Spontaneous spinal epidural haematoma in a youngster. The case of an 8-year-old boy with spontaneous spinal epidural haematoma and complete post-operative recovery is described. A review of the literature with particular emphasis on the paediatric age-group is given. The clinical presentation and aetiology are discussed.


Early surgical intervention in posthemorrhagic hydrocephalus. 10 premature infants were found to have posthemorrhagic hydrocephalus (PHH) over a 4-year period. All weighed less than 2,500 g. The diagnosis was established by ventricular puncture and ventriculogram. Ventriculo-peritoneal (V-P) shunt placement was done in each infant soon after establishing the diagnosis, while the cerebrospinal fluid (CSF) was bloody. The mean age of infants at surgery was 21.4 days (range 16-33 days). Shunt obstruction occurred in 3 infants and infection in 1. No other complication was observed. All infants improved markedly in the immediate postoperative period, especially respirator-dependent infants. All survived; preliminary follow-up revealed that 3 were severely retarded and 2 mildly retarded; 5 infants were normal. Our preliminary experience with early V-P shunt procedure in the treatment of PHH has been favorable and follow-up appears encouraging.


Successful surgical treatment of pontine vascular malformation in a 3-year old. Case report. A 3-year-old male presented with progressive right hemiparesis. Clinical and radiological studies suggested the diagnosis of brain stem glioma. At surgery, a small vascular malformation of the pons was encountered. Although rare, vascular malformations must be considered in the differential diagnosis of brain stem masses.


Subtemporal decompression for the slit-ventricle syndrome after shunting in hydrocephalic children. During the past 2 years, we have performed subtemporal decompression to deal with recurrent shunt obstruction in 22 hydrocephalic patients with the slit-ventricle syndrome. 13 patients have been followed-up for more than 1 year and 2 others for more than 2 years. The frequency of hospitalization for shunt-revision has been greatly reduced. These results confirm that subtemporal craniectomy is useful in the treatment of patients in whom this syndrome develops.


Hydrocephalus and elevated intracranial venous pressure. Case report. An infant with a large arteriovenous malformation and communicating hydrocephalus is described. The author suggests that the hydrocephalus is caused by impaired absorption of cerebrospinal fluid due to increased intracranial venous pressure.


On the pathology of experimental hydrocephalus induced by artificial increase in endoventricular CSF pulse pressure. In lambs, acute and chronic communicating hydrocephalus has been created by merely increasing the amplitude of the intraventricular cerebrospinal fluid (CSF) pulsations, leaving the mean CSF pressure unchanged. The pathologic changes occurring in this type of hydrocephalus do not differ qualitatively from those observed in obstructive hydrocephalus. Beside demonstrating a direct role of ventricular pulsations in the ventricular dilation, the observed findings indicate that an increase in mean CSF pressure is not a necessary condition for the occurrence of the hydrocephalus. Consequently, the hypothesis of abnormal high intraventricular CSF pressure oscillations at the basis of the ventricular dilation of normal pressure hydrocephalic patients, receives a significant support. Further support is provided is provided by considering the recorded experimental pathological lesions are quite similar to those observed in normal pressure hydrocephalic patients with patent subarachnoid CSF pathways.


Teratocarcinoma of the brain -- a treatable disease? Teratocarcinoma of the brain is a rare teratoid tumor. Less than 100 cases have been previously reported and only 4 were patients with tumors in the lateral ventricular system. At the Children's Hospital of Philadelphia, 2 patients with teratocarcinoma of the lateral ventricle of the brain have been treated in the past 5 years and both are long-term survivors. A third patient with a recurrence at University Hospital is now receiving chemotherapy. Our experience suggests that a combination of radical surgery, supervoltage radiotherapy and chemotherapy may be successful in controlling these neoplasms. Furthermore, since there is increasing evidence that they have a strong histologic relationship to the embryonal carcinomas that arise in the testes and elsewhere, it appears logical to apply chemotherapeutic strategies to these intracranial neoplasms based on experience with such tumors located in other regions of the body.


[Hypothyroid, iodine-deficient goitre in neonates (author's transl)]. Within one year 1750 mature neonates were examined for congenital hypothyroidism. The region of Göttingen is known to be an iodine-deficient one. Sixteen goitrous, hypothyroid neonates with a low total thyroxine, and raised serum TSH concentration were first diagnosed through this screening programme. As a result of the intra-uterine iodine deficiency, serum triiodothyronine concentration was elevated while urinary iodine excretion was reduced compared with euthyroid neonates. During iodine treatment the size of the thyroid became normal within eight days, TSH after 3.8 days and serum thyroxine after three days. Serum triiodothyronine concentration remained elevated for several weeks, presumably as a result of the persisting iodine deficiency. The results indicate that a neonatal screening programme for hypothyroidism is as essential as adding iodine to table salt for avoiding neonatal and foetal maldevelopment.


[Results of reconstruction of the carotid artery (author's transl)]. In 90 patients a total of 100 reconstructions of the carotid artery had been performed between 1972 and 1977. No patient died from the operation, 3 suffered central nervous complications, and 33 had transitory defects of cranial nerves. These defects were reversible in all but two patients. After an average observation period of 20 months 11 patients had died (6 from cardiac infarction, 2 from stroke, and 3 from unknown reasons). Of the 31 patients with clinical stage II, 24 were without symptoms, 5 had only rare transient ischaemic attacks, and two patients had the same frequency of attacks as preoperatively. Among the 39 stage III patients 35 showed complete or partial regression of symptoms whereas 4 had had repeat strokes. 55 of the 59 corrected stenoses were patent as shown by Doppler ultrasound. In contrast attempts to correct complete internal carotid artery occlusions showed unsatisfactory results.


[The saralasin test in the diagnosis of hypertension (author's transl)]. The saralasin test was performed in 68 hypertensives. A clear-cut dependence of the test results on initial plasma-renin concentration and particular sodium balance was demonstrated. Because of this dependence the saralasin test should be performed only under constant conditions. A mild stimulation of the renin-angiotension system by salt restriction to a mean sodium excretion of 50 mmol daily and 80 mg furosemide by mouth 12 hours before the test seems best. In this way essential and renovascular hypertension could be distinguished with considerable reliability (P less than 0.001). Among patients with essential hypertension one could clearly separate those with high plasma-renin concentration from those with a normal or low one. Among patients with renovascular hypertension those with haemodynamically significant renal artery stenosis could with high probability be distinguished from those with non-effective stenosis. A positive saralasin test without testing the function of the normal contralateral kidney does not provide an indication for operation.


[Clinically unrecognised tuberculosis in autopsy material (author's transl)]. Among 18,724 unselected postmortems performed between 1955 and 1975 811 (4.33%) cases of post-primary tuberculosis were found among which 370 (45.6%) were active and 441 (54.4%) inactive. Occurrence of death involved tuberculosis in 32% of all cases discovered at autopsy. The age group over 50 years was mainly affected and males were clearly more frequently involved, particularly in pulmonary tuberculosis. 333 cases of tuberculosis (41.1%) had been recognised clinically and had mainly consisted of chronic pulmonary forms. In the older age group the proportion of cases diagnosed prior to death decreased steadily. Tuberculosis was associated with other diseases in most patients, particularly with cardiovascular disease and malignant tumours. During the investigation period the number of patients with active tuberculosis decreased. The number of cases of miliary tuberculosis among the active forms remained approximately the same.


[Hairy cell leukaemia (author's transl)]. Among 12 patients with hairy cell leukaemia two died without splenectomy and two more 11 months and 7 1/2 years after the operation. In all patients removal of hypersplenism led to improvement of haematological criteria over several months up to many years. This was particularly true for platelets and granulocytes and thus for the proneness to infection and haemorrhagic diathesis. In two patients normalisation of all haematological parameters including histologically scarcely detectable hairy cells was observed. Differential diagnosis from prolymphocyte leukaemia is difficult. Nuclear morphology permits recognition of different degrees of maturation of hairy cells. In cases where tartrate resistant acid phosphatase (TRAP) cannot be demonstrated cytologically, demonstration of isoenzyme 5 is sometimes possible using gel electrophoresis. Demonstration of TRAP remains valuable for the diagnosis of hairy cell leukaemia, however only in connection with other criteria indicating this form of leukaemia.


[Hair growth, liver function and light sensitivity during oral retinoid therapy for psoriasis (author's transl)]. In a multicentre, cooperative study into the treatment of extensive psoriasis with a new aromatic retinoid (Ro 10--9359) trichogram, liver function tests and the light erythema threshold were investigated. In some of the patients hair loss occurred, usually in the fifth to eighth week after a total dose of 1.9 g retinoid. In all cases this improved an average of six weeks after dose reduction or cessation of treatment. The trichogram in 27 patients showed a diffuse toxic hair loss. In 70% the effluvium was telogenic, in 22% telogen-dystrophic. GPT, GOT, alkaline phosphatase and prothrombin index showed no significant alterations during retinoid treatment. However, in individual cases there was a rise in GOT and GPT up to 80 U/l. Furthermore there was a statistical tendency in rising bilirubin levels. Finally there was no evidence for an increase in light sensitivity after three weeks of retinoid treatment. Measurement of the erythema threshold showed rather more a reduction in light sensitivity under treatment.


[Treatment of open cavernous pulmonary tuberculosis (author's transl)]. In two groups, each of 40 patients, with previously untreated cavernous pulmonary tuberculosis the following treatment schemes were compared in a randomised clinical study: isoniazid, ethambutol, and rifampicin (control group) and isoprodian with rifampicin (study group). Bacteriologic and radiographic parameters showed that in the study group the results were at least as good as in the control group. Symptoms of drug intolerance were equally rare in both groups.


[On the question of cortisol administration in adrenocortical insufficiency (author's transl)]. The question whether clinically normal adrenal cortical function can be satisfactorily checked by laboratory studies during substitution treatment with adrenocortical hormones was investigated in 39 patients with adrenal insufficiency. The routine measurement of serum cortisol levels (2 to maximally 7 hours after the morning dose of hydrocortisone or cortisone acetate) revealed marked individual variations which were relatively independent of the morning dosage. Similar extreme variations were found for free urinary corticoids in 24-hour urine. Both values could not be correlated with the clinically individualised and satisfactory long-term substitution dosage and cannot be used, therefore, in the customary manner to assess a normal hormonal state. It appears unlikely that such information can altogether be obtained from plasma levels.


[Myelography with metrizamide (author's transl)]. Metrizamide is a non-ionised, water soluble, and completely absorbable contrast medium which can be used to demonstrate all sections of the spinal canal. The previous strict separation between water soluble, ionised media for the lumbal canal and oily media for the lumbosacral junction as well as the thoracic and cervical canal is no longer necessary. Experience in 110 patients shows that if a suitable concentration is chosen all segments of the spinal canal can be adequately investigated using the lumbal route. The application of contrast media via a lateral cervical entry or suboccipital puncture was not necessary in a single case. Side effects in the form of adhesive arachnoiditis were not observed.


