Polymyxin resistance in Agrobacterium tumefaciens and its effect on crown gall tumor induction.

Polymyxin-resistant (PBLr) mutants of Agrobacterium tumefaciens A6, B6, and B6M were isolated from polymyxin-sensitive (PBLs) parent strains in a defined medium containing 600 microgram of polymyxin B sulfate per millilitre. The weight and number of tumors induced by PBLr mutants on a variety of host plants such as carrot, potato, and pinto bean were 45--75% less than those induced by PBLs wild types. The crude cell envelopes (CCE) prepared from both PBLs and PBLr bacteria were inhibitory for tumor initiation when they were applied before or during the inoculation of viable tumorigenic bacteria, but not when they were applied 30 min after the inoculation of infectious bacteria. The potency to inhibit the tumor initiation by the CCE prepared from PBLs cells was approximately 50% higher than that by the equal amount of the CCE prepared from PBLr cells. The concentration of CCE preparations required to reduce tumor induction 50% in carrot and pinto bean was determined to be 2.6 mg/mL and 4.0--6.2 mg/mL for the CCE derived from PBLs and PBLr cells, respectively. These data suggest that the envelope structure or composition of PBLs and PBLr cells is distinct, and that the acquisition of resistance to polymyxin by agrobacteria modifies envelope structure or components which are essential for tumor initiation.


Cyclic adenosine 3', 5'-monophosphate in Neisseria gonorrhoeae.

Intracellular cyclic adenosine 3', 5'-monophosphate (cAMP) was measured in two laboratory strains of Neisseria gonorrhoeae. Decreasing the glucose content of a defined media from 33 mM to 5.5 mM glucose resulted in an 11-to 25-fold increase of intracellular cAMP.


Infectious bursal agent vaccination of chicks from infectious bursal agent-vaccinated dams.

Chicks from infectious bursal agent-vaccinated broiler breeders were vaccinated with a commercial infectious bursal agent vaccine at intervals after hatching. Bursas from some of these chicks were examined for infectious bursal agent-specific fluorescence four days after vaccination and bursas from others were examined for histological lesions of infectious bursal disease 21 days after vaccination. Serological studies were conducted to determine if active immunity to infectious bursal agent followed vaccination. Chicks failed to develop immunity if their levels of maternally-derived serum neutralizing antibody were in excess of approximately log(2) 7 at the time of vaccination. When antibody titres fell below this level, vaccination usually resulted in infectious bursal agent virus replication in the bursa and consequential bursal damage but was followed by development of active immunity.


Tumor promoter 12-O-tetradecanoylphorbol-13-acetate is not a prostaglandin E-type agonist.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), proposed to fit a prostaglandin type receptor, does not compete with [3H] prostaglandin E1 (PGE1) for the established PGE1 receptor of fat cells.


Simultaneous production of casein and mammary tumor virus in mouse mammary epithelial cells grown on floating collagen gels.

Simultaneous production of casein and mammary tumor virus (MTV) was analyzed in monolayer cultures of mammary epithelial cells from pregnant BALB/cfC3H mice. A comparison of the 2 cell culture substrata, plastic culture dish and floating collagen gel, showed that the latter supported a much higher degree of simultaneous casein and MTV production in the presence of insulin, cortisol and prolactin in serum-free culture medium. Importance of floating collagen gel was further shown by delaying the flotation of gels. When the release of gels was delayed, there were concomitant delays in the increase of casein and MTV production. These results indicate that hormones, nature of substratum and flotation regulate the degree of differentiation of mammary epithelial cells in vitro.


Initiation-promotion skin carcinogenesis: inhibition by cyclic and non-cyclic nucleotides.

The effect of nucleotides on initiation-promotion skin carcinogenesis in Swiss mice was investigated. Cyclic AMP was given before initiation with DMBA, between initiation and promotion, and at the same time as promotion with croton oil. Cyclic AMP was more effective in inhibiting tumor development when injected at the same as promotion with croton oil. 5'-adenosine-monophosphate (5'-AMP) and cyclic GMP were as effective as cyclic AMP in inhibiting tumor development under these conditions. However, adenosine, dibutyryl-cyclic AMP and 5'-guanosine-monophosphate (5'-GMP) were ineffective.


Dissociation between cardiac cyclic AMP and myocardial contractility induced by verapamil, calcium and magnesium ions.

Isolated, perfused rat hearts were used to study the effects of verapamil, excess calcium ions or excess magnesium ions on changes in heart cyclic AMP and myocardial force of contraction. Verapamil caused a dose-dependent decrease in force of contraction and a nondose-related reduction in cyclic AMP. Perfusion of hearts with medium containing 5 mM calcium produced a significant rise in cyclic AMP, but no change in contractile force. The depressant effects of verapamil on contractility and cyclic AMP were reversed by 5mM calcium; excess calcium in the perfusion fluid also containing verapamil prevented the myocardial depressant effects of verapamil. Acutely elevating the magnesium concentration in the perfusion medium decreased force of contraction, accentuated the negative inotropic effect of verapamil, but did not decrease cyclic AMP or enhance the verapamil-induced reduction in cyclic AMP.


[Oncological cytodiagnosis of the mammary gland].

Oncological mammary cytodiagnosis concerns a secretion, imprint, and punction. It is an integral and unsubstitutable part of the team cooperation in the counselling and prophylactic medical activity. Its pros are made use of in the field of prebioptic selection. Together with clinical and X-ray examination it forms a diagnostic whole which can be used in active prophylaxis as a method of screening. The arbiter of suspect and positive findings is biopsy.


The role of antitumor antibiotics in current oncologic practice.

The antitumor antibiotics have thus made a major impact on oncologic practice. The continued search for productive strains of these organisms should be encouraged; in addition, the activity and toxicity spectrum suggests the need for vigorous analogue development. An active anthracycline devoid of cardiotoxicity, a bleomycin with no effect on pulmonary tissue, an analogue of streptozoticin devoid of nephrotoxicity -- these would be advances of inestimable benefit to the cancer patient of the future.


Glycosylated hemoglobin assay and oral glucose tolerance test compared for detection of diabetes mellitus.

We compared the oral glucose tolerance test (I) as evaluated by six commonly used scoring methods and total glycohemoglobin assay (II) with respect to their value in the diagnosis of diabetes mellitus. Depending on the evaluation method used for I, 16.7 to 64.3% of those subjects diagnosed as diabetic or borderline by this test were judged to be normal by II. The best agreement was between II and the Unger evaluation method. High-density-lipoprotein cholesterol, which showed an inverse correlation with II, was decreased in subjects judged to be diabetic by the Unger method. We conclude that the utilization of II measurement as a screening method for diabetes mellitus is consistent with a conservative approach to the diagnosis of diabetes.


Simplified immunonephelometric quantitation of apolipoprotein B in hyperlipoproteinemic serum.

An immunonephelometric procedure for determination of apolipoprotein B in human serum [Clin. Chem, 25, 221-226 (1979)] was modified by using the detergent octadecenylamine-polyoxyethylene, which may bind selectively to the phospholipid moiety of lipoproteins. Preincubation of hyperlipemic serum samples with lipases could then be omitted.


Simple and rapid method for the diagnosis of chronic granulomatous disease, measuring hydrogen peroxide and superoxide anions released from leukocytes in whole blood.

A simple quantitative method to measure release of hydrogen peroxide and superoxide anions from leukocytes using less than 10 microliter blood is described. Whole blood instead of a leukocyte suspension was used. The release of hydrogen peroxide was measured fluorometrically by adding a blood sample to an assay mixture containing homovanillic acid, peroxidase and azide. The release of superoxide anions was measured spectrophotometrically with a dual wavelength spectrophotometer by adding a blood sample to an assay mixture containing cytochrome C. The normal values of the releasing activities are given.


Serum angiotensin converting enzyme activity in patients with chronic renal failure on long term hemodialysis.

Serum angiotensin converting enzyme activity was assayed by a spectrofluorometric method in 19 patients with chronic renal failure on long term hemodialysis and 19 control subjects. Converting enzyme activity was increased in 11 of 19 (58%) patients compared with the controls (p less than 0.005). There was no correlation between serum converting enzyme activity and plasma renin activity or blood pressure in the patients. Possible mechanisms responsible for the increased converting enzyme activity are discussed.


Lipoprotein spectrum analysis of uremic patients maintained on chronic hemodialysis.

It is known that patients maintained on chronic hemodialysis have elevated plasma lipids. In order to establish whether the type of kidney pathology is related to a specific lipoprotein abnormality, we measured plasma lipoprotein in eight patients with glomerulonephritis, two patients with polycystic kidney disease and nine patients that had been surgically nephrectomized. The concentration and composition of plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) in patients were compared to plasma lipoproteins in a control group. In all patient groups, the lipoprotein alterations appeared identical. VLDL were elevated 3 to 4 fold, LDL were decreased 15--35% and HDL were decreased 30--45% when compared to values of our control group. Since no differences in the lipoprotein spectrum were found among the patient groups, it appears that the hypertriglyceridemia of chronic uremia is due to the uremic state per se and is not related to a specific pathology of the kidney.


Properties of a membrane-bound phosphatase activity in normal and abnormal red blood cells.

A neutral, membrane-bound, phosphatase activity was characterized in normal red blood cells, using p-nitrophenylphosphate as substrate. Its specific activity was 1.59 nmol mg-1 min-1. The kinetics were of the Michaelis type: KM,app = 2.5 X 10(-3) M. It was stimulated by K+ and inhibited by ouabain, a behaviour reminiscent of (Na+ + K+)-ATPase. In 10 patients with homozygous sickle cell disease and in 11 patients with unidentified congenital hemolytic anemias, the specific activity was significantly increased. In general, the phosphatase retained Michaelis-Menten kinetics. However, in four patients from the same family with an unidentified hemolytic anemia, the kinetics yielded a biphasic curve instead of a rectangular hyperbola, a change consistent with the existence of an inhibition by substrate excess. From detailed analysis of the curve, the apparent inhibitor constant for pNPP was determined: Ki,app approx. 2.5 X 10(-2) M. This novel abnormality of the red cell membrane might be the distinctive feature of a given type of congenital hemolytic anemia.


