomen in bumblebees. 1. The narrow passage within the petiole between thorax and abdomen is anatomically constructed so that counter-current exchange should retain heat in the thorax despite blood flow to and from the cool abdomen. 2. However, the counter-current heat exchanger can be physiologically circumvented. Exogenously heated bumblebees prevented overheating of the thorax by shunting heat into the abdomen. They also regurgitated fluid, which helped to reduce head temperature but had little effect on thoracic temperature. 3. Temperature increases in the ventrum of the abdomen occurred in steps exactly coinciding with the beats of the ventral diaphragm, and with the abdominal 'ventilatory' pumping movements when these were present. The ability to prevent overheating of the thorax by transport of heat to the abdomen was abolished when the heart was made inoprative. 4. At low thoracic temperatures the ventral diaphragm beat at a wide range or rates and with varying interbeat intervals, while the heart beat at a high frequency relative to the ventral diaphragm, but at a very low amplitude. However, when thoracic temperature exceeded 43 degrees C the amplitudes of both were high, and the interbeat intervals as well as the beating frequencies of the two pulsatile organs became identical in any one bee. Furthermore, heated bees engaged in vigorous abdominal pumping at the same frequency as that of their heart and ventral diaphragm pulsations. 5. The results indicate that the anatomical counter-current heat exchanger is reduced or eliminated during heat stress by 'chopping' the blood flow into pulses, and the blood pulses are shunted through the petiole alternately by way of a switch mechanism.


Evidence for a polyspermy block at the level of sperm-egg plasma membrane fusion in Urechis caupo. The results of sperm binding experiments reveal no change in the sperm binding properties of the egg surface coat at fertilization of Urechis caupo eggs. When fertilized eggs are reinseminated, sperm continue to attach to the egg surface coat. The acrosomal tubules of supernumerary sperm are observed in the perivitelline space closely apposed to the egg membrane. Thus, the polyspermy block in Urechis eggs involves neither alteration of sperm binding sites nor inhibition of the acrosome reaction. Our results suggest that the block is at the level of sperm-egg membrane fusion.