Frequency facilitation and post-tetanic potentiation of a unitary synaptic potential in Aplysia californica are limited by different processes. Post-tetanic potentiation (PTP) of the monosynaptic and unitary excitatory postsynaptic potential (EPSP) recorded in cell R15 of the abdominal ganglion of Aplysia californica was observed after repetitive stimulation of the right visceropleural connective. PTP at this synapse developed after a few pulses (about 20) and after trains of low frequency stimulation (1/2 sec) under normal physiological conditions of media and temperature. No phase of post-tetanic depression was observed. Evidence is presented that the PTP is due to an increase in transmitter release. The amplitude of the PTP was a function of the frequency and number of stimuli in the preceding train. The PTP was observed to decay, with a single exponential time course, to the size of an isolated EPSP. The rate constant of PTP decay depended upon both the frequency and number of stimuli in the preceding train. The magnitude of the various types of synaptic plasticities seen at this junction, i.e., synaptic depression, frequency facilitation and PTP, correlated with the size of an isolated EPSP as well as with each other. Based on the analysis of the data in terms of a flow model of transmitter release, it is concluded that: (a) during a train of repetitive stimulation the net rate of transmitter supply into the immediately available pool (net transmitter mobilization) increases, the efficiency of the release mechanism (fractional release) increases, and the pool of immediately available transmitter depletes; (b) upon the cessation of the train, as the peak amplitude of PTP is approached, the increased but diminishing rate of net transmitter mobilization refills the available pool to its equilibrium size, while the fractional release is still elevated; (c) during the PTP period after the peak potentiation, the elevated fractional release slowly decays with a single exponential time course; (d) the size of the facilitated EPSPs during the train is limited by the net rate of transmitter supply, although the efficiency of release is also increased; while the size of the EPSPs during the falling phase of the PTP period is determined solely by an increased efficiency of the release mechanism; and (e) the rising phase of the PTP observed in the period shortly after termination of the train is produced by the refilling of the depleted pool of available transmitter in the presence of an elevated release efficiency.


Dopamine, serotonin and related compounds: presynaptic effects on synaptic depression, frequency facilitation, and post-tetanic potentiation at a synapse in Aplysia californica. Dopamine, serotonin and related compounds (referred to collectively as biogenic amines) were found to modify transmission at the presumably cholinergic synapse made by an axon in the right visceropleural connective onto cell R15 of the abdominal ganglion of Aplysia californica. (1) With chronic application, dopamine hyperpolarizes R15, and serotonin depolarizes R15. Both actions upon the membrane potential desensitize in 10 min. All the actions described below were studied with chronic perfusion of the biogenic amines after desensitization of this postsynaptic action. (2) The biogenic amines drastically reduce the size of the EPSP evoked at the synapse under investigation; but they do not alter the ACh potential evoked in the soma of R15. (3) The biogenic amines reduce the amplitude of synaptic depression. The relationship between the effects of the amines on the size of an isolated EPSP and on synaptic depression differed from this relationship as affected by post-tetanic potentiation (PTP) or by changes in the Ca2+-Mg2+ balance. (4) The biogenic amines increase frequency facilitation, when the latter is defined as the ratio of the facilitated to the isolated EPSP. However, the absolute magnitude of the facilitated EPSP is always reduced at long times after introduction of the agent; shortly after introduction of the biogenic amines the absolute magnitude of the facilitated EPSP is unaffected in most preparations.


Heterosynaptic inhibition modifies the presynaptic plasticities of the transmission process at the synapse in Aplysia californica. The monosynaptic and unitary excitatory postsynpatic potential (EPSP) observed in cell R15 of the abdominal ganglion of Aplysia californica upon minimal stimulation of the right visceropleural connective exhibits several presynaptic plasticities (synaptic depression, frequency facilitation, post-tetanic potentiation). We studiied effects of branchial nerve stimulation (heterosynaptic stimulation) on these plasticities of the homosynaptic (right connective) path. A burst of heterosynaptic stimulation (20 pulses at 4/sec) decreased the amplitude of an isolated homosynaptic EPSP. The rate of recovery from heterosynaptic inhibition (HSI) was a function of the rate of stimulation of the homosynaptic path so that at a stimulus frequency of 1 pulse/sec to the right connective (RC) the HSI lasted less than 20 sec while at a RC stimulus frequency of 1/10 sec the HSI persisted for more than 60 sec. While the frequency facilitated EPSP (during homosynaptic stimulation at 1/sec) was only transiently affected by heterosynaptic stimulation the effect on the subsequent post-tetanic potentiation was much more pronounced and longer lasting (more than 30 min). This suggests a specific effect of HSI on the rate constant of decay of elevated fractional release, as observed upon bath applications of biogenic amines. Heterosynaptic stimulation also reduces synaptic depression but the reduction in the depression is more than would be caused by comparable reduction of the first EPSP of a pair of high Mg2+, low Ca2+ or the addition of carbachol to the perfusion medium. The duration of the effect on synaptic depression was the same as the effect on EPSP1.


Failure of procaine penicillin and zinc bacitracin to modify the response of the fowl to stressors. 1. The effect of either procaine penicillin or zinc bacitracin (25 mg active base/kg food) on the response of young birds to three different stressors - withdrawal of food for 18 h, exposure to cold for 24 h or treatment with ACTH- has been examined. 2. Neither antibiotic had any consistent effect on the stress responses. 3. It is concluded that neither penicillin nor bacitracin has any stress-ameliorating activity at least under the conditions of the test.


[Subcellular distribution of the protein kinase activity in the normal and neoplastic thyroid tissue of the rat]. Thyroid tissue has been fractionnated by centrifugation (105 000 q) of its homogenate. Protein-kinase activity in presence of histone is distributed in nuclei (11.5%) mitochondira (22.8%), microsomes (9.8%) and soluble fraction (56%); it is activated by cyclic AMP and GMP, mostly in soluble and nuclear fractions. Protein-kinase activity of total homogenate of neoplasic thyroid (strain 1-5G Wollman) in presence of histone is 3 times higher than in normal tissue and more activated by cAMP. In absence of histone, protein-kinase activity is the more important in mitochondrial and microsomal fractions of normal thyroid and in soluble and nuclear fraction of neoplastic tissue.


[Presence of various protein kinases and of partially copurified endogenous substrates in the soluble fraction of an experimental tumor of the rat thyroid gland]. Various fractions containing protein-kinases have been partly purified from supernatant (105 000 g) of an experimental rat's thyroid cancer (1-8 Wollman). These fractions are activated at different levels by cyclic nucleotides and present an unequal affinity for protamine; they phosphorylate an endogenous substrate apparently copurified with the enzyme fractions and adorbed with them on calcium phosphate gel.


[Effects of chloramphenicol and thiamphenicol on sleep of the mouse]. The oral administration of 0.25-2.0 g/kg of chloramphenicol at 9h selectively suppresses paradoxical sleep in mice for a duration of 2-4 hours. A dose of 1 g/kg given at 17h suppresses paradoxical sleep for 7 hours. Slow wave sleep decreased for 2 hours after administration. Thiamphenicol (1 g/kg) has no effect under the same conditions.


[Secretions of GH, FSH and LH during sleep of the normal child and the child with retarded growth]. In normal children the major GH release begins during NREM sleep of first cycle. At puberty secretion of gonadotropins is enhanced and secretion of LH occurs with the same periodicity as the sleep cycles. Two groups of dwarfish are seen: the first lacks both GH secretion during sleep and the increase of gonadotropins at puberty. The second group exhibits GH, LH and FSH secretion patterns similar to normal children. Study of secretion patterns of GH, FSH and LH during sleep in children can document the degree of maturation of the hypothalamic pituitary hormonal system.


Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase. Sarcolemmal membranes isolated from guinea pig heart ventricles contained an ATP-dependent calcium-sequestering activity. Sarcolemmal calcium accumulation but not binding was enhanced by preincubation of membranes with exogenous protein kinase, with cyclic AMP, or with isoproterenol. Protein kinase (EC 2.7.1.37) increased the V of Ca2+ accumulation by sarcolemma without any significant effect on the affinity for Ca2+. The endogenous protein kinase activity present in isolated sarcolemma affected membrane phosphorylation. Cyclic AMP increased the endogenous kinase activity modestly, whereas histone increased it significantly. Exogenous protein kinase also catalyzed phosphorylation of these membranes. Endogenous and exogenous kinase-catalyzed phosphorylation of sarcolemma was hydroxylamine-insensitive. Ca2+-dependent ATPase (EC 3.6.1.3) (extra ATPase) activity of sarcolemma was also increased by protein kinase.


The interaction of ligands with chemically modified phosphorylase b. Phosphorylase b which had been inactivated with 5-diazo1H-tetrazole was specifically labelled with 4-iodoacetamidosalicylic acid (a fluorescent probe) or with N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)iodoacetamide (a spin label probe) so that the binding of ligands and accompanying conformational changes could be determined by fluorescence or electron spin resonance changes, respectively. The allosteric effector, AMP, causes conformational changes similar to those caused in the native enzyme. The affinity of binding of phosphate or AMP to the inhibited protein is the same as for the unmodified protein. The heterotropic interactions between glucose-1-phosphate or glycogen and AMP are much less in the inactivated enzyme than in unmodified phosphorylase. Using a light scattering assay, it is shown that the modified enzyme binds to glycogen less strongly than the native protein. Phosphorylase b which had been inactivated by carbodimide in the presence of glycine ethyl ester, resulting in the modification of one or more carboxyl groups, was labelled with the spin label probe described above. The modified enzyme has an affinity for AMP similar to that of the native enzyme. AMP binding to the modified enzyme is tightened by glycogen, weakened by glucose-6-phosphate and is unaffected by glucose-1-phosphate. The actions of 5-diazo-1H-tetrazole and carbodimide on phosphorylase are discussed in the light  of the above observation.


Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes. A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholi-date into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2'-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.


Inhibition by saccharin of glucose-6-phosphatase: effects of alloxan in vivo and deoxycholate in vitro. Inhibition by saccharin of rat liver glucose-6-phosphatase (EC 3.1.3.9) generally decreased as the pH increased in the range pH 4-8. This pattern was exhibited by homogenates from control and alloxan-treated animals assayed each in the absence and presence of 0.2% (w/v) deoxycholate. Saccharin inhibited in competitive fashion with respect to glucose-6-phosphate (glucose-6-P). There was a small increase in Km (glucose-6-P) but not K1 (saccharin) values in alloxan-treated rats when assays were conducted in the absence of deoxycholate. In the presence of this detergent there was no significant difference in these kinetic parameters between the alloxan-treated and control groups. Deoxycholate decreased Km (glucose-6-P) and increased K1 (saccharin) values. Calculations using these kinetic parameters indicate that, under usual hepatic glucose-6-P concentrations and relatively high levels of saccharin in liver, the inhibition by saccharin of glucose-6-phosphatase is unlikely to be of major significance in vivo.