[Biological availability of digoxin and beta-acetyldigoxin after single-dose administration (author's transl)]. The biological availability of digoxin tablets (Lanicor) and beta-acetyldigoxin tablets (Novodigal) was tested after single-dose administration. Plasma levels over 48 hours, the area under the blood-level curves and the cumulative urinary excretion over seven days served as a measure of biological availability. The area under the blood-level curve after 1.0 mg digoxin by mouth was 40.7 +/- 1.7 ng . ml-1 . h and after 1.0 mg beta-acetyldigoxin by mouth 39.1 +/- 1.4 ng . ml-1 h, compared with 56.1 +/- 1.4 after 1.0 mg digoxin intravenously. Seven days later 0.67 +/- 0.12 mg digoxin of the orally administered digoxin, 0.68 +/- 0.12 mg digoxin of the orally administered beta-acetyldigoxin and 0.81 +/- 0.08 mg of the intravenously administered digoxin were excreted in the urine. There was no significant difference in the biological availability of the two drugs (P greater than 0.05).


[Diagnostic significance of lactate concentration in CSF in patients with meningitis (author's transl)]. White-cell count and differential blood count, total protein, lactate dehydrogenase activity (E.C. 1.1.1.27) and lactate concentration were determined in 496 CSF samples, obtained by lumbar puncture. Lactate was measured with a new enzymatic test. Reference values were determined for lactate and lactate dehydrogenase (90% limits: 1.2-2.1 mmol/l and 6-26 U/l, respectively). Lactate content proved to be best in the differential diagnosis between acute bacterial and abacterial meningitis: concentrations of 3.5 mmol/ml and above were found exclusively in bacterial meningitis. As for other diseases, markedly increased lactate concentrations were measured only in patients with brain tumour or cerebrovascular disease (up to 9.3 mmol/l). Determination of lactate concentration alone would have led to a misdiagnosis of acute bacterial meningitis in 3% of cases. If the CSF contains 3.5 mmol/l lactate or more and the leucocyte cell count is above 800/microliter, brain tumour or cerebrovascular disease can be practically excluded and the diagnosis of bacterial meningitis made with a high degree of reliability.


[Prodromal symptoms of acute myocardial infarction (author's transl)]. 75 out of 100 patients who had survived myocardial infarction, had had prodromal symptoms. 62 patients reported specific prodromi such as first manifestations of angina pectoris or increase in duration and intensity of angina pectoris which had existed for some time previously, and in some cases had been accompanied by general complaints. A further 13 patients had only general symptoms such as onset of dyspnoea, palpitations, or a drop in performance during the 3 months prior to the infarct. The primary manifestation of symptoms consisted of exercise-induced angina pectoris at the beginning of the defined prodromal period in most cases, whereas later in the course manifestations occurred in the form of exercise-independent and spontaneous angina pectoris. In 9 patients symptomatology changed and exercise-induced angina transformed either into spontaneous angina pectoris or permanent pectanginous pain. In 8 of these 9 patients infarction occurred within less than a week after the change of symptoms. Prodromi occurred with approximately the same frequency in anterior and posterior wall infarction. The same applied to the distribution between transmural and non-transmural infarction.


[A comparison of percutaneous transhepatic cholangiography and endoscopic retrograde cholangiopancreatography (author's transl)]. Results of percutaneous transhepatic cholangiography (PTC) with the Chiba needle were compared with those obtained by endoscopic retrograde cholangiopancreatography (ERCP). PTC was used in 102 cases, most often after ERCP had been inconclusive or had failed. Overall success rate of PTC was 79%. Contrast medium could be injected in only 66% of cases with normal-sized bile passages, but in 98% of those with enlarged passages. The most frequent side effect was temporary pain in the right upper abdominal quadrant. One patient had transitory severe peritoneal irritation with shock. Emergency operation was never required. PTC is technically simpler, takes less time and causes less stress to the patient, but it is less informative than ERCP. If, therefore, both methods are mastered technically, ERCP should be employed first. If it fails to provide the diagnosis, PTC can be performed at once. Biliojejunal anastomoses and pancreatic pseudocysts are absolute indications for the primary use of PTC to investigate the cause of biliary stasis. On the other hand, allergies to iodine or contrast medium and disorders of coagulation are absolute indications for ERCP.


[Candida coxitis (author's transl)].

[Candida coxitis (author's transl)]. Following drug-induced agranulocytosis and antibiotic and steroid treatment, sepsis due to candida albicans together with bilateral fungal coxitis developed in a 41-year-old female patient. Satisfactory eradication of the inflammatory process was achieved with combined treatment with amphotericin B and 5-fluorocytosine, so that mobilisation was possible after surgical fitting of bilateral total endoprothesis.


[A simple method for determining blood-glucose levels in diabetic out-patients (author's transl)]. 24 hour blood-sugar profiles were obtained in 25 diabetic out-patients. The samples were obtained by the patients themselves even when at work, the glucose-level measurements in the laboratory. The relative differences between duplicate values was less than 10%. The results obtained made it possible to alter the treatment regimen so that, on average, there was a marked reduction in blood-sugar variations during the day.


[Demonstration of left-ventricular contraction anomalies with the two-dimensional sector-scanner (author's transl)]. Echocardiographic findings were compared with those obtained by cardiac catheterisation in 134 patients. Echocardiography was performed in the standard plane of the long and short axes, as well as from the cardiac apex. After dividing the left ventricle into several segments wall movement was measured in them and compared with those obtained by ventricular angiography in the right and left anterior oblique positions. It was found that in patients with left-ventricular aneurysm, proven by left ventricular angio, there was complete conformity with regard to localisation and extent of the aneurysm between the two methods of investigation. In this group 79% of the demonstrated segments could be adequately assessed and, in 91%, agreed with the radiological findings. In the group of patients without aneurysm only 68% of the segments could be adequately assessed, and 77% correctly assessed wall movement when compared with the laevocardiogram. Tracing recorded from the cardiac apex gave especially good results in demonstrating the ventricle and its segments.


[Resorption, distribution and excretion of the Bulgarian preparation, adepren, in experimental animals]. The authors carried out studies of the original Bulgarian preparation Adepren on male rats Wistar with a weight of 170-200 gm. Single groups of animals were treated singly with venous doses respectively 2,5 and 10 mg/kg of body weight, but other three groups with oral administration of 10, 100 and 200 mg/kg of body weight. The content of the preparation was determined after decapitation of the rats at various intervals of time in plasma, brain, liver and kidneys. Excretion was examined in urine and feces for a period of four days and nights (in metabolic cages). There was a slow resorption of Adepren in the gastro-intestinal tract. The preparation underwent biotransformation in the organism of the rats after oral usage and the basic metabolite was 4-aminocholine, excreted in urine. Adepren and its metabolites were excreted in rats mainly through urine.


[Morphological changes in the myocardium after thymectomy]. The authors followed in dynamics the systolic arterial blood pressure and morphometric changes in the myocardium (cardiac, index, mean number of myocardial cells per unit of area c I mm2, mean diameter of the myocardial cells of the papillary muscles and subendocardial laer of the left ventricle) in thymectomized and sham thymectomized rats. The studies were made after thymectomy and sham thymectomy on the second, sixth, twelfth and sixteenth week. The systolic arterial blood pressure in thymectomized rats was elevated gradually and reached up to 170 mmHg on the sixteenth week, but it was in normal ranges in sham thymectomized animals. The cardiac index and mean diameter of the myocardial cells were larger in thymectomized rats, but the mean number of cells was smaller in comparison with sham thymectomized animals-results showing development of myocardial hyperthrophy after thymectomy. The induced myocardial changes were probably of adaptive character and could be due to neurohormonal mechanisms and hemodynamic loading of the heart becuase of the elevated arterial blood pressure after thymectomy.


[Arterial scleroproteins in atherosclerosis and hypertension (experimental studies)]. The authors examined neosynthesis of fiber proteins (scleroproteins) in the aorta of rats with genetic hypertonia and with experimental atherosclerosis after application of 3H-proline and 3H-lysine and subsequent determination of radioactivity of collagenous and elastic in the aortic wall. There was a great increase in incorporation a labelled precursors of collagen and elastin in the aorta of hypertonic and atherosclerotic animals in comparison with the control rats-a manifestation of increased "de novo" synthesis of fiber proteins in rats with these arterial diseases. Furthermore the increased collagenosis dominated over that of elastogenesis. The irregularity in the activation of biosynthesis of both sclero-proteins in rats with hypertonia and atherosclerosis caused remodeling of macromolecular structure of the aretrial wall with a predominance of collagen over the remaining components of the connective tissue matrix. The resulting fibrosis of the arterial wall favoured the fixation of hypertonia and progression of atherosclerosis.


[Changes in the transverse striated musculature during treatment with the pesticide, Neguvon, and TMB-4 reactivator]. The authors found that the contractile activity of isolated myofibers as-well as superprecipitation of myosin B were reduced after intoxication of white rats with 1/2 of dose of LD50 of neguvon. The reaktivator of cholinesterase TMB-4 (20 mg/kg) recovered the contractile capability of myofibres even on the third day after treatment, but superprecipitation of myosin B-after the twentieth day.


[Disturbances in the processes of division and differentiation of the spermatopoietic epithelium in chronic Ramrod poisoning]. The authors examined histomorphologic changes in the spermatopoietic epithelium in white rats during a chronic a 4-and 6-month intoxication with the herbacide ramrod, used in doses of 1/20, 1/100 and 1/200 of LD50. The authors found that the preparation, known as moderate toxic pesticide, inhibited spermatopoesis at the phase of the formation of spermatids, caused disorganization of seminal epithelium and occurrence of irregular dividing forms. There was dystrophic-degenerativ changes in sex tissue during longer intoxication as well as occurrence of multinuclear gigantic cells. Disturbance in the kynetics of the mitotic division of spermatogonium and blockade of meiosis at its early phases were found as well. The authors assumed that damages in spermatopoiesis were due to distrubed metabolism of the whole organism. An important role was given to the vascular changes, which induced secondary enhancement of the metabolic disturbances in the gonads.


[Change in ion transport across the epithelium of the frog skin isolated from the body]. The authors carried out measurements of ionic transport through the epithelium on preparations of isolated and nonisolated skin by the method of Ussing and Zerahn (short-circuiting technique). The blood and nervous connection with the body was preserved in some of the skin preparations (nonisolated) during the experiment. For the purpose the two chambers of Ussing were modified in such a way that this connection could not be interrepted becuase of compression of blood vessels and nerves. Comparative measurements showed that isolation of the skin from the body of a frog caused significant reduction of active transport of ions through the skin epithelium. After addition of acethylcholine, eserine or dipterex to the solution of the internal or external surface of the preparation the nonisolated skins revealed statisticaly not proven tendency to greater reactivity.


