Experimental intraamnionic exposure of bovine fetuses with subgroup 2, type 7 Adenovirus. Three third trimester fetuses were exposed to a subgroup 2, type 7 adenovirus (adeno 7) by intraamnionic infection. The virus caused preterm delivery of two clinically ill calves and one stillbirth. The two premature calves died 12 and 72 hours after birth. An elevated serum neutralizing antibody titer (1:256) to adenovirus 7 was found in one principal calf at birth. Adenovirus 7 was recovered from several tissues of the live calves and the spleen of the stillborn calf. Fetuses exposed by intraamniotnic injection with virus carrier only, were born healthy after normal gestational periods and no viruses were isolated from the tissues. Clinically ill calves were weak, severely depressed and unable to stand and nurse. Gross postmortem lesions were nonspecific and consisted of petechial and ecchymotic hemorrhages and edema of the gastrointestinal tract. Histopathological lesions included vasculitis, necrosis of the mucosa of the forestomach, mild gastroenteritis and acute, nonsuppurative focal necrosis of the liver, kidney and adrenal gland. Intranuclear inclusion bodies were seen in pericytes, macrophages, hepatocytes, epithelial cells of adrenal cortical sinusoids of the zona glomerulosa and zona fasciculata and renal tubular epithelium.


Acetaldehyde affects mammalian neuromuscular transmission without observable postsynaptic effects. Acetaldehyde, the first metabolite of ethanol, caused a reversible block of the end-plate potential (EPP) in the rat and mouse phrenic nerve--hemidiaphragm preparation. Decrease and block of the EPP occurred over a bath concentration range from 3 to 25 mM. The phrenic nerve compound action potential was blocked along with the EPP, and this block was not reversed by high bath Ca2+ concentration. The muscle action potential was unaffected even at concentrations up to 50 mM. Over the same concentration range (3--25 mM), miniature end-plate potential (MEPP) frequency sometimes decreased a few minutes after application, and over the ensuing 10--20 min would steadily increase to as much as 11 times the base-line frequency, particularly with higher doses. However, the shape of averaged MEPPs remained unchanged after acetaldehyde application, suggesting that this aldehyde does not have post-synaptic effects.


Characterization of the inotropic response induced by stimulation of beta-adrenergic and H1 histaminergic receptors in guinea pig left atria. The inotropic response induced by beta-adrenergic and H1 histaminergic receptor stimulation was characterized in guinea pig left atria by obtaining dose-response relationships for isoproterenol and histamine under various experimental conditions. Conditions (hypothermia, high frequencies of stimulation, and large extracellular calcium concentrations) which enhanced the ability of cardiac muscle to develop force also increased the sensitivity of the left atrium to isoproterenol while decreasing its efficacy. On the other hand, conditions which enhanced the ability of cardiac muscle to develop force depressed the efficacy of histamine to such an extent that the sensitivity to histamine was also decreased. In addition, conditions which markedly depressed the ability of cardiac muscle to develop force also decreased the efficacy and sensitivity to histamine. The data indicate that while beta-adrenoceptor stimulation results in an inotropic response under all conditions studied, stimulation of H1 histaminergic receptors results in an inotropic response only within a narrow range of experimental conditions.


Photoaffinity labeling of the angiotensin II receptor; pharmacology of the labeling peptides in the dark. The biological activities of photoaffinity labeling analogs of angiotensin II (ATII) and their precursors were measured in rabbit aorta strips in the dark. Most of the analogs behave as reversible, specific agonists, one as a competitive inhibitor. The activities are discussed in line with the current view of structural requirements. The modifications consisted of substitutions on the aromatic nuclei of Tyr4 and Phe8 in [Sar1]ATII with (4'-NO2) Phe, (4'-NH2)Phe, (4'N3)Phe, (4'-N2 +)Phe, and (4'-NH2-3', 5'-I2)Phe. It is shown that the affinity of the ATII analogs modified in position 4 depends on the electronegativity and not on space-filling properties of the aromatic residue; rising electronegativity lowers the affinity, i.e. [sar1, (4'-NO2)Phe4]ATII has no more measurable activity. Substituting the aromatic side chain in position 8 of [Sar1]ATII gives well-binding analogs with intrinsic activities from 0 to 100% and activity seems to depend only on stereochemical requirements. Agonists and partial agonists bear rather small groups like -NH2, -N3, -NO2, and -N2 +. The only antagonist [Sar1, (4'-NH2-3',5'-I2)Phe8]ATII resembles the antagonist E1Sar1, Leu8]ATII in competitivity and binding.


Stereospecific presynaptic inhibitory effect of delta9-tetrahydrocannabinol on cholinergic transmission in the myenteric plexus of the guinea pig. delta9-Tetrahydrocannabinol (THC) is very lipid soluble, as are many anesthetic agents. The action of anesthetics is nonspecific; isomers are equieffective. THC is optically active; therefore, the effects of its stereoisomers were studied on the electrically and chemically stimulated longitudinal muscle strip of guinea pig ileum. The results demonstrate that both isomers depress the response to electrical stimulation in a dose-related manner. The maximum effect is gradually reached in approximately 20 min. The (-) isomer is 24.6-fold more active than the (+) isomer (ED50 for (-) THC is 1.25 X 10(-7) M, for (+) THC, 3.08 X 10(-6) M) and the site of action appears to be presynaptic because responses to ACh are not significantly depressed. The depressant effects are relatively nonreversible. Membrane concentrations calculated at the ED50 values for the (-) isomer are of the order of 0.5 mM/kg dry membrane, well within the range for anesthesia. Thus THC may be regarded as a partial anesthetic since some of its actions are similar to those of the classical anesthetics, yet it possesses selective action at the neuronal membrane or tissue level.


Effect of ovine prolactin on serum somatomedin bioactivity in hypophysectomized female rats. Ovine prolactin (oPRL) increased serum somatomedin (SM) bioactivity in hypophysectomized female rats. ACTH, in small but not large doses, augmented this oPRL effect. These results suggest that in the female rat PRL may regulate SM production. Adrenal factors may variably modulate SM production or serum SM bioactivity.


5-Fluoroorotate-resistant mutants of Salmonella typhimurium. Spontaneously occurring mutants of Salmonella typhimurium resistant to 5-fluoroorotate (5-FOA) were isolated. One class of mutant showed marked derepression of pyrimidine biosynthetic enzymes and had the unusual property of being unable to grow on nutrient agar. However, when the osmotic strength of nutrient agar was increased, the mutants were able to grow. The genetic basis for the osmotic fragility and elevated pyr enzyme synthesis was the result of mutations affecting pyrH, encoding the enzyme uridine 5'-monophosphate kinase.


Construction of an Escherichia coli strain which excretes abnormally large amounts of adenosine 3',5'-cyclic monophosphate. It has previously been shown that an Escherichia coli CRP- strain 5333 accumulates abnormally large amounts of adenosine 3',5'-cyclic monophosphate (cAMP). Using P1 transduction, the CRP- character was transferred to E. coli Crookes strain which is deficient for cAMP phophodiesterase (CPD-). The resulting strain HY22 (CRP-, CPD-) accumulates greater amounts of cAMP both intracellularly and extracellularly than does 5333. In glucose minimal medium, an HY22 cell accumulates 100 times more cAMP intracellularly and excretes cAMP 150 times faster than does a wild-type E. coli cell.


Automatic quantitative analysis of the electromyogram in partially denervated distal muscles: comparison with motor unit counting. Using the ANOPS-101 minicomputer, an automatic analysis of muscle action potentials (MAP) was performed on a total of 53 extensor digitorum brevis (EDB) and abductor pollicis brevis (APB) muscles in patients with other evidence of denervation. The results were compared with observations made on 27 control subjects and they were also matched against estimates of functioning motor units made by the incremental stimulating technique of McComas et al (1971). Although the incidence of abnormal MAP parameters was relatively low in both muscles, it was possible to improve the diagnostic yield by combining the results for individual muscles. The relative merits and disadvantages of motor unit counting and automatic MAP analysis are discussed.


Accumulation and removal of Hg203 in different regions of the rat brain. We have studied the brain regional distribution of methyl mercury following intravenous administration of CH3 203HgCl in rat. Early peak levels were obtained in cerebellum, medulla oblongata and midbrain. The efficacy of removal of 203Hg by different chelators is also region dependent. The most efficient chelator for brain mercury proved to be mesodimercaptosuccinic acid.


Virology and histopathology of the trigeminal ganglia of Americans and Japanese. Herpes simplex virus in the trigeminal ganglia of humans was studied in Philadelphia, Pennsylvania, United States of America, and in Kyoto, Japan. The prevalence of recurrent herpes labialis and of clinically latent herpes simplex virus within trigeminal ganglia was determined in inhabitants of the two cities. In addition, a comparison was made of the prevalence of mononuclear cell infiltration in the trigeminal ganglia of Americans and Japanese. Recurrent herpes labialis was found to be significantly less common in the Japanese city than in the American city. Herpes simplex virus was rescued less commonly from the trigeminal ganglia of cadavers in Japan than in America. The difference was significant. The frequency of mononuclear cell infiltration in the trigeminal ganglia of Americans and Japanese is not significantly different. These observations, as well as previously reported serological studies, suggest that despite the ubiquitous nature of herpes simplex virus in America and Japan, the Japanese have less clinically overt disease caused by this virus.


Anabolic steroid therapy and intrahepatic cholangiocarcinoma. A 47-year-old man who had been treated with an anabolic steroid for refractory anemia developed an intrahepatic cholangiocarcinoma with metastases to abdominal lymph nodes and lungs. Microscopically, the tumor showed mucin production and was devoid of hepatocellular elements. Previous reports have suggested a possible relationship between anabolic steroid therapy and hepatocellular carcinoma. In many such cases, there are doubts about the histological diagnosis, malignant potential, and the nature of the association between the steroids and the tumors. The presence of distant metastases attests to the malignant nature of the tumor in the present case. Despite the apparent temporal eligibility of the steroid as an etiologic agent, a causative relationship between therapy and tumor is not established.


Severe pulmonary hemorrhage following lymphography. The hemoptysis which occurs following lymphography typically consists of one or more episodes of blood-tinged sputum, and generally requires no treatment. In contradistinction to this "mild" form of hemoptysis, the current report describes a case of hemoptysis following lymphography--hemoptysis so severe as to require multiple transfusions. Time course and possible etiologic factors are discussed.


Myxoid variant of malignant fibrous histiocytoma: ultrastructural observations. The ultrastructural findings in 4 cases of the myxoid variant of malignant fibrous histiocytoma (MFH) are described. Although this neoplasm is characterized by a high rate of local recurrence, the overall prognosis is considerably better than the usual nonmyxoid MFH. The neoplasm is composed of an admixture of round, stellate and multinucleated giant cells within a myxoid and well-vascularized stroma. Electron microscopy demonstrated four principal cell types: a primitive mesenchymal cell, spindle cells of fibroblastic and histiocytic nature, and multinucleated giant cells. These observations complement the light microscopic features but probably are insufficient to differentiate critically this entity from other myxomatous lesions of mesenchymal soft tissue such as myxoma, pseudosarcomatous fasciitis, myxoid liposarcoma. The polymorphic cellular composition lends support to the concept that the neoplasm is probably derived from a primitive multipotent mesenchymal cell capable of structural and functional modulation toward more cytodifferentiated forms.


The problem of carcinoma developing in a fibroadenoma: recent experience at Memorial Hospital. Fourteen new cases of unsuspected carcinoma developing in fibroadenomas are reported with a detailed analysis of their preoperative findings; histopathology, the results of varying surgical procedures and a three month to twenty-six year follow-up. The majority of lesions were lobular carcinoma in situ (71%) and 29% of all cases were found to have carcinoma of the contralateral breast. Our study suggests that for invasive carcinoma within a fibroadenoma complete mastectomy is warranted in virtually all instances while noninvasive disease treated by complete mastectomy is essentially curative. Contralateral breast biopsy at the time of diagnosis with a careful life-time follow-up are appropriate because of the high risk of contralateral invasive coarcinoma. There seemed to be no evidence of striking or unusual epithelial hyperplasia in the breast tissue adjacent to fibroadenomas that contained carcinoma suggesting that the carcinomas are not intrinsically different from those not related to fibroadenomas.


Scar cancer of the lung: increase over a 21 year period. In a review of 1,186 cases of lung cancer found amoung 7,629 autopsied cases over a 21 year period a total of 82 peripheral cancers related to scars were found, constituting 1% of the autopsied cases and 7% of the lung tumors. 15% of all lung tumors were peripheral (vs. bronchogenic) and the percentage rose from less than 7 in the time period of 1955 to 1960 to a little more than 23 in the 1970 to 1976 time period. 45% of all peripheral lung cancers originated in a scar. Less than 2% of all lung cancers were found associated with scars in the 1955 through 1959 time period. This increased to nearly 16% in the 1970 through 1975 time period. 72% of the scar cancers were adenocarcinomas and 18% were of squamous cell type. The rest were large cell undifferentiated carcinomas and none was oat cell or small cell type. Over three-quarters of these scar cancers were found in the upper lobes and more than half were related to infarcts. Less than a quarter were related to tuberculosis scars. No relationship was found between smoking habits and scar cancer.


Prolactin receptors in human breast carcinoma. A preliminary investigation into prolactin receptors in human breast carcinomas provided strong evidence that specific binding of prolactin was occurring in at least three of the nine specimens examined (eight human breast carcinomas and one scalp metastasis). These "prolactin receptor positive" tumors were all from premenopausal patients. Three of the tumors of postmenopausal women also suggested the occurrence of specific prolactin binding but, as saturation of the receptors had not been achieved in these assays, the results require confirmation. Some general trends in the binding characteristics of the tumors of premenopausal and postmenopausal patients were also observed.


Endometrial findings in postmenopausal women receiving long-term estrogen therapy. An histological study of the endometrium of 43 postmenopausal women under the chronic effect of cyclically administered conjugated estrogens was performed. Four atypical hyperplasias and one adenocarcinoma in situ were found. The frequency of these findings is similar to the one found in a sample of the population consulting the gynecologic clinic of our Hospital that never received estrogen therapy.


Genetic mechanisms in cancer predisposition: report of a cancer family. A family is described in which four children developed cancer affecting different organs:lymphoma, meningeal sarcoma, osteogenic sarcoma, and adenocarcinoma of the cecum. Since there was only one other case of cancer in previous generations of this family, an hypothesis is put forth to explain this unusual aggregation on the basis of recombination of common genes. It is postulated that each parent carried a different combination of genes which, though not associated with increased cancer predisposition in the combinations in which they were present in the parents, due to independent assortment resulted in a combination producing cancer susceptibility in half of the offspring. Such genetic loci could include factors similar to an oncogene which is normally held in control by genes at another locus; thus the dominant oncogene without the dominant controlling genes would make for cancer susceptibility, while the controlling genes without the oncogene would be associated with cancer resistance since two mutations would then be required for malignant development. To explain the occurrence of lymphoma in one of the children in this family, a third set of genes is included in this model--genes affecting immunocompetence, in which the normal allele is dominant. This three locus model has the advantage of being able to explain not only the occasional cancer family, but also the distribution of cancer susceptibility and resistance in the general population.


The triad of hemochromatosis, hepatoma and erythrocytosis. The triad of hemochromatosis, hepatoma and erythrocytosis is a rare combination. Hemochromatosis is often not recognized until the patient presents with the symptoms of hepatocellular carcinoma and erythrocytosis, and the development of erythrocytosis is an important clue to the under-lying hepatoma. The high serum iron concentration and the high saturation of the iron-binding protein, as well as the typical bone marrow hemosiderin pattern, are important aids in the recognition of hemochromatosis. To date, all patients with this triad have been elderly males. The clinical course is usually one of rapid deterioration and death. The seven previously reported cases have been reviewed and the relationship of the erythrocytosis to the increased production of erythropoietin is discussed.


Primary oat cell carcinoma of the larynx: a case report and review of the literature. A case of an extrapulmonary oat cell carcinoma arising in the larynx of a 45-year-old male is described. Ultrastructural study demonstrated numerous neurosecretory granules in the tumor cells identical to those described in oat cell carcinomas of the lung. Primary oat cell carcinomas of the larynx are highly malignant neoplasms. Of six previously reported acceptable cases of this rare entity, with adequate followup, all but one has died of tumor within 14 months of diagnosis. The present patient is alive without evidence of tumor 15 months following diagnosis. Aggressive therapy consisting of a combination of surgery, radiation therapy and systemic chemotherapy was utilized in this case. The use of such aggressive combination therapy appears indicated in cases of primary oat cell carcinoma of the larynx if improved survival is to be achieved.


The value of percutaneous needle biopsy in the management of primary bone tumors. Thirty-four suspected primary bone neoplasms were evaluated by needle biopsy at M. D. Anderson Hospital and Tumor Institute between September 1976 and February 1978. Adequate material for evaluation was obtained in 31 cases. The accuracy rate of the procedure was 93%. Needle biopsy is a rapid, effective and safe method of evaluating unsuspected primary lesions of bone. The indications, contraindications, and technique are emphasized. The benefits over open biopsy are indicated.


Role of superoxide dismutase in cancer: a review. Diminished amounts of manganese-containing superoxide dismutase have been found in all the tumors examined to date. Lowered amounts of the copper-zinc-containing superoxide dismutase have been found in many, but not all, tumors. At the same time, tumors have been shown to produce superoxide radicals. It is shown how diminished enzyme activities along with radical production may lead to many of the observed properties of cancer cells. The apparent exploitation of the differences between normal and cancer cell superoxide dismutase activity in the treatment of cancer is discussed.


Effect of 1-methyl-1-nitrosourea on poly(adenosine diphosphate-ribose) polymerase activity at the nucleosomal level. The stimulation of poly(adenosine diphosphate ribose) [poly(ADP-ribose)] polymerase activity at the nuclear level after damage of HeLa cells by 1-methyl-1-nitrosourea has been previously reported. We have observed a similar activation of the enzyme after treatment of cells with MNU at the nucleosomal level of chromatin (greater than 1N). This stimulation of enzyme activity did not occur through an inhibition of the glycohydrolase enzyme which cleaves poly(ADP-ribose), or elongation of poly(ADP-ribose) chains, or an increased biosynthesis of enzyme protein. The increased activity appears to be a consequence of the generation of more acceptor sites on nuclear proteins for initiation of poly(ADP-ribose) synthesis. The data indicate that MNU increased the accessibility of nucleosome core histones for modification by poly(ADP) ribosylation.


Nitrosourea interaction with chromatin and effect on poly(adenosine diphosphate ribose) polymerase activity. Poly(adenosine diphosphate ribose) polymerase, a chromatin-bound enzyme, was stimulated 150 to 200% after treatment of HeLa cells with methylnitrosourea (MNU). In contrast, a slight inhibitory effect on enzyme activity was observed after treatment of cells with various concentrations of chloroethylnitrosoureas. To define precisely the differential effects of nitrosoureas on the enzyme activity, their interactions with chromatin substructure were studied. A nonrandom, in vivo alkylation of chromatin DNA by equimolar concentrations of MNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was revealed by digestion of nuclei from drug-treated cells with micrococcal nuclease and DNase I. [methyl-14C]MNU interacted preferentially with the more accessible regions of chromatin, the internucleosome linkers, whereas, the [chloroethyl-14C]CCNU alkylated the nucleosomal core DNA to a greater extent. These two drugs also differed in their extent of covalent modification of histone and nonhistone chromosomal protein. The binding of MNU to histones was greater than of CCNU.  CCNU mainly affected nonhistone proteins. This difference in the reactivity of methyl and chloroethyl nitrosoureas with chromatin may relate to their differential effect on poly(adenosine diphosphate ribose) polymerase activity, as well as to their carcinogenic and antitumor properties.


New side effect of the hypoxic cell sensitizer, misonidazole. As part of a phase II study with the hypoxic cell radiosensitizer, misonidazole, we have encountered two cases of dermatitis. When all drugs were withdrawn and the patients were challenged with a small single dose of misonidazole, identical adverse reactions reappeared demonstrating a drug-related skin hypersensitivity.


Selective pancreatic vein catheterization for hormone assay in endocrine tumors of the pancreas. To evaluate its potential as an alternative to angiography in the diagnosis of pancreatic tumors, we performed selective vein catheterization for hormone assay on 11 patients with islet cell tumors and islet cell hyperplasias. In all patients, the abnormal pancreatic tissue was correctly localized preoperatively by this method.


Ultrastructure and cytochemistry of the yolk syncytial layer in the alevin of trout (Salmo fario trutta L. and Salmo gairdneri R.) after hatching. II. The cytoplasmic zone. In contrast to the vitellolysis zone which is involved in the degradation of the yolk, the cytoplasmic zone of the yolk syncytial layer, composed of many cytoplasmic organelles, is implicated in a secretory process. The granular endoplasmic reticulum of the latter is extremely well developed and organized into trabeculae. The yolk nuclei show a RNA positive reaction. The Golgi apparatus is implicated in the elaboration of very low density lipoproteins (VLDL) and of acid phosphatase. The numerous mitochondria seem to suggest an important energy metabolism in the solubilisation area. The appearance of certain special structures (crystalline bodies, pseudovesicular structures, lipochondria) as well as their relation with the organelles of the cytoplasmic zone, re-inforces the impression of a layer with a secretory nature and suggests its participation in the remodelling of the yolk products produced in the vitellolysis zone. The results of the investigations concerning the acid phosphatase activity suggest that this enzyme plays a role on the one hand in the degradation of certain platelets which penetrate into the cytoplasmic zone, and on the other hand in the regulation of VLDL, in the lysis of secretory products elaborated by the cytoplasmic zone. The possible presence of microperoxisomes is discussed as well as the positive detection of glucose-6-phosphate dehydrogenase.


Localization of corticotropin- and endorphin-related peptides in the intermediate lobe of the rat pituitary. The question is examined whether alpha-melanocyte stimulating hormone (alpha-MSH), adrenocorticotropic hormone (ACTH), met-enkephalin and beta-endorphin are detectable by enzyme immunocytochemistry in the cells of the intermediate lobe (PI) of the rat pituitary. By applying antibodies against alpha-MSH, ACTH and beta-endorphin on light microscopic sections, intense immunostaining was found in all PI-cells. At the ultrastructural level, after treatment of consecutive serial sections with these three antibodies the immunoreactivity was localized in the same secretory granules. No specific metenkephalin immunoreactivity could be detected in the cells of the intermediate lobe.


Immunofluorescence on avian sarcoma virus-transformed cells: localization of the src gene product. The localization of the avian sarcoma virus src gene product (termed p60src) was examined by indirect immunofluorescence in cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D (SR-RSV-D). Antiserum to p60src was obtained from rabbits bearing SR-RSV-D-induced tumors, and immunofluorescence was performed on chicken embryo fibroblasts (CEF) transformed with SR-RSV-D, as well as normal rat kidney (NRK) cells transformed by the same virus (termed SR-RK cells). Both acetone and formaldehyde fixation were used for the immunofluorescence tests. The specificity of the anti-tumor serum was first demonstrated in both cell systems by gel electrophoresis of immunoprecipitates prepared from 35S--methionine-labeled cells. Anti-tumor serum precipitated p60src from SR-RSV-D-transformed CEF but not from CEF infected with a transformation-defective mutant of SR-RSV-D. All viral structural proteins and precursors contained in these immunoprecipitates could be eliminated by competition with unlabeled virus. Similar experiments on SR-RK cells indicated that no viral proteins other than p60src were expressed in these cells, and this observation was supported by immunofluorescence tests using antiserum to whole virus. For immunofluorescence localization of p60src, reactions with viral structural proteins were blocked with unlabeled virus. This presaturation step, obligatory for p60src detection in the SR-RSV-D-transformed CEF, was unnecessary when antitumor serum was tested on SR-RK cells, since p60src was the only viral protein detectable in these cells. With acetone-fixed cells, p60src-specific immunofluorescence revealed a characteristic fluorescence pattern which was similar in both cell systems. The principal pattern was diffuse and situated in the cytoplasm. A clear nuclear fluorescence was never observed. Immunofluorescence on formaldehyde-fixed cells also indicated the cytoplasmic location of p60src and revealed a specific subcytoplasmic concentration of the fluorescence. With both fixation methods, an additional fluorescence pattern was seen between cells in contact, and was also found in both SR-RK cells and SR-RSV-D-transformed CEF. Immunofluorescence on viable cells suggested that p60src was not on the surface of these transformed cells. The fluorescence patterns were specific for avian sarcoma virus-transformed cells and were not found in uninfected cells, cells infected with a transformation-defective mutant of SR-RSV-D or cells transformed by an antigenically unrelated murine sarcoma virus. Furthermore, anti-tumor serum did not contain antibodies to proteins of the microtubules or intermediate filaments.


Characterization of cross-reacting antigens on the Epstein-Barr virus envelope and plasma membranes of producer cells. A rabbit antiserum has been prepared against the B95-8 transforming strain of EBV. The antiserum has a high virus neutralizing titer (approximately 1:1000) against both the marmoset B95-8 EBV and the human P3HR-1 EBV. The neutralizing antibodies may be absorbed completely with EBV producer cell lines, but not with nonproducer cell lines or producer cell lines treated with phosphonoacetic acid (PAA) so as to be nonproducer. After repeated absorption with PAA-treated B95-8, the serum remains reactive with the membranes of producer cell lines as judged by immunofluorescence or the 125I--Staphylococcal protein A radioimmunoassay. Thus the neutralizing antigens are expressed on the membranes of producer cell lines and may be purified from this source using the serum and 125I--Staph A binding as an assay. The ability of the serum to differentiate between producer and nonproducer cells by means of cell surface determinants has been exploited to achieve a separation of these two populations from the same culture. Immunoprecipitation by the protein A technique shows that the serum recognizes two polypeptides from producer cells of approximate molecular weights 150,000 and 75,000.


RNA sequencing provides evidence for allelism of determinants of the N-, B- or NB-tropism of murine leukemia viruses. Previous genetic and biochemical studies identified three large RNAase T1-resistant oligonucleotides, each associated with either the N-, B- or NB-tropism of murine C-type viruses of BALB/c origin. These oligonucleotides were shown to lie in the 5' third of the oligonucleotide maps of their respective viruses. We sequenced the three oligonucleotides and found that they share a 10 base sequence. Together these observations provide good evidence that the determinants of N-, B- or NB-tropism monitored by the three oligonucleotides are allelic. The oligonucleotides associated with N- and B-tropism differ in sequence at four of sixteen nucleotides, while the B- and NB-tropism-associated oligonucleotides differ in sequence by only one base out of sixteen. These results are consistent with the possibilities that B-tropic viruses may arise from N-tropic viruses by recombination, while NB-tropic viruses may arise from B-tropic virus by mutation. An unexplained finding was that a 10 base sequence present in the oligonucleotide associated with N-tropism is also found in the 3' third of the genomes of the N-, B- and NB-tropic viruses studied.


DNA of noninfectious and infectious integrated spleen necrosis virus (SNV) is colinear with unintegrated SNV DNA and not grossly abnormal. The cleavage sites of eight restriction endonucleases in linear spleen necrosis virus (SNV) DNA were mapped, and the map was oriented with respect to viral RNA. With the aid of this map, several structural features of the viral DNA were elucidated: unintegrated linear SNV DNA is terminally redundant; the majority of SNV DNA molecules integrated in chicken DNA, which were previously shown to be present in many sites in cellular DNA, are colinear with unintegrated viral DNA; no tandem integration of proviral molecules is detectable; and the majority of integrated SNV DNA molecules, including integrated SNV DNA molecules previously shown to be noninfectious, do not have an altered restriction enzyme digestion pattern.


[Opiate receptor sites in neuronal and glial cell lines in culture]. A stereospecific saturable high affinity binding of 3H-naloxone has been found in 3 glial and 2 neuronal cell lines. Kinetic study of binding seems to indicate only one class of receptor sites in the 5 cell lines. Acute exposure to morphine (1 X 10(-5)M) concurrent with PGE1-induced stimulation of adenylate cyclase did not result in a decrease of the cAMP level in any cell line tested.


[Experimental dissociation between hippocampal theta activity in wakefulness and hippocampal theta activity in paradoxal sleep in the rat]. The effects of electrolytic lesion of the septum on the theta activity of the dorsal hippocampus were studied in the chronically implanted Rat during wakefulness and paradoxical sleep. The experimental results show that depending on its localization, septal lesion can either: (1) eliminate the wakefulness theta rhythm without suppressing that of paradoxical sleep; (2) eliminate the paradoxical sleep theta rhythm without suppressing that of wakefulness. These results suggest that there are two kinds of theta activity having different anatomophysiological bases and a different functional significance: one associated with wakefulness and the other with paradoxical sleep.


[Epidemiological study of pulmonary hydatid cyst in the Ivory Coast. 3 cases]. No cases of echinococcus have been reported up to this time in Ivory Coast, where it has been considered non-existant in either pulmonary or hepatic form. The three cases of pulmonary echinococcus reported here show the classic paucity of clinical symptoms  associated with classical radiological findings. The epidemiological research confirms in one case definite infection occurring in Ivory Coast and the other two either in the Ivory Coast or in closely neighbouring areas.


[Cyclic AMP and control of ion exchanges in the fish intestine]. In isolated intestine of the European Flounder, cyclic AMP and theophylline induce a reduction of the negative transmural potential difference and short circuit current, increase severalfold the passive permeability for chloride and abolish the active transport of this ion. These effects are very different from those described in mammals.


Toluene di-isocyanate-induced asthma. I. Reactions to TDI, MDI, HDI and histamine. A group of twenty four workers handling di-isocyanates and with respiratory disease were investigated by occupational-type bronchial provocation tests for sensitivity to toluene di-isocyanate (TDI), to which all were exposed, and to diphenylmethane di-isocyanate (MDI) and hexamethylene di-isocyanate (HDI). Sixteen gave asthmatic reactions to TDI and eight of these also reacted to MDI. Four of the eight TDI and MDI reactors had histories of exposure only to TDI, and of them two reacted also to HDI. Of nine subjects tested with HDI, three gave asthmatic reactions, and all three also reacted to TDI and MDI. Thus reactions to MDI and HDI were elicited only in the TDI reactors. The possibility of specific sensitivity to these and other di-isocyanates requires tests in subjects exposed to them and not to TDI.


Toluene di-isocyanate-induced asthma. II. Inhalation challenge tests and bronchial reactivity studies. In sixty-three workers exposed to toluene di-isocyanate (TDI), no overall differences in bronchial reactivity to histamine inhalation and to exercise testing were found between the total groups of positive and negative TDI reactors to provocation tests. A subgroup of TDI highly sensitive subjects reacting to very low concentrations (less than or equal to 0.001 p.p.m.) were more sensitive to both histamine and exercise than the group who were less sensitive to TDI, and who reacted to higher concentrations (0.002--0.02 p.p.m.) than the group of non-reactors. There were, however, in the last group a number of subjects with high degrees of histamine reactivity who did not react to the TDI. These findings suggest that, on the one hand, the asthmatic reactions to TDI cannot be attributed solely to non-specific mechanisms and, on the other, that in subjects with high degrees of specific sensitivity non-specific mechanisms may also be playing a part.


Renal function studies using 99mTc-dimercaptosuccinic acid. 99mTc-dimercaptosuccinic acid (DMSA), provides both a well defined renal image via preferential cortical accumulation, and a quantitative assessment of separate kidney function, correlating well with renal plasma flow obtained from the 131I-Hippuran renogram of each kidney. 99mTc-DMSA is capable of labeling the functioning nephrons in the cortex without interference by the activity of urine flow, suggesting that DMSA should be termed a cortical tubular marker. 99mTc-DMSA scintigraphy has advantages over conventional methods in that it can be performed repeatedly with little discomfort to the patients, as a routine clinical examination.


Treatment of pompholyx due to nickel allergy with chelating agents. Ingestion of nickel is important for the chronicity of hand eczema in nickel-sensitive patients. We have tried the effect of chelating agents. Diethyldithiocarbamate (DDC) and tetraethyl thiuramdisulfide (TETD) have proved effective in the treatment of nickel carbonyl poisoning. A 66-year-old woman suffering from severe nickel dermatitis has been treated in three periods, first with DDC 100 mg Q. I. D. for 20 days, and later with TETD 100 mg T. I. D. for two  periods. An improvement has been observed in all three experiments.


Hypothalamic-pituitary-adrenal function in extrinsic asthma. Hypothalamic-pituitary-adrenal function in a well-defined, carefully selected group of 25 patients with extrinsic asthma was assessed by measuring plasma levels of adrenocorticotropic hormone (ACTH) and of 11-deoxycorticol after administration of metyrapone and by measuring the level of cortisol following stimulation with cosyntropin. No difference was demonstrated between asthmatic subjects and 20 normal age-matched controls. In addition, neither the response of the level of ACTH nor of 11-deoxycortisol correlated with the duration of asthma or the severity as assessed in 23 patients by tests of pulmonary function. We conclude that there is no abnormality in hypothalamic-pituitary-adrenal function in patients with extrinsic asthma, and we suggest that previous data suggesting such an abnormality may reflect heterogeneous groups of patients, inaccurate methods, and the variability of normal responses to ACTH and stimulation with metyrapone.


Pulmonary malignant fibrous histiocytoma. Light and electron microscopic studies of one case. A malignant fibrous histiocytoma (MFH) arising in the lungs of a 51-year-old man was studied by light and electron microscopy. Features observed were identical to those of MFHs which occur in the skin and subcutaneous tissue and less commonly in other deep locations. By light microscopy, a storiform pattern with admixture of fibroblasts and histiocytes, as well as xanthomatous and giant cells, was noted. Undifferentiated tumor cells along with fibroblasts and histiocytes in different degrees of differentiation were identified ultrastructurally. These findings lend support to the concept that MFH is a sarcoma of primitive mesenchymal cell origin. The addition of the lung as another primary site for the development of this tumor is consistent with the view that MFHs may potentially arise in any part of the body.


Urinary excretion of mecillinam by volunteers receiving film-coated tablets of pivmecillinam hydrochloride. Mecillinam is a new amidinopenicillin which is taken orally as its ester pivmecillinam. 10 healthy volunteers were each given 200 mg of pivmecillinam hydrochloride four times daily and all urine was collected and cooled to -20 degrees C to minimize antibiotic degradation. 43% of the ingested antibiotic was recovered from the urine in its microbiologically active form, during the first 12 h and 34% during the subsequent 24 h. The corresponding figures for 10 volunteers receiving double this dose of antibiotic was 30 and 34%. The mean half life of mecillinam in urine due to spontaneous breakdown at 37 degrees C was 17 h. There was evidence that stearate-film-coated pivmecillinam hydrochloride tablets may be less likely to cause dyspepsia than gelatine capsules.


Effect of thyrotoxicosis on gluconeogenesis from alanine in the perfused rat liver. The influence and mechanism of action of T4 treatment on hepatic gluconeogenesis from alanine was studied in isolated rat livers. Conversion of alanine into glucose was increased markedly in livers of thyrotoxic rats compared to that in normal rats. Estimation of metabolic intermediates of the gluconeogenic pathway showed that T4 treatment produced forward cross-over between pyruvate and phosphoenolpyruvate, which suggests that this point is a control site. Alanine transport into the liver cells was apparently active since the alanine level was higher within the hepatocytes than in the extracellular space. Furthermore, T4 treatment stimulated the transport of alanine into the liver cells. alpha-Aminoisobutyric acid uptake was significantly higher by liver of thyrotoxic animals than of normal rats. The hepatic level of cAMP was also higher in experimental livers than control livers. These studies suggest that T4 stimulates hepatic gluconeogenesis from alanine and the underlying mechanism involves an increase in both the transport of amino acid into the liver cell and the conversion pyruvate to phosphoenolpyruvate.


Reversal of dexamethasone inhibition of adrenocorticotropin release in a mouse pituitary tumor cell line either by growing cells in the absence of dexamethasone or by addition of hypothalamic extract. Release of ACTH by a mouse pituitary tumor cell line )AtT-20/D-16v) is inhibited by 10(-8)-10(-6) M 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha, 21-trihydroxy-1,4-pregnadiene-3,20-dione (dexamethasone). Dexamethasone does not inhibit cell growth in this concentration range. Cortisol and corticosterone are almost as potent as dexamethasone in inhibiting ACTH release, whereas 17 beta-estradiol and testosterone have no effect. In rapidly growing cultures of tumor cells removal of dexamethasone leads to complete reversal of the inhibitory effect of the steroid on ACTH accumulation in culture medium within 4-5 days (3.5-4 generation times). The extent of reversal of the dexamethasone effect in slowly growing cultures (generation time 96-150 h) and in rapidly growing cultures (24-30 h) is proportional to the amount of growth that takes place in the absence of dexamethasone. Addition of hypothalamic extract to dexamethasone-treated cultures and to untreated cultures stimulates the release of ACTH 4- to 8-fold. The response occurs within 15 min after the addition of the extract and is dependent on the dose of the extract.


Stimulation of prostaglandin accumulation in preovulatory rat follicles by adenosine 3',5'-monophosphate. LH stimulates an increase in prostaglandins in vitro in preovulatory follicles from rats pretreated with PMS gonadotropin. The role of cAMP in this action of LH was examined by incubating preovulatory follicles with various substances and determining the resultant prostaglandin (PG) E accumulation by radioimmunoassay. LH (5 microgram/ml) increased PGE accumulation to approximately 4 times the control (181 +/- 23 to 886 +/- 83 pg/follicle). The addition of 20 mM cAMP also stimulated PGE accumulation, and the addition of 20 mM cAMP in the presence of 0.5 mM 1-methyl-3-isobutylxanthine was as effective as LH. Other nucleotides such as ATP, ADP, 3'-AMP, 5'-AMP, cGMP, and O2'-monobutyryl-cAMP did not stimulate PGE accumulation. On the other hand, (Bu)2cAMP, 8-bromo-cAMP, and N6-monobutyryl-cAMP produced an increase in PGE accumulation similar to that observed with LH. In addition, 10 microgram/ml cholera toxin was shown to increase both cAMP and PGE accumulation in preovulatory follicles. These results indicate that the prostaglandin response of follicles is specific for cAMP-like nucleotides or substances capable of increasing intracellular cAMP. The data support the concept that cAMP mediates the effect of LH on PGE accumulation in preovulatory follicles in the rat.


Specificity of the immunocytochemical luteinizing hormone-releasing hormone receptor reaction. Affinity-purified anti-LHRH, affinity-purified LHRH-anti-LHRH complex, as well as antisera depleted of anti-LHRH by solid phase immunoabsorption were used to test the specificity of the immunocytochemical approach for detection of receptor-bound LHRH. The solid phase immunoabsorbent was prepared by attaching LHRH to Sepharose via an RNase spacer. Purified anti-LHRH was eluted from the immunoabsorbent by acidification. Unabsorbed antisera as well as purified antibody conferred moderate immunocytochemical staining to the secretion granules of rat pituitary gonadotrophs. Staining intensity became greatly increased upon treatment of the electron microscopic sections with LHRH before immunocytochemical staining. All gonadotroph staining was abolished when absorbed antisera were used. The specificity of the immunoabsorbent was tested on mixtures of anti-LHRH and anti-ACTH17-39. On absorption of such mixtures with insolubilized LHRH, all gonadotroph staining disappeared, but the optical density indices of ACTH staining remained unaffected. It is concluded that gonadotroph secretion granules contain, in addition to an LHRH receptor reacting with LHRH in vitro, stable complexes of endogenous LHRH with receptor.


Effect of gonadal steroids on the luteinizing hormone and follicle-stimulating hormone response to 8-bromo-adenosine 3',5'-monophosphate in anterior pituitary cells in culture. -8-Bromo-cAMP stimulated the release of LH (approximately 20-fold) and FSH (approximately 2-fold) in control rat anterior pituitary cells in primary culture. 8-Bromo-cAMP-induced LH release was 2-3 times greater in cells preincubated for 48 h in the presence of 10(-8) M 17 beta-estradiol (E2). The LH response to 10(-10) M LHRH was similarly increased by E2 pretreatment whereas progesterone (P), which had no effect on the response to 8-bromo-cAMP, led to a reduction of the stimulatory effect of E2 on the LH response to the neurohormone. Preincubation with 10(-8) M testosterone (T) decreased the LH response to 8-bromo-cAMP by about 40%, whereas the response to LHRH was 80% inhibited. 8-Bromo-cAMP-induced FSH release was slightly increased in cells preincubated with E2 but was greatly augmented in cells treated with E2 + P. Basal FSH release was slightly increased (40%) after preincubation with P or T whereas the response to 8-bromo-cAMP was not significantly affected. The sensitivity of the FSH response to LHRH was increased by E2 whereas P and T led to a stimulation of the maximal FSH response to LHRH with no significant effect on the LHRH ED50 value. The present data indicate that P and E2 exert their effects on LH and FSH release at steps before and after cAMP formation, respectively. T appears to act at both steps on the release of the two gonadotropins.


Effects of thyrotropin on thyroglobulin exocytosis and iodination in the rat thyroid gland. Rats, pretreated with thyroxine for 2 days, were given one or two iv injections of 500 mU of TSH; in some groups the second TSH dose was replaced by 0.75 micronmol isoproternol. The effects of the thyroid stimulators on the following parameters were studied: the number of exocytotic vesicles in the follicle cells; the incorporation of 125I into thyroid proteins, measured over periods of 5 min; and the thyroidal cAMP contents. At 2 h after TSH administration, a second dose of TSH failed to stimulate iodination while at 8 h the iodination response was "normal". Two hours after TSH the follicle cells contained practically no exocytotic vesicles but at 8 h they had a full supply of vesicles, and this was emptied by the second TSH injection. THE CAMP content was less increased by the second TSH injection than by the first one, but the stimulatory effect of the second TSH dose on cAMP was the same at 2 h and at 8 h; this indicates that the lack of iodination response at 2 h was not simply due to blocking of TSH receptors. Isoproternol, which acts on other receptors than does TSH, cause a similar cAMP increase incontrols and at 2 h and 8 h after TSH, but stimulated iodination only in controls and at 8 h after TSH; this supported the conclusion that the lack of iodination response to a second TSH dose at 2 h was not due to impairment of the adenylate cyclase-cAMP system. These observations taken together strongly indicate that a rapid iodination response to TSH depends on stimulated exocytosis which, in turn, requires a pool of exocytotic vesicles in the follicle cells. Such a coupling between exocytosis and iodination seems appropriate since by exocytosis uniodinated thyroglobulin and membrane, showing peroxidase activity histochemically, are delivered to the site of iodination, the apical cell surface.


Enriched populations of rat pituitary thyrotrophs in monolayer culture. Rat anterior pituitary cells were dissociated and subjected to 4 h of unit gravity sedimentation. Eighty-five 8-ml fractions were collected, and nine pooled fractions were placed in monolayer culture for 1 week. The attached cells were immunocytochemically stained for TSH using an antiserum previously shown to be specific for the beta subunit of TSH and all culture media saved for subsequent assay of TSH. Thyrotrophs were localized both immunocytochemically and by radioimmunoassay to 1-2 fractions from the top of the sedimentation chamber. Typically, it was found that 60-80% of the cells in these fractions were immunocytochemically identified as thyrotrophs. Electron microscopic observations indicated that such cells displayed the classical morphological features associated with thyrotrophs. The enriched thyrotroph cultures responded to 1.0 mM dibutyryl cyclic AMP with an increased release of TSH indicating that the intracellular secretory machinery was not altered by the procedures employed. In all cases, the basal TSH secretion decreased with time in culture. In addition, the fractions containing the most TSH in the culture media were usually one fraction lighter (lower sedimentation rate) than the fractions which were found immunocytochemically to contain the highest percentage of thyrotrophs. The results suggest the possibility of two functionally distinct thyrotroph cell types as has been suggested for the pituitary somatotrophs.


Uncoupling by cooling of secretion and adenosine 3',5'-monophosphate accumulation in stimulated dog thyroid slices. Thyroid metabolism undergoes general activation in the presence of TSH. Secretion, [1-14C]-glucose oxidation, protein iodination, and cyclic AMP (cAMP) accumulation are stimulated. In intact tissue, it is difficult to dissociate effects of cAMP itself and effects secondary to cAMP-induced secretion. We report herein that cooling to 20 C dissociates the action of TSH on secretion from all its other effects. At this temperature, TSH stimulation of secretion was completely inhibited whereas cAMP accumulation was enhanced. The TSH effect on [1-14C]glucose oxidation was maintained and its action on protein iodination was diminished but not abolished. The ATP content was not decreased. The mechanism of action of cooling was investigated. No pseudopod formation of intracellular colloid droplets were observed by scanning electron microscopy and transmission electron microscopy, thus indicating that the inhibition affects the first step of secretion, i.e., colloid phagocytosis. Microtubules were not seen at 2 C but were normally present at 20 C. The data are compatible with the hypothesis that inhibition of phagocytosis at 20 C could be a consequence of a lipid phase transition in the membrane.


Angiotensin II receptors and aldosterone production in rat adrenal glomerulosa cells. Specific receptors for angiotensin II (A II) were demonstrated in membrane fractions and collagenase-dispersed cells from the zona glomerulosa of the rat adrenal gland. The equilibrium association constant (Ka) of the A II binding sites was similar in particulate fractions (2.0 +/- 0.4 (SE) X 10(9) M-1) and intact glomerulosa cells (1.8 +/- 0.3 X 10(9) M-1). Specific binding of [125I]iodo-A II was enhanced by increasing sodium concentration, and in the presence of dithiothreitol, EDTA, and EGTA. Plasma membrane fractions prepared by density gradient centrifugation showed increased binding of [125I]iodo-A II, and were correspondingly enriched in adenylate cyclase and sodium-potassium-dependent ATPase. Steroid production by collagenase-dispersed adrenal glomerulosa cells was highly responsive to A II and ACTH. Significant increases in aldosterone and corticosterone production were elicited by A II concentrations as low as 3 X 10(-11) M, equivalent to normal blood levels of A II in rats (5 X 10(-11) M). The maximum increase in aldosterone production, of 6--7 times the basal value, was obtained at 10(-9) M A II. Dispersed capsular cells were also highly sensitive to ACTH, responding to concentrations down to 3 X 10(-12) M with increased aldosterone production, reaching a maximum aldosterone response of 20-fold above the basal value. The magnitudes of the aldosterone and corticosterone responses to A II in capsular and fasciculata-reticularis cells were commensurate with the distribution of A II receptors, which were 11-fold more concentrated in capsular cells. The ability of A II to evoke aldosterone production at physiological concentrations, and the correspondence between A II binding and steroidogenesis in capsular cells, demonstrate the functional importance of A II receptor sites in the zona glomerulosa of the rat adrenal cortex.


Effects of a thyroid hormone analog on fetal rat hepatocyte ultrastructure and microsomal function. The effects of an analog of thyroxine, 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT), on fetal rat hepatocyte ultrastructure and microsomal function were investigated by using the techniques of quantitative electron microscopy and enzyme assays. Rats were injected with DIMIT (10 microgram/100 g BW) or vehicle daily from the 15th through the 19th day of pregnancy. Fetuses were sacrificed on the 20th day of gestation. In comparison with controls, DIMIT-treated livers 1) were devoid of glycogen; 2) contained smaller hepatocytes; 3) contained a greater number of hepatocytes; 4) had an increased volume density of mitochondria; and 5) had increased NADPH-cytochrome c reductase and glucose-6-phosphatase activities. Surface areas of rough and smooth surfaced endoplasmic reticulum were unaffected by the hormone analog, and cytochrome P-450 was not induced. All of the changes that were produced by DIMIT in the 20-day-old fetal rat, as well as smooth endoplasmic reticulum and cytochrome P-450 development, are observed in normal animals within the first 3 days after birth. The data suggest that thyroid hormone may be a physiological stimulus for certain aspects of early hepatic development, but that it acts in combination or in sequence with other factor(s) to produce the full complement of structural and functional changes that occur perinatally in the rat.


Tetanus toxin interactions with the thyroid: decreased toxin binding to membranes from a thyroid tumor with a thyrotropin receptor defect and in vivo stimulation of thyroid function. Normal rat thyroid membranes adsorb neurotoxicity when incubated with purified tetanus toxin. Membranes from a rat thyroid tumor with a thyrotropin receptor defect adsorb very little neurotoxicity when similarly evaluated. This inability of the tumor membranes to adsorb neurotoxicity is correlated with a defect in their ability to bind both 125I-labeled tetanus toxin and [125I]iodothyrotropin. The effect of tetanus toxin on the release of radioiodine from the thyroids of appropriately prepared mice has been measured by adapting methods used for the bioassay of thyrotropin. One minimum lethal dose of tetanus toxin given sc caused a significant release of radioiodine into the blood of mice 48 h after injection. In mice subjected to the stress of prior bleedings or anesthesia, the release of radioiodine from the thyroid by tetanus toxin was accelerated, i.e., the increase in blood radioiodine could be measured 24 h after injection. These results again suggest that tetanus toxin may interact with thyrotropin receptors on thyroid plasma membranes. The "sympathetic overactivity syndrome" seen in some patients with tetanus and the syndrome characterized as "thyroid storm" in patients with Graves' disease are discussed as they may relate to these observations.


Impaired inner medullary production of prostaglandin E2 in the kidney of the myxedematous rat. Renal cAMP concentration and renal prostaglandin (PG) E2 and PGF2alpha production were determined in control rats and rats with surgically induced myxedema. Slices from the kidneys of each group were incubated in vitro. cAMP concentration of inner medullary slices from the kidneys of myxedematous rats was decreased in comparison to slices from control rats. There was no difference between the two groups when slices from outer medulla and cortex were compared for cAMP level. In myxedema, there was decreased inner medullary production of PGE2, but not PGF2alpha. The difference in PGE2 production between myxedema and control rat kidneys was not corrected by addition of either arachidonic acid (170 micronM) or phospholipase A2 (5 microgram/ml). Arachidonic acid, however, increased inner medullary cAMP concentration in myxedema kidneys to a value not measurably different from control. Thus, there appears to be a decreased PGE2 production in the renal inner medulla of rats with myxedema. In view of the fact that PGE2 has previously been shown to increase inner medullary cAMP content, the reduced inner medullary content of cAMP would appear to be attributable to reduced PGE2 production.


Effects of thyrotropin on iodine metabolism of dog thyroid cells in tissue culture. When grown in the presence of thyrotropin, dog thyroid cells in culture from follicle-like structures, take up labeled iodide, and iodinate macromolecular components in the cell. When grown in the absence of thyrotropin, dog thyroid cells in culture form a monolayer, take up only 6% of the iodide of follicular cells, and do not iodinate macromolelcular components in the cell. The iodide uptake in monolayer cells does, however, reflect an incorporation process unique to thyroid cells because hepatocytes and fibroblasts do not have the capacity of the monolayer cells to take up iodide. Thyrotropin stimulation of monolayer cells for a prolonged period (3-8 days) causes the cAMP levels of these cells to return to levels identical to those in follicular cells. The increased cAMP levels are not due to the induction of an adenylate cyclase enzyme, because homogenates of monolayer cells have a thyrotropin-stimulable adenylate cyclase activity. The low level of cAMP, thus, seems to be a problem of receptor coupling to the adenylate cyclase enzyme. The return of cAMP to normal levels is accompanied by an increase in iodide uptake and by macromolecular organification; the return of cAMP levels to normal values is not accompanied by follicular development. The majority (75%) of the iodinated macromolecular product accumulated by follicular thyroid cells, by monolayer thyroid cells stimulated with thyrotropin for a prolonged period, or by thyroid cells treated with dibutyryl cAMP from the onset of culture has the characteristics of 19 S thyroglobulin. The remainder appears to be low mol wt material which may be thyroglobulin-related i.e., be either precursor or biodegraded material.


Characterization of adult bovine adrenocortical cells throughout their life span in tissue culture. The characteristics of adult bovine adrenocortical cells were studied throughout their life span of 55-65 generations in monolayer culture. Over this period, the cells maintained the capacity to synthesize steroids when tested with repeated maximal doses of ACTH, prostaglandin E1, monobutyryl cAMP, or cholera toxin. Prostaglandin E1 stimulated cAMP production and steroidogenesis, and inhibited DNA synthesis, as measured by incorporation of [3H]thymidine, with dose-response characteristics that did not vary over the first 50 generations in culture. In contrast, the maximal rate of cAMP production stimulated by ACTH declined exponentially at a rate of 7% per generation. In primary and secondary cultures, ACTH stimulated steroidogenesis maximally and inhibited [3H]thymidine incorporation into DNA completely at a half-maximal effective concentration (ED50) of 0.08 nM which was two orders of magnitude less than the ED50 of 8 nM for stimulation of cAMP production. As the ACTH-stimulated maximal rate of cAMP production fell with increasing generation number, the ED50 for ACTH stimulation of steroidogenesis and inhibition of DNA synthesis increased. From about the 20th generation onward, the ability of ACTH to inhibit DNA synthesis maximally declined so that by the 40th generation, cells were completely resistant to the growth-inhibitory effects of ACTH. High-dose ACTH continued, however, to stimulate steroid production maximally over the 50 generations studied. In late passage cells, the ED50 for ACTH stimulation of steroidogenesis was 8 nM, identical to that for cAMP production. Although ACTH-stimulated cAMP production was related to both stimulation of steroidogenesis and inhibition of DNA synthesis, higher cAMP levels appeared required for inhibition of DNA synthesis than for stimulation of steroidogenesis. Mitogenic responses to fibroblast growth factor and to angiotensin II were retained throughout long term growth in culture. The progressive loss of ACTH-responsiveness was specific and a function of aging of bovine adrenocortical cells in culture.


Independence of steroidogenic capacity and luteinizing hormone receptor induction in developing granulosa cells. The relationship between FSH-induced acquisition of LH/hCG receptors and the steroidogenic capacity of granulosa cells from estrogen-primed hypophysectomized rat ovaries has been examined. Granulosa cells harvested from the immature preantral follicles of animals not treated with FSH (controls) displayed negligible specific human [125I]iodo-hCG binding and produced only minimal amounts of progesterone during 48 h of culture in vitro. Addition of highly purified hFSH or prostaglandin-E2 (PGE2) to the culture medium elicited substantial increases in progesterone production which were not accompanied by measurable increases in [125I]iodo-hCG binding. Treatment with oFSH in vivo for 24 h led to the initiation of antrum formation in many follicles and was accompanied by an 8-10-fold increase in hCG binding by freshly isolated granulosa cells. Basal, hFSH-, and PGE2-stimulated progesterone production during culture was also greater than controls. In contrast, cells from animals receiving oFSH in vivo for only 12 h showed no increase in hCG binding either before or after culture, yet basal and stimulated progesterone production in vitro was significantly greater than controls, indicating that the initiation of steroidogenesis was antecedent to LH/hCG receptor induction. Only those cells obtained after the 24-h in vivo treatment with oFSH produced elevated amounts of progesterone when incubated in the presence of hCG, thereby showing that the observed increases in [125I]iodo-hCG binding reflected the induction of functionally active LH/hCG receptors. Pharmacological stimulation of steroidogenesis by cell suspensions with N,O'-dibutyryl cAMP resulted in consistently high levels of progesterone production irrespective of previous treatment with FSH in vivo. This uniform expression of in vitro steroidogenic capacity occurred in the complete absence of measurable increases in LH/hCG receptors, suggesting that these two fundamental developmental processes are independent phenomena which may be under separate regulation in vivo.


Evidence that the hypothalamus mediates endotoxin stimulation of adrenocorticotropic hormone secretion. The site of action of Escherichia coli endotoxin in inducing ACTH secretion was studied in vivo and in vitro. Hypophysectomized rats, bearing two to three transplanted pituitaries under the kidney capsule and "primed" with exogenous ACTH, responded to 2.0-7.5 microgram/100 g BW ip or iv endotoxin with a several-fold increase of plasma corticosterone. This response was markedly reduced by hypothalamic lesions and completely abolished by removing the entire forebrain. Endotoxin added directly to cultured rat adenohypophyseal cells in a concentration up to 10 microgram/ml did not induce significant ACTH secretion. We conclude that endotoxin-induced ACTH secretion from heterotopically transplanted pituitaries is mediated primarily by the hypothalamus, presumably through hypothalamic CRF that reaches the transplanted pituitaries via the systemic circulation.


Decreased Leydig cell responsiveness in the testicular feminized male rat. The Stanley-Gumbreck pseudohermaphrodite or testicular feminized male (tfm) rat exhibits a decreased Leydig cell sensitivity to human CG (hCG) measured by androgen and cyclic-3',5',-adenosine monophosphate production in vitro. These changes were associated with an 80% reduction in the number of LH receptors in the tfm testis, when compared on the basis of equivalent amounts of testis particle protein or per 10(6) isolated Leydig cells. Androstenedione and not testosterone is the major androgen secreted by the tfm Leydig cell and androstenedione secretion is, therefore, a more appropriate end point than testosterone secretion for Leydig cell function in tfm animals. A dose of hCG (3 ng/2 ml) which elicited a near maximal response in androgen production from the decapsulated testes and Leydig cell suspensions of normals rats, did not significantly stimulate androgen production from Leydig cells of the tfm animals. A much higher dose of hCG (200 ng/2 ml) gave a response from the tfm Leydig cells which was comparable to that obtained with 3 ng from Leydig cells of normal littermates. This indicates that the small number of LH receptors on the tfm Leydig cell membrane are functional and that the reduction in receptor number results in a decrease in the sensitivity of response to LH rather than a reduction in the maximum steroid response.


Interaction of cyanoketone and other steroid nitriles with cytochrome oxidase, hemoglobin, and cytochrome P-450. The inhibitory activity of cyanoketone (CNK; 2alpha-cyano-4,4,17alpha-trimethyl-17beta-hydroxy-5-androsten-3-one), was investigated for enzymes of the respiratory chain and cholesterol side chain cleavage (CSCC). In bovine corpus luteum mitochondria incubated with [26-14C]cholesterol, 500 micron CNK caused 90% inhibition of pregnenolone synthesis. Comparable results were obtained with adrenal and placental mitochondria. Addition of CNK to bovine corpus luteum mitochondria or to cytochrome P-450 purified from this source elicited a concentration-dependent, reverse type I difference spectrum with an absorption maximum at about 423 nm and a minimum at about 395 nm, confirming binding to oxidized cytochrome P-450. This spectral change resembles those of steroids which inhibit CSCC. In mitochondrial preparations, CNK induced a second peak at about 445 nm. This peak was similar to that elicited by the interaction of potassium cyanide with cytochrome a3 when the former is added to rabbit heart mitochondria which are devoid of P-450. Like cyanide, CNK block mitochondrial respiration at the cytochrome oxidase site, and induced spectral changes in human hemoglobin. Therefore, this peak at 445 nm probably represents the interaction of CNK with oxidized cytochrome a3. Several other steroid nitriles had little, if any, effect on CSCC activity, nor did they induce spectral changes with cytochrome oxidase or hemoglobin. It appears that the steroid configuration of CNK is responsible for the binding to P-450 and inhibition of CSCC, whereas the binding to cytochrome a3 and hemoglobin and the inhibitory effect on electron transfer are probably related to the cyano group of CNK.


Regulation of 18-hydroxydeoxycorticosterone in the rat. Simultaneous measurements of plasma 18-hydroxydeoxycorticosterone (18 OH-DOC), corticosterone, and aldosterone were performed in the rat by using RIA. Under basal conditions, 18 OH-DOC levels averaged 11.3 +/- 3.7 (SE) ng/ml. Plasma concentrations were increased 8-fold (86.8 +/- 8.0 ng/ml) 1 h after 1 U sc ACTH. Dexamethasone suppressed 18 OH-DOC to less than 1.5 ng/ml, irrespective of Na+ intake. Neither Na+ depletion nor Na+ loading had any influence on 18 OH-DOC levels. Excellent correlation between 18 OH-DOC and corticosterone (r = 0.90, P less than 0.001) was observed, whereas there was none between 18 OH-DOC and aldosterone (r = 0.061, P greater than 0.6). In the rat, 18 OH-DOC is an ACTH-dependent steroid which does not appear to be under the influence of the renin-angiotensin system.


Hypothyroidism-induced changes in triiodothyronine binding to nuclei and cytosol-binding proteins in rat liver. A tracer dose of [125I]T3 was given iv to normal, thyroidectomized, and propylthiouracil-fed rats and the distribution of radioactivity in serum and liver fractions was studied over 1 h. Total liver homogenate and serum 125I were higher at all times in hypothyroid rats and, in all groups, showed a continuous fall over the period studied. Hepatic nuclear 125I was maximal at 20 min in all and was greater in hypothyroid rats; there was more 125I in the hepatic cytosol of normal rats than in that from either thyroidectomized or propylthiouracil-fed animals. Binding studies with [125I]T3 and purified hepatic neclear preparations in vitro indicated that both the association constant, Ka (1.08-9.0 x 10(9) M-1) and the capacity (500-600 pg/mg DNA) in thyroidectomized and goitrogen-treated rats were similar to those obtained with normal animals. Cytosol, on the other hand, showed a decrease in binding capacity without change in affinity in livers of hypothyroid rats. Analysis of binding data by Hill plots indicated the presence of both positive and negative cooperativity in binding of T3 by rat liver cytosol proteins. In the in vitro experiments, higher serum radioactivity alone could not account for increases in the hepatic nuclear 125I in the hypothyroid rats because cytosol 125I (presumably in dynamic exchange with both blood and nuclei) was less. Consequently, cytosol T3-binding proteins may regulate the free T3 concentration in the cell and, thus influence the distribution of the hormone in other cellular compartments.


Inhibitory and facilitatory areas of the ventral midbrain mediating release of corticotropin in the cat. To examine the role of the ventral midbrain in the control of release of ACTH, we stimulated electrically 92 sites in the mesencephalon of 15 cats anesthetized with chloralose/urethane. Responses of arterial pressure could not account for change of release of ACTH. Three active areas were identified. First, in a dorsal facilitatory area that includes the dorsal longitudinal fasciculus, electrical stimulation led to changes in ACTH of +106, +117, and +90 pg/ml at 1.5, 3.5,  and 6.5 min, respectively (P less than 0.05). Second, in a more ventral inhibitory area that includes the mammillary peduncle, electrical stimulation led to changes in ACTH of -63, -72, and -47 pg/ml, respectively (P less than 0.05). Third, in a ventral facilitatory area that includes the ventral tegmental area of Tsai, electrical stimulation led to changes in ACTH of +57, +56, and +59 pg/ml, respectively (P less than 0.01). The inhibitory and facilitatory areas of the ventral midbrain appeared to be continuous, respectively, with the inhibitory and facilitatory areas mediating control of ACTH in the dorsal rostral pons and in the hypothalamus. Anatomical evidence indicates projections from these ACTH-active areas of the midbrain and of the pons to ACTH-active areas of the hypothalamus. Thus, the present results suggest that the midbrain areas identified may represent pathways from ACTH-active areas of the pons to the hypothalamus.


Effect of piperoxane on serum prolactin: possible role of epinephrine-mediated synapses in the inhibition of prolactin secretion. Intravenous administration of 2,5 mg/kg piperoxane produced a rapid and significant increase in serum PRL concentrations in four non-human primates. This PRL increase was maximal 15 min after piperoxane infusion and significant, when compared with baseline levels, in the +15, +30, +45, +60, and +90-min samples. The iv administration of 5 mg/kg piperoxane also produced a rapid and significant increase, whereas saline 0.5 mg/kg or 1.0 mg/kg, did not change serum PRL levels. The iv administration of 10 microgram/kg clonidine, but not saline, produced a rapid and significant reduction in serum PRL levels. PRL levels were significantly reduced +15, +30, and +60 min after the clonidine infusion. Pretreatment with a bolus of 10 microgram/kg clonidine at -15 min caused a significant attenuation of the piperoxane-induced elevation in serum PRL in two monkeys. These data support the hypothesis that alpha-adrenergic receptors are involved in the inhibition of PRL secretion. These data are compatible with noradrenergic or adrenergic mechanisms which remain to be defined.


Membrane receptor function and the loss of glucagon-stimulated adenylate cyclase activity in hepatomas. Plasma membranes were prepared from homogenates of two well differentiated hepatomas (Morris rat 7787 and Dalton mouse 9815), two poorly differentiated hepatomas (Morris rat 7288-C and Dalton mouse 129), and normal liver. Adenylate cyclase activity and [125I]iodoglucagon binding were measured in the plasma membrane preparations over a wide range of glucagon concentrations. Nether glucagon-stimulated adenylate cyclase activity nor [125I]iodoglucagon binding could be detected in the poorly differentiated hepatomas. Fluoride and epinephrine stimulated adenylate cyclase activity in all hepatomas. Maximum activity of glucagon-stimulated adenylate cyclase and maximum binding of glucagon in the wall differentiated hepatomas were less than those of normal liver. Plasma membranes from liver and hepatomas were solubilized with Lubrol-PX and, after reducing the concentration of detergent, were incubated with [125I]iodoglucagon and then chromatographed on a column of Bio-Gel A 1,5 m. Two peaks containing both protein and [125I]iodoglucagon were found for normal liver but not for the poorly differentiated hepatomas. Fractions from the Bio-Gel column containing the greatest concentration of protein were also subjected to a binding microassay. Material from the poorly differentiated tumors did not bind glucagon in this system, whereas the solubilized normal liver membranes bound up to 1.4 pmol [125I]iodoglucagon/mg protein. This indicates that there is no detectable glucagon receptor in these undifferentiated tumors.


Relaxin-dependent adenosine 6',5'-monophosphate concentration changes in the mouse pubic symphysis. The amount of cAMP in the pubic symphyses of estrogen-primed mice increases after injection of the hormone relaxin. This relaxin-induced increase in symphyseal cAMP was observed in immature, mature, and ovariectomized mature mice and could be prevented by prior ip injection of rabbit antibodies to porcine relaxin or by treatment of relaxin with dithiothreitol. The highest level of cAMP was measured 30 min after relaxin injection; the level of measurable cMAP then diminished rapidly. Estrogen-priming of the mice was not a prerequisite for a relaxin-induced response to occur. Relaxin administration did not increase the level of cAMP in a non-target tissue such as liver, nor could an increase in cAMP in the pubic symphysis be elicited by injection of insulin, a protein of similar size and structure, or glucagon, a known stimulator of liver cAMP levels.


Failure of angiotensin II to inhibit corticotropin-stimulated cortisol secretion. In view of the reported inhibitory effect of angiotensin II on cortisol secretion in human subjects, the effect of local angiotensin infusions on steroid secretion maintained by ACTH was examined by using sheep with cervical autotransplanted adrenal glands. During sustained submaximal stimulation by exogenous ACTH (40--80 microunit/min), the addition of local infusions of angiotensin II (1.6--160.0 ng/min) caused increased aldosterone and smaller increments in cortisol secretion in most experients. There was no evidence of inhibition of cortisol secretion by angiotensin. When similar experiments were undertaken during maximum stimulation by ACTH (16.6 mU/min), increments in aldosterone, but not in cortisol secretion, were observed. These studies exclude an acute inhibitory effect of angiotensin on cortisol biosynthesis, at least in ovine adrenal glands, during stimulation by ACTH.


The biphasic stimulatory and inhibitory effects of concanavalin A on thyroid activation induced by thyrotropin. Concanavalin A (Con A) was tested for its ability to affect thyroid activation induced by TSH in mouse thyroid lobes. Pretreatment of thyroid lobes with Con A at concentrations from 1.55--400 microgram/ml was found to have biphasic stimulatory and inhibitory effects of the TSH-induced accumulation of cAMP and formation of colloid droplets. Low concentrations of Con A potentiated TSH activation of thyroidal formation of cAMP and endocytosis. In contrast, higher concentrations of Con A markedly inhibited these TSH effects. The inhibitory effects observed after preincubation with Con A were abolished by the addition of alpha-methyl-D-glucoside to the incubation medium. A high concentration of Con A also inhibited cAMP formation induced either by prostaglandin E2 or the long-acting thyroid stimulator. However, the basal and TSH-stimulated glucose oxidation in mouse thyroid lobes was not depressed by a high concentration of Con A. Uptake of 125 I-labeled Con A by thyroid tissues increased with time up to 1 h and was directly proportional to tissue weight. These findings suggest that the specific interaction between Con A and its receptors may lead to conformational changes in the structure of the membranes of the thyroid follicular cells which facilitate TSH-induced thyroid hormone secretion via the adenylate cyclase-cAMP system.


Granules and Golgi vesicles with differential reactivity to ACTH antiserum in the corticotroph of the rat anterior pituitary. Slices of unembedded rat anterior pituitaries, fixed with a periodate-lysine-paraformaldehyde (PLP) fixative, were incubated with guinea pig antiserum to ACTH and stained with a peroxidase-conjugated IgG