Prostaglandin E2 and cyclic AMP in the coronary vasodilatation due to cardiac hyperactivity. In the isolated perfused rat heart, the dose-related cardiostimulation produced by norepinephrine (NE) or calcium chloride (Ca2+) was followed by a corresponding increase in coronary flow (CF) and in the cardiac level of adenosine 3',5'-cyclic phosphate (cAMP). Prolonged prostaglandin E2 (pge2) infusion did not change the basic force of contraction, CF, or cAMP level but when NE or Ca2+ were administered, only the responses of the CF and the cAMP were diminished. A phosphodiesterase inhibitor, diazoxide (Dx), caused insignificant increase in the basal cAMP, without affecting the force of contraction or CF. With NE or Ca2+, during Dx both the changes in CF and cAMP were augmented compared to the nontreated hearts. The inhibitory effects of NE or Ca2+ remained unchanged. Propranolol abolished the NE but not the Ca2+ effects. It is suggested that PGE2 modulates the cardiac cAMP level and that the latter plays an important role in the adaptive regulation of the CF. It is also postulated that changes in cAMP levels may be brought about by the hyperactivity per se produced by a variety of cardiostimulating agents.


Loss of ACTH biological activity in plasma during extraction with a commercial radioimmunoassay kit. The process used for extracting adrenocorticotropic hormone (ACTH) from plasma in the Searle ACTH immunoassay kit involves a loss of 70-100% of the biological activity of the ACTH.


Is cyclic guanosine monophosphate the internal 'second messenger' for cholinergic actions on central neurons? The most consistent effects produced by intracellular injections of guanosine 3',5'-cyclic monophosphate (cGMP) (but not 5'-guanosine 5'-monophosphate in spinal motoneurons of cats are a rise in membrane conductance, acceleration in time course of spike potentials, and accentuation of the post-spike hyperpolarization. Associated changes in resting potential are smaller, less constant, and more often in the depolarizing than hyperpolarizing direction, cGMP tends to increase electrical excitability but reduces excitatory post-synaptic potential amplitudes. Most of the effects of intracellular cGMP are quite different from, or indeed opposite to, those of either extra- or intracellular applications of acetylcholine and therefore not consistent with the proposal that cGMP is the internal mediator of muscarinic actions.


Temperate phages of Clostridium perfringens type C1. Four phages isolated from carrier strains of Clostridium perfringens type C belong to two classes. The three phages of class I, c1, c3, and c4, and homoimmune and serologically closely related. The phage of class II, c5, is heteroimmune to the class I phages and not related to them serologically. Transduction experiments with several of the phages were negative. Mutants of the indicator strain with surface alterations occurred spontaneously in stock cultures. Electron micrographs show the phages of each class to be distinct yet similar, having polyhedral heads of about the same diameter 55 nm, and long, flexible tails without sheaths or collars. Phages c4 and c5 were characterized for their lysogenic properties. Phage c4 was inducible with mitomycin C. Both c4 and c5 were temperate viruses by the test of stability of their respective lysogens to phage-specific antisera.


Enumeration of sublethally heated staphylococci in some dried foods. The effect of 45 substances to restore the salt tolerance of sublethally heat-injured Staphylococcus aureus was tested. Sodium pyruvate, yeast extract, L-histine, casitone (Difco), adenosine triphosphate, and acetylphosphate were effective. For enumeration a repair medium was first used, containing sodium pyruvate and penicillin in 1% skim milk. This step was followed by counting on Baird-Parker agar with penicillinase. This method was selective; fewer than 100 staphylococci/g food could be enumerated and it gave counts about 8 times higher than the method of Giolitti and Cantoni used as a five-tube most probable number technique. Heat injury sensitized S. aureus to polymyxin.


Further studies on the relative antiviral efficacies of interferons induced by poly I:C and Mengo virus. Mouse serum interferons induced by polyI:C, vesicular stomatitis virus (VSV), reovirus, and Mengo virus were assayed in monolayers of mouse L-929 cells by the plaque-reduction method using both VSV and Mengo as challenge viruses. Titers obtained with Mengo virus as challenge were all lower than with VSV. With the interferons induced by VSV, reovirus, and ployI:C, the reductions were of the order of two- to three-fold. With Mengo virus-induced interferon the reduction was much greater (about 17-fold). This offers an explanation for the observation that, unit for unit (measured by the plaque reduction of VSV), Mengo virus-induced interferon is only about 1/10 as effective as polyI:C-induced interferon in protecting mice against lethal infection with Mengo virus. The data are consistent with the hypothesis that an interferon antagonist is produced in the serum of mice infected with Mengo virus. This antagonist, which is not produced in mice inoculated with polyI:C, or reovirus, effectively blocks the antiviral action of interferon during Mengo virus infections, both in vivo and in vitro.


Mannitol production in fungi during glucose catabolism. The levels of phosphofructokinase (EC 2.7.1.11) and mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) have been determined in a number of Mucor and Penicillium species. Mannitol-1-phosphate dehydrogenase was found in only one species of mucor, Mucor rouxii, and this with a specific activity much lower than that found in Penicillium species. All of the fungi tested in the Ascomycetes class exhibited mannitol-1-phosphate dehydrogenase activity. Interference from both mannitol-1-phosphate dehydrogenase and NADH oxidase (EC 1.6.99.5) caused some difficulty initially in detecting phosphofructokinase in Penicillium species; the Penicillium phosphofructokinase is very unstable. Penicillium notatum accumulates mannitol intracellularly; detection of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase (EC 3.1.3.22) activity in cell-free extracts indicates that the mannitol is formed from glucose via fructose-6-phosphate and mannitol-1-phosphate; no direct reduction of fructose to mannitol could be detected. The mannitol-1-phosphate dehydrogenase was specific for mannitol-1-phosphate and fructose-6-phosphate; NADP+(H) could not replace NAD+(H). The phosphatase (EC3.1.3.22) exhibited a distinct preference for mannitol-1-phosphate as substrate; all other substrates tested exhibited less than 25% of the activity observed with mannitol-1-phosphate.


Wilms' tumour: adjuvant treatment with actinomycin D and vincristine. In an unselected series of 49 children with Wilms' tumour treated in 1969-74 the 5-year relapse-free survival and survival rates were 78% and 81%, respectively, whereas in the series of children treated in 1963-68 the corresponding rates were 49% and 70%. The significant improvement in the relapse-free survival rate was a result of adjuvant treatment with actinomycin D and vincristine (AMD + VCR), which, in some patients, eradicated occult metastatic disease. In the treatment of lung metastases the combination of whole-lung irradiation and maintained chemotherapy with AMD + VCR proved excessively toxic: in 5 of 11 patients acute diffuse pneumonitis developed, and it was fatal in 3. Adjuvant AMD + VCR therapy is advocated in all patients with Wilms' tumour except children less than 12 months old with a tumour of moderate size, limited to the kidney and completely resectable.


Chorionic gonadotropin.

Chorionic gonadotropin. One of the most exciting and useful developments in modern oncology has been the recognition that certain biochemical tags can be used as cell markers in diagnosis, in monitoring the effects of anti-tumor therapy, and in follow-up to detect tumor recurrence. The measurement of chorionic gonadotropin (CG) in women with choriocarcinoma and/or gestational trophoblastic disease was the first test that could be relied upon entirely for this purpose. Furthermore, the amount of functional neoplastic tissue could be approximated by determining the production rate of this hormone which in normal physiologic conditions is only produced by syncytiotrophoblasts. The measurement of CG is now similarly used for the management of patients (both male and female) with choriocarcinoma originating in the tests and ovary. More recently a wide variety of nontrophoblastic malignant neoplasms have also been shown to produce CG, presumably from primitive tumor cells. Work is currently underway to ascertain the extent and potential usefulness of this observation in a wide variety of tumor systems, in the hope that improved tumor monitoring might be achieved.


Hormones and gynecologic cancer.

Hormones and gynecologic cancer. Available basic information about the regulation of rates of production, blood levels, intracellular metabolism, and action of steroid hormones and prolactin is reviewed and related to high-risk factors and therapeutic procedures applicable to gynecologic cancer.


Viruses and cancer of the lower genital tract. The importance of viruses as oncogenic agents in animals is well established. Recent work suggests that viruses may also be etiologically related to some human cancers. Herpes simplex virus type 2 (HSV-2) and the genital wart virus are prime suspects in carcinomas involving the female lower genital tract. In particular, a close association has been found between HSV-2 infection and cervical neoplasia in cytohistopathologic and seroepidemiologic studies. Preliminary results of prospective studies show further that women with genital herpetic infection are at increased risk of developing cervical neoplasia. Additional studies are also in progress on animal models, including subhuman primates, and efforts continue in the attempt to confirm the presence of viral genetic material or its expression in human cervical cancer cells. The possibility that human wart viruses have an oncogenic potential is suggested by clinicopathologic and electron microscopic observations. Further research is needed to ascertain the precise role of viruses in genital cancer.


Current status of treatment of gestational trophoblastic disease. A brief summary is given of the developments leading to the current status of treatment of gestational trophoblastic neoplasms, both nonmetastatic and metastatic. Hydatidiform mole has been identified as the precursor type of pregnancy in approximately 50% of those women developing metastatic disease. The other half developed this as consequences of either a full-term delivery or some type of abortion. Sensitive assays for human chorionic gonadotropin (HCG) are needed in the diagnosis, management, and follow-up of these patients. Current therapy is outlined: nonmetastatic disease receives single agent chemotherapy with methotrexate or actinomycin D, with approximately 100% cure and 90% retention of reproductive function; metastatic disease, "low risk", receives single agent chemotherapy with the same drugs, with an expected cure rate of 95-100%; metastatic disease, "high risk", receives initial therapy with multiple agent chemotherapy, with a cure rate of approximately 75%. Current unresolved questions are discussed briefly.


Herpes simplex virus-specific antigens in exfoliated cervical cells from women with and without cervical anaplasia. By the indirect immunofluorescence technique the presence of herpes simplex virus-specific antigens was investigated in cervical cells of 530 women with normal cervical epithelium, 175 with bland disorders, 52 with dysplasias, and 38 with invasive cervical carcinomas of the uterine cervix. Antigens were present in 9% of samples from women with normal cervical epithelium; they were present in 41% of the samples from women with bland disorders, 61% of those from dysplasia patients, and 94% of those from invasive carcinoma patients. The testing of 3 consecutive imprints of 68 antigen-positive and 232 antigen-negative women at 6-month intervals revealed that, in cervical cells, herpesvirus-specific antigens persisted throughout the 1-year period of the follow-up.


Variation in glycosaminoglycan components of breast tumors. The correlation between the content of individual glycosaminoglycans and the histological patterns are studied on breast tumor tissues. The myxomatous stroma of intracanalicular fibroadenoma contained a large amount of glycosaminoglycans, which were mainly hyaluronic acid. The chondroitin 4- and 6-sulfate level was also high. As the supporting stroma of this tumor became denser and more fibrous, the level of hyaluronic acid content was reduced. In the case of pericanalicular fibroadenoma, glycosaminoglycans were small in amount and the levels of hyaluronic acid and chondroitin sulfate were low, but the ratio of dermatan sulfate content was higher. In the case of gynecomastia, the conent was almost the same as that of pericanalicular fibroadenoma. Scirrhous carcinoma tissues contained a relatively large amount of hyaluronic acid and chondroitin sulfate. No remarkable differences in heparan sulfate content were observed in any one of the breast tumors tested. Dermatan sulfate-chondroitin sulfate copolymers were detected in all the tumors. The presence of dermatan sulfate seemed to have an intimate relation with the fibrogenesis in the interstitial stromal element of the tumor tissues.


Biochemistry and enzyme induction in MC-29 virus-induced transplantable avian hepatoma. For the biochemical characterization of a new transplantable hepatoma derived from the MC-29 virus-induced liver tumor, the macromolecular content and the inducibility of glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, and aryl hydrocarbon hydroxylase were compared in chicken liver and in this hepatoma. The alteration of the nucleocytoplasmic ratio was deduced from measurements of DNA, RNA, protein, and phospholipid contents of the whole cell homogenate and cell fractions. The increased nuclear and decreased cytoplasmic content of macromolecules suggests a dominancy of the nuclei in the tumor cells. Glucose-6-phosphatase and aryl hydrocarbon hydroxylase activities were lower by 60 and 80%, respectively, in the highly proliferating hepatoma than in the liver. In contrast, glucose-6-phosphate dehydrogenase activity increased in the hepatoma. However, enzyme inducers, such as methylcholanthrene, hydrocortisone, and insulin, were able to enhance the activity of these enzymes in the liver but had no stimulating effect on the hepatoma.


Electrophysiological properties of ethylnitrosourea-induced, neoplastic neurogenic rat cell lines cultured in vitro and in vivo. A comparative analysis was performed on the electrophysiological properties of 11 neoplastic neurogenic cell culture lines and five other cell lines of different origin (HV1C, rat bile duct carcinoma; BICR/M1RK, rat mammary tumor; HeLa, human cervix carcinoma; 3T3, mouse embryo; REe, rat embryo). Neurogenic lines were derived either from N-ethyl-N-nitrosourea-induced neoplasms of the nervous system or from cultured fetal rat brain cells that had undergone neoplastic transformation in vitro after exposure to N-ethyl-N-nitrosourea in vivo. Electrical membrane excitability was lacking in all neurogenic cells analyzed. Their membrane potential and input resistance values were similar to those of the nonneurogenic lines. Intercellular ionic coupling was consistently observed between cells of a fibroblastoid shape or cells bearing multiple cytoplasmic processes (i.e., all neurogenic lines HV1C, BICR/M1RK, and 3T3). Epithelioid cells (i.e., HeLa, REe, an NV1C subpopulation, and a GV1C1 variant) showed no such intercellular communication. In vivo monolayer cultures on glass coverslips were obtained by a modified i.p. diffusion chamber technique. Under these conditions, the cells (with the exception of a glioma-derived cell line) retained the morphological appearance and electrophysiological properties observed in vitro.


Kinetic properties of alcohol dehydrogenase in hepatocellular carcinoma and normal tissues of rat. It was previously reported that the properties of alcohol dehydrogenase of a rat hepatocellular carcinoma (Becker H-252), a tumor of intermediate growth rate, were different from those of the liver enzyme, suggesting different isozymes. To determine whether the degree of differentiation affected the isozyme of alcohol dehydrogenase, a fast-growing, poorly differentiated tumor and one that is well differentiated and of intermediate growth rate were studied. Alcohol dehydrogenase from Morris hepatoma 7288ctc, a fast-growing, poorly differentiated tumor, had properties similar to those found with the Becker-H-252 tumor, including a high Km for ethanol and acetaldehyde and the absence of substrate inhibition. By contrast, alcohol dehydrogenase from the well-differentiated Morris hepatoma 5123C had properties similar to those of the liver enzyme. Thus, alcohol dehydrogenase is another example of an enzyme the isozyme composition of which changes with neoplastic de-differentiation. Further studies, including gel electrophoresis, substrate specificity patterns, and interaction with antibodies to alcohol dehydrogenase, are required to determine the factors responsible for the biochemical defect that occurs at the molecular level during carcinogenesis and whether the alcohol dehydrogenase isozymes in the Becker H-252 and Morris 7288ctc hepatomas are identical. A survey of several normal rat tissues revealed that only the stomach contains this unique isozyme of alcohol dehydrogenase.


Binding of cyclic nucleotides with proteins in malignant and adenosine cyclic 3':5'-monophosphate-induced "differentiated" neuroblastoma cells in culture. The binding of adenosine cyclic 3':5'-monophosphate (cyclic AMP) with soluble (100,000 X g supernatant), pellet, and total homogenate proteins from cyclic AMP-induced "differentiated" mouse neuroblastoma cells increased by about two-fold. The extent of binding with soluble proteins was higher than that with pellet proteins. The binding of cyclic AMP with soluble proteins from 5'-adenosine monophosphate-treated, serum-free medium-treated, sodium butyrate-treated, 6-thioguanine-treated, or X-irradiated neuroblastoma cells did not significantly change. When the soluble proteins containing bound cyclic [3H]AMP were filtered through a Sephadex G-25 column, the relative amount of protein-bound cyclic [3H]AMP in differentiated cells was greater than that in malignant cells, but the amount of free cyclic [3H]AMP was correspondingly less. The electrophoretic characteristics of cyclic AMP-binding proteins of differentiated and malignant cells were identical. There were two binding peaks, but the extent of binding at each peak was relatively high in differentiated neuroblastoma cells. An increase in cyclic AMP binding occurred 24 hr after treatment of neuroblastoma cells with prostaglandin E1. This increase was completely blocked by cycloheximide but not by actinomycin D. The binding was heat labile and sensitive to protease action. These data indicate that the increase in binding in differentiated cells is due to an elevation in the levels of binding proteins. The binding of cyclic AMP with soluble proteins from rat glial cells and mouse L-cells did not significantly change after treatment with prostaglandin E1 or an inhibitor of cyclic AMP phosphodiesterase. Cyclic AMP and guanosine cyclic 3':5'-monophosphate bind with the same proteins, but cyclic AMP has about 10-fold higher binding affinity than does guanosine cyclic 3':5'-monophosphate.


Differences between pyrimidine nucleoside monophosphate kinase from rat Novikoff ascites hepatoma and rat liver. Pyrimidine nucleoside monophosphate kinase [deoxycytidine monophosphate:adenosine triphosphate (dCMP:ATP) phosphotransferase. EC 2.7.4.14] has been purified from rat Novikoff ascites hepatoma and rat liver, each to a single major band appearing on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Differences exist in regard to efficiency and regulation of enzymatic activities. The Km values of the tumor kinase for cytidine monophosphate (CMP) (0.0053 +/- 0.0008 MM) and dCMP (0.715 +/- 0.068 MM) are approximately one-fourth the Km values of the rat liver kinase, for CMP (0.030 +/- 0.007 MM) and dCMP (2.77 +/- 0.39 MM). The tumor dCMP kinase exhibits a lower Km for ATP (0.134 +/- 0.008 MM) than the rat liver kinase (0.68 +/- 0.09 mM). Moreover, the dCMP:CMP kinase activity ratio for the tumor enzyme is 1.12, while that for the rat liver enzyme is 0.45. The uridine monophosphate:CMP kinase activity ratio for the tumor enzyme is 1.93, while that for the rat liver enzyme is 2.68. Lower concentrations of dithiothreitol are required for 50% reactivation of the tumor dCMP kinase (1.00 mM) and CMP kinase (0.10 mM) than rat liver dCMP kinase (2.20 mM) and CMP kinase (0.57 mM). Thus, the kinase from Novikoff hepatoma exhibits properties of increased efficiency and relaxed regulation of activity which render it more suitable for a tumor, in which active DNA synthesis is ongoing.


Pathology of lymphosarcoma in sheep induced with bovine leukemia virus. Sixty-nine sheep were infected with bovine leukemia virus from bovine lymphosarcoma materials. Twenty-four developed lymphosarcoma and died from 13 to 66 (average, 29) months later. Circulating lymphocytes were increased to leukemia levels (70,000 to 403,000/cu mm blood) in only eight sheep within 2 to 3 months of death. Various lymph nodes and visceral organs including heart, abomasum, uterus, kidneys, and urinary tract were commonly affected as in cattle with the adult form of lymphosarcoma. In one sheep the skin was involved. The liver was involved in only one case. This was in contrast to more frequent involvement reported in literature for naturally occurring lymphosarcoma. The neoplasms in experimental sheep are regarded as a mixture of reticulum or histiocytic cells and lymphoid cells with transitional forms supported by a usually sparse and diffuse fibroplasia and a web of silver-staining reticulin fibers.


Experimental hepatocellular carcinogenesis. The sequential development of lesions in the liver leading to hepatocellular carcinoma in experimental animals appears to follow a similar pattern irrespective of the carcinogen. The speed with which the lesions develop is related to the chemical, the dose and dosing schedule, and the diet fed. In studies with aflatoxin B1 there is a predictable series of morphological expressions beginning with focal areas of hydropic degeneration (Stage 1); hyperplastic basophilic cells, singly or in close association with Stage 1 (Stage 2); nodular hyperplasia of parenchymal cells which become progressively more abnormal (Stage 3); transitional cell changes (Stage 4); and, ultimatley, hepatocellular carcinoma (Stage 5).


Sex hormones and hepatic neoplasia.

Sex hormones and hepatic neoplasia. The recent increase in hepatocellular neoplasms in women of child-bearing age taking oral contraceptives and in individuals of both sexes taking anabolic androgenic steroids is a phenomenon that requires urgent attention. Although most of the lesions are benign, reports of carcinomas in a few of these individuals and our own observation of an adenoma that shows borderline malignant transformation suggest that some of the adenomas are premalignant lesions. The pathology of our own cases and those reported in the literature are reviewed, and hypotheses are offered for a carcinogenic role of synthetic steroid sex hormones. These speculations are based on known toxic and metabolic actions of the drugs and are amenable to experimentation.


Preneoplastic lesions in murine mammary cancer. The current model for murine mammary tumorigenesis indicates that discrete, morphologically identifiable preneoplastic lesions precede and give rise to mammary tumors. The hyperplastic alveolar nodule is the primary lesion that precedes and gives rise to mammary tumors in mice infected with the mammary tumor virus (Bittner) or its variants. In mice fed 7,12-dimethylbenzanthracene or urethan, hyperplastic alveolar nodules are present but infrequent, and the major mammary dysplasias present are ductal hyperplasias. The ductal hyperplasias can be divided into four general morphological types: (a) simple terminal duct hyperplasia; (b) lobular hyperplasia; (c) papillary hyperplasia; (d) end-bud hyperplasia. All types of ductal hyperplasias are characterized by intraductal epithelial hyperplasia and have been shown to give rise to mammal adenocarcinomas in situ or by transplantation into the mammary gland-free fat pads of syngeneic mice. Our current hypothesis is that murine mammary cancer can evolve through one of several intermediate stages. The intermediate stages can be either alveolar or ductal hyperplasias. The latter resemble, in many instances, ductal lesions seen in human breast cancer.


Effects of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide and its metabolites on Novikoff hepatoma cells. Studies were undertaken to determine the effects of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) and its metabolites on the growth and macromolecular synthesis of Novikoff hepatoma cells in culture. DTIC (3.0 mM) in light decreased the viable cell count by 90% within 96 hr. DTIC protected from light, 2-azahypoxanthine, dimethylamine, and 5-aminoimidazole-4-carboxamide, all at 3.0 mM, reduced the rate of cellular proliferation. 5-Diazoimidazole-4-carboxamide (1.0 mM) and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (3.0 mM) decreased the viable cell count by 99%. Effects on macromolecular synthesis were determined by the rate of incorporation of the appropriate 3H-labeled precursor. Results after 6 hr are given as percentage of controls. DTIC (1.0 mM) in light inhibited DNA (8%), RNA (41%), and protein (63%) synthesis. DTIC (1.0 mM) protected from light inhibited DNA (12%) and RNA (57%) synthesis. 5-Diazoimidazole-4-carboxamide (0.1 mM) inhibited DNA (1%), RNA (9%), and protein (1%) synthesis. 5-(3-Methyl-1-triazeno)imidazole-4-carboxamide (1.0 mM) inhibited DNA (72%) and protein (65%) synthesis but stimulated RNA (127%) synthesis. 2-Azahypoxanthine (1.0 mM) inhibited DNA (43%), RNA 82%) and protein (28%) synthesis. 5-Aminoimidazole-4-carboxamide (3.0 mM) stimulated DNA (354%) and RNA (266%) synthesis. These data show that DTIC is able to generate several toxic metabolites that may be responsible for its biological effects.


Autogenous immunity to mouse mammary tumor virus in mouse strains of high and low mammary tumor incidence. Specific radioimmune precipitation assays were utilized to demonstrate the presence of precipitating antibodies to mouse mammary tumor virus (MMTV) in the high-spontaneous mammary tumor strains of mice: C3H/HeN+, GR/N, BALB/cfC3H, and C57BL/6 X C3H F1 (hereafter called B6C3F1). Antibody titers in C3H/HeN+ mice increased with age, with highest titers observed in tumor-bearing animals. MMTV-precipitating antibodies were not detectable by radioimmune precipitation assay in low-mammary tumor strains (AKR, BALB/c C57BL/6, and C3H/HeN-) but were detectable in MMTV-inoculated BALB/c mice. Appearance of antibodies preceded palpable tumor formation, and antibody titers were directly correlated to virus dose. Natural antibody to MMTV in C3H/HeN+ and B6C3F1 mice coexists with the murine leukemia virus natural antibody as determined by competition radioimmunoassays.


Changes in transfer RNA's in human malignant trophoblastic cells (BeWo line). Ten aminoacyl transfer RNA's prepared from human malignant trophoblastic cells (BeWo line) were compared with the corresponding aminoacyl transfer RNA's from normal human chorionic tissue by cochromatography on a RPC-5 column. Phenylalanyl transfer RNA (Phe-tRNA) of BeWo cells had, in addition to the single species of Phe-tRNA found in normal chorionic tissues, an early eluting component. When Phe-tRNA from the chorion was exposed to mild acid, which selectively excises the Y base, it eluted in the same position as the early eluting Phe-tRNA of BeWo cells. Therefore, the BeWo Phe-tRNA is partially undermodified. Tyrosyl transfer RNA of BeWo cells exhibited a broad-based peak which eluted later than the normal and probably consists of two or more tyrosyl transfer RNA's. Seryl transfer RNA of BeWo cells showed two peaks of acceptor activity, while seryl transfer RNA of normal chorion had a third peak that eluted at a higher salt concentration. In addition, in an early eluting methionyl and lysyl transfer RNA and in a late eluting arginyl transfer RNA from BeWo cells and normal charion, quantitative alterations were detected. The remaining four transfer RNA's, leucyl, aspartyl, valyl, and histidyl, from the two sources did not show any significant differences in elution profiles. These alterations of the chromatographic profile appeared to be due to new or altered species of transfer RNA. They were not due to differences in the aminoacyl transfer RNA synthetase. The transfer RNA methyltransferase capacity of the enzymes from BeWo cells was 2-fold higher than that of the enzymes extracted from the chorion.


Characteristics of cell membranes from somatic cell hybrids between rat hepatoma and mouse L-cells. Cell membranes from mouse L-cells (L-B82), rat hepatoma (HTC-H1), and three clones of their somatic cell hybrids (07, V4a, and V5) showing different degrees of density-dependent inhibition of growth were analyzed by polyacrylamide gel electrophoresis. The membrane polypeptides of the hybrid clones were all similar and all showed higher proportions of polypeptides with molecular weights of 56,000 and 45,000 than their parents of their normal counterparts. The major glycoprotein form cell hybrids appeared to be identical with that of rat liver or rat hepatoma cells and different from that of L-cells. One hybrid showed density-dependent inhibition growth; the other two, like both parents, did not. All produced tumors in nude mice, although tumor production by the hybrids was delayed. A large external protein (M.W. 240,000) iodinated by lactoperoxidase-catalyzed reaction was virtually missing in the parents but was present at high levels in all their hybrid clones. Thus, there was a lack of correlation between the presence of this protein, growth control in vitro, and tumorigenicity. Furthermore, no correlation was seen between agglutination of these cells by concanavalin A and tumorigenicity. The factors controlling these membrane properties thus are independent of density-dependent inhibition of growth and of those controlling the expression of cancer.


Studies on the selective action of cyclophosphamide (NSC-26271): Inactivation of the hydroxylated metabolite by tissue-soluble enzymes. The hypothesis that selective action of cyclophosphamide, compared to other nitrogen mustards, is due to a balance between enzymatic formation of inactive metabolites and chemical formation of the alkylating product was studied in view of previous observation in our laboratory. Metabolite analysis, inhibition of growth of tumor cells in culture, and kinetic analysis of relevant enzyme activities were used in this investigation. The effect of tissue-soluble enzyme fractions on biochemically prepared aldophosphamide, aldophosphamide analogs, and phosphoramide mustard showed: (a) a range of deactivation abilities with aldophosphamide (liver greater than kidney greater than intestinal mucosa greater than tumor greater than spleen = bovine serum albumin solution); (b) the formation of different amounts of carboxyphosphamide from aldophosphamide; and (c) only comparatively small reductions in the toxicity of phosphoramide mustard and of 4-hydroxy-4methylcyclophosphamide. Correlations were found between NAD+-dependent aldehyde dehydrogenase activity and the deactivation ability of tissue-soluble enzyme fractions. Blockage (by C4 substitution) or inhibition (by disulfiram) of secondary oxidation of aldophosphamide, mediated by aldehyde dehydrogenase, resulted in diminished deactivation ability in vitro and reduced selectivity in vivo.


The effect of cyclic adenosine 3',5'-monophosphate on the uptake of estradiol by the neonatal mouse uterus: an autoradiographic study. Uterine tissue from neonatal mice was incubated in vitro at 0 degrees C for 1 h in a medium containing 1 X 10(-8) M 3H-estradiol with or without 1 X 10(-4) M dibutyryl cyclic AMP. In some incubations the temperature was raised to 37 degrees C for 15 min after incubation in the cold, in others the temperature was kept at 0 degrees C during this 15 min period. The tissue was frozen in liquid propane cooled in liquid nitrogen, sectioned at 2 or 4 microns, and autoradiograms prepared according to the dry-mount procedure. cAMP increases the cellular uptake of 3H-estradiol in uterine tissue. After rising the temperature to 37 degrees C, grains appeared over the nuclei. cAMP at low temperature increased the cellular uptake of 3H-estradiol, but the grains were not associated with the nuclei. In the autoradiograms the grain number above the epithelium was markedly less than above the stroma.


[Denitrifying bacteria of genus Alcaligenes isolated from soil]. The organism isolated is a small non-sporulating Gram-negative rod, motile by means of peritrichous flagellae. It is an oxidase-positive chemo-organotroph utilizing O2,NO-3,NO-2 and N2O as resiratory substrates. Primary alcohols as well as numerous organic and amino acids are utilized as carbon and energy sources. Carbohydrates are not assimilated. Poly-beta-hydroxybutyrate is accumulated intracellularly. The bacterium is assigned to the genus Alcaligenes and its phenotype characteristics are compared with those of A. faecalis.


Calcium accumulation and enzymatic activities of subcellular fractions from aortas and ventricles of genetically hypertensive rats. Subcellular fractions were obtained from aortas and ventricles of 6-month-old spontaneously hypertensive and normotensive Wistar rats by the use of differential and sucrose density gradient centrifugation. These preparations were studied to determine what alterations in calcium accumulation and enzymatic activities might be associated with hypertension. The total amount of calcium accumulation (in the presence of ATP and 17 muM free calcium) by the plasma membrane-enriched fraction from hypertensive rat aortas significantly less than that from normotensive rats (11.3 +/- 0.4 vs 16.2 +/- 1.6 mumol of calcium/g of protein, n = 8). In contrast the specific activities of the plasma membrane marker enzymes, 5'-nucleotidase and phosphodiesterase I, were 80% and 40% greater, respectively, in the hypertensive than in the normotensive fractions. On the other hand, various fractions from ventricles of the two types of rats were generally similar in enzyme activities and calcium accumulation. The decreased rate of relaxation of aortas from spontaneously hypertensive rats may be caused by the decreased rate of calcium transport demonstrated in this study.


Evaluation of 99mtechnetium stannous pyrophosphate as an imaging agent in acute myocardial infarction. Serial 99mTc pyrophosphate scintigrams were obtained 7 hr to 15 days after experimental acute myocardial infarction produced by permanent or transient coronary occlusion. Scintigrams were interpreted visually and the increased radioactivity in the precordial image was quantitated and compared to extent of infarction found histologically. Results of these studies indicate: 1) 99mTc pyrophosphate imaging is an extremely sensitive for detection of acute myocardial infarction, i.e., infarction in excess of 1% of the left ventricular mass was detected. 2) Early detection of acute infarction is a function of blood flow since 7 hr after infarction scans were negative after permanent occlusion but were strongly positive after transient occlusion. 3) Radioactivity in the precordial image was inversely related to extent of infarction after permanent occlusion and directly related to extent of infarction after transient occlusion. 4) 99mTc pyrophosphate localized in areas with significant histologic infarction but the distribution of radioactivity was not proportional to extent of infarction.


Asthma due to non-occupational exposure to toluene (tolylene) di-isocyanate. The production of asthma by toluene di-isocyanate, aspirated into the ventilating system and emanating from the exhausts of a neighbouring factory, was established in three otherwise non-exposed clerical workers. Occupational-type exposure tests with a polyurethane varnish/toluene di-isocyanate mixture elicited vigorous and prolonged non-immediate asthmatic reactions, thus enabling identification of the otherwise, obscure etiological cause of their asthma.


Chromatography on Florisil in the quantitative estimation of urinary and other porphyrins. We describe the use of Florisil chromatography to purify and fractionate porphyrins into uro-, copro-, and protoporphyrins before their measurement by quantitative fluorometry. Porphyrinogens in fresh urine may be rapidly oxidized to porphyrins by chloranil, or slowly in weakly alkaline solution, before the sample is applied to the Florisil column. Aliquots of the separate ammoniacal eluates may be lyophilized for additional fractionation by direct thin-layer chromatographic analysis if desired. The behavior of delta-aminolevulinic acid, porphobilinogen, and other pyrrole compounds in this system is also described.


Evaluation of adenosine 5'-monophosphate and fluoride as adenylate kinase inhibitors in the creatine kinase assay. Adenylate kinase (EC 2.7.4.3) interferes positively in the serum creatine kinase (EC 2.7.3.2) assay when the rate of ATP production is monitored by a coupled enzyme system. A dual assay, measuring creatine kinase and adenylate kinase activity, was used to evaluate AMP and other possible adenylate kinase inhibitors that would permit specific measurement of creatine kinase activity in the presence of adenylate kinase. We found that AMP, routinely included in the creatine kinase assay system to inhibit adenylate kinase, partially inhibits both human serum creatine kinase and purified creatine kinase from rabbit muscle. The amount of creatine kinase inhibition is related directly to the AMP concentration and inversely to the substrate (ADP) concentration. We found that 25 mmol/liter of fluoride inhibits adenylate kinase without measurable effect on creatine kinase activity. We developed a serum creatine kinase assay including fluoride, and compared it with the dual assay system and with two commercial assay kits. Other halides or adenosine 2'-monophosphate did not selectively inhibit adenylate kinase.


Effect of reaction initiator on human lactate dehydrogenase assay. Human lactate dehydrogenase isoenzymes I and V have decreased activities when the reaction is initiated with lactate. No loss in lactate dehydrogenase I activity was found when the reaction was initiated with enzyme or NAD+. For lactate dehydrogenase V an NAD+-initiated reaction, as compared to an enzyme-initiated reaction, yields lower activity in sodium pyrophosphate buffer but higher activity in tris(hydroxymethyl)aminomethane buffer. Both isoenzymes have higher lactate-to-pyruvate activity when assayed in the latter buffer than when assayed in the former. Human lactate dehydrogenase V (but not I) exhibited different activities when assayed with lactate from two different commercial sources. Human lactate dehydrogenase assayed by the pyruvate-to-lactate reaction is not affected by the choice of reaction initiator.


Apoprotein profile of plasma and chylous ascites lipoproteins. Plasma and chylous ascites lipoproteins were compared in a rare case of exudative enteropathy associated with stenosis of the thoracic duct. All ascites lipoproteins separated by ultracentrifugation showed a much higher triglyceride/protein ratio and a lower cholesterol ester content than their plasma counterparts. Polyarcylamide gel electrophoresis of apoproteins before and after fractionation on Sephadex G-200 essentially showed, compared to normal plasma, a reduction of apoprotein C in VLDL and HDL particularly obvious for apoCIII1 and CIII2. Ascites lipoproteins were characterized by an increased apoA content in chylomicrons, VLDL and LDL and a reduced percentage of apo CII and CIII1, mostly in chylomicrons, VLDL and HDL. The differences in composition between plasma and ascites might be explained by different origins for the various apoprotein subfractions. The transsudation of chyle into the abdominal cavity might divert some peptides of intestinal origin from their normal entry into the circulation.


Hepatic collagen proline hydroxylase activity in alcoholic liver disease. The activity of hepatic collagen proline hydroxylase was examined in biopsy samples as a factor in collagen synthesis in 77 patients with alcoholic liver disease. The urinary excretion of peptide bound hydroxyproline was also measured in most of the patients, as an index of collagen degradation. The highest activities of collagen proline hydroxylase were found in the patients with alcoholic hepatitis. Enzyme activity was markedly increased in patients with non-specific changes on liver biopsy, whereas, patients with fatty infiltration had only mild elevations, and those with inactive cirrhosis had normal enzyme activity. Urinary hydroxyproline was elevated only in patients with alcoholic hepatitis and inactive cirrhosis. Follow-up determinations in 16 patients with alcoholic hepatitis, after 4 to 5 weeks, revealed a decrease in enzyme activity, but no change in urinary hydroxyproline. We conclude that among the types of alcohol-related liver diseases, alcoholic hepatitis is associated with the greatest turnover of hepatic collagen.


Diagnosis of dyslipoprotein aemia by molecular sieve chromatography. Agarose gel molecular sieve chromatography has been used as a tool in the diagnosis of dyslipoproteinaemias. The elution profile from control plasma contained three peaks, corresponding to very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). These accounted for 9.5%, 23.5% and 67% of the total lipoprotein absorption at 280 nm. Elution patterns from dyslipoproteinaemic serum showed alterations, not only in peak area, but also in peak migration rate through the agarose gel. These variations were reproducible and of such magnitude as to permit the use of molecular sieve chromatography as a tool in the diagnosis of dyslipoprotein-aemias of Type I, II (a + b), III, IV and V.


Application of nicotinamide-adenine dinucleotide analogs for clinical enzymology: a spectrophotometric method for clinical analysis of lactate dehydrogenase patterns with the use of a nicotinamide-adenine dinucleotide analog. The activities of porcine lactate dehydrogenase (LHD) isoenzymes were analyzed using nicotinamide-adenine dinucleotide (NAD) or its analogs as a cofactor, with varying concentrations of L-lactate from 13 to 530 mM. The greatest differences between H4-type and M4-type isoenzymes in reaction rates were observed when their activities were compared in a reaction mixture containing 530 mM lactate and NAD, and also in a system of 13 mM lactate with thionicotinamide-hypoxanthine dinucleotide as a cofactor. The ratio of the LDH activity exerted in the former reaction mixture to that exerted in the latter was termed the N/T value. The N/T values of porcine H4 and M4 isoenzymes were 0.49 and 9.33, respectively. The N/T values of other three isoenzymes (H3M1, H2M2 and H1M3) were calculated by assuming that the single subunits H1 and M1 contribute one-fourth of the values of 0.49 and 9.33, respectively, and a given isoenzyme which is a combination of four subunits of H and M comprises the sum of their values. The calculated values agreed fairly well with the experimental results. The N/T value method was found to be applicable to human LDH isoenzymes, and sera from various patients were analyzed in comparison with hormonal sera. The method is particularly suitable for the numerical expression of LDH isoenzyme profiles.


Application of nicotin amide-adenine dinucleotide analogs for clinical enzymology: alcohol dehydrogenase activity in liver injury. The activities of alcohol dehydrogease(ADH) in serum and in the subcellular fractions of rat liver were determined with n-amyl alcohol or ethanol as substrate and thionicotinamide-adenine dinucleotide as coenzyme. It was found that the enzyme's activity ratio on the amyl alcohol and ethanol(A/E value) of serum and on the particulate fractions of the liver were different, but the A/E value of the soluble fraction was similar to that of serum. The A/E value of the particulate fractions were higher than that of the soluble fraction. From the results of experimental liver damage in the rat, it seems that estimation of the A/E value of ADH activity in serum is a useful parameter for the diagnosis of active liver injury. Since the A/E values of patients' sera differed from those of the normal subjects, the estimation of the A/E value of serum may give diagnostic information on liver injury, especially in chronic liver injury.


The determination of silicates in urinary calculi. The determination of silica and silicates in urinary calculi is rendered difficult by the common occurrence in such material of high concentrations of calcium, magnesium and orthophosphate. The problem has been studied, and manual and semi-automated procedures for the determination are described. 145 stones from the urinary tracts of patients with spinal cord injury were analysed, but in all of them the silicon content was below the limit of detection of 0.7 gSiO2 per 100 g calculus.


Neonatal metabolism and endocrinology studied by exchange transfusion. The work reviewed here illustrates how a therapeutic procedure, exchange transfusion of newborn infants, may be used to gather information which is both of practical value to infants treated in this way and also of value in the study of human neonatal physiology. The scientific deductions that can be drawn are weakened by the uncontrolled nature of the subjects available for study but this problem can be mitigated by changing one variable at a time between two groups of clinically similar infants undergoing transfusion and paying attention only to large changes in whatever is measured. In this way it has been possible to show that the glucose of ACD blood stimulates insulin and GH secretion and that the stimulation of insulin secretion is less, and that of GH more, if the transfusion is performed via the umbilical artery rather than via the vein. Arterial transfusions may be more stressful than venous ones since they are associated with greater growth hormone, ACTH and glucocorticoid release. Citrate, the other additive in ACD blood, causes a fall in ionised plasma calcium levels resulting in a stimulation of PTH secretion and mobilisation of calcium and phosphorus. Transfusion with heparinised blood is therefore preferred by some because normoglycaemia is preserved during and for three hours after transfusion, whereas post-transfusion hypoglycaemia may occur after ACD transfusion. However, heparin transfusion causes a marked rise in plasma FFA levels which may interfere with the binding of bilirubin by albumin. In either type of transfusion the side-effects may be minimised by feeding the baby afterwards, as soon as practicable. Thyroid hormones are washed out of the infant during transfusion but normal thyroid balance is restored quickly afterwards. The temperature of the donor blood does have thermal effects on the baby but these are less than might be expected due to the rapid equilibration of donor blood temperature with that of the room. The metabolic consequences of transfusion with cold blood are less than might be anticipated due in part to the glucose infusion that is part of an ACD transfusion.


Prenatal diagnosis of homoxygous familial hypercholesterolemia: investigation of a case at risk. Cultivated amnion cells obtained from a pregnancy at risk for the homozygous form of familial hypercholesterolemia were analyzed, as were fibroblasts from normal, heterozygous and homozygous controls. Three different methods were employed in order to compare their diagnostic value: i. Acetate incorporation into the cellular 3beta-OH-sterol fraction; ii. LDL-binding to the cell surface receptor; and iii. Oleate incorporation into the cholesterylester pool of the cells after addition of LDL to lipoprotein-deficient growth medium. The best discrimination between normal, heterozygous and homozygous cells was achieved using the third technique. On the basis of the acetate incorporation analysis, we concluded that the child is not homozygous, but probably completely unaffected. This diagnosis was confirmed by repeated determinations of plasma cholesterol levels during the first 11 months of life. Our investigations further substantiate the specularion that prenatal diagnosis of this disorder is possible.


Lp(a) lipoprotein/pre-beta1-lipoprotein, serum lipids and atherosclerotic disease. With appropriate electrophoretic techniques and fresh serum samples, the Lp(a) lipoprotein/pre-beta1-lipoprotein is demonstrable as a distinct zone in the area between beta-lipoprotein and ordinary pre-beta-lipoprotein, when sera which are strongly positive with respect to the Lp(a) antigen are analyzed. The Lp(a) lipoprotein is a genetically determined normal serum component. The phenotype Lp(a+) was found significantly more frequently in two series of patients with coronary heart disease (CHD) than in appropriate controls. The frequency difference between patients and controls was particularly pronounced for the Finnish samples studied, 55% of the patients having the phenotype Lp(a+), as opposed to only 31% of the healthy controls. As judged from electrophoresis strips, hibh concentrations of Lp(a) lipoprotein/pre-beta1-lipoprotein were positively correlated with coronary score as determined by angiography. This correlation was highly significant. Total serum cholesterol value was slightly higher in Lp(a+) than in Lp(a-) persons from two of the four population samples studied, but no statistically significant difference was found. Serum triglyceride levels exhibited a statistically insignificant trend towards higher values in Lp(a-) than in Lp(a+) individuals, in three of the four samples tested. The strong association between the phenotype Lp(a+) and CHD, as well as the correlation between high amounts of Lp(a) lipoprotein/pre-beta1-lipoprotein and coronary score on one hand, and the weak correlation between presence of Lp(a) lipoprotein/pre-beta1-lipoprotein and lipid values on the other, make it highly unlikely that the increased frequency of the Lp(a+) phenotype in CHD patients merely reflects an over-all increase of the intravascular pool of LDL and/or VLDL reflected in increased serum levels of cholesterol and/or triglycerides. By the same token, it is unlikely that the insignificant effect on lipid values can, on its own, explain the correlation between Lp(a) phenotype and CHD.


Pulmonary metastases in Wilms' tumour.

Pulmonary metastases in Wilms' tumour. In a series of 87 patients with a diagnosis of Wilms' tumour seen at the Great Ormond Street Hospital for Sick Children between 1960 and 1973, 15 patients had pulmonary metastases at the time of diagnosis and a further 22 patients developed pulmonary metastases within 18 months of their initial nephrectomy. The incidence of later development of metastases was very much greater in those patients with locally advanced primary tumours than in those with a completely resectable, encapsulated tumour. In both groups of patients the disease-free survival rate was closely related to initial local tumour staging. In the whole series there were 11/37 (42-5%) patients with pulmonary metastases who survived disease-free for two or more years following treatment. The survival rate was highest in those patients who had a solitary pulmonary metastasis which was surgically resected (5/6); this type of lesion was seen only in those patients who had a localised primary tumour. In those patients with multiple pulmonary metastases, treated with irradiation to the whole lungs and single-agent chemotherapy, there were only 6/31 disease-free survivors; this group of patients all had advanced primary tumours and there was a high incidence of associated abdominal recurrence. The need for multiple agent chemotherapy in conjunctin with surgery and radiotherapy, both as prophylaxis against and in the treatment of metastases from Wilms' tumour is stressed.


Evaluation of gallium-67 in the diagnosis of bronchial carcinoma. Twenty-four patients with carcinoma of the bronchus had lung scans performed with gallium-67. The results were compared with corresponding chest radiographs. In four patients who had been treated with radiotherapy or cytotoxic drugs, there was no uptake of isotope in the lesion. The isotope, however, identified the neoplasm accurately in 17 out of the 20 cases (85%). The relative distribution of isotope in neoplastic tissue and adjacent inflammatory reaction is discussed as well as those cases in which either the radiograph or the scan failed to demonstrate the malignant lesion. Gallium scanning in this study failed to indicate the extension of growth into the mediastinal glands.


The spectrophotometric determination of human serum carboxypolypeptidase with angiotensin converting enzyme-like activity. 1. A colorimetric method was developed for the direct chemical assay of human carboxypeptidase A (carboxypolypeptidase; EC 3.4.12.2) with angiotensin converting enzyme-like activity in serum or plasma, with the substrate analogue glycyl-L-histidylglycine and the angiotensin converting enzyme substrate angiotensin I (A-I). This method was based on the spectrophototometric determination of histidylglycine and histidyl-leucine, products of the hydrolysis of glycyl-L-histidylglycine and A-I respectively. omicron-Phthalaldehyde reacted with the imidazole moiety of nu-terminal histidyl peptides to produce a yellow chromophore. 2. A large number of inhibitors were tested for their effects on carboxypolpeptidase activity. The hydrolysis of Gly-His-Gly and A-I was inhibited by histidyl-leucine and angiotensin II, both products of the hydrolysis of A-I. Bothrops jararaca venom extract, EDTA, rho-chloromercuribenzoate, 8-hydroxyquinoline and 2,3-dimercaptopropanol, previously reported as converting enzyme inhibitors, also inhibited carboxypolypeptidase activity. 3. Angiotensin converting enzyme activity in the serum of sixty-six adults ranged from 10 to 37 nmol of glycyl-L-histidylglygine hydrolysed in 10 min by 10 mu1 of serum at 37 degrees C and pH 7-25.


Effect of diphosphonates on adenosine 3':5'-cyclic monophosphate in mouse calvaria after stimulation by parathyroid hormone in vitro. 1. The diphosphonates, disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) and disodium dichloromethylene diphosphonate (Cl2MDP), inhibit bone resorption in animals and in explanted bone in tissue culture. The possibility that these effects might be due to inhibition of skeletal adenylate cyclase has been studied. 2. EHDP and Cl2MDP, added for 30 min to the incubation medium at concentrations known to inhibit bone resorption, had no effect on basal content of adenosine 3':5'-cyclic monophosphate (cyclic AMP) of mouse calvaria incubated in vitro, nor did they inhibit the rise in cyclic AMP induced by bovine parathyroid hormone. 3. Pretreatment of mice for 3 days with Cl2MDP also had no effect on cyclic AMP under basal conditions or after incubation of explanted calvaria with parathyroid hormone in vitro. EHDP under similar conditions slightly inhibited the increase induced by parathyroid hormone but had no effect on basal concentrations of cyclic AMP. 4. It is suggested that the inhibition of adenylate cyclase is not an essential feature of the reduction of bone resorption by diphosphonates, which may act by direct inhibitory effects on the dissolution of hydroxyapatite and perhaps by other unidentified effects on bone cells. Key words: adenosine 3':5'-cyclic monophosphate, bone, dichloromethylene diphosphonate, diphosphonates, ethane-1-hydroxy-1,1-diphosphonate, parathyroid hormone.


The effect of insulin on adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate concentrations in human plasma. 1. The action of insulin on plasma cyclic nucleotide concentrations in normal human subjects has been studied after intravenous injection, alone and in combination with glucagon. 2. After injection of insulin alone there was an initial small, though not significant, decrease in plasma cyclic AMP at 15 min followed by an increase to more than twice the initial concentration at 30 min. The increase was absent when hypoglycaemia was lessened by infusion of glucose after insulin injection. 3. Injection of insulin caused no significant change in plasma cyclic GMP concentration, whether or not glucose was infused after the hormone. 4. Glucagon (3-300 nmol, 10-1000 mug), caused a dose-dependent increase in plasma cyclic AMP concentration. The rise in plasma cyclic AMP produced by 3 or 30 nmol of glucagon was not significantly modified by simultaneous injection of insulin (44 nmol; 6 units).


Effects of prednisolone on the small intestinal mucosa of the rat. 1. The effects in rats of an oral pharmacological dose of prednisolone on mucosal function, enzymology, lysosomal membrane fragility, morphology and cell kinetics have been examined in proximal jejunum and distal ileum. 2. The maximum absorptive capacity for galactose was significantly greater in both the jejunum and the ileum of the steroid-treated animals. This was due to an increase in carrier-mediated transport in the individual enterocytes and not to a change in the cell population. The Michaelis constant for galactose was not significantly altered by prednisolone. 3. Activities of brush-border and mitochondrial enzymes and of RNA were increased in isolated enterocyte preparations from the jejunum and ileum of the steroid-treated group. 4. Lysosomal membrane fragility was unaltered in the prednisolone-treated group. 5. Morphometrical observations confirmed that the size of the enterocyte population was unaltered by prednisolone. Studies on cell kinetics indicate that the effects of prednisolone are due to a direct action on the enterocyte and not secondary to changes in migration rate.


Diseases associated with herpesviruses.

Diseases associated with herpesviruses. Human herpesviruses have been associated with numerous diseases throughout history (Table 3), but their ability to induce latent and recurrent infections, alongwith their oncogenic capabilities, is only beginning to be understood. Accordingly, our ability to deal with the diseases induced by herpesviruses is severely limited by our lack of information concerning the basic processes of virus-host cell interactions and systemic host factors; especially immune factors that are involved in the disease process.


The abnormal brain scan: specificity of descriptive parameters. When a brain scan is abnormal, one can often describe the abnormality in terms of its location, shape, sharpness, brightness, and other descriptive parameters. Certain types of abnormality have been linked to certain specific diagnoses: for example, a crescent-shaped lesion would suggest subdural hematoma to many people, a wedge-shaped lesion would suggest cerebral vascular accident, etc. Some features thought to be characteristic of certain diseases are actually quite nonspecific. For example, the "doughnut" sign--at first believed to be characteristic of brain abscess--has also been found in primary and metastatic tumors, CVA, and subdural hematoma. The "crescent" sign that was at first thought to be specific for subdural hematoma occurs also in meningitis, scalp or skull trauma, meningioma en plaque, etc. Some features of abnormalities are highly specific for certain diseases; e.g., wedge- or flame-shaped lesions are rarely seen with disorders other than CVA, and lesions in the midline or in the posterior fossa are almost invariably tumors. This article reviews the features of abnormalities on brain scans that in the literature have been reportedly associated with specific types of disease and explores the strength and validity of the associations.


Poly-hormonal evaluationof pulsatile adenohypophysis incretory activity during sleep in normal, experimental and pathologic conditions. Secretory patterns of some anterior pituitary hormones were recorded during sleep, and related to EEG-phases. The same observations were carried out in conditions in which the normal sleep course was intentionall (i.e., repeated arousals, administration of tryptophan) or pathologically (i.e. chronic schizophrenia) modified. Plasma gonadotropins (lh, fsh), growth hormone and ACTH were assayed. In basal conditions, the well-known peak of HGH, which corresponds to the delta phase of sleep, was confirmed. This peak is strictly linked to the number and duration of stage 4 sleep, and follows it in its experimental and/or pathological disturbances. The secretion of ACTH, on the other hand, seems to be completely independent from the morphology of sleep. The secretion of this hormone is typically phasic; along with this phasic secretion, a background secretion is detectable, which increases steadily during the sleep span, with maximal values in the early morning hours. Gonadotropin secretion is pulsatile, episodic and completely independent from the sleep phases, in basal as well as in experimental and pathologic conditions.


Circadian rhythm and cell cycle: possible entraining mechanisms. Circadian rhythms and cell cycles are endogenous self-sustained oscillations. They differ in several characteristics of the rhythmicity such as temperature dependency, reaction to external stimuli and the impact of protein synthesis. In multicellular organisms, the cell cycle very often is entrained by a circadian rhythm as has been analyzed in rats and mice, in particular. The same seems to hold true in cell cultures: Yoshida-ascites hepatoma cells, human embryonic fibroblasts, rat liver and rat hepatoma cells show circadian changes in the percentage of cells in G1-, S- and G2 + M-phases. The different phases within the cell cycle were determined by applying impulse cytophotometric methods. Some hypothetical mechanisms of entrainment of the cell cycle by circadian rhythms are discussed. Possible entrancing signals (Zeitgebers) are membrane and transport functions, cyclic nucleotides, nuclear non-histone proteins and their phosphorylation, and RNA synthesis. The maxima of the circadian rhythms of most of these functions in rat liver can be arranged in a certain temporal sequence.


[VIP and GIP-producing pancreatic tumour: relationship to the Verner-Morrison syndrome]. A 59-year-old man had explosive watery diarrhoea, tendency towards collapse, flushes and aphonia. Pre-operative serum concentrations of vasoactive intestinal polypeptide (VIP) were up to 1030 ng/l, those of gastric inhibitory polypeptide (GIP) up to 2675 ng/l, as measured by radioimmunoassay. Cross-reaction by antisera used in the radioimmunoassay were excluded. Pancreatic tumour was diagnosed by ultrasound and by elective coeliac arteriography. After excision the abnormal fidings disappeared as did the symptoms. Biological half-life of plasma-VIP (determined during removal of the tumour from plasma samples by radioimmunoassay) was about 45 minutes. The tumour produced both VIP and GIP.


[Late effects of tumour treatment. Structural changes in the spinal column seen on X-rays]. Cure rates are increasing in pediatric oncology; simultaneously the numbers of late effects of therapy are also increasing. 64 children with malignant neoplastic disease in whom the spine had been partially or totally exposed during X-ray treatment were re-investigated for radiographically visible irradiation effects on the vertebral column. 140 different abnormalties were found in 56 children of which scolioses were the most important. Irradiation for Wilms' tumour produced more numerous changes than direct irradiation of the vertebral column. In spite of the unexpectedly high number of growth defects rigorous X-ray treatment is indicated in certain circumstances. The growth abnormalities were of only minor clinical significance.


[Dynamics of sleep and the waking state after labyrinthine deafferentation]. The author carried out studies on Awake state and sleep (slow and paradoxal) after lybyrinth deafferentation. The states were evaluated by behavior, EEG, myogram and eye movements. The labyrinth defferentation was performed in combination (chemical and mechanical). Bilateral labyrinth deafferentation reduced the duration of the awake state from 30% to 17%, p less than 0.001. The slow sleep was increased at the same time but there was no statistical significance. The parodoxal sleep was due both to the increased number of episodes of parodoxal sleep and to the increased of their mean duration. The latent period of the first appearance of the episode of paradoxay sleep was shortened.


[Mechanism of action of lithium in manic-depressive psychoses: current research results]. The mechanism of action lithium in the treatment of manic-depressive illness is far from being known. A great progress will be achieved when it is known wherther lithium has an actual anti-depressive action, which implies to determine whether all the "endogenous" depressive states belong to the same pathogenic group. Lithium salts have a real therapeutic action on manic states which may be put together with the decrease of available Norepinephrine that lithium salts bring about on cerebral receptors. This decrease concords with the aminergic theory of affective disorders. Many actions of lithium salts on the physiologic systems have been brought to evidence but the uncertainties that remain concerning their interactions prevent us to formulate any coherent assumption.


Estrogen-binding parameters of cytoplasmic and nuclear receptors in an established rat endometrial cell line and tumor. We have consistently found receptors for estradiol in both the cytosol and nuclear extracts of a rat endometrial cell line and in transplantable tumors derived from this cell line. The equilibrium dissociation constants (Kd) and the rate constants for the receptor-estradiol interaction in these cells and tumors did not differ significantly from those of the cytosol receptor in the rat uterus. A mean Kd of 3 x 10(-10) M with a rate of association (Ka) of 3 x 10(5) M-1sec-1 and a rate of dissociation (Kd) of 1.5 x 10(-5) sec-1 were obtained for nuclear and cytosol receptors for both tumors and cells. For uterine cytosol, a Kd of 8 x 10(-10) M, ka = 2.8 x 10(5) M-1sec-1 and kd = 1 x 10(-5) sec-1 were obtained. Although no differences were seen in equilibrium and kinetic parameters for estradiol-17beta binding between the nuclear and cytosol receptors of tumors and cells, an apparent difference in the relative affinities of nuclear and cytosol receptors for estrone was detected. This suggests that the binding site in nuclear receptors may have been modified. Implications of this observation with regard to receptor translocation and the mechanism of action of sex hormones are being considered.


125I-LH binding to rat testes at various ages and posthypophysectomy. Binding of 125I-LH by the rat testes was investigated during various stages of sexual maturation and in mature animals following hypophysectomy. Hormone binding per mg testicular tissue increased with age and was shown to be due to larger receptor concentration rather than greater binding affinity. This observation cannot be accounted for by changes in the relative number of Leydig cells and suggests, therefore, that Leydig cells acquire additional receptors during sexual maturation. Binding of 125I-LH to mature testes declined after hypophysectomy. Three days following pituitary removal the LH-receptor concentration decreased to one half of normal control value, then remained unchanged until the 37th post-operation day. Replacement therapy with LH, FSH or testosterone propionate failed to maintain 125I-LH binding at prehypophysectomy level.


Elevation of cyclic AMP levels and adenylate cyclase activity in duodenal mucosa from vitamin D-deficient rats by 1alpha,25-dihydroxycholecalciferol (1alpha,25-(OH)2D3). A single 270 ng dose of 1alpha,25-(OH2D3 rpoduced elevations in cyclic AMP content and adenylate cyclase activity in duodenal mucosa from previously vitamin D-deficient rats. No changes in jejunal or ileal cyclic AMP levels or duodenal cyclic GMP levels were observed. Since 1alpha,25-(OH)2D3 increased both baseline and NaF-stimulated adenylate cyclase activity, it is possible that the vitamin leads to enhanced enzyme synthesis. While parallel changes in duodenal cyclic AMP levels and active calcium absorption in response to 1alpha,25-(OH)2D3 were observed at 6,12,24 and 48 hr after treatment, increases in calcium absorption were observed at 3 hr in duodenum and at 48 hr in ileum in the absence of changes in cyclic AMP levels. Further studies will be required to determine whether or not the changes in duodenal cyclic AMP levels are direct or indirect effects of 1alpha,25-(OH)2D3 administration, and to determine the role, if any, of this nucleotide in the hormones' effect on intestinal calcium absorption.


Binding of 125I-HCG by rat testis homogenates in early neonatal period. The binding of 125I-HCG by rat testis homogenates in early neonatal period was studied. The specificity of this binding was demonstrated by competition of unlabeled HCG and LH for binding sites and by failure of such binding to occur in specimens obtained from control tissues. FSH and ACTH were not able to compete significantly for 125I-HCG binding. The HCG-LH receptor is present in the testis during the whole postnatal period. The uptake of 125I-HCG by the testis of young rat does not differ during first two weeks of life and the rise of binding occurs at about the third postnatal week.


Binding of testosterone, dihydrotestosterone and progesterone to rat prostate cytosol: application of microcalorimetry. The binding capacity of rat prostate cytosol for dihydrotestosterone, testosterone and progesterone was studied in vitro. The thermodynamic parameters obtained by microcalorimetry point to the importance of the observation of energy changes occuring during the hormone-receptor complex formation.


Radioligand- receptor assay of LH-RH.

Radioligand- receptor assay of LH-RH. Characteristics of LH-RH interactions with the plasma membrane of the anterior pituitary were utilized to develop a radioligand- receptor assay for the hypothalamic decapeptide. Plasma membranes were prepared from rat adenohypophyses and association of 125I-LH-RH with the cell membrane was found to reach a plateau after 30 min incubation time. Sensitivity of the present competitive protein binding assay was 10 ng. The system was shown to be sensitive to structural changes in the LH-RH molecule. No cross-reactivity of (3--10) LH-RH, pyro-His-Trp-SerOMe and TRH was recorded with LH-RH, whereas slight cross-reaction was observed for (Lys8) LH-RH and (Ala4) LH-RH showed marked cross-reaction. Although the present system is not sensitive enough to estimate physiological levels of LH-RH it seems to be a valuable approach for setting up an LH-RH assay system.


LH-RH-sensitive adenylate cyclase in isolated plasma membranes of rat adenohypophyses. The adenylate cyclase system in isolated plasma membranes of rat adenohypophyses was studied. Formation of cAMP was found to be dependent on plasma membrane protein. Production of cAMP could be stimulated by the addition of LH-RH. This process was shown to be a function of LH-RH concentration. Basal and stimulated cAMP could be stimulated by NaF. The results of the present experiments suggest that cAMP formation is time dependent. The synthesis of cAMP is a possible effector of LH-RH actions.


Milk-ejecting and uterotonic activities of oxytocin analogues in rats. The milk-ejecting activity of oxytocin analogues modified in the aminoterminal part of the molecule was determined in experiment on rats in vivo. As compared with oxytocin, the analogues did not have significantly higher milk-ejecting activity and there was no dissociation of the two basic oxytocin-like activities.


Effect of prolactin on LH receptor in rat luteal cells. This investigation was undertaken to determine whether prolactin might act as a luteotropic hormone in the rat by increasing the receptor for LH. Immature female rats were treated with 5 SC injections of oFSH beginning at 0900 h on day 25; luteinization was induced with a single SC injection of oLH at 1500 h on day 27. In this model, LH-receptor activity, assessed by specific binding of [125I]iodohCG to ovarian membrane fractions, increased steadily (greater than 7-fold) from day 27 to day 33; specific [125I]iodoprolactin binding rose greater than 3-fold between days 27 and 31. Serum progesterone measured at 1500 h rose from greater than 10 ng/ml on day 27 to greater than 130 ng/ml on day 34. 2-Bromo-alpha-ergocryptine (CB-154, [EC]; 35 mug) given 2 X/day beginning at the time of LH injection, blocked nocturnal increases in serum prolactin measured at 0200 h on days 29, 30, and 31, and resulted in reduced binding of [125I]iodohCG on days 31 and 33; serum progesterone was similarly reduced on days 31 and 34 in EC-treated rats. Simultaneous treatment with EC and prolactin completely reversed the effects of EC with regard to both binding of [125A]iodohCG and serum progesterone. Changes in ovarian uptake of [125I]iodohCG in vivo were similar to those of the in vitro experiments described above. EC and/or prolactin treatment did not affect binding of [125I]iodoprolactin. In summary, the data indicate that LH in the absence of prolactin can induce the formation of corpora lutea which have an increased number of prolactin receptors but which have a few LH receptors and a reduced capacity to produce progesterone. If prolactin is present during the early luteinization process, corpora lutea develop an increased capacity to bind LH and to produce progesterone. It is possible that the increase in LH-receptor and progesterone production occur as independent events both mediated by the luteotropic action of prolactin. Alternatively, LH receptor might be limiting for LH stimulation of progesterone production.


Development of insulin and glucagon binding and the adenylate cyclase response in liver membranes of the prenatal, postnatal, and adult rat: evidence of glucagon "resistance". Although plasma glucagon levels in the rat fetus are in the adult range, hepatic glycogen is present in far greater abundance in the fetus than in the adult. To explain this paradox, adenylate cyclase response to glucagon was studied in partially purified membranes of rat livers obtained throughout perinatal life and at 3 months of age. The adenylate cyclase response to glucagon (10(-9) M) was only 7% of the adult response at day 15 of fetal life and 20% on the 21st day. No until after the 30th day postpartum did not reach maturity. Yet, the adenylate cyclase response to stimulation by NaF was comparable to the adult response throughout fetal life. The binding of [125I]iodoglucagon (2 X 10(-9) M) by these membrane preparations was only 1% of the adult level at day 15 of fetal life and increased to 23% at the 21st day, and, like the adenylate cyclase response to glucagon, did not reach maturity until after the 30th day of postnatal life. In contrast, insulin binding on the 15th day of gestation was 11% of the adult level and on the 21st day 45% of the adult level, reaching adult levels by the 30th postnatal day. An increase in membrane-associated particles, reflecting intramembranous protein, was observed during prenatal life, but the mean particle number per mum2 reached adult levels on the 21st day of fetal life, indicating that subsequent changes in hormone binding were clearly independent of non-specific changes in the number of particles. The findings suggest that the fetal liver is less sensitive to glucagon action than the adult liver, and that this glucagon "resistance" is mediated by a reduced capacity of the hepatocyte to bind glucagon at a time when substantial binding of insulin is demonstrable. Selective discrimination against glucagon may be important in promoting the anabolic processes required for normal fetal development.


Sex-related differences in the regulation of cytoplasmic estrogen receptor levels in responsive tissues of the rat. Cytoplasmic estrogen receptors were measured in the anterior pituitary and hypothalamus of intact adult Holtzman male and female rats at various time intervals following injection of 10 mug of 17beta-estradiol. Following equivalent depletion of receptors at 1 h in either sex, replenishment of cytosol receptor levels progressed at a greater rate and to a higher level in the male than in the female. Cycloheximide only partially inhibited replenishment, and its blocking effect was similar in males and females. The administration of 5 mug of 17beta-estradiol to adult males elicited less depletion than did 10 mug, and a completely cycloheximide-sensitive replenishment phase. Patterns of depletion and replenishment in immature rats following 0.1 mug of 17beta-estradiol were the same for males and females. In an analysis of the effects of neonatal androgenization, Charles River rats were used and it was initially demonstrated that no sex differences in receptor levels existed between normal males and females. After an injection of an androgenizing level of testosterone propionate on day 3 of age, a full normal complement of receptors was found in estrogen-responsive tissues at 90--100 days of age. The functional dynamics of receptor depletion and replenishment under the influence of exogenous estrogen, however, indicated that the anterior pituitary of the androgenized female responded differently from that of the untreated female, and similarly to that of the male. The hypothalamic receptor dynamics in the male were unaltered by early androgen administration, but estrogen-induced depletion of receptors in the female hypothalamus was considerably less extensive in the androgenized animal than in the untreated control. Whereas cytoplasmic estrogen receptor levels are essentially independent of sex in the rat, the present results indicate a sex-linked difference in the dynamics of receptor turnover in the anterior pituitary and hypothalamic response to 17beta-estradiol.


Androgen and estrogen receptors in brain cytosol from male, female, and testicular feminized (tfm/y hermaphrodite) mice. Specific binding of [3H] 5alpha-dihydrotestosterone (DHT) and [3H] estradiol by cytoplasmic extracts from whole brain of castrated male, female, and androgen-insensitive, testicular feminized (tfm/y male-female), mice has been investigated using glycerol gradient centrifugation and charcoal assay. Mouse brain cytosol contains macromolecules with the characteristics of steroid hormone receptors, binding preferentially with high-affinity androgens or estrogens. Both DHT- and estradiol-receptor complexes migrate at 8-9 S in gradients at low ionic strength and at 4-5 S in gradients containing 0.5M KCl. KD's (mean +/- SE) for DHT binding by brain cytosol from castrated males, females, and tfm/y male-female are 1.1 +/- 0.4, 0.9 +/- 0.4, and 0.8 +/- 0.1 X 10(-9)M, respectively. DHT binding activity in brain cytosol from tfm/y male-female mice is reduced to about 20-30% of that from their normal littermates, as is the case for tfm/y male-female kidney cytosol. The residual androgen receptor in tfm/y male-female brain cytosol has normal sedimentation properties. Unlike the situation for androgen binding, the number of estradiol binding sites is comparable in brain cytosol from male, female, and tfm/y male-female mice. KD's (mean +/- SE) for estradiol binding are 1.6 +/- 0.5 X 10(-10)M for castrated males, 2.4 +/- 0.4 X 10(-10)M for females, and 1.8 +/- 0.4 X 10(-10)M for tfm/y male-female. Cross-competition experiments with unlabeled estradiol, DHT, or testosterone, have shown a difference in the degree of specificity of the androgen and estrogen receptors, the estrogen receptor having considerably more specificity. For the interaction of estradiol with the androgen receptor, the Ki is 8-9 X 10(-9)M. The decrease in the number of DHT binding sites in the brain of tfm/y male-female mice without a concomitant decrease in estradiol binding sites, and the different specificities of the two sites, point to the existence of distinct androgen and estrogen receptor molecules in mouse brain cytosol.


Activation of thyroid adenyl cyclase by antisera to thyroid plasma membrane preparations. Adenyl cyclase was assayed in a series of plasma membrane fractions prepared from bovine thyroids using sucrose density gradient ultracentrifugation. The activity of adenyl cyclase in the plasma membranes was found to be stimulated several-fold by antisera raised in rabbits against the plasma membranes and by purified IgG prepared from these antisera. The data indicate that the antibodies to thyroid plasma membrane manifest properties similar to those of the immunoglobulins long-acting thyroid stimulator (LATS) and human specific thyroid-stimulating immunoglobulin (HTSI) found in patients with Graves' disease. It is suggested that, in these patients, the antigen which leads to the production of LATS and HTSI is most likely situated in the thyroid plasma membrane.


Studies on the corticotrophin-releasing activity of vasopressin, using ACTH secretion by cultured rat adenohypophyseal cells. CRF activity of synthetic vasopressins and pitressin was studied in an in vitro system of cultured rat adenohypophyseal cells using direct measurement of ACTH by radioimmunoassay. Pitressin (posterior pituitary extract) induced a dose-related secretion of ACTH whereas synthetic arginine or lysine vasopressin were devoid of CRF activity, even with the largest tested dose (4 mug/ml). No potentiation of the CRF activity of hypothalamic extract was observed with any vasopressin preparation studied. We concluded that: 1) the CRF activity of posterior pituitary extract is not due to vasopressin, and 2) the ACTH secretion induced by vasopressin administration in vivo is unlikely to be due to a direct effect of vasopressin on adenohypophyseal cells.


Studies on fast feedback mechanisms by endogenous glucocorticoids. Negative feedback in the hypothalamic-pituitary-adrenal axis by exogenous glucocorticoids has been well established, but it is still in doubt whether ACTH-releasing factor (CRF) activity in the hypothalamus is inhibited by endogenous glucocorticoids under conditions of stress. In our first experiment, CRF activity in the hypothalamus of rats was significantly increased, 2 min after onset of immobilization, for 2 min; a rapid drop of CRF activity followed immediately. On the other hand, the concentration of plasma corticosterone changed more slowly; it was increased at 5 min, and showed a progressive further increase until 17 min after the administration of the immobilization stress. A high plateau level persisted for the next 8 min, after which the concentration of plasma corticosterone fell, reaching the control level at 40 min. In order to test for negative feedback inhibition by endogenous glucocorticoids in the hypothalamic-pituitary-adrenal axis, a 2nd immunobilization stress was administered to rats 5 or 23 min after the 1st stress, and hypothalamic CRF activity and plasma corticosterone were examined after the 2nd stress. When the 2nd stress was applied 5 min after the start of the 1st stress, during the phase of a gradual increase of endogenous glucocorticoids, CRF activity was not increased 2 min after the 2nd stress. In contrast, an increase of CRF activity and plasma corticosterone was observed when the 2nd stress was administered 23 min after the onset of the 1st stress, during the phase of a high plateau level of endogenous plasma corticosterone. These data suggest that CRF activity in the hypothalamus is governed by a fast feedback, rate-sensitive mechanism.