[Method of determining tissue renin activity using heterologous serum]. The authors described a method for determination of tissue renin activity with heterologous substrate. The preparation of the substrate was performed at several stages: salting with amonium sulfate; dialisis of the precipitate till complete separation of amonium sulfate molecules; distruction of angiotensinases by interchangeble souring and alcalization of the medium; lyophylization of the pure substrate. The obtained renin-substrate was preserved in ampules and its usage had a series of advantages--duration, economic, a possibility for standartization of the determination, etc., which were described in details in the article. The described in details also the quantitative determination of the renin activity in the tissues (renal and cerebral) with the help of the obtained substrate as the moments, modiied by the authors, were indicated.


[Pharmacokinetics of the original Bulgarian preparation, tempidon, in experimental animals]. The authors carried out a pharmacokynetic study on the Bulgarian preparation Tempidone in rats. They examined resorption, distribution and excretion of the preparation in urine, bile and feces of rat after venous and oral administration. The experiments were carried out on 207 white male rats of the Wistar strain, weighting from 160 to 180 gm. Blood samples were obtained at various intervals from the onset of venous administration of the preparation in a dose of 100 mg/kg of body weight. After oral administration of the dose of 125 mg/kg of body weight in addition to blood samples other samples from brain, liver, stomach and intestines (small and large) were obtained as well. Excretion of Tempidone through the bile was examined after intraperitoneal administration of 250 mg/kg of body weight. They proposed pharmakynetic models after venous and oral administration of Tempidone. The obtained results gave evaluation for the rate, with which the distribution processes in the organism of rats occurred. Pharmacologic action of Tempidone coinceded in time with the creation and support of effective plasma concentrations. The wide distribution and preservation of Tempidone in the tissues suggested that it underwent the so called tissue binding.


Characterization of cholinergic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system. Acetylcholine and nicotine stimulated vasopressin (VP) release from the organ-cultured rat hypothalamo-neurohypophyseal system (HNS). Nicotinic antagonists, hexamethonium, tetraethylammonium chloride, and trimethaphan blocked VP release in response to acetylcholine and nicotine. A muscarinic agonist, methacholine, was ineffective in eliciting VP release from HNS explants at a molar concentration equal to the maximally effective concentration of acetylcholine (10(-5) M). Atropine, a muscarinic antagonist, was an ineffective blocking agent for acetylcholine. These data indicate that the cholinergic receptor in the HNS explant is nicotinic rather than muscarinic in character.


Steroid metabolism by the lung: conversion of dihydrotestosterone to 5 alpha-androstan-3 alpha, 17 beta-diol by rat lung tissue in vitro. The potential of lung tissue of adult male rats metabolize dihydrotestosterone (DHT) in vitro was examined. Within 3 min, a homogenate of 100 mg lung tissue, cleared of blood by perfusion before homogenization, metabolized 90% of the [3H]DHT substrate. Appoximately 80% of the DHT was converted to 5 alpha-androstan- alpha, 17 beta-diol. The amount of 5 alpha-androstan-3 alpha, 17 beta-diol formed during a 5-min incubation increased linearly, with substrate concentrations ranging from 3.3 x 10(-8) to 3 x 10(-6) M. Thus, the capacity of rat lung tissue to metabolize DHT in vitro and the rate of 3 alpha-reduction of DHT are sufficiently great to consider the possibility that lung may be responsible for the rapid clearance of DHT from the circulation in this species.


Early effects of thyrotropin on ribonucleic acid transcription in the thyroid. Previous studies on the effects of TSH on the incorporation of labeled precursors into RNA have demonstrated an increase in the percentage of label in the phosphorylated nucleotide precursors of RNA. To avoid this nonspecific effect of TSH, we chose to measure RNA transcription in a system which uses exogenous phosphorylated precursors. Isolated nuclei were prepared from the thyroids of dogs which had been injected 90 min earlier with TSH. TSH caused a mean increase of 168% above control in total transcriptional activity, 193% in polymerase II-mediated (alpha-amanitin-sensitive) activity, and 155% in RNA polymerase I- and III-mediated (alpha-amanitin-resistant) activity. To determine whether part of this early effect of TSH was due to increased activity of RNA polymerase, incubations were also carried out in the presence of actinomycin D, which blocks transcription of endogenous template but does not inhibit transcription of added synthetic template poly deoxyadenylic-deoxythymidylic acid (dA.dT). Under these conditions, about half of the TSH-induced increase in the transcription of endogenous template could still be detected. Thus part of the early effects of TSH on RNA synthesis appears to be mediated through increased polymerase activity as well as enhanced template activity.


Effect of human chorionic gonadotropin and prolactin on 20 alpha-hydroxysteroid dehydrogenase activity in granulosa cells of immature rat ovary. The effects of FSH, hCG, and PRL on the activity of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) of separate ovarian components of hypophysectomized, diethylstilbestrol-treated rats were studied. Enzyme activity was found to reside mainly in granulosa cells. FSH induced an increase in enzyme activity. A preparation of FSH was purified by adsorbing its LH contamination on rat corpora lutea membranes and by further neutralizing LH traces with an antiserum to the beta-subunit of LH. This purified FSH retained the ability to induce a 6-fold increase in specific enzyme activity in granulosa cells of hypophysectomized, diethylstilbestrol-treated rats. Administration of hCG to rats treated with purified FSH, further enhanced 20 alpha-SDH activity in granulosa cells up to 11.5-fold above control. PRL, which is known to inhibit 20 alpha-SDH activity in regressing rat corpora lutea, suppressed the FSH-induced increase in enzyme activity in the granulosa cells.


Prolactin and zinc effects on rat vascular reactivity: possible relationship to dihomo-gamma-linolenic acid and to prostaglandin synthesis. Ovine PRL at low concentrations potentiated pressor responses to norepinephrine and angiotensin in an isolated perfused rat mesenteric vascular preparation. Higher concentrations inhibited these pressor responses. Pressor responses to potassium which depend on extracellular calcium entry into the muscle were unaffected by PRL at any concentration. Either cortisol or lithium could completely block the PRL effect. Dihomo-gamma-linolenic acid (DHGL) and prostaglandin E1 had effects similar to those of PRL in that they potentiated norepinephrine responses at low concentrations, inhibited at high ones, and had no effect on potassium responses. Arachidonic acid and prostaglandin E2 potentiated both norepinephrine and potassium responses and had no inhibitory effects at high concentrations. Neither lithium nor cortisol blocked the effects of DHGL or arachidonic acid. Zinc had actions similar to those of PRL and DHGL, but which could be blocked only by lithium and not by cortisol. These results are consistent with the concept that PRL increases synthesis of the 1 series of prostaglandins by mobilizing DHGL. They provide further evidence that zinc may play a role in some actions of PRL.


Increases in mitochondrial steroidogenesis after short term incubation of porcine ovarian follicles with luteinizing hormone. [4-14C]Cholesterol side chain cleavage, progesterone synthesis, and cytochrome oxidase activities were measured in mitochondria from unincubated and short term incubated large (8-10 mm) follicles isolated from porcine ovaries. Compared to the activity of mitochondria from unincubated follicles, specific [4-14C]cholesterol side chain cleavage activity in mitochondria from follicles incubated with LH (0.05 microgrogram/ml) did not change significantly after 12 h, but increased almost 2-fold after 18 h and 5-fold after 24 h. Also, specific mitochondrial progesterone synthesis activity increased dramatically after incubation of follicles for 24 h with LH. In comparison, mitochondria prepared from follicles incubated without LH showed no significant change in specific [4-14C]cholesterol side chain cleavage or progesterone synthesis activities after 18 h of incubation. While both of these activities increased after incubating follicles 24 h without LH, the values were significantly lower than those observed for preparations from follicles incubated with LH. In contrast to these changes in mitochondrial steroidogenesis, specific cytochrome oxidase activity in mitochondria did not change after incubation of follicles without or with LH. It is concluded that incubation of follicles with LH stimulates the development of mitochondrial steroidogenesis but initially does not affect some components of the respiratory chain.


Progesterone receptor in the hypothalamic cytosol of female rats. A progesterone (P)-binding component with a sedimentation coefficient of 8S has been demonstrated in hypothalamic cytosol from ovariectomized, estrogen-primed rats using both [H]P-containing sucrose-glycerol gradient analysis and dextran-coated charcoal adsorption of the preisolated 8S component. The association rate constant (K+1) was determined to be 1.90 +/- 0.38 (SD) X 10(6) M-1 min-1 at 0-2 C. The dissociation rate constant (K-1) was 1.86 x 10(-2) min-1, as calculated from the half-dissociation time [37.0 +/- 7.3 (SD) min]. The apparent dissociation constant (Kd) at 0-2 C was determined to be 6-10 nM by Scatchard plot analysis of data obtained from either direct [3HA]P binding or competition of the [3H]P binding by nonradioactive P and by calculating from K-1/K+1. The 8S binding component was protein in nature, and the concentration of binding sites was 12 fmol/mg cytosol protein. On a per U cytosol protein basis, the relative capacities of the specific 8S binding components were: uterus greater than pituitary greater than hypothalamus greater than hippocampus/amygdala greater than cerebral cortex. Competition studies showed a high specificity for P and 5 alpha-dihydroprogesterone. Corticosterone (C), although competing for the binding, had an affinity 8-fold less than P. Implantation of C in adrenalectomized, ovariectomized, estrogen-implanted rats suppressed the 8S binding of [3H]C without affecting the [3H]P binding. The binding of [3H]P to the cytoplasmic 8S component of hypothalamus was greater than that of combined hippocampus and amygdala, while the reverse was observed for the binding of [3H]dexamethasone. These results demonstrate in rat hypothalamic cytosol a tissue and hormone-specific, high affinity, 8S progesterone-binding protein which has many of the properties expected of a hormone receptor.