Acromegaly due to production of a growth hormone releasing factor by a bronchial carcinoid tumor.

We have studied growth hormone production in a patient with a bronchial carcinoid and acromegaly. The absence of growth hormone from the carcinoid tumour was demonstrated by extraction, cell culture and immunoperosidase techniques. Using a linked perfusion culture system, effluent from the bronchial carcinoid tumour culture stimulated a rapid release of growth hormone from a rat pituitary monolayer. This is the first time evidence of growth hormone releasing activity by a bronchial carcinoid has been demonstrated in a production.


Activation of the alternative pathway of complement by Entamoeba histolytica.

A cytopathogenic effect was observed when Entamoeba histolytica was exposed to human sera from individuals with no clinical history or laboratory evidence of amoebiasis. Absorption studies showed that the effect was not due to natural antibodies. Studies performed using ethylenediamine tetracetic acid (EDTA), cobra venom factor (CoF) and heat-inactivation at 56 degrees C, indicated that the cytopathogenic effect was complement dependent. Furthermore, by using ethylene glycol tetracetic acid (EGTA) and Mg++, zymosan, heat-inactivation at 50 degrees C to destroy the activity of factor B of the alternative pathway, as well as electrophoretic studies with anti-human factor B, it was possible to determine that E. histolytica activated the properdin pathway. Finally, complement determinations indicated that incubation of E. histolytica with normal human serum consumed complement. The diminution in CH50 correlated with a consumption of C3 but not of C1, C4 and C2. It was concluded from these results that trophozoites of E. histolytica activate the alternative pathway of the human complement system.


Epstein-Barr virus-related antibody patterns in ataxia-telangiectasia.

Epstein-Barr virus-related antibody titres were determined in twenty-seven patients with ataxia-telangiectasia (AT) and twenty-two healthy members of their families, in twenty-two patients with other diseases, among them ten with Behçet's disease and ten with various primary immune deficiencies, and fifteen healthy members of their families, and in twenty-three unrelated healthy controls. The AT patients showed an increased incidence (55.6%) of high antibody titres (greater than or equal to 1:320) to viral capsid antigen (VCA), and also a high incidence (48.2%) of antibodies to Epstein-Barr virus (EBV) induced early antigens (EA), but low titres (less than 1:10) of antibodies to the EBV-associated nuclear antigen (EBNA) in 44.2% of the cases. The geometric means of anti-VCA were three-to four-fold higher, and of anti-EBNA six-fold lower, than those of the control groups. The patients with the other diseases did not differ significantly from the controls except for a higher incidence of anti-EBNA titres of less than 1:10 (38.1% vs 4--5%). AT patients with low anti-EBNA titres tended to have more advanced T cell deficiencies than AT patients with moderate anti-EBNA titres, as detected by counts of total lymphocytes and E-rosetting cells, and skin test responses. The results support the hypothesis that a functioning T cell system is required to release EBNA from EBV genome-carrying cells for initial and maintained production of anti-EBNA.


LRC Coronary Prevention Trial: Baltimore.

Intense, initial efforts to enroll 250 individuals in the Coronary Prevention Trial at Johns Hopkins University through referrals from private physicians and from commercial laboratories were largely unsuccessful. Not until a mass-screening effort was initiated in the community were we able to contact sufficient nunbers of potential study candidates. The time needed to acquire the trial candidates was 2.5 times as long as our original estimate, and 35,000 individuals were tested in the process.


Treatment of Cushing's disease with adrenal blocking drugs and megavoltage therapy to the pituitary.

Eighteen patients with Cushing's disease were seen over a 40-month period and considered for treatment by pituitary irradiation and adrenal blocking drugs. Fourteen patients entered the study and each received megavoltage therapy to give a mean dose of 4600 rad to the pituitary over 31 days. Each patient was treated for one (two patients) or two (12 patients) years with one or both of the adrenocortical enzyme inhibitors, metyrapone or aminoglutethmide to suppress cortisol secretion. Doses were adjusted to maintain urinary free cortisol secretion below 300 nmol/24 h. One patient failed to complete the trial. Normal urinary free cortisol excretion and plasma cortisol concentration were maintained after treatment in eight of the remaining 13 patients after therapy. Only one patient required cortisol replacement and normal menstrual function was restored in five of the six women. The remaining five patients relapsed and four were subsequently treated by total adrenalectomy. It was noted that the patients who responded to treatment were substantially younger than the therapeutic failures. It is suggested that this treatment is most useful in the management of younger patients.


Combined total body irradiation and local radiation therapy in oat cell carcinoma of the bronchus.

Use of total body irradiation (TBI) as a systematic therapy has been evaluated in controlling subclinical and established metastases in oat cell bronchus carcinoma. Preliminary results suggest that patients without clinical evidence of distant metastases do better than those with metastases. No patient in this series developed brain metastases. This could be related to TBI but may again be just a coincidence. The booster radiation dose (1000 rad) given to the liver seems to be inadequate in preventing metastases there, as all the deaths were due to liver metastases. In view of this, a higher dose to the liver is recommended.


Non-function in nephroblastoma (Wilms's tumour): a report on the excretory urography of nine cases.

Non-function of a kidney invaded by nephroblastoma has not been extensively documented and may be featured only when a relatively large series is reviewed. It occurred in nine (15.5%) of 58 cases seen over an eight year period. Two of these nine cases had bilateral involvement and one showed curvilinear calcification. The mechanism of non-function appears to be due to replacement of most of the kidney substance by tumour, and this may be augmented by invasion of the renal pelvis causing malignant hydronephrosis. Ultrasonography has become increasingly utilised in confirming the diagnosis of renal tumours, but arteriography may still be the critical examination in the 'unusual' case.


Effects of glucocorticoid deficiency on renal medullary cyclic adenosine monophosphate of rats.

1. The effects of adrenalectomy on the adenylate cyclase--adenosine 3':5'-cyclic monophosphate (cyclic AMP) system of rat renal medulla were examined to evaluate the mechanism of the impaired water diuresis in glucocorticoid deficiency. 2. Concentrations of cyclic AMP in medullary tubules from adrenalectomized rats were significantly higher than in the tubules from control animals both in the presence and absence of antidiuretic hormone. 3. This abnormality was corrected by the treatment in vivo of the adrenalectomized rats with dexamethasone, but addition of this drug to the incubation medium did not abolish the differences in cyclic AMP between tubules from adrenalectomized and normal rats. 4. The activity of adenylate cyclase or cyclic AMP phosphodiesterase in vitro was not affected by adrenalectomy. 5. In glucocorticoid deficiency, the concentration of cyclic AMP in medullary tubules is increased both with and without antidiuretic hormone. This abnormality may render medullary tubules more permeable to water and may underlie the impaired water diuresis in glucocorticoid deficiency.


Clinical trial with testosterone undecanoate for male fertility control.

The newly available orally effective testosterone undecanoate (TU) was investigated as a possible means for male fertility control. One of 7 normal volunteers exposed to 80 mg TU three times a day for 10-12 weeks became azoospermic, the remaining showed slightly suppressed or unaffected sperm counts. The insufficient suppression of spermatogenesis in 6 out of 7 subjects may be due to the fact that testosterone levels are only sufficiently high to suppress gonadotropins for some hours after ingestion of the drug.


Pituitary hormones and amnesia.

Pituitary hormones profoundly influence behavior through direct actions on the brain. One of these behavioral effects is the attenuation of experimental amnesia. Traditionally, amnesia is considered as a "loss of memory." Memory comprises at least 2 stages: input (memory consolidation) and output (memory retrieval). Theoretically, disturbance of either aspect of memory may be the cause of amnesia. Also, it is possible that amnesia is based on a factor or factors not related to memory. Data and theories on amnesia in man were reviewed. Some salient features were mentioned: (1) amnesia can be induced by a variety of agents; (2) amnesia covers periods ranging from seconds to years; (3) amnesia gradients can be established; (4) amnesia is to a large extent reversible. From this survey, it seems possible that amnesia is not a homogeneous phenomenon and that even in one person a disturbance of both memory consolidation and memory retrieval may be produced by one and the same event. Animal studies in general have confirmed these conclusions. We have developed an animal model in order to study the effects of pituitary peptides on amnesia. This model is based on CO2-induced amnesia for a one-trial passive avoidance response in rats. This amnesia could be attenuated by treatment with ACTH-analogs 1 hour before the retrieval test. This anti-amnesic effect of ACTH-analogs was not dependent on the nature of the behavioral response or the amnesic treatment. The vasopressin-analog DGLVP similarly exerted an anti-amnesic effect when injected before the retrieval trial. In contrast to ACTH-analogs, however, it also reduced the amnesia when injected before acquisition. These results suggest that amnesia may comprise a "faulty-consolidation" and a "faulty-retrieval" component, which may be amended by different pituitary hormones. The study of the anti-amnesic activity of peptides therefore not only serves to characterize the nature of the behavioral effect of these peptides but may also prove to be helpful of the unraveling of processes involved in amnesia.


Regional asssignments of the loci AK3, ACONS, and ASS on human chromosome 9.

Experiments are described in which human cells carrying balanced reciprocal translocations involving four different regions of chromosome 9 were fused with a Chinese hamster cell line and the resulting hybrids used to obtain subchromosomal assignments of the loci ASS, AK3, and ACONS. ASS was localized on the distal portion of the long arm of chromosome 9, in the region 9q34 leads to 9qter, and AK3 and ACONS on the short arm, in the region 9pter leads to 9p13.


[Early auditory evoked potentials, triggered by a sine-wave stimulus (author's transl)].

Within the first 10 ms after a sine-shaped sound wave (tone pip) seven small-amplitude potentials can be recorded in persons with normal hearing and normal brainstem functions. These components, in the nanovolt range, correspond to the electrical activity of various pathways of the auditory tract. In accordance with this view the resulting potentials were assigned to the following structures in the region of the periphery and the brainstem: component I corresponds to the cochlea or acoustic nerve (receptor, II to the cochlear nucleus (medulla), III to the upper olive (caudal pons), IV to the lateral lemniscus (rostral pons), V to the inferior colliculus (midbrain), VI to the medial geniculate body (diencephalon), VII to the acoustic radiation (cortex). Clinically well defined lesions of the acoustic nerve and brainstem indicate that there is a close topographical relationship between the clinical localisation and absence or delay of the individual components.


Photosensitive epilepsy in Papio papio as a model for drug studies.

Photically-induced epilepsy in Papio papio provides a convenient model for assessing the efficacy and the acute neurological toxicity of new anticonvulsant drugs prior to clinical trial. Its drug sensitivity is broadly comparable to that of generalized seizures in man. It also shares some specific properties with syndromes of reflex epilepsy in man.


The physiological interpretation of electrical stimulation of the nervous system.

The physiological basis of the responses of cells in the nervous system to electrical stimulation is described by reference to the strength-duration curve and to the characteristics of ion-carriers in the cell membranes. The effects of the electrode systems used to deliver the electrical stimuli are considered briefly, followed by an account of the relative electrical excitability of nervous elements in peripheral nerves, spinal cord and the cellular nuclei of the nervous system. The responses of more complex physiological systems to both peripheral and central electrical stimulation are then considered, with more detailed reference to neural mechanisms involved in analgesia at peripheral, spinal and brain-stem levels.


Functional electrical stimulation in control of motor output and movements.

In patient with damaged upper motor neurones we show the therapeutic effect of electrical stimulation (called FES) of peripheral mixed nerves on the restoration of motor activity and movements. The results of neurophysiological, kinesiological and clinical observations are presented. We discuss the possible mechanisms, especially the spinal ones, which are fundamental for such a rhythmic activity as gait. We discuss them also from the point of view of activation of proprioceptive feedback mechanisms and of achieved sensory reinforcement influencing the spinal reflex mechanisms as well as the preserved supraspinal integrated activity which contributes to the long-term FES effect. The stimulation modes, the control of stimuli in relation to the needs of individual patients (hemiplegia in adults, paraparesis, cerebral palsy in children and multiple sclerosis) as well as the motor deficit are discussed. We conclude that the electronic system used for this purpose represents a functionally active orthotic aid with therapeutic effects.


Polyneuropathy. Facts and fancies.

Some conclusions are drawn from findings in 167 consecutive patients with the ordinary "garden variety" of polyneuropathy; the aetiology was unknown in 15%. Histological findings in sural nerves were related to clinical and electrophysiological abnormalities. In some patients with discrete clinical abnormalities, sensory and motor conduction and amplitudes of evoked sensory and muscle action potentials were normal, whereas the nerve biopsy showed slight but definite abnormalities. The reverse, abnormal nerve conduction and normal histological findings, did not occur. Histological findings were rarely, and electrophysiological findings were not, specific for the aetiology or type of a neuropathy. Thus, neither conduction studies nor conventional or single fibre electromyography can identify the underlying pathology: loss of large myelinated fibres (greater than 7 micrometers) was equally prominent in nerves with de- and re-myelination as in those without them. Paranodal and segmental demyelination in less than 20% of the teased fibres occurred as often in nerves with as in those without disproportionate slowing in conduction. When the recorded conduction velocity was equal to that to be expected from the fibres with the largest diameter, slowing in conduction could be explained by axonal degeneration ("proportionate" slowing, 79% of the nerves). When the recorded velocity was disproportionately slower than that expected from fibre diameter (21% of the nerves), causes other than loss of the largest fibres must be assumed to explain the slowing in conduction. Myelin abnormalities in more than 50% of the teased fibres were found only in nerves from patients with the hypertrophic type of peroneal muscular atrophy and in postgastrectomy neuropathy and can probably explain the marked disproportionate slowing in conduction. The material contained, however, only one patient with acute idiopathic polyradiculoneuropahy. In diabetic neuropathy, segmental demyelination was present in only 8 of 502 teased fibres (9 nerves), remyelination was present in 135 fibres, and could not explain the disproportionate slowing in conduction. The mechanism of disproportionate slowing, when it is not due to demyelination, is still obscure.


Structure-function correlations in myasthenia gravis and a new myasthenic syndrome.

The physiologic hallmark of MG is the small amplitude of the mepp. This can be correlated with a deficiency of postsynaptic AChR. The AChR deficiency is caused by antiAChR antibodies. Antibody dependent, complement mediated lysis of the postsynaptic membrane contributes significantly to the AChR deficiency. The abundance of immune complexes localized at the end-plate correlates with the amount of AChR remaining at the end-plate and with the mepp amplitude. The physiologic hallmarks of the new myasthenic syndrome are a small quantum content of the end-plate potential due to a decreased store of immediately releasable quanta, repetitive response of the muscle to a single nerve stimulus and refractoriness to anticholinesterase drugs. The findings are explained by a marked decrease of the size of the nerve terminal and by the total absence of AChE from the end-plate.


Corticoliberin (CRF) activity of the rat neurohypophysis.

Corticoliberin (CRF) activity of the rat stalk-median eminence region (SME) and neural lobe of the pituitary (NL) was tested in vitro using cultured cells of anterior pituitary. The activity of NL was found to be more than 50% lower than that of SME. The parallelism of dose-response curves suggests that corticotropin releasing substances originating from NL and SME may be qualitatively identical. Thus, neurosecretory cells producing corticoliberin seem to form a continuous neurohemal system consisting of the median eminence, the stalk of the neural lobe.


Basal levels of pituitary ACTH and plasma corticosterone after complete or frontal cuts around medial basal hypothalamus.

Plasma corticosterone level and corticotropin (ACTH) content of the anterior pituitary (AP) were measured after various cuts around the medial basal hypothalamus (MBH). Seven to eight days after placing a complete cut around MBH there was no change in plasma corticosterone or ACTH content of AP; the slight rise in ACTH concentration was offset by the decreased weight of AP. Twenty eight days after a frontal cut there was no change in plasma corticosterone and in bioactive or immunoreactive ACTH concentration of AP.


A case of pituitary adrenocorticotropin-dependent Cushing's syndrome in the horse.

In the horse, a syndrome of hirsutism, hyperglycemia, glucosuria, polydipsia, polyuria, polyphagia, and progressive debilitation has been recognized. Most often the syndrome has been associated with adenomas of the pars intermedia of the pituitary and bilateral adrenal hyperplasia or nodular hyperplasia involving primarily the zona fasciculata. Previously, the syndrome has been ascribed to compression of the hypothalamus by an expanding but functionally inactive pituitary neoplasm. In the present case, with RIA determination of plasma ACTH concentrations, the syndrome was ascribed to pituitary ACTH-dependent hyperadrenocorticism and likened to human Cushing's disease.


Low density lipoprotein receptors in bovine adrenal cortex. I. Receptor-mediated uptake of low density lipoprotein and utilization of its cholesterol for steroid synthesis in cultured adrenocortical cells.

Functioning bovine adrenocortical cells in monolayer culture were shown to obtain cholesterol for steroid synthesis from plasma low density lipoprotein (LDL). When grown in medium devoid of lipoproteins, the cells developed a minimal enhancement in steroid secretion in response to ACTH or cholera toxin. However, when LDL was available, steroid secretion was stimulated 4- to 9-fold. To determine the mechanism for this effect, we used LDL in which the protein component was labeled with 125I and the cholesteryl ester component was labeled with [3H]cholesteryl linoleate. These studies demonstrated that the cells derived cholesterol from LDL by binding the lipoprotein at a high affinity receptor site, internalizing it, and hydrolyzing its cholesteryl esters within lysosomes. The resultant free cholesterol was used for steroid synthesis and also acted to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis within the cell. LDL receptor activity was enhanced several-fold by treatment of the cells with ACTH or cholera toxin. High density lipoprotein, which did not bind to the LDL receptor, was not degraded with high affinity by the cells and did not support steroid synthesis. The current data suggest that the bovine adrenal cortex can obtain cholesterol for steroid hormone secretion from circulating LDL by means of a high affinity LDL receptor pathway. In a subsequent paper in this series, a similar high affinity LDL-binding site is demonstrated in membranes prepared from fresh bovine adrenocortical tissue.


Low density lipoprotein receptors in bovine adrenal cortex. II. Low density lipoprotein binding to membranes prepared from fresh tissue.

Low density lipoprotein (LDL)-binding activity was measured in whole homogenates and membranes prepared from fresh bovine adrenal cortex by an ultracentrifugation assay. The binding site for 125I-labeled LDL in isolated membranes shared the properties of the LDL receptor previously demonstrated in intact monolayers of cultured bovine adrenocortical cells. The amount of high affinity [125I]iodo-LDL-binding activity in the adrenal cortex was 6- to 12-fold higher than in the medulla of the same glands. Large amounts of high affinity [125I]iodo-LDL-binding activity were also present in the ovarian corpus luteum but not in the ovarian interstitium. Lesser amounts of high affinity binding activity were observed in 14 other bovine tissues. These results lend support to the concept that cells in the bovine adrenal cortex can obtain cholesterol for steroid hormone synthesis through the receptor-mediated uptake of plasma LDL.


Effects of diamide on basal and thyrotropin-stimulated thyroid metabolism.