The testicular estrogen receptor system in two strains of mice differing in susceptibility to estrogen-induced Leydig cell tumors. Cytosol obtained from cryptorchid testes of tumor-susceptible BALB/c and resistant C3HBi (Z) mice both bound 17 beta-estradiol (E2) and diethylstilbestrol (DES) specifically. The dissociation constant (Kd) of this binding component (RE) for E2 was determined to approximate 5 x 10(-9) M. Gel filtration of cytosols resulted in a significant increase in the binding constant (Kd approximately 3 x 10(-10) M) with the majority of the complex migrating in the 7-8S area after sucrose gradient centrifugation. Incubation of either untreated or gel-filtered cytosol with [3H]DES resulted in considerable nonspecific binding appearing in the 4S region in a low salt sucrose gradient. This 4S binding of [3H]DES was not inhibited by the addition to the incubation mixtures of a 100-fold excess of either E2 or DES, while the lesser peak at 7-8S as well as the major 7-8S peak formed with E2 were inhibited by both. In vitro translocation of the cytosol RE to the nucleus was demonstrated in both mouse strains using either estrogen. Quantitation of the in vivo translocation, employing the exchange method after a single injection of 2.5 micrograms E2/mouse, revealed a rapid increase in cytoplasmic receptor content accompanied by a concomitant increase in nuclear receptor content. Greater nuclear receptor content was identified in nuclei from BALB/c mice than in those from Z animals 45 min after injection of E2. The binding behavior of E2-RE complexes to nuclei was studied by the KCl extraction method. The percent extracted from the nuclei in the Z strain was significantly greater than that in the BALB/c at all concentrations of KCl tested. Essentially 100% of the RE was extracted from nuclei of Z animals at 0.4 M KCl, while nuclei of BALB/c mice retained 35-40% even in 2 M KCl. Cross-over experiments in a cell-free system suggested that the difference in binding was due to differences in chromatins rather than in nuclear  estrogen-receptor complexes. The greater nuclear receptor content and stronger binding of nuclear receptor to chromatin might explain why estrogen-induced phenomena, including neoplastic transformation occur to a much greater degree in the BALB/c strain than in the Z strain of the mouse.


The availability of cortisol in amniotic fluid to the fetus and chorionic and amniotic membranes. This study examined whether cortisol, present in amniotic fluid (AF), could reach the fetal blood, and might be available to various fetal organs and to the fetal membranes and placenta. [3H]Cortisol ([3H]F) was injected into the AF of five sheep at 130--143 days gestation. Samples of AF and fetal blood were collected for 120 min, at which time the fetuses were delivered and fetal organs and fluid were collected for measurement of the [3H]F content. [3H]F disappeared from AF with an initial half-life of about 40 min and was detected in fetal blood by 30 min after intraamniotic injection. At 120 min after injection, [3H]F was widely distributed within the pregnant uterus, and the highest concentrations of radioactivity were found in allantoic fluid, fetal membranes, and placental tissue. The concentration of [3H]F in the umbilical vein was higher than in umbilical arterial blood in four of five sheep. [3H]F and [3H]cortisone were found in several fetal tissues, including adrenal, kidney, pancreas, lung, and liver. [3H]F in tracheal and stomach fluid was generally less than in fetal blood. We conclude that 1) cortisol can reach the fetus from AF, 2) a major route of entry may be via the vasculature of the fetal membranes, 3) a considerable proportion of [3H]F is retained by the fetal membranes, and 4) interconversion of [3H]F and [3H]cortisone may occur in some fetal tissues.


Relationship of insulin binding to amino acid transport by cultured 14-day embryonic chick heart cells. The characteristics of insulin receptors were studied in cultured embryonic chick heart cells which demonstrated insulin-responsive amino acid transport. Binding of [125I]iodoinsulin was time dependent, reversible, saturable, species specific, and proportional to cell number. Optimum binding occurred at pH 7.8 in the presence of 0.1% bovine serum albumin. Curvilinear Scatchard plots were found for chicken and bovine insulin binding at 15 and 30 C. Equilibrium association constants (Kas) and maximum capacities were calculated based on a two-receptor model. When studied at 15 C, chicken insulin was bound with Kas of 5.0 and 0.026 nM-1 for the high and low affinity receptors, respectively. Bovine insulin bound with Kas of 2.1 and 0.03 nM-1. The binding capacities of 600 and 9000 molecules/cell for the high and low affinity receptors, respectively, were the same for both species of ligand. At 30 C, the Ka of the high affinity chicken insulin receptor interaction decreased to 1.6 nM-1, whereas the low affinity Ka was not changed. In competitive binding assays, chicken insulin was 4 and 250 times more potent than bovine and guinea pig insulin, respectively. Human GH and mouse epidermal growth factor did not compete with chicken insulin. Although mouse epidermal growth factor did not compete with insulin, it did stimulate 2-aminoisobutyric acid accumulation. The maximal stimulation by this hormone was less than and additive to that produced by the maximal stimulation of insulin. These data indicate that insulin and epidermal growth factor acted through different receptors to stimulate amino acid transport. When insulin-stimulated functional response was compared to calculated receptor occupancy in this two-receptor model, stimulation of amino acid transport paralleled occupancy of the low affinity receptor such that at half-maximal transport stimulation, approximately 50% of these receptors were occupied. Half-maximal stimulation of 2-aminoisobutyric acid transport occurred at 18- and 20-nM concentrations of bovine and chicken insulins, respectively. Guinea pig insulin failed to stimulate 2-aminoisobutyric acid uptake even at 350 nM. Both the affinity of binding in the low affinity class and the insulin concentration for half-maximal stimulation of 2-aminoisobutyric acid transport were independent of temperature in the temperature ranges studied. We conclude that 14-day embryonic chick heart cells possess at least two classes of receptors which bind insulin. Occupancy of the lower affinity class of insulin receptors correlates quantitatively with insulin stimulation of 2-aminoisobutyric acid transport.


Development of glycoprotein hormones and their alpha- and beta-subunits in bovine fetal pituitary glands. I. Quantitation of thyrotropin, follicle-stimulating hormone, and luteinizing hormone by radioligand receptor assays. The development of TSH, FSH, and LH in bovine fetal pituitary glands was studied by the respective radioligand receptor assays. The content of pituitary TSH increased gradually in fetuses of both sexes throughout gestation, and no significant difference was observed for the pituitary concentration of TSH between male and female fetuses at different stages of gestation. The pituitary content of FSH increased gradually in male fetuses, but increased dramatically in female fetuses after midgestation. Similarly, the concentration of pituitary FSH was 2- to 3-fold higher in female than male fetuses after midgestation. The pituitary content of LH increased gradually and proportionally with the age of the fetus, and no significant difference was observed between sexes. However, the concentration of pituitary LH remained relatively constant for both sexes throughout gestation.


Plasma vasopressin in conscious goats after cerebroventricular infusions of angiotensins, sodium chloride, and fructose. Using a sensitive RIA, the levels of plasma arginine vasopressin (pAVP) were determined from jugular venous blood of conscious goats given cerebroventricular (c.v.) infusions of angiotensins, saralasin, NaCl, and fructose. In hydrated goats, c.v. angiotensin II (0.1--1.0 microgram) caused a dose-dependent rise of pAVP, drinking, and antidiuresis. The same responses were obtained after angiotensin III (1.8 microgram) and hypertonic NaCl (0.5 M), but the effect on water intake was less striking. [des1,2]Angiotensin II hexapeptide and isotonic NaCl (0.15 M) failed to affect these variables. In nonhydrated goats, there were no changes in drinking, diuresis, or pAVP after c.v. infusions of saralasin (5.0 microgram) and isotonic NaCl (0.15 M). Fructose (0.3 M) infusions lowered the pAVP, apparently by reducing the cerebrospinal fluid (CSF) Na+ concentration, while the renal free water clearance turned positive. Angiotensin III thus carries the minimal structural requirements for pAVP release via central nervous receptors in the goat. Lack of a saralasin effect suggests that, in the nonhydrated goat, angiotensin II may not regulate pAVP via receptors accessible to the CSF. Sodium-sensitive cells monitoring the Na+ concentration of the CSF seem to control the pAVP.


The effect of hypothyroidism on growth, serum growth hormone, the growth hormone-dependent somatomedin, insulin-like growth factor, and its carrier protein in rats. To study the possible mechanisms involved in growth retardation associated with hypothyroidism, serum T4, GH, the GH-dependent somatomedin, insulin-like growth factor (IGF), and its carrier protein (CP) were measured in hypothyroid rats and their age-matched controls. Three groups of rats were studied: infant, immature, and adult. Marked hypothyroidism (serum T4, less than 1 microgram/dl) was produced in experimental animals by providing them with drinking water containing 0.05% propylthiouracil. Infant and immature hypothyroid rats weighed markedly less than normal controls and had significantly reduced serum levels of GH, IGF, and CP. Normal adult rats, treated with propylthiouracil for 60 days, also weighed considerably less than control animals and exhibited a significant drop in serum GH, IGF, and CP during this period. The administration of bovine GH to hypothyroid adult rats for 7 days did not restore either IGF or CP levels to normal, indicating that their decrease in serum was, in part, a direct result of hypothyroidism per se. These results indicate that serum levels of GH, IGF, and CP are at least partly under thyroid hormone control. Furthermore, these studies suggest that the growth retardation associated with hypothyroidism may be mediated through somatomedin activity.


Glucocorticoid receptors in thymocytes of fetus, newborn, and adult CBA mice. [3H]Dexamethasone binding was studied in vitro in cell suspensions of thymus from fetal and newborn mice. The number of glucocorticoid receptors appeared to be identical in fetal, newborn, and adult CBA mice. The affinities of these receptors, calculated by Scatchard analysis, were similar in the three groups of animals. The extent of in vitro steroid-induced inhibition of [3H]uridine incorporation by fetal and adult thymocyte suspensions was very similar. These results suggest that no significant variations in glucocorticoid receptors occur in thymic tissue during the neonatal period.


Delineation of the glucocorticoid-sensitive period of intestinal development in the rat. Jejunal sucrase has been used as a marker for intestinal development. The effects of sequential adrenalectomy and sequential administration of hydrocortisone have led to the conclusion that the glucocorticoid sensitivity of the jejunum ceases abruptly at a postnatal age of 17--18 days. Adrenalectomy on day 17 or earlier resulted in significant depression of the usual developmental rise of sucrase activity, whereas adrenalectomy on days 18, 21, or 28 or in adults had no effect on sucrase activity. In contrast, the effect of adrenalectomy on body weight was similar to all ages studied. When hydrocortisone (50 microgram/g BW) was administered to intact animals on day 15 or 16, it caused significant elevation of sucrase activity but, when administered on day 17, 18, or 28, there was no difference between control and treated animals. Since adrenalectomy on day 15 delayed weaning, it was possible that the glucocorticoid dependence of the younger animals was mediated by effects on feeding behavior. However, a further study showed that complete prevention of weaning did not depress sucrase activity between days 15--21. Thus, at early ages, when the tissue was sensitive to glucocorticoid, it was independent of dietary regulation. In contrast, at later ages (days 25 and 27), prevention of weaning caused significant depression of jejunal sucrase activity.


Mechanism of action of a new antialdosterone compound, prorenone. Two new aldosterone antagonists, K-prorenoate [potassium 3(17 beta-hydroxy-6 beta, 7 beta-methylen-3-oxo-4-androsten-17 alpha-yl)propionate] and prorenone [3(17 beta-hydroxy-6 beta, 7 beta-methylen-3-oxo-4-androsten-17 alpha-yl) propionic acid gamma-lactone], its lactonic form, were studied in rat kidney using in vitro systems. Study of [3H]prorenone binding by a recently developed computer method indicated a high affinity, low capacity class of sites which are, seemingly, mineralocorticoid receptors. In competition experiments performed on [3H]aldosterone- and [3H]dexamethasone-binding sites, prorenone appeared to be a good competitor for mineralocorticoid-binding sites and a poor competitor for glucocorticoid-binding sites. The specificity of this molecule was further confirmed by its poor ability to displace [3H]dihydrotestosterone from rat prostate androgenic receptors compared to spironolactone [3-(3-oxo-7 alpha-acetylthio-17 beta-hydroxy-4-androsten-17 alpha-yl) propionic acid gamma-lactone]. In the same experiments, K-prorenoate demonstrated a very low affinity for the two types of receptors. The behavior of [3H]prorenone cytosolic complex was also studied in kidney mince experiments, which showed that the [3H]prorenone complex was not able to translocate into the nucleus. Prorenone inhibited the binding of [3H]aldosterone to the receptor and, consequently, the nuclear binding of aldosterone was not observed.


Circannual variation and genetic regulation of hepatic testosterone hydroxylase activities in inbred strains of mice. Hydroxylation of testosterone by hepatic microsomes at positions 6 alpha, 6 beta, 7 alpha, 15 alpha, and 16 alpha has been studied in C57BL/6J, 129/J, and A/J mice. Differences in hydroxylase activities between the C57BL/6J and A/J strains and between the C57BL/6J and 129/J strains were investigated using standard genetic breeding protocols. In the C57BL/6J X A/J line, 6 alpha- and 7 alpha-hydroxylase activity appeared to be under polygenic control in both sexes, as did 15 alpha-hydroxylase activity in females. The lack of 16 alpha-hydroxylase activity observed in 129/J females behaved as a recessive, autosomal, single locus, sex-limited trait. In several instances, the level of hydroxylase activity at one position appeared to be affected by the level of hydroxylase activity at a second position; however, no clear-cut pattern was discerned. Over a period of a year, a remarkable cycle in total hydroxylase activity for all mice was observed; the average activity was greatest in December and least in April.


Antipsychotic drugs inhibit prolactin release from rat anterior pituitary cells in culture by a mechanism not involving the dopamine receptor. Bromocriptine, a dopamine agonist, inhibited secretion of PRL and did not affect GH release from rat anterior pituitary cells in culture. The reversal of this inhibition of PRL release by butaclamol, a dopamine antagonist, was stereospecific; 10 nM d-butaclamol completely reversed the inhibition caused by 10 nM bromocriptine, while l-butaclamol had no effect at concentrations up to 10 microM. However, both enantiomers at 10 microM inhibited PRL release to 30% and GH release to 91% of control values. Two other dopamine antagonists also inhibited hormone release. Haloperidol (10 microM) inhibited PRL release to 23% of control values and did not affect GH release; 3.3 microM pimozide inhibited PRL and GH release to 18% and 38% of control values, respectively. These data indicate that, the inhibition of PRL by antipsychotic drugs is not mediated through the dopamine receptor.


Purification and characterization of rat relaxin. Two highly purified forms of relaxin, designated CM1 and CM2, were obtained from rat ovaries collected on day 20 of gestation. The isolation procedure consisted of aqueous extraction, followed by fractionation with Sephadex G-50 and ion exchange chromatography. The yields of CM1 and CM2 were approximately 140 microgram/geq ovarian fresh tissue. No difference in biological potency between CM1 and CM2 was found when they were bioassayed with mouse pubic symphysis bioassays. Physicochemical analyses indicated that CM1 and CM2 were similar but not identical. The molecular weights of CM1 and CM2 were approximately 6000, as determined by ultracentrifugation. Analytical acrylamide disc gel electrophoresis at pH 4.3 demonstrated that CM1 and CM2 had different electrophoretic mobilities. Electrofocusing indicated the isoelectric points of CM1 and CM2 were pH 7.6 and pH 9.4, respectively. The amino acid compositions of CM1 and CM2 were similar but not identical. Slab gel electrophoresis in polyacrylamide gel with sodium dodecyl sulfate showed that both reduced rat relaxin and reduced porcine relaxin migrated farther than their unreduced forms. This observation supports the view that rat relaxin, like porcine relaxin, consists of two chains linked by disulfide bonds.


Development of a homologous radioimmunoassay for rat relaxin. Highly purified rat relaxin has been radioiodinated to specific activities of approximately 100 micro Ci/microgram with the Bolton and Hunter reagent [N-succinimidyl 3-(4-hydroxy-5-[125I]iodophenyl) propionate]. A rabbit antirat relaxin serum, applicable in a final dilution of 1:100,000, was developed in a rabbit using unconjugated highly purified rat relaxin. A specific and precise double antibody RIA for rat relaxin sufficiently sensitive to routinely measure from 32--2000 pg rat relaxin was developed. Using this RIA, relaxin immunoactivity levels in extracts of pregnant rat ovaries were found to rise from 0.8 microgram/geq ovarian fresh tissue on day 8 of pregnancy to 723 microgram/geq ovarian fresh tissue on day 20 of pregnancy and then to drop precipitously to 6 microgram/geq ovarian fresh tissue on day 1 of lactation. Consistent with the occurrence and relative levels of relaxin in the ovarian extracts, levels of relaxin in pregnant rat serum were less than 2 ng/ml on day 10 of pregnancy, approximately 150 ng/ml on days 20 and 22 of pregnancy, and 12 ng/ml on day 2 of lactation.


Effect of lithium on bone metabolism in organ culture. The effects of lithium on bone metabolism were determined using different organ culture models for the study of resorption and collagen synthesis. At concentrations of 3 X 10(-3)--10(-2) M, lithium blocked the stimulation of 45Ca and lysosomal enzyme release by parathyroid hormone (PTH) but did not affect bone resorption in control cultures in the absence of PTH. Lithium inhibition was observed in continuous culture with PTH or when bones were exposed to both agents for only 8 h. Lithium was less effective in inhibiting resorption of bones pretreated with PTH, and pretreatment with lithium did not prevent subsequent stimulation of bone resorption by PTH. Lithium did not inhibit the incorporation of labeled proline in collagen and noncollagen protein or of labelled thymidine into DNA.


Thiourea and cyanamide as inhibitors of thyroid peroxidase: the role of iodide. Thiourea, methylmercaptoimidazole, propylthiouracil, and thiouracil are all potent inhibitors of thyroid peroxidase (TPO)-catalyzed iodination. Unlike the cyclic thioureylenes, thiourea at 5 mM has no effect on guaiacol oxidation. If iodide is added to guaiacol assays containing thiourea, enzyme activity is lost. The latter observation may be explained as follows. In the presence of iodide, the iodinating species [TPO.Ioxid], oxidizes thiourea to formamidine disulfide. This product decomposes to cyanamide at neutral pH. We have shown cyanamide to be an inhibitor of the peroxidative and iodinating functions of TPO. Studies in rats demonstrate that doses of thiourea which completely inhibit in vivo protein-bound iodine formation have no irreversible effect on TPO, as measured by guaiacol peroxidation after removal of the thyroids. The major in vivo action of cyanamide is similar to that of thiourea. The data suggest that the primary in vivo and in vitro mode of action of thiourea is the reversible Ioxid-trapping mechanism. The anomalous inhibition of guaiacol peroxidation seen in the presence of thiourea plus iodide derives from the formation of formamide disulfide, followed by its nonenzymic decomposition to cyanamide.


Interactions of gonadotropins with corpus luteum surface membranes. V. Differential effects of digitonin on the buoyant densities of light and heavy rat ovarian membrane fractions. Previous studies have indicated that rat luteal cells at certain stages of development can be fractionated so as to obtain two plasma membrane fractions with different densities and different profiles of marker enzymes. The light membrane fractions (density 1.13) contain the majority of hCG-binding sites and little or no cyclase enzyme, while the heavy membranes (density 1.17) contain the majority of cyclase enzyme and lesser quantities of hormone-binding sites. These membrane fractions were further compared with respect to their susceptibility to perturbation by digitonin. The buoyant density of luteal cell light membrane fractions, as marked by [125I]iodo-hCG binding, Mg2+-dependent ATPase, and 5'-nucleotidase, were highly perturbable by digotonin (delta density, greater than 0.05), while adenylate cyclase activity and phosphodiesterase activity associated with this fraction were only slightly perturbed (delta density, less than 0.02). The buoyant density of luteal cell heavy membrane fractions, as marked by adenylate cyclase, ATPase, and nucleotidase, was not significantly perturbed by digotonin. The hCG binding associated with the heavy membrane fraction was not perturbed by digitonin. From these studies, we conclude that the adenylate cyclase activity associated with light membrane fractions is due to contamination by heavy membranes, while the hCG-binding activity in heavy membrane fractions is intrinsic to that membrane. Except for the lysosomal marker (glucuronidase), which was solubilized by digitonin, the detergent had no significant effect on the density of mitochondrial, Golgi, GERL (Golgi, endoplasmic reticulum, and lysomal), or endoplasmic reticulum membranes. Plasma membranes from isolated granulosa cells and ovaries obtained 24 h after priming with PMS gonadotropin-hCG behaved as heavy membranes (density, 1.17) which contained hCG-binding sites, adenylate cyclase, nucleotidase, and Mg2+-dependent ATPase. These were not significantly perturbed by digitonin. The appearance of light membranes and the segregation of adenylate cyclase from the majority of hCG-binding sites is a development feature of the luteal cell.


Effect of prostaglandins (F2 alpha, E1, and E2) on blood pressure and oxytocin-induced intramammary pressure responses in rats. In lactating rats, vasoactive prostaglandin (PG) doses of F2 alpha (4 and 8 microgram/kg), E1, and E2 (2 and 4 microgram/kg each) reduced the intramammary pressure response to standard iv doses of 300 microU oxytocin by 50--80%. Adrenergic blockers, phenoxybenzamine and/or propranolol (1 mg/kg each sc) did not influence the blood pressure response to PGF2 alpha, PGE1, or PGE2. The oxytocin-antagonistic action of a single iv PGF2 alpha dose (4 microgram/kg) could not be altered by adrenergic blockers. In contrast, the oxytocin-antagonistic effects of PGE1 and PGE2 (2 microgram/kg each) were completely eliminated after alpha-receptor blockade, while the activity of oxytocin was augmented. Under beta-receptor or alpha- and beta-receptor blockade, the oxytocin-antagonistic effects of PGE1 and PGE2 were almost abolished. alpha-Receptor blockade reduced the oxytocin-antagonistic action of infused PGF2 alpha (8 microgram/kg.min for 15 min) by 38%. beta- or alpha- and beta-receptor blockade had no effect. The oxytocin-antagonistic actions of PGE1 and PGE2 (4 microgram/kg.min for 15 min each) were greatly reduced under alpha-receptor blockade. beta-Receptor blockade had no influence on the oxytocin-antagonistic activities of PGE1 or PGE2; under alpha- and beta-receptor blockade, the inhibitory actions of PGE1 and PGE2 were reduced by 60--70%. Mechanisms of PG-induced inhibition of the oxytocin response may involve mammary vasoconstriction and/or alterations in myoepithelial activity of cAMP and cGMP.


The cholesterol side chain cleavage system of the rat adrenal cortex and its relationship to the circadian rhythm. The adrenal mitochondrial cytochrome P450 systems were examined at the low and high points of the circadian rhythm in female rats maintained on a light cycle from 0600--1800 h. The rate of association of cholesterol with the cholesterol side chain cleavage form of cytochorme P450 (P450scc) was measured by light absorption spectrometry. Cholesterol binding to P450scc was as high in rats killed at the beginning of the dark period as it was in stressed rats at the beginning of the light period. Stressing rats at the beginning of the dark period did not result in a further increase in the rate of cholesterol association with P450scc despite a further increase in serum corticosterone levels. This suggests that other reactions of cholesterol metabolism in the adrenal cortex are contributing in a significant way to the increased rate of corticosteroidogenesis seen in the stressed animals.


Somatostatin analogs inhibit somatostatin release. To determine if, like insulin, somatostatin inhibits its own secretion from the pancreas, nonimmunoreactive analogs of somatostatin were perfused in an isolated dog pancreaticoduodenal preparation using a nonrecirculating system. [D-Trp8-D-Cys14]somatostatin, at a concentration of 200 ng/ml, blocked the response of somatostatin-like immunoreactivity (SLI) to cholecystokinin and arginine. When perfusion of the analog was discontinued, SLI release increased. At a concentration of 0.1 ng/ml, des Asn5-[D-Trp8]somatostatin lowered SLI levels significantly without significantly reducing glucagon levels. At a concentration of 1 ng/ml, des Asn5-[D-Trp8]somatostatin significantly inhibited SLI as well as insulin and glucagon release. Perfusion of glucagon at a concentration of 10 ng/ml failed to overcome the blockade of SLI and insulin release caused by 50 ng/ml des Asn5-[D-Trp8]somatostatin. The results are compatible with a direct inhibitory effect of somatostatin analogs upon SLI release and raise the possibility of a self-inhibiting action of the native hormone.


The mineralocorticoid antagonist activity of an 11 beta,18-oxidopregnane. Removal of the 18-hydroxy group of the hemiacetal form of aldosterone transforms its activity from that of pure agonist to predominant antagonist. The 18-deoxy derivative possesses one third of the binding affinity of aldosterone for the cytoplasmic mineralocorticoid receptor of rat kidney and exhibits an approximate 2:1 antagonist to agonist ratio in both toad bladder and adrenalectomized rat bioassay systems. The promising properties of the 11 beta,18-oxidopregnane tested included very low androgen receptor affinity and approximately equal effectiveness in vitro and in vivo in displacing aldosterone from mineralocorticoid-binding sites in the rat.


Aldosterone, deoxycorticosterone, corticosterone, and prolactin changes during the lifespan of chronically and spontaneously hypertensive rats. Male and female spontaneously hypertensive rats (SHR), which develop hypertension spontaneously with maturation, were autopsied at select time intervals from weaning to 28 months. Their blood pressure began to rise steeply at 4--5 weeks, reaching a zenith of 180--240 mm Hg after 4 months. Elevated blood pressures were maintained in both sexes. After 20 months, the male SHR began to die of myocardial infarction and hypotensive crisis. Heart and adrenal gland weight increased progressively not only during the phase of rapidly rising blood pressure but also during the period of plateaued but sustained high blood pressure. RIA of plasma levels of aldosterone, deoxycorticosterone, corticosterone, and PRL, under both quiescent and mildly stressful conditions, demonstrated that the pituitary-adrenal axis of SHR progressively increases its propensity to respond to stress with maturation. This capacity to respond to stress was maintained despite the severe high blood pressure and the attainment of relative old age, i.e. 2 yr. An incremental change in circulating PRL, corticosterone, and aldosterone as early as 2 months of age, when blood pressure levels are beginning to rise, suggests that there may be some connection between the genetically programmed pathogenesis of the spontaneous hypertension and the progressively increasing (with age) sensitivity of the pituitary-adrenal axis to stress.


The ontogony of the human placental glucocorticoid receptor and inducibility of heat-stable alkaline phosphatase. The human placenta was found to contain a cytosol receptor for glucocorticoids. The concentration of this receptor in term placenta was 27-fold higher than that found in cytosol from first trimester placenta. The levels of cytosol glucocorticoid receptor in three trophoblastic cell lines (JAr, BeWo, and JEG) were also determined and all were found to be low. The ability of prednisolone, a potent glucocorticoid, to stimulate heat-stable alkaline phosphatase activity found in these cells was tested. Although control experiments demonstrated that the conditions were adequate to stimulate HeLa cell alkaline phosphatase, none of the trophoblastic lines responded to prednisolone administration. This result may be explained by the observation that the JAr cells lacked any detectable glucocorticoid receptor and the receptor levels in cytosol prepared from JEG and BeWo cells were 12% and 2%, respectively, of those measured in HeLa cytosol. Our studies also suggest that the increase in serum levels of heat-stable alkaline phosphatase observed during pregnancy may reflect increasing placental sensitivity to glucocorticoids as a result of increased receptor levels.


Suppression of vasopressin secretion by clonidine: effect of alpha-adrenoceptor antagonists. Studies were performed in anesthetized dogs to determine if the diuretic effect of clonidine results from inhibition of vasopressin secretion. Intravenous clonidine (30 microgram/kg) decreased plasma vasopressin concentration (as measured by RIA) from 10.9 +/- 1.5 to 5.0 +/- 1.1 ng/ml (P less than 0.01) in association with a transient increase in arterial blood pressure and a decrease in heart rate. Intravenous administration of two alpha-adrenoceptor antagonists, piperoxane and phentolamine, virtually abolished the pressor effect of clonidine but did not prevent the suppression of plasma vasopressin concentration. Clonidine decreased plasma vasopressin concentration from 11.9 +/- 3.1 to 3.3 +/- 1.0 pg/ml in the phentolamine-treated dogs (P less than 0.01) and from 18.1 +/- 4.5 to 12.4 +/- 3.6 pg/ml in the piperoxane-treated dogs (P less than 0.05). These results provide direct evidence that the diuretic effect of clonidine results from inhibition of the secretion of vasopressin. This inhibition does not appear to be a consequence of the pressor effect of the drug but may result from a direct action in the central nervous system.


Immunoreactive somatostatin in rat hypophysial portal blood. Somatostatin levels have been determined by RIA in hypophysial portal blood of pentobarbital-anesthetized male rats. In most animals, immunoreactive somatostatin (SRIF) levels were higher in hypophysial portal blood than in systemic blood. In euthyroid rats, the mean level was 158 +/- 27 pg/ml (n = 8); SRIF was undetectable (less than 30 pg/ml) in systemic blood of these rats. It is suggested that endogenous SRIF was not degraded during the collection of stalk blood, since synthetic SRIF is stable when incubated in rat serum during 4 min at 37 c and 2 h at 0 C, i.e. under the conditions the blood was kept during the collection. SRIF in hypophysial portal plasma had the same immunoreactivity with a specific antiserum against SRIF as did synthetic SRIF. Gel filtration of hypophysial portal plasma revealed two immunoreactive peaks, the major one corresponding to synthetic SRIF, the smaller one representing a larger molecular form. Thyroidectomy and excess of T4 did not modify the levels of SRIF in hypophysial portal blood, suggestinc SRIF is stable when incubated in rat serum during 4 min at 37 C and 2 h at 0 C, i.e. under the conditions the blood was kept during the collection. SRIF in hypophysial portal plasma had the same immunoreactivity with a specific antiserum against SRIF as did synthetic SRIF. Gel filtration of hypophysial portal plasma revealed two immunoreactive peaks, the major one corresponding to synthetic SRIF, the smaller one representing a large molecular form. Thyroidectomy and excess of T4 did not modify the levels of SRIF in hypophysial portal blood, suggesting that the feedback of thyroid hormones on TSH secretion does not involve changes in the secretion of SRIF by the hypothalamus.


Changes in the testosterone to dihydrotestosterone ratio in plasma and testes of maturing rabbits. Testosterone (T) and dihydrostestosterone (DHT) were radioimmunologically assayed in the testes and plasma of rabbits at 1, 10, 20, 40, and 60 days of age and at 3, 4, 5, and 8 months. In the testis (nanograms per 100 mg testis) and plasma (picograms per ml), both hormones are low at birth, reach their maxima between 60--90 days, and then decline and remain low after 120 days. In the testes, the T:DHT ratio is very high from birth (4.2 +/- 0.4) to 60 days (8.8 +/- 1.1). A significant decrease (P less than 0.001) occurs between 60 (8.8 +/- 1.1) and 90 (1.2 +/- 0.1) days. The T:DHT ratio is always less than or equal to unity after 90 days. From 1--60 days, T is the dominant testicular hormone, while the levels of DHT are greater than or equal to those of T after 60 days. The mean value of the plasma T:DHT ratio is always greater than unity from birth (1.7 +/- 0.4) to adulthood (2.6 +/- 0.7). The dominant circulating hormone, at all ages, is T. Our results show that sexual maturation in rabbits is characterized by an inversion of the T:DHT ratio in the testes but not in the plasma.


The interaction between dietary calcium and gonadal hormones in their effect on plasma calcium, bone, 25-hydroxycholecalciferol-1-hydroxylase, and duodenal calcium-binding protein, measured by a radioimmunoassay in chicks. Male chicks, fed low, normal, or high calcium- and cholecalciferol-containing diets for 14 days, were given three combined injections of 17 beta-estradiol and testosterone (7 and 2.4 mg/kg/dose, respectively) or the vehicle alone, at 3-day intervals. The hormonal treatment resulted in increased plasma calcium and medullary bone calcium concentrations, independently of the dietary calcium intake. Kidney 25-hydroxycholecalciferol-1-hydroxylase and duodenal calcium-binding protein were increased in response to gonadal hormones. The magnitude of this response markedly diminished with increased calcium intake and almost completely disappeared in chicks fed the high calcium diet. The results suggest that the increases in plasma calcium and medullary bone formation due to gonadal hormones are independent of calcium intake while the effect of hormones on duodenal calcium-binding protein and the 25-hydroxycholecalciferol-1-hydroxylase activity appears to be mediated through the change in calcium needs due to medullary bone formation.


Effects of calcitonin and other hormones on bile calcium excretion in thyroparathyroidectomized rats. The effects of calcitonin (CT) and other hormones on the bile calcium excretion after a single intraperitoneal administration of calcium (4.0 mg/100 g BW) was investigated in thyroparathyroidectomized rats. Porcine, salmon, and synthetic eel CT (80 MRCmU/100 g BW, respectively) markedly increased the bile calcium excretion in comparison with that of calcium-administered rats. Tetragastrin (7.5 microgram/100 g BW), and insulin (50 mU/100 g BW) did not alter significantly the bile calcium excretion, while epinephrine (100 microgram/100 g BW), glucagon (50 microgram/100 g BW), and parathyroid hormone (25 U/100 g BW) increased significantly. The increasable effect of CT on the bile calcium excretion was significantly prevented by epinephrine (10 and 100 microgram/100 g BW). The present results suggest that the bile calcium excretion may be increased by the hormones which causes the elevation of cyclic AMP in the liver cells of rats.


Chromatin transcription by progesterone-receptor complex in rabbit uterus. This study was designed to investigate the chromatin transcription in vitro by progesterone-receptor complex in the estrogen-primed rabbit uterus. RNA synthesis by the uterine chromatin was stimulated when progesterone-uterine cytosol complex was bound to the chromatin and the stimulation was dependent upon the dose of the bound progesterone-cytosol 8S complex and upon the incubation time of the complex in the presence of the chromatin. Either norethindrone- or dydrogesterone-uterine cytosol complex also stimulated the uterine chromatin transcription but the extent was less than that of progesterone complex. These results indicate that progesterone as well as synthetic progestogens can directly regulate chromatin transcription via progesterone receptor in the rabbit uterine cells.


A case of insulin resistant diabetes with possible antibodies to insulin receptors. A case of a 19-year-old, non-obese female with insulin resistant diabetes mellitus and polycystic ovary syndrome was reported. The maximal insulin requirement attained 360 units per day, but a satisfactory control of diabetes did not follow. The patient's serum contained not only anti-insulin antibodies, but also possible anti-insulin receptor antibodies which were demonstrated by the 125I-insulin binding test using insulin receptors derived from human placental plasma membrane. The insulin resistance in this case was assumed to be caused primarily by possible blocking antibodies to insulin receptors and partly by anti-insulin antibodies because of the following observations. First, high serum free insulin (165 microunits/ml) without hypoglycemia indicates the presence of insulin resistance due to other factors than antiinsulin antibodies. Second, the titer of 125I-insulin binding capacity of serum was not unusually higher than those seen in chronically insulin-treated diabetics. Third, immunologically heterospecies insulin (fish insulin) was also ineffective. The clinical features such as absence of ketoacidosis and association with polycystic ovary syndrome resemble those of an unique diabetic syndrome reported previously though acanthosis nigricans and endogenous hyperinsulinemia were not found in this case. Her insulin resistance remitted spontaneously and over the next 18 months' observation, her diabetes remained regulated without insulin therapy.


Studies on the effect of neonatal hypermetabolism on hypothalamo-pituitary-thyroid axis in adult rats. Neonatal rats which had received a daily injection of 50 microgram of 2,4-dinitrophenol (DNP) or 30 microgram of L-thyroxine (T 4) for 7 days beginning on the day of birth were compared as to the late effect of the hypothalamo-pituitary-thyroid axis with the neo saline control. Neo DNP rats and neo T 4 rats revealed the retardation of growth compared with neo saline rats. The plasma level of TSH in both groups presented its low response following TRH administration. Furthermore, plasma TSH levels following the challenge of PTU were depressed in both neo DNP and neo T 4 rats compared with neo saline control rats. A small dose of T 4 injection, however, did not bring any difference on plasma TSH levels between neo T 4 and neo saline control rats while neo DNP rats showed a little blunted response of pituitary compared with neo T 4 and neo saline rats. Pituitary contents of TSH in neo T 4 rats decreased, but not in neo DNP rats. These results suggest that neonatal hypermetabolism causes the hypofunction of pituitary-thyroid axis through adult life and that the alteration of hypothalamus may be more obvious in neo T 4 rats than in neo DNP rats.


Effects of oral administration of trypsin inhibitor and repeated injections of pancreozymin on the insulin and glucagon contents of rat pancreas. Rats were given soybean trypsin inhibitor or repeatedly injected with pancreozymin (daily 40 I.D.U./kg) for 7 days, and the insulin and glucagon contents of the pancreas were measured. The insulin and glucagon contents were markedly increased after these treatments and this effect was especially conspicuous after injections of large doses (daily 120 I.D.U.) of depot-type pancreozymin. Insulin content thus reached 1.9 times, and glucagon content 2.4 times as much in control values. This result is compatible with our previous histological finding that not only the exocrine pancreas but also islet cells undergo the trophic effect of endogenous and exogenous pancreozymin.


Half life of gastrointestinal glucagon-like immunoreactive materials. The composition, half life and hyperglycemic action of the porcine gastrointestinal glucagon-like immunoreactive materials were examined. Glucagon immunoreactivity (GI) measured using specific antiglucagon serum was more abundunt in the extract from the gastric fundus than in the one from the small intestine. When the extract from the gastric fundus was injected in dogs, the half life (T1/2) of total glucagon-like immunoreactivity (total GLI) measured using nonspecific antiglucagon serum was 9.5 +/- 1.1 min (mean +/- SEM), which was longer than that of crystalline pancreatic glucagon, 3.4 +/- 0.2 min, but shorter than that of the extract from the small intestine, 15.9 +/- 1.3 min. On the other hand, T1/2 for GI from the gastric fundus was 5.1 +/- 0.9 min, which was not significantly different from that of crystalline pancreatic glucagon. Blood sugar levels were significantly increased from the basal by 25 +/- 4 mg/100 ml at 10 min and 19 +/- 4 mg/100 ml at 15 min following an injection of the extract from the gastric fundus, but such a change in blood sugar levels was not demonstrated when the extract from the small intestine was injected. These results suggest that GI of the gastric fundus is close to pancreatic glucagon in respect of its metabolism and hyperglycemic activity.


Human plasma C-peptide immunoreactivity: its correlation with immunoreactive insulin in diabetes, and chronic liver and renal diseases. The correlation between plasma C-peptide immunoreactivity (CPR) and immunoreactive insulin (IRI) was investigated during the oral glucose tolerance test in 20 normals, 127 diabetics, and 39 non-diabetics with chronic liver or renal disorders. When all subjects were included, the increment of CPR 30 minutes after glucose load (deltaCPR) correlated well with that of IRI (deltaIRI) (r = 0.66, p less than 0.001), but the return of CPR towards the basal level was delayed as compared with IRI. The positive correlation was also observed between the sum of 6 IRI and that of 6 CPR values during the glucose tolerance test in diabetics and controls (r = 0.53, p less than 0.001). deltaCPR/deltaBS (30 min.) was also well correlated with deltaIRI/deltaBS (30 min.), and was specifically low in diabetics. Insulin-treated maturity-onset diabetics showed low but considerable CPR responses while no CPR responses were observed in insulin-treated juvenile diabetics. In each plasma sample, CPR always exceeded IRI on the molar basis. At fasting CPR/IRI ratio was 15.6 +/- 1.7 (mean +/- SE) in normals and 14.9 +/- 1.3 approximately 16.9 +/- 1.0 in diabetics. In chronic liver diseases IRI response was augmented while CPR response was not different from that of controls, and the molar ratio of CPR/IRI was significantly low (9.5 +/- 1.1). On the contrary, it exceeded that of normals in chronic renal diseases (35.7 +/- 14.9). It is concluded that, first, the plasma CPR response appears to be a valuable indicator of pancreatic B-cell function, and second, it is, nevertheless, modified in chronic liver or renal disorders.


Studies on the nuclear binding of steroid hormone-receptor complex; binding of liver and thymus dexamethasone-receptor complex and prostate dihydrotestosterone-receptor complex to nuclei from various tissues. To examine the binding specificity of steroid hormone-cytoplasmic receptor complexes to nuclei, binding of 3H-dexamethasone (Dex)-liver, 3H-Dex-thymus and 3H-dihydrotestosterone (DHT)-prostate receptor complexes to nuclei from liver, prostate, thymus, spleen and kidney was studied. It was observed that a significant amount of steroid-receptor complexes was bound to any nuclei used in the present study and the extent of the binding of receptor complexes to nuclei from homologous tissues was not always greater than that to nuclei from heterogenous tissues. However, a significant portion of the 3H-Dex-liver and 3H-DHT-prostate receptor complexes was not absorbed by nuclei from kidney, spleem, and thymus, and the unabsorbed complexes were efficiently bound to liver and prostate nuclei. The results obtained indicate that two types of receptor complex with regard to nuclear binding were present in cytosols of liver and prostate; one binds to nuclei from kidney, spleen, thymus, liver and prostate and the other does not bind to nuclei from kidney, spleen and thymus but does bind to nuclei of liver and prostate. The latter type of receptor complex was not observed in the cytosol from the thymus.


Stability of isoenzymes of alkaline phosphatase in various buffer systems. The stability of isoenzymes of alkaline phosphatase from liver, bones and small intestine was compared after addition to inactivated serum in the buffer systems: glycine, 2-amino-2-methyl-1-propanol, diethanolamine and 2-amino-2-methyl-1,3-propandiol at 37 degrees C. The mentioned isoenzymes were inactivated to different extents in glycine and 2-amino-2-methyl-1-propanol buffers. In diethanolamine and 2-amino-2-methyl-1,3-propandiol buffers sufficient stability of isoenzymes is obtained so that only these buffers are suitable for activity determinations of alkaline phosphatase at 37 degrees C.


Copper regulation of ceruloplasmin in copper-deficient rats. In copper-deficient rats, oral intubation of copper increases the rate of ceruloplasmin synthesis without affecting general synthesis of plasma or liver proteins. It also restores the enzyme from half to full activity. Copper given by injection at doses commonly employed has additional nonspecific effects on protein synthesis and in some strains of rats produces severe hemolysis. In contrast to deficient rats, in normal rats copper does not elevate plasma ceruloplasmin unless hemolysis also occurs. Thus, at least in deficiency, copper availability controls the rate of synthesis, acitvation, and plasma concentration of ceruloplasmin.


Glucose-6-phosphate dehydrogenase from lactating rat mammary gland and R3230AC adenocarcinoma. A number of properties of glucose-6-phosphate dehydrogenase from lactating rat mammary gland and R3230AC rat mammary adenocarcinoma are compared. The main electrophoretic forms of the enzyme from these sources are indistinguishable with respect to charge and molecular weight whereas the minor forms show differences in these properties. The subunit molecular weight and steroid inhibition of the enzymes from the lactating gland and tumor are not significantly different. These results are contrasted with similar studies in mice.


Isatin-enzyme interactions. VII. Mechanism of inhibition of rat kidney alkaline phosphatase. Isatin has been found to inhibit rat kidney alkaline phosphatase (EC 3.1.3.1). The inhibition is dependent on isatin concentration and is of un-competitive type. The hydrolysis of disodium phenyl phosphate by the enzyme at different temperatures (17--37 degrees C) obeys the Arrhenius equation. Energy of activation in the absence and presence of isatin has been found to be 9.84 and 10.24 kCal/mol. The hyperbolic profile of isatin inhibition; the lowering of both Km and Vmax in the presence of isatin, and, small changes in enthalpy, free energy and entropy in the presence of isatin suggest a non-allosteric un-competitive inhibition of the enzyme.


Changes in pyruvate kinase isozymes of rat small intestine during development and the synergistic effect on them of thyroid and glucocorticoid hormones. Pyruvate kinase isozymes in rat small intestine changed markedly during the postnatal period. The activities of type M2 subunits rapidly increased before weaning, while those of type L subunits decreased slightly. The administration of hydrocortisone induced normal changes prematurely, whereas the administration of thyroxine increased their extent. On simultaneous administration, the two hormones had synergistic effects.


Studies on the mechanism of the rate-enhancing effect of heparin on the thrombin-antithrombin III reaction. The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several mechanisms for this effect are possible. To study the problems commercial heparin was fractionated into one fraction of high anticogulant activity and one of low anticoagulant activity by affinity chromatography on matrix-bound antithrombin III. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed immunoelectrophoresis technique. As was to be expected, the high activity fraction was strongly bound to antithrombin III while the low activity fraction was weakly bound. In contrast, thrombin showed equal binding affinity for both heparin fractions. The ability of the two heparin fractions to catalyse the inhibition of thrombin by antithrombin III was determined and was found to be much greater for the high activity heparin fraction. A mechanism for the reaction between thrombin and antithrombin III in the presence of small amounts of heparin is suggested, whereby antithrombin III first binds heparin and this complex then inhibits thrombin by interaction with both the bound heparin and the antithrombin III.


Phase behaviour of cord factor and related bacterial glycolipid toxins. A monolayer study. C-6 esters of methyl alpha-D-glucoside and C-6, C-6' 'diesters of alpha, alpha'-D-trehalose with C18 and C32 threo and erythro mycolic acids (from chemical source) and of C80-erythro-mycolic acid (from natural source) have been synthesized. Esters of a C32 deoxy analogue were prepared as well. Throughout a monolayer study at the air-water interface, these glycolipids are shown to form well organized phases in which the two hydrocarbon chains of mycoloyl residues must be in interaction. Compression isotherms of C32 esters suggested a transition between liquid-expanded and liquid-condensed states. Latent heats Qc and entropy changes delta S associated with these phase transitions as well as the critical temperature at which they occur have been measured. Within the monolayer, the molecular packing of these glycolipids depends on the presence of the hydroxyl group of mycoloyl residues and on its stereochemistry. In particular intermolecular hydrogen bonds between these groups are postulated in the case of the bis(C32-erythro-mycoloyl)-trehalose. On the other hand, short chain C18 esters form fluid phases (t greater than 10 degrees C) whereas very long chain C80 mycoloyl esters of trehalose exist in a condensed state (t = 20 degrees C). These glycolipids were found to interact strongly with dipalmitoylphosphatidylcholine and egg yolk lecithins (3-sn-phosphatidylcholine). Their phase behaviours are discussed in connection with hypotheses concerning the way they can interact with mitochondrial membranes.


Fluctuations in proline and other free amino acids during the mitotic cycle of the myxomycete Physarum polycephalum. 1. During synchronous growth of the acellular slime mould Physarum polycephalum the free amino pool had two maxima, one of 650 units [nmol/plasmodium dry weight (mg)] at metaphase and the other of 780 units in mid G2 with minima of 550 units before and after mitosis. 2. Proline formed 20--25% of the total pool with aspartic acid, glutamic acid, threonine, valine, leucine, lysine and arginine making up 55% of the pool. 3. The fluctuation of proline during the mitotic cycle was quite different from that of the other amino acids and was transiently very low during telophase.


Purification and characterization of 3,4-dihydroxyphenylalanine decarboxyase from pig kidney. A procedure for 3,4-dihydroxyphenylalanine decarboxylase from pig kkdney purification is described in detail. The preparation has no detectable impurity on electrophoresis and on ultracentrifugation and authors. However two significant differences are observed: a different stimulation of activity by added pyridoxal 5'-phosphate and a nearly complete decarboxylation of L-3,4-dihydroxyphenylalanine in absence of added coenzyme. Absorption, fluorescence and circular dichroism properties of the coenzyme-apoenzyme interaction are also described. The results are consistent with the existence of at least four coenzyme-apoenzyme complexes, three of them active.


The polysomal proteins of L cells. Discrimination between the structural ribosomal proteins, the exchangeable ribosomal proteins and the non-ribosomal proteins by two-dimensional dodecylsulfate electrophoresis and autoradiography. Three groups of proteins can be clearly discriminated in the total protein of L cell polysomes by selective labelling in the presence of low doses of actinomycin D and two-dimensional polyacrylamide/dodecylsulfate gel electrophoresis followed by autoradiography: (a) structural ribosomal proteins which are not labelled in the presence of actinomycin D and form stained non-radioactive spot in gels; (b) exchangeable ribosomal proteins which are labelled in the presence of actinomycin D and stained radioactive spots; (c) non-ribosomal proteins which are detectable only by autoradiography of gels. The large and small subunits of L cell ribosomes contain respectively 45 and 34 ribosomal proteins with molecular weights less than or equal to 50 000; seven of the large subunit proteins and nine of the small subunit proteins are exchangeable. Most of the non-ribosomal proteins migrate in the region of the related to the separation of the ribosomal proteins of mammalian cells and the possible significance of the presence of non-ribosomal proteins in polysomes are discussed.


Crystal structure analysis of the tetragonal crystal form are preliminary molecular model of pig-heart citrate synthase. The crystal structure of pig heart citrate synthase was analyzed at 0.35-nm resolution. Chain tracing was possible and an initial molecular model constructed. The dimensions of the dimer molecule (located on a crystallographic diad) are 7.5 x 6.0 x 9.0 nm. The chain folding is characterized by the predominance of helices and the absence of sheet structure. The electron density accounts for 355 residues per monomer, so that about 80 residues must be disordered in the crystal. The disordered segment in probably N-terminal. The ordered part consists of two closely associated domains, a large domain with 300 residues and a C-terminal domain of 55 residues consisting of 3(anti)parallel helices. The large domain is built from 12 helical segments, some of which are buried in the interior of the molecule. Inhibitor binding studies with citrate and CoA revealed citrate binding sites but showed no electron density for CoA. It is suggested that CoA binds to the disordered, flexible N-terminal domain. Experiments of limited proteolysis with trypsin showed that under conditions a segment of Mr 9000 is cleaved off selectively. The remaining 35 000-Mr part is dimeric.


Identification of the amino acid residue modified in Bacillus stearothermophilus alcohol dehydrogenase by the NAD+ analogue 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine. 4-(3-Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl-14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme-coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C-labelled coenzyme analogue and subseqeuntly carboxymethylated with unlabelled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys-38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analogue. This residue is homologous to Cys-43 in yeast alcohol dehydrogenase and Cys-46 in the horse liver enzyme but, unlike the latter two, Cys-38 is not reactive towards iodoacetate in the native bacterial enzyme.


Purification and characterization of a chymotrypsin-like enzyme (protease-S) in thermally injured rat skin. Protease-S was extracted from thermally injured rat skin, and partially purified by column chromatography using Sephadex G-50, CM-Sephadex (A-50), Sephadex G-75 gel filtration. The optimum pH of this enzyme was 8.6--8.8, and the molecular weight determined by Sephadex G-75 gel filtration was approximately 30 000. This enzyme is active on the N-acetyl-L-tyrosine ethyl ester, N-succinyl-L-phenylalanine-p-nitroanilide (of chymotrypsin substrate) but not N-tosyl-L-arginine methyl ester, N-benzoyl-L-arginine-p-nitroanilide. Also, protease-S was completely inhibited by diisopropylfluorophosphate (1 mM) or phenylmethylsulfonylfluoride (10 micrometer), and N-tosyl-L-phenylalanine chloromethylketone (1 mM). These results are very similar to those obtained with bovine chymotrypsin. But the enzyme is not identical with the chymotrypsin-like proteases in mast cells and leukocyte granules. When proteases-S was measured during the inflammatory reaction in vivo, maximal activity was found after 8 h, at the end of inflammation.


The complete amino-acid sequence of histone H2B from the mollusc Patella granatina. 1. From the marine mollusc, Patella granatina, a histone has been isolated. Its primary structure has been established and it has been designated histone H2Bpatella. It consists of a polypeptide chain of 121 amino acids. 2. In the carboxy-terminal two thirds of the molecule there is a highly degree of sequence homology to the corresponding region in calf histone H2B with identical residues in 95% of the positions. 3. In the N-terminal 22 amino acids histone H2Bpatella differs considerably from the mammalian histone H2B and it is shorter by four residues.


The effect of Mg2+ on some properties of nucleotide-free elongation factor Tu from Bacillus stearothermophilus. The nucleotide-free elongation factor from Bacillus stearothermophilus provides a means to study the effect of Mg2+ ions on various reactions of the protein. The binding of GDP to the protein is stimulated by Mg2+. From comparative studies with other metal ions, particularly Mn2+, it appears that this stimulation is due to the formation of a metal - GDP complex which is bound to the protein. Protection against proteolysis by trypsin is afforded by both Mg2+ and Mg - GDP, but not by GDP alone. The rate of substitution of the sulphydryl group associated with aminoacyl-tRNA binding, either 5,5'-dithio-bis(2-nitrobenzoic acid) or N-ethylmaleimide is reduced in the presence of Mg2+ - All these observations show that Mg2+ not only is involved in GDP binding but also has a direct effect on the tertiary structure of the protein.


Property and activity of mycoloyl esters of methyl glucoside and trehalose. Effect on mitochondrial oxidative phosphorylation related to organization of suspensions and to acyl-chain structures. Methyl alpha-D-glucoside and alpha, alpha'-D-trehalose acylated on their primary hydroxyl group by alpha-alkyl-beta-hydroxy acids (mycolic acids) with 18, 32 and more than 80 carbon atoms were studied. These cord factors and analogues when dispersed in water gave hydrated and organized phases which bore resemblances and differences to phospholipid liposomes. Phase transition temperature was determined by fluorescence for the C32 derivatives and by differential scanning calorimetry for the C80 derivatives. A narrow temperature interval between the gel and the fluid states was observed, indicating that this transition was a strongly cooperative phenomenon. The activity on mitochondria of these diacyl trehaloses was studied with succinate as substrate. The fluid suspensions were more active than the rigid ones. There was no influence of the relative configuration of the mycoloyl residue. Acyl chains with 18, 32 and more than 80 carbon atoms had rather similar activities on phosphorylation, but differences appeared when considering effects on respiration. Electron microscopy suggested that cord factor weakened the peripheral part of the inner membrane, but cristae looked unchanged. These observations, together with the results of our previous monolayer studies of these glycolipids, led us to propose a model for interaction of cord factor with mitochondria.