The effect of diamide on basal and TSH-stimulated thyroid metabolism was studied using bovine and dog thyroid slices. Diamide (10 mM) inhibited basal and TSH-stimulated net cAMP production, basal and cAMP-stimulated protein kinase activity, and stimulation by TSH of colloid droplet formation and organification of iodide and abolished basal and TSH-stimulated uptake of 32P into phospholipids. In thyroid slices incubated with 0.1 mM diamide, none of these activities, whether basal or TSH-stimulated, was affected. However, 0.1 mM diamide increased the net basal production of cAMP and potentiated the effect of TSH on this process. These results demonstrate that the previously reported inhibition of protein kinase by 2-20 mM diamide is not specific and that this compound cannot be used to determine which of the metabolic effects of TSH are dependent upon cAMP activation of protein kinase. While 0.1 mM diamide increased cAMP in thyroid slices, it did not reproduce any of the other effects of TSH.


Somatostatin secretion from monolayer cultures of neonatal rat pancreas.

Monolayer cultures of neonatal rat pancreas have been characterized as an in vitro system for studying SRIF secretion. Marked 12- and 6-fold potentiation of SRIF release occurred with N-2-O-dibutyryl cAMP monosodium salt and theophylline, respectively. High glucose (300 mg/dl) stimulated SRIF release, whereas galactose was without effect. Exogenous insulin did not alter SRIF release, and the SRIF responses to theophylline and glucose were unaffected by the addition of antiinsulin serum to neutralize the insulin released by these agents. Arginine evoked a significant 2-fold increase in SRIF release. Exogenous glucagon produced slight but not significant stimulation of SRIF release. However, after exposure of the cultures to antiglucagon serum to diminish the concentration of glucagon in contact with the SRIF cells, exogenous glucagon produced a marked enhancement of SRIF secretion. These data suggest that glucose, arginine, glucagon, N-2-O-dibutyryl cAMP monosodium salt, and theophylline stimulate SRIF secretion, probably by direct effects on D cells or through mechanisms other than increased insulin secretion. Monolayer cultures of rat pancreas should provide a powerful in vitro system for studying pancreatic SRIF physiology.


Inhibition of the pituitary-adrenal response to stress during deprivation-induced feeding.

Plasma corticosterone and plasma and pituitary ACTH concentrations were determined during feeding and after application of an acute stress at various times after food and water presentation to male rats maintained on a restricted feeding and watering schedule. Both plasma corticosterone and ACTH concentrations fell after the presentation of food and water, and this fall was accompanied by increased levels of ACTH in the pituitary gland. In addition, a rise in plasma levels of ACTH was inhibited in response to an acute stress applied at 0--5 min after presentation of food and water, but ACTH synthesis was not. This inhibition of ACTH and corticosterone secretion in response to stress was transient and dissipated as a relatively linear function of the interval between food presentation and application of the stress. The results suggest that this feeding-induced, corticosteroid-independent inhibition of pituitary-adrenal activity involves active inhibitory mechanisms operating initially on ACTH secretory processes of the pituitary and later on the synthesis of ACTH or on the secretion of hypothalamic corticotropin-releasing factor.


Molecular weight forms of adrenocorticotropic hormone secreted by primary cultures of mouse anterior pituitary.

Extracts of mouse anterior pituitary cells in monolayer culture were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate the molecular weight forms of ACTH. The gels were sliced and each segment was eluted. The eluates were assayed for ACTH immunoactivity. Approximately 10% of the immunoactivity in the extracts was found to be present as 20,000--32,000 mol wt ACTH. The remainder of the immunoactivity was equally distributed between two forms of ACTH with apparent molecular weights of 11,800 and 4,500. This distribution is very similar to that found in extracts of mouse anterior pituitary. Mouse anterior pituitary cultures were incubated for 2 h in serum-free tissue culture medium. ACTH was concentrated from the medium and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The medium was found to contain predominantly the 4,500 and 11,800 forms of ACTH. When vasopressin was added to these cultures (100 ng/ml), the rate of secretion of ACTH was more than doubled in serum free medium. Analysis of the medium from vasopressin-stimulated cultures showed that the 4,500 and 11,800 mol wt forms of ACTH were again the predominant forms present.


Mechanism of action of prolactin on adrenocortical steroid secretion in hypophysectomized female rats.

Studies were carried out to determine the actions of PRL on adrenocortical function in hypophysectomized female rats in the presence and absence of ACTH. PRL administration alone decreased 5 alpha-reductase activity but did not significantly affect the rates of corticosterone secretion or peripheral plasma corticosterone concentrations. The activities of several steroidogenic enzymes (cholesterol desmolase, 11 beta-hydroxylase, and 21-hydroxylase) were also unaffected by PRL. Adrenal steroidogenesis was increased by ACTH treatment, as expected, resulting in an increase in corticosterone secretion. However, since adrenal 5 alpha-reductase activity was higher in ACTH-treated hypophysectomized rats than in normal animals with intact pituitary glands, large amounts of 5 alpha-dihydrocorticosterone (DHB) and 3 beta, 5 alpha-tetrahydrocorticosterone (THB) were also secreted. PRL, when administered in combination with ACTH, potentiated the effecte levels. PRL did not affect cholesterol side chain cleavage, 11 beta-hydroxylation, or 21-hydroxylation in ACTH-treated rats. However, administration of PRL to ACTH-treated rats lowered adrenal 5 alpha-reductase activity, decreasing DHB and THB secretion. The decrease in DHB and THB secretion approximated the increase in corticosterone output. The results indicate that, in the presence of ACTH, PRL increases corticosterone secretion by decreasing intraadrenal degradation of corticosterone and not by enhancing steroidogenesis.


Antiestrogen inhibits the induction of progestin receptors by estradiol in the hypothalamus-preoptic area and pituitary.

Estradiol treatment of ovariectomized-adrenalectomized rats produced an increase in cytosol progestin binding in the hypothalamus-preoptic area (HPOA), pituitary, and uterus. In the HPOA and pituitary, this induction of progestin receptors by estradiol was inhibited by the antiestrogen CI-628 under a variety of dose and time conditions. In the uterus, inhibition of the full effect of estradiol on progestin binding was observed after 3 days of injection of antiestrogen and estradiol. In the absence of estradiol, the antiestrogen produced a slight induction of progestin receptors in the HPOA and pituitary and a substantial induction of progestin receptors in the uterus. The results suggest that the induction of progestin receptors could be a key intermediate step in some behavioral and neuroendocrine actions of estradiol.


Inhibitory effects of long term treatment with a luteinizing hormone-releasing hormone agonist on the pituitary-gonadal axis in male and female rats.

Following previous observations of the potent antifertility effects of acute treatment with LHRH or its agonistic analogs in male and female rats, this study describes the effect of long term (up to 12 weeks) treatment with an LHRH agonist, [D-Ala6, des-Gly-NH210]LHRH ethylamide (LHRH-A), on testicular and ovarian gonadotropin receptor levels and function in the rat. Treatment of adult male rats with 100 ng LHRH-A every third day led to a progressive decrease of testis, seminal vesicle, and prostate weight up to 12 weeks of treatment, while the inhibitory effect (70%) on LH receptor levels was already maximal at 1 week. When adult female rats were injected daily with 5 microgram LHRH-A for 12 weeks, ovarian LH, FSH, and PRL receptor levels were reduced 50--90%, while plasma estradiol and progesterone levels were inhibited 60% (compared with diestrus day 1). The effect of chronic administration of a low dose (5 IU) of hCG in the male rat was transient. By contrast, no sign of resistance developed up to 12 weeks of treatment with the LHRH agonist in either male or female animals.


Immunohistochemical localization of adrenocorticotropin in the rat brain.

ACTH and some of its fragments have been shown to play a role in a variety of adaptative mechanisms. To clearly identify the nervous structures containing ACTH in the rat brain, an immunohistochemical localization of this peptide was conducted at both light and electron microscopic levels. Nervous fibers staining for ACTH were found to be largely distributed throughout regions of hypothalamus, thalamus, and midbrain. Positive fibers could also be occasionally observed in the spinal cord. Immunostained neuronal cell bodies were only detected in the arcuate nucleus. Essentially, the same results were obtained 2 and 8 weeks after hypophysectomy. In animals pretreated with colchicine, the intensity in the staining of cell bodies was markedly increased, making possible the detection of a larger number of cell bodies. At the electron microscopic level, it was demonstrated that ACTH is contained in dense core vesicles present in nervous fibers and endings. These results indicate that ACTH of nonpituitary origin is synthesized in the central nervous system and could probably be considered as a neurotransmitter of still undefined function.


Glucocorticoid regulation of prolactin receptors on mammary cells in culture.

Mouse mammary epithelial cultures were examined for the ability to specifically bind [125I]PRL after cultivation on floating collagen gels. Corticosterone, particularly hydrocortisone, were effective in increasing the ability of mouse mammary cells to bind [125I]PRL. The absence of a glucocorticoid in the medium resulted in a loss of PRL binding during the 3 days in culture. 17 beta-Estradiol, progesterone, and aldosterone at equal molar concentration had no or only a small effect in increasing [125I]PRL binding.


Luteinizing hormone-releasing hormone inactivation by purified pituitary plasma membranes: effects of receptor-binding studies.

Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [125I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (approximately 10(-6) M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Gly10, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The enhanced biological activity of the analog, therefore, may be due to its resistance to inactivation by enzymes on the pituitary cell surface. The membrane-associated inactivating enzyme could play an important role in vivo in determining the concentration of intact LHRH available at the receptor site which initiates gonadotropin release.


An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.


Comparative study of cyclic AMP-generation system, steroid biosynthesis and lipid metabolism in vitro in ACTH responsive and unresponsive adrenal tumors.

The responses of the cyclic AMP-generation system and corticosteroids biosynthesis to ACTH and angiotensin II and cholesterol and other lipid contents in adrenal tissues were estimated in the in vitro experiments in 3 cases of Cushing's syndrome due to ACTH-responsive and unresponsive adenomas, one case of Cushing's disease (diffuse hyperplasia), one case of primary aldosteronism and one normal subject. The responses of cAMP accumulation and corticosteroids production to ACTH in in vitro studies were quite in good agreement with the in vivo responses of plasma cortisol by ACTH infusion test. The adenylate cyclase activity decreased and the phosphodiesterase activity increased in the case of hyperplasia and in one case of ACTH-responsive adenoma, whereas the basal cyclic AMP content was slightly more in ACTH-responsive adenoma and maximal in hyperplasia compared with that of the normal adrenal tissue. The characteristic features observed in ACTH-unresponsive adenoma were the largest amount of the basal corticosteroids production and esterified cholesterol content, and the lowest content cAMP. These results indicate that there was not always the consistent correlation between the cAMP-generation system, corticosteroids and aldosterone production, and conversion of cholesterol to pregnenolone by the stimulation of ACTH and angiotensin II in adrenal tumors.


Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.


Liver and kidney glucose-6-phosphatase levels in carbon tetrachloride- and DDT-administered mice.

Mouse liver and kidney glucose-6-phosphatase levels were found to be decreased 24 h after administration of various doses of carbon tetrachloride (CCl4) when compared to controls. Liver glucose-6-phosphatase levels were always decreased to a greater extent than the kidney enzyme in mice given the same amount of CCl4. Administration of p,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl) ethane (p,p'-DDT) to mice did not significantly alter the glucose-6-phosphatase levels of liver or kidney.


Localization of the high-affinity ATP site in adenosine-3':5'-monophosphate-dependent protein kinase type I. Photoaffinity labelling studies with 8-azidoadenosine 5'-triphosphate.

8-Azido-adenosine 5'-triphosphate (n8(3)ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3':5'-monophosphate (cAMP). It competes with ATP for the high-affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction. n8(3)ATP incorporated into the holoenzyme is covalently bound irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high-affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n8(3)ATP is bound to the catalytic subunit. After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [gamma-32P]n8(3)ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low-affinity catalytically active ATP site into a high-affinity inactive site.


Distribution of the messenger RNA coding for the common precursor of corticotropin and beta-lipotropin within the bovine pituitary.

The distribution of the mRNA coding for the common precursor of corticotropin and beta-lipotropin among different parts of the bovine pituitary has been investigated by quantifying the mRNA activity with the use of a cell-free protein-synthesizing system. The results obtained have demonstrated that this mRNA activity is located both in the anterior lobe and in the intermediate lobe, while it is essentially not detectable in the neural lobe nor in the stalk. The structural identity of the translation products of corticotropin/beta-lipotropin mRNA from the anterior and from the intermediate lobe has been indicated by their molecular weight as well as by the electrophoretic patterns of the peptide fragments formed from them upon partial enzymatic proteolysis or upon cyanogen bromide cleavage. The specific activity of corticotropin/beta-lipotropin mRNA in the intermediate lobe is about 20-fold higher than that in the anterior lobe, and the total activity of this mRNA in the former is about 2-fold higher than that in the latter. In the intermediate lobe, the translation product of corticotropin/beta-lipotropin mRNA amounts to almost one-third of the products encoded by total translatable mRNA. These results indicate that corticotropin/beta-lipotropin mRNA represents a major mRNA species in intermediate lobe of the pituitary, thus suggesting that this lobe may perform a highly specialized function in producing a large amount of the common precursor of corticotropin and beta-lipotropin.


Sialic-acid content of low-density lipoproteins controls their binding and uptake by cultured cells.

The (high-affinity receptor)-mediated uptake of homologous low-density (low-rho) lipoproteins by cultured human arterial smooth muscle cells or human skin fibroblasts is controlled by the sialic acid content of low-rho lipoprotein particles. This conclusion is derived from the following results. 1. Gangliosides incubated with native low-rho lipoproteins associate with low-rho lipoprotein particles. Low-rho lipoproteins modified by associated GLac1, GGtet1, and GGtet2b + GGtet3 gangliosides are internalized by arterial smooth muscle cells at a rate up to 80% lower than native low-rho lipoproteins or those preincubated with desialized gangliosides. 2. The inhibitory effect of gangliosides is specific for high affinity uptake and not detectable on skin fibroblasts deficient in low-rho-lipoprotein receptor. 3. Desialyzed low-rho lipoproteins are internalized by smooth muscle cells up to 100% faster than native low-rho lipoproteins, the enhancement of uptake corresponding to the degree of desialization.


Affinity labelling of yeast and liver alcohol dehydrogenases with the NAD analogue 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine.

The NAD analogue 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine inactivates alcohol dehydrogenases from horse liver and yeast by modification of amino acid side chains at the active sites of the proteins. In the presence of excess inactivator the reaction is pseudo first order. The stoichiometry is one male inactivator incorporated per mole enzyme subunit. The liver enzyme is inactivated by ketoalkylation of the essential cysteine residue at position 46. No intermediate reactions of other residues are detected, and added cysteine does not influence the modification. In contrast, the labelling results with the yeast enzyme depend on cysteine treatment. The only radioactive peptide isolated is labelled on the essential cysteine residue 43.


Hydrophobic interactions of cytochrome c oxidase. Application to the purification of the enzyme from rat liver mitochondria.

The binding of rat liver cytochrome c oxidase to phenyl-Sepharose and various alkyl and omega-aminoalkyl agarose gels has been studied. Deoxycholate-solubilized cytochrome c oxidase was tightly bound to hexyl, octyl, omega-aminohexyl, omega-aminooctyl agarose as well as to phenyl-Sepharose. This hydrophobic interaction was used for the purification of cytochrome c oxidase. The enzyme which was eluted from phenyl-Sepharose was devoid of NADH (NADPH)-acceptor reductase activities. The heme a content was 15.4 nmol per mg of protein. The purified enzyme was resolved into seven polypeptides upon polyacrylamide gel electrophoresis in sodium dodecylsulfate with molecular weights of 40,000, 23,200, 21,500, 14,500, 12,600, 8900, and 4900. Antibodies raised in rabbits against the pure enzyme did not cross-react with cytochrome c oxidases from either beef heart or yeast mitochondria. Cytochrome c oxidase bound to octyl-Sepharose or phenyl-Sepharose exhibited a very low catalytic activity. The possible modes of interaction of cytochrome c oxidase with the hydrophobic ligands are discussed.


Tonic foot: clinical and neurophysiological study of a case.

A case of involuntary plantar flexion of the toes and sole with no other neurological pathological sign is reported. The electromyographic activity of some muscles of the involved limb. the behavior of the phenomenon during the sleep-wakefulness cycle and spinal reflex excitability have been studied. According to the clinical and neurophysiological findings, the phenomenon appears to be a dystonia. Such a dystonia may occur as the only pathological neurological sign or together with the other signs of central nervous system involvement. Particular care has to be taken to distinguish 'tonic foot' from hypertonus of sole flexor muscles occurring almost always in all spastic hemipareses.


Sleep electroencephalograms and sleep stages in hypoparathyroidism.

2 patients with hypoparathyroidism, one idiopathic and the other pseudoform, were studied by overnight polygraphic recording of the sleep EEG and related activities before and after start of treatment to investigate their sleep EEG patterns and states, with the results as follows. (1) Conspicuous abnormalities were noted in the sleep record, namely, disappearance of humps, spindles and hill waves, emergence of the mitten pattern and rather monotonous delta rhythm bursts. The EEGs, both awake and asleep, showed findings suggestive of a possible disorder of the desynchronizing mechanism of the brain stem. Normal sleep EEG patterns were restored after the serum calcium level had returned to the normal range. (2) In the course of overnight sleep, disturbance in the cyclic changes of sleep EEG pattern and absence of REM sleep were observed. Restoration to the normal course of sleep was achieved following the normalization of serum calcium level. Divalent calcium ion is considered to have intimate bearing upon the mechanism whereby synaptic chemical transmitters are activated. When viewed with reference to the monoamine hypothesis of Jouvet, the disturbance in the course of sleep observed in the present cases, especially the absence of or disturbed REM sleep, is inferred to be related to the hypocalcemia.


Involvement of pre- and postsynaptic receptors in catecholaminergic control of paradoxical sleep in man.

In an attempt to clarify the roles of pre- and postsynaptic catecholaminergic (CA) receptors in the regulation of paradoxical sleep (PS) in man, the effects of various doses of chlorpromazine (CPZ) and clonidine (CLN) have been investigated. The action of CPZ was biphasic, small doses enhancing and larger doses depressing the production of PS. However, the effect of CLN was monophasic, showing no modification after small doses, and a decrease in production of PS after moderate doses. In addition, a small dose of CLN, ineffective by itself, completely abolished the effect of a small dose of CPZ. These observations can be explained by preferential blockade of presynaptic alpha-receptors by a small dose of CPZ. They show, too, that it is possible to investigate in man the functional consequences of pharmacological manipulation of pre-synaptic receptors on cerebral CA neurons.


Opiate receptor binding affected differentially by opiates and opioid peptides.

The potencies of various opiates in displacing several 3H-opiate ligands' binding to rat membranes vary depending on the nature of the ligand. Whereas opiate antagonists, as well as the opioid peptides and some agonists (etorphine, levorphanol and phenazocine) display similar affinities in displacing either 3H-opiate or 3H-methionine enkephalin binding, other agonists (such as morphine and oxymorphone) are considerably (20-50 times) weaker in displacing 3H-enkephalin than 3H-dihydromorphine binding. These agonists also compete for 3H-enkephalin binding with shallow displacement curves, and are greatly weakened in displacing 3H-naloxone binding in the presence of sodium. These agonists differ from the other opiate classes by possessing a relatively hydrophilic component in their C-ring moieties which may provide a basis for the differential interactions of drugs with the opiate receptor.


Neurotensin: contractile activity, specific binding, and lack of effect on cyclic nucleotides in intestinal smooth muscle.

Nuerotensin contracted the isolated longitudinal smooth muscle strip of the guinea-pig intestine. It induced a biphasic response (relaxation followed by contraction) when the muscle was contracted by histamine. Tetrodotoxin blocked the contraction and the contraction phase of the biphasic response. Dose responses of the relaxing effect of neurotensin and its analogs D-Arg8-neurotensin and D-Arg 9-neurotensin on tetrodotoxin-treated muscle gave EC50 values of 5 nM, 5.5 nM and 110 nM for the three peptides, respectively. [3H]Neurotensin bound specifically to membranes prepared from isolated longitudinal muscle strips of the guinea-pig intestine. In binding studies using [3H]neurotensin and unlabelled peptides, the KDs of neurotensin, D-Arg 8-neurotensin and D-Arg9 neurotensin were 4 nM, 2 nM and 300 nM, respectively, values which are in close agreement with the EC50 of the three peptides. Neurotensin had no effect on the cAMP and cGMP contents in longitudinal intestinal muscle strips of the guinea pig, an observation which suggests that cyclic nucleotides are not involved in the mechanism of action of neurotensin in this muscle preparation.


Classification of cat retinal ganglion cells into X- and Y-cells with a contrast reversal stimulus.

A contrast reversal (alternating phase) stimulus was used to study the responses of 150 retinal ganglion cells from 15 adult cats. Because the majority of the cells did not show perfect linear spatial summation, a ratio of the firing rates at two time periods was used to express the degree of nonlinearity. Y-cells showed a high degree of nonlinearity, and their mean null ratio was significantly lower than that of X-cells. With the stimulus at the null position, X-cells had an unmodulated discharge rate which was significantly higher than maintained activity, while the firing rate of Y-cells was lower than maintained activity. With the stimulus placed at an eccentric position in the receptive field, X-cells responded in a sustained manner, while Y-cells respond transiently. Because of these observations, we conclude that X-cells correspond to the sustained cells, while Y-cells correspond to the transient cells.


Transynaptic transport of procion yellow in the central nervous system.

Intracellular recordings were performed in slices of the lateral geniculate body, the visual cortex, and the hippocampus of guinea pigs. After recording, Procion Yellow was iontophoretically injected. During the injection period the neurones were activated antidromically and/or transynaptically. Up to 6 h after dye injection slices were fixed in a buffered formaldehyde/sucrose solution and later histologically processed. In the lateral geniculate body, after orthodromic stimulation across the optic tract, optic tract fibers were stained secondarily. Orthodromic activation across intrageniculate connections led to secondarily stained geniculate neurones. In the visual cortex orthodromic activation of the injected neurones across white matter led to secondarily stained fibres travelling in the direction to the white matter or to secondarily stained neurones in the vicinity of the injected neurone. In the hippocampus anti- and orthodromic activation across the alveus or the mossy fibre pathway led to a secondary staining of neighbouring pyramidal neurones and to a secondary staining of fibres which where afferent to the injected neurone. All slices where Procion Yellow was detected in the extracellular space were rejected. These findings led to the conclusion that Procion Yellow must have been transported across chemical synapses from the post- to the presynaptic side. The transport occurred only if the concerned synapses were activated.


Alterations of beta-adrenoceptor-density and cAMP-synthesis in rat-erythrocytes after stress erythropoiesis.

During the maturation of red blood cells from rats after stress erythropoiesis, adenyl cyclase activity and beta-adrenoceptor density (pmoles/mg protein) decrease at distinctly different rates suggesting a different turnover of these membrane units.


[Behaviour of the prolyl hydroxylase to collagen synthesis in KB cells].

KB cells, which synthetized collagen at a low rate, shown a prolyl hydroxylase activity at the same rate that fibroblast. The relationship between collagen synthesis and prolyl hydroxylase activity in these cells was discussed.


The detection of superoxide anion from the reaction of oxyhemoglobin and phenylhydrazine using EPR spectroscopy.

The low temperature EPR spectrum of a quickly reacted mixture of oxyhemoglobin and phenylhydrazine was studied. With the use of a computer, the spectral contribution of methemoglobin in the region of g = 2 was  subtracted. The remaining spectrum was that of an axial free radical (g perpendicular = 2.00, g parallel = 2.06) having the magnetic parameters of superoxide anion. In the presence of superoxide dismutase, this axial radical was not seen, confirming that superoxide anion is indeed generated by the reaction.


[Effect of large doses of phenamine after previous administration of 5-hydroxytryptophan and para-chlorophenylalanine on the spontaneous behavior of cats and the effects induced by caudate nucleus stimulation].

In non-immobilized male cats the stereotypy behavior evoked by threshold doses of phenamine was registered cyclographically along with the motion arrest reaction, circular reaction and their electrographic "adjuncts" (cortical caudate-induced spindles and desynchronization). Against the background of 5-Hydroxytryptophan (5-HT), a serotonine precursor, and p-chlorphenylalanine (PCPA), an inhibitor of the mediator synthesis, phenamine produced stereotype in the formerly administered dose, but its picture changed. Under the effect of 5-HT the amplitude of the stereotypy movements declined, they would become better organized, whereas after administration of PCPA they would, on the contrary, assume a chaotic "unshackled" character. The ability of phenamine to upset the behavioral and electrographic characteristics of the caudate nucleus activity was somewhat attenuated by 5-HT and potentiated by PCPA. It is suggested that the release of serotonin in the ventral region of the caudate nucleus intensified by phenamine may be responsible for the limitation of the amplitude and regulating the nature of the stereotype movements evoked by it.


Diet, central nervous system, and aging.

A variety of morphological, structural, and chemical changes have been described in the central nervous systems of aging humans and animals. Brain size and volume decline during senescence, and the brain atrophy is accompanied by changes in the number, size, and ultrastructural characteristics of nerve and glial cells. Moreover, recent evidence suggests that the ability of central nervous system cells to communicate with one another via the release of neurotransmitter compounds might be impaired in the elderly. Nutritional factors may play important roles in the aging process of the central nervous system by influencing brain neurotransmission, or by accelerating or retarding geriatric changes in central nervous system structure.


Prolactin receptors in the ovary.

The binding of prolactin (PRL) to the plasma membranes of bovine and human ovaries was investigated using both homologous and heterologous 125I-prolactin. Saturation and Scatchard analysis demonstrated that human prolactin binds to human ovarian membranes with a Kd of 2 x 10(-10) M; to bovine ovarian membranes with a Kd of 1.9 x 10(-10) M; and to bovine corpora lutea membranes with a Kd of 1.9 x 10(-10) M. The concentrations of binding sites in bovine and human ovaries were 2.9 x 10(-15) moles/mg of protein and 2.0 x 10(-15) moles/mg of protein, respectively. The number of bindings sites in the bovine corpora lutea was 1.5 x 15(-15) moles/mg of protein. Specificity studies with bovine PRL, ovine PRL, human luteinizing hormone, human follicle-stimulating hormone, and bovine growth hormone showed this binding to be specific. Comparison of binding of PRL to membranes of other target and nontarget tissues suggests that the ovary is one of the primary target tissues. These data suggest that prolactin plays a role in the ovarian cycle.


[Denaturation changes in the proteins of the membrane structures of the brain during deprivation of the paradoxical stage of sleep].

24--96-hour REM-sleep deprivation entailed a rise of SH-group content in the proteins of the rat diencephalon and mesencephalon tissue homogenates, whereas SH-group content of the homogenates of the cortex, pons with medulla oblongata, and cerebellum remained unchanged. Neither did the content of SH-groups change in the soluble protein fraction nor the content of low-molecular thiol substances in the brain parts under study. From among the subcellular fractions of the brain-stem homogenate, the increase of SH-group content after 24-hour REM-sleep deprivation occurred in the synaptosomal and nuclear fractions but not in the mitochondrial one. The REM-sleep deprivation did not affect the --S--S-group content in these fractions. In the anterior parts of the brain-stem only REM-sleep deprivation led to conformational changes of structural proteins towards their denaturation but without rupture of the disulfide bonds.


[Effect of blockade of the adrenergic structures of the hypothalamus on thermoregulatory reactions].

The blockade of alpha-adrenergic receptors of medial preoptic area detained the appearance of vascular thermoregulatory reaction and increased the threshold of the organism central temperature. The blockade of beta-adrenergic structures induced opposite effects. The excitatory efferent impulses from the thermosensitive neurons of the hypothalamus to thermogenic effectors and to the vasomotor system seem to be realized through the alpha-adrenergic structures of the brain, and the inhibitory--through the beta-adrenergic structures.


[Changes in muscular thermogenesis in cold adapted rats following beta-adrenoreceptor blockade].

The administration of beta-adrenergic blocking agent propranolol to cold adapted rats entailed some decrease of the total metabolic reaction and body temperature as well as an additive (compensatory) increase of electrical muscle activity. The compensatory effect was more obvious in postural-tonic groups of muscles (m. trapezius, m. masseter) and in deeper portions of muscles (m. tibialis ant., m. trapezius), mainly presented by the red fibers. An adaptive increase of non-shivering thermogenesis and temperature effect of muscular contraction seem to be mainly controlled by beta-adrenergic mechanisms.


[Fabry disease: clinical, biochemical and electron microscopical studies (author's transl)].

Biochemical investigations on blood-plasma and serum as well as well as electron microscopic investigations on skin lesions biopsy specimens were carried out in one case of morbus Fabry 35 years of age distinctly showing cardio-renal symptoms. The diagnosis of Fabry disease was made when the patient was hospitalized for treatment of a chronic glomerulonephritis. The patients' serum alpha-galactosidase activity was reported to be 10 to 13% of normal controls, whereas the values of trihexosyl-ceramids for blood-plasma and serum were increased. Electron microscopic studies revealed that the endothelial cells and pericytes of the cutaneous vessels as well as the fibroblasts contain numerous liposome-like cytoplasmic inclusions of different size showing an internal lamellar structure with exact periodizity of 65 A. These inclusion are not surrounded by a membrane structure, however, it is possible that the arise from defect lysosomes which accumulate trihexosyl-ceramids owing to insufficient of alpha-galactosidase activity.


Twenty-one years of follow-up studies of familial epidermodysplasia verruciformis.

21 years of follow-up study of a family with epidermodysplasia verruciformis (e.v.) have shown that members of one family can be infected with different human papillomaviruses (HPVs), either HPV 3 or HPV 4, and sometimes with both. The clinical picture resembled disseminated flat warts in cases induced by HPV 3, whereas in those caused by HPV 4 there were flat red or red-brownish plaques and depigmented pityriasis versicolor-like lesions. Malignancies developed only in family members infected with HPV 4, whereas the cases due to HPV 3 ran a more benign and slowly progressive or stationary course. There were also abortive and regressive cases, and the 3 children in whom the wart-like lesions did not recur after removal had an unimpaired cell-mediated immunity (CMI). In all cases of e.v., irrespective of the inducing virus, CMI was low, which seems to be an important factor in the pathogenesis of the disease. Humoral antibodies directed specifically against HPV 3 were present in the majority of the cases, mainly those infected with HPV 3.


[Xanthogranuloma in adults. Clinico-pathological study of a case].

The authors report a case of naevoxanthoendothelioma in a 58-year-old woman. 24 cases of naevoxanthoendothelioma in adults have already been reported in the literature. If the age of the patient is considered, nosologic problems may arise, especially with the strictly cutaneous reticulohistiocytosis of Senear and Caro. Biological and ultrastructural features allow the authors to develop some hypotheses about pathogenesis of the disease.


Herpes simplex virus isolation from pyoderma gangrenosum lesions in a patient with chronic lymphatic leukemia.

Herpes simplex virus type 2 was isolated and identified from the vesicular border of pyoderma gangrenosum lesions on the genital region of a patient with chronic lymphatic leukemia. Dramatic relief of pain as well as quick disappearance of the vesicular margin of the lesions and of the inflammatory halo around them occurred as a result of local treatment with a solution of zinc acetate. A careful search for a viral agent should be done in every case of pyoderma gangrenosum occurring in a patient with a hematological malignancy or/and impaired immunity, especially if the lesions are on the face or in the genital region.


Theophylline: biochemical pharmacology and pharmacokinetics.

Theophylline and its derivatives, such as aminophylline, have an established role as bronchodilators, although their mode of action in man is not clear. There is circumstantial evidence that therapeutic doses of theophylline may have a phosphodiesterase inhibiting effect, thus potentiating the effects of cyclic AMP. However, it remains to be established whether this is the primary mode of action of theophylline at the biochemical level. The pathways of theophylline metabolism have been clarified, although most of the enzymes involved have not been characterized. Hepatic microsomal enzyme induction by polycyclic hydrocarbons will increase the rate of theophylline elimination. There are a number of factors which influence theophylline clearance in adults, which is known to be highly variable. These factors include obesity, smoking habit, diet and the presence of such diseases as hepatic cirrhosis, acute pulmonary oedema, cor pulmonale and viral respiratory infection. There is a good correlation between plasma theophylline level and bronchodilator effect. This can be demonstrated at plasma levels as low as 5 microgram/ml, although optimal levels are usually greater than 10 microgram/ml. Unacceptable toxicity usually occurs in association with plasma levels greater than 20 microgram/ml. The maintenance of adequate plasma theophylline levels by the use of a sustained-release aminophylline tablet is discussed.


Failure of severe maternal stress or ACTH during pregnancy to affect emotionality of male rat offspring: implications of litter effects for prenatal studies.

Daily maternal neck restraint, whole body restraint, hyperthermia, and ACTH treatment during the last 3rd of gestation had no reliable effect on open-field and cage-emergence behavior in male Sprague-Dawley offspring. Many of these treatments, however, produced considerable maternal pathology and evidence for maternal adrenocorticoid release. Significant litter effects were found for almost every morphological and behavioral measure. Failure to control for the litter variable may account for many previously reported effects of prenatal stress on emotionality in rats. Female rats showed greater activity and less defecation than males on postpubertal open-field and cage emergence tests.


Insulin receptors in AH-66 ascites hepatoma cells and liver of tumor-bearing rats.

Properties of insulin receptor on plasma membranes isolated from AH-66 ascites hepatoma cells and liver of tumor-bearing rats were studied. Specific binding (total binding minus nonspecific binding) of 125-I-labeled insulin to plasma membranes from AH-66 tumor cells and liver of normal and tumor-bearing rats were 33, 31, and 16% of a fixed amount of labeled insulin (1 x 10(5) cpm) added to each membrane preparation, respectively. Using Nisonoff plot, these membranes were found to possess at least two types of insulin receptors with a high affinity-low capacity and a low affinity-high capacity, as has been shown in normal liver. Total number of binding sites (high affinity plus low affinity sites, 8.4 x 10(-12) mol/mg protein) in hepatoma cells was more than that (7.0 x 10(-12) mol/mg protein) in normal rat liver. However, kinetic constants of binding in receptors of two types on tumor cells and tumor-bearing rat liver were similar to those of membrane receptors from normal rat liver. Insulin receptors of the hepatoma cells were considered to be highly specific for insulin from the results of competition with other peptide hormones. Inhibition of insulin binding with the tumor cell membranes by concanavalin-A, a competitive inhibitor for insulin receptor sites, did not differ very greatly from that of normal liver.


Isolation of a sarcoma virus from a spontaneous chicken tumor.

A spontaneous tumor was obtained from a hen of White-Leghorn stock raised in a local farm in Yamaguchi City, in December 1973. The tumor had characteristics of fibro- or myxofibro-sarcoma and could be maintained by transplantation in chickens of similar flocks. This tumor was proved to produce sarcoma virus at 33rd passage and the transforming virus was designated as Y73 sarcoma virus. Biological studies suggested that the virus recovered from the tumor belonged to avian leukosis-sarcoma complex having subgroup A specificity. The possibility of defectiveness of this virus will be discussed.


Effects of alcohols on the actions of VIP and secretin on acinar cells from guinea pig pancreas.

In dispersed acini prepared from guinea pig pancreas, ethanol inhibited the increase in amylase secretion caused by cholecystokinin, carbachol, secretin, or vasoactive intestinal peptide. Ethanol did not alter binding of [125I] vasoactive intestinal peptide to pancreatic acinar cells or the inhibition of binding cause by secretin or vasoactive intestinal peptide. Ethanol potentiated the increase in adenylate cyclase activity and cellular adenosine 3':5'-monophosphate caused by secretin or vasoactive intestinal peptide. This potentiating action was reversible and could also be detected with straight-chain alcohols having fewer than seven carbon atoms. At sufficiently high concentrations, straight-chain alcohols having more than two carbon atoms inhibited the action of secretin or vasoactive intestinal peptide on adenylate cyclase activity, and this and this action was irreversible.


The ultrastructural localization of transport ATPase in the rat liver at non-bile canalicular plasma membranes.

Na,K-ATPase in rat livers was localized cytochemically at the ultrastructural level. The Ernst technique, a method using p-nitrophenylphosphate (pNPP) substrate, was used to demonstrate ouabain-sensitive, K-dependent phosphatase, an enzyme of the Na,K-ATPase reaction sequence. Reaction product was localized predominantly on the sinusoidal and non-bile canalicular (intercellular) surfaces. This localization contrasts with previous histo-chemical studies using ATP substrate and with models that have considered the transport enzyme to be localized at the canalicular surface. If Na,K-ATPase is of importance in bile salt independent flow, a significant presence of the enzyme at sites other than the canalicular membrane suggests that a paracellular movement of sodium and water into the canaliculus must be considered.


Hepatocellular carcinoma in primary biliary cirrhosis: report of four cases.

Of 98 patients dying with primary biliary cirrhosis only four developed hepatocellular carcinoma. It is suggested that the development of hepatocellular carcinoma is uncommon in this type of chronic liver disease because of its known female preponderance, and the fact that cirrhosis develops late in the course of the illness.


Histochemistry and cytochemistry of 3,3'-diaminobenzidine. A review.

The review presents applications of 3,3'-diaminobenzidine (DAB) to histochemistry and cytochemistry, including demonstration of oxidative enzymes and hemoproteins, tracer experiments, immunocytochemistry and methods based on non-enzymic DAB oxidation. A special attention is paid to methodological problems concerning the specifity and sensitivity of DAB reactions.


Studies on the physiology of pollen and pollen tube growth. IV Eschscholtzia californica Cham.

Eschscholtzia californica stigmas with germinating pollen at different stages of development were the subject of histochemical studies which aimed the localization of several enzymes like phosphorylase, leucine amino peptidase, nonspecific esterase, cytochrome oxidase, aldolase, alpha-glycerophosphate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, monoamine oxidase, alpha-galactosidase, beta-glucosidase and beta-galactosidase. Pollen and pollen tubes were shown to contain starch, lipid, proteins and soluble sugars as the storage products. These storage products were utilized during germination and tube growth. The role of different enzymes in the process of germination and tube growth is discussed. From the distribution of oxidoreductases it is inferred that respiration plays an essential role in the tube growth. During pollen germination probably the reserve proteins were transported to pollen tube tip. The increase of activity of alpha-and beta-galactosidase in pollen tubes indicates on their involvement in carbohydrate metabolism. The role of alpha-galactosidase in the metabolism of galactolipids is also inferred. Similarly, the reaction catalysed by beta-glucosidase resulted in the production of aglycon and glucose; of these the former possibly act as a substrate of peroxidase. Some of the glycosidases diffused out of pollen wall on the stigma and participated in the release of free sugars of the female tissue.


Clinical studies using radiocolloid in chyluria.

Very little radioactive colloid, injected subcutaneously is excreted in urine by the normal kidney. In chyluria patients, however, radiocolloid injected subcutaneously (not venously) is passed through in larger quantities in the urine to the control patients, because of the lymphatic communication with urinary tract. As far as we investigated, measurement of the radiocolloid in urine is a valid clinical test for chyluria patients.


Primary liver tumors in the pediatric age group: an angiographic challenge.

Eleven children, ranging in age from three months to fifteen years, with primary liver tumors were examined by large volume direct magnification arteriography (2.5--3 ml of contrast material per 1 kg body weight for a single selective study). The following tumors were found: hepatoblastoma (1), hepatocellular carcinoma of the adult type, hepatoma (3), infantile hemangioendothelioma (4), hemangiosarcoma (1), focal nodular hyperplasia (1) and cystic mesenchymal hamartoma (1). Some of the tumors have a quite typical angiographic appearance as infantile hemangioendothelioma, hemangiosarcoma, cystic mesenchymal hamartoma. The other highly vascular neoplasms show unspecific signs of vascular malignant tumors; subtile angiographic signs may, however, be present and help in the differential diagnosis.


Effects of adrenalectomy and hypercorticism on the ACTH content of the anterior and posterior pituitary in rats with inherited diabetes insipidus (Brattleboro strain).

The effects of adrenalectomy (Adx) and hypercorticism on the ACTH content in the anterior (AH) and the neurointermediate lobe (NIL) of the pituitary in Long Evans (+/+), heterozygous (+/DI) and homozygous (DI/DI) Brattleboro rats were determined using dispersed adrenal cells bioassay. Adx decreased the NIL-ACTH content in +/DI and DI/DI rats and left it unchanged in the +/+ rats. Adx increased the AH-ACTH content in the three groups. Hypercorticism had a delayed decreasing effect both in the AH and in the NIL in all rats, with one exception for the NIL in DI/DI rats. Conversely to what appeared in Wistar rats, in Long Evans and Brattleboro rats the corticosterone administered in drinking water was unable to reduce the increase in AH-ACTH activity. These data suggest that Brattleboro, and, to a lesser extent, Long Evans rats from which the former are derived present some particularities in the regulation of their corticotropic function at the AH and the NIL level. We also observed that NaCl (0.9%) added to drinking water and hypercorticism are two factors able to increase diabetes insipidus in homozygous rats without modifying the water intake in Long Evans and heterozygous rats.


Effect of chlorphentermine on hormone content and function of the adrenal cortex in rats.

Amphiphilic drugs like chlorphentermine induce a generalized lipid storage disease upon chronic application. The adrenal cortex is among the organs most heavily affected. We therefore determined the urinary corticosterone excretion during the treatment of rats with chlorphentermine and the corticosterone content of the adrenals and its blood level at the end of the treatment period. In addition, the responsiveness of adrenal cortex was tested by application of ACTH. During treatment, the corticosterone excretion declined considerably. Both the corticosterone content of the adrenals and the plasma level were found depressed at the end of a treatment period of 8 weeks. The ACTH evoked response was also diminished. The results indicate that the chlorphentermine-induced lipidosis is associated with a reduced corticosterone production of the adrenal cortex of rats. At present it cannot be decided whether the cortical insufficiency is causally related to lipidotic alterations of the cortical cells, or whether it is caused or additionally influenced by alteration at a higher level, e.g. hypothalamic centers or anterior pituitary.


The carbohydrate moiety of human chorionic gonadotropin: lack of competition with HCG for testicular receptors and anti-HCG-serum.

A glycopeptide fraction has been prepared from human chorionic gonadotropin (HCG) by digesting the reduced, S-carboxymethylated hormone with pronase and fractionating the digest by gel exclusion chromatography. The glycopeptide fraction was estimated to contain (w/w) 29% sialic acid, 31% hexose, 23% hexosamine, and 17% amino acids and/or peptides; thus, the glycopeptide mixture is 83% carbohydrate compared to intact HCG which is about 30% carbohdyrate. There was no cross-reactivity of the glycopeptide fraction with an antiserum directed against HCG. Also, when corrected for minimal non-specific effects, the fraction failed to displace 125I-HCG from a rat testicular preparation even when tested at a 10,000-fold (w/w) excess. Thus, any model involving carbohydrate effects in gonadotropin action must include the protein moiety as a necessary component.


Effect of bromocriptine on the control of plasma aldosterone diurnal variation in normal supine man.

In order to investigate the role of prolactin in the control of the circadian rhythm of plasma aldosterone (PA), plasma renin activity (PRA), cortisol (PC), aldosterone and prolactin (PRL) levels were determined in samples at 4-hour intervals from 5 normal supine men over a period of 24 h under basal conditions and subsequently over a period of 24 h during suppression of prolactin release by bromocriptine (CB-154). After suppression of prolactin, statistically signific1nt circadian rhythms in PC and PA have been detected with a moderate decrease of PA concentration, while the PC level remained unalterated. PRA rhythmicity persisted with a significant shift of acrophase and remarkable reduction of plasma levels. Moreover, during CB administration a significant correlation was obtained between PA and PC, while no correlation was detected between PA and PRA. These data are consistent with the following concepts: (a) the prolactin does not play a significant role in the regulation of circadian rhythm and concentration of plasma aldosterone in normal supine men, and (b) bromocriptine induces a remarkable reduction of PRA and a variable decrease in plasma aldosterone, but it does not influence the secretion of cortisol in normal subjects.


Interaction of aging with in vitro adrenocortical responsiveness to ACTH and cyclic AMP.

In vitro adrenal accumulation of cyclic 3'5'-adenosine monophosphate (cyclic AMP) and release of corticosterone in response to adrenocorticotropic hormone (ACTH), cyclic AMP or theophylline was assessed in 60- and 340-day-old male rats. Adrenal tissue from mature animals secreted significantly smaller quantities of corticosterone in response to ACTH, theophylline or cyclic AMP. Additionally, mature tissue accumulated significantly less cyclic AMP after treatment with ACTH or a combination of ACTH and theophylline. The data suggest an age-related refractoriness of adrenal cortical tissue to ACTH which may in part be related to decreased availability of and/or sensitivity to cyclic AMP.


13C-NMR spectroscopy of human serum high density lipoprotein enriched with labelled phospholipids.

Native human serum high density lipoprotein (HDL) (d = 1.063--1.21g x cm-3) was enriched with phosphatidylcholines labelled with 13C in the polar head group ([N-13CH3]choline) and in the fatty acyl chains ([26-13C]cholesterol) and its linoleic acid ester using the previously described exchange method (Stoffel et al. 1978). The properties of the HDL particles with the exchanged lipid classes were the same as those of the native particles (Mr, CD, fluorescence, lipid and apoprotein stoichiometry, electrophoretic mobility). The T1-times were very similar to those obtained previously with recombined apolipoprotein-[13C]lipid complexes and further support our proposals concerning lipid and apoprotein interactions in the HDL particle.


Response of platelets exposed to potassium tetraperoxochromate, an extracellular source of singlet oxygen, hydroxyl radicals, superoxide anions and hydrogen-peroxide.

When potassium tetraperoxochromate (K3CrO8) is added to platelet suspension media it decomposes to the oxygen species hydrogen peroxide, superoxide radicals, hydroxyl radicals, and singlet oxygen. K3CrO8 induces a reversible shape change and aggregation of human platelets and, in the presence of Tris or sucrose, also the release of serotonin. Its effect on shape change and aggregation is due to the long-lived species hydrogen peroxide and is abolished by indomethacin and acetylsalicylic acid. Superoxide radicals, which are formed from K3CrO8 in HEPES-containing media do not evoke a platelet response. The release of serotonin depends on an interaction of hydroxyl radicals with Tris or sucrose and is associated with excessive formation of thiobarbituric acid-reactive material from platelets. Other scavengers of hydroxyl radicals such as mannitol, dimethylsulfoxide, EDTA or histidine prevent the release and the formation of thiobarbituric acid chromogen. Interaction of hydroxyl radicals with Tris or sucrose most likely results in the generation of short-lived intermediates which may act on platelets to produce thiobarbituric acid chromogen and to promote serotonin release. These effects on platelets are not inhibited by acetylsalicylic acid or indomethacin. Therefore the highly reactive hydroxyl radical and singlet oxygen, when generated extracellularly, do not mediate their effects via the enzyme-catalyzed prostaglandin pathway, in contrast to those evoked by the less reactive hydrogen peroxide.


Studies on cytochrome c oxidase, IV[1--3]. Primary structure and function of subunit II.

The amino acid sequence of polypeptide II from beef heart cytochrome c oxidase is described. Comparision of this primary structure with those of azurins, plastocyanins and stellacyanins reveals clear homologies among them. Thus subunit II of the oxidase is a member of this copper protein family. The sequence homology indicates a copper binding site consisting of two invariant histidines and two sulfur-containing amino acids. Thus subunit II is like a blue copper protein with type I copper.


Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick Disease.

Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%. Sphingomyelinase activiy was separated into three peaks (I-III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced. Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.


Hormone interactions in the Sertoli cells.

The effects of follicle-stimulating hormone (FSH) and testosterone in rat Sertoli cells were investigated in vitro by means of isolated cell populations. The Sertoli cells selectively bind FSH, and respond to FSH stimulation with increased accumulation of endogenous cyclic AMP and secretion of androgen-binding protein (ABP). FSH binding and cyclic AMP response in the Sertoli cells change dramatically during sexual maturation. Cyclic AMP response decreases despite an increase in FSH-binding receptors per cell. Evidence has been provided for the existence of cytoplasmic and nuclear androgen receptors and chromatin acceptor-sites that specifically bind the androgen-receptor complex in the Sertoli cells. A model has been proposed for the hormonal interactions in the seminiferous tubule and the possible role of Sertoli cells in mediating the hormonal effects on spermatogenesis.


Cytolytic activity in vitro of lymph node cells after exposure in vivo to tumor cells suspended in blocking sera.

The in vivo effect of blocking sera (bs) on both tumor growth and subsequent in vitro cytolytic activity of regional lymph node cells was determined following injection of hepatoma cells suspended in normal sera (ns) or bs into the hind footpads of guinea pigs. Tumor growth was unaffected by bs but the primary response to tumor by LNC draining tumor/bs sites was significantly lower in 4/6 experiments as compared to cells from lymph nodes draining tumor/ns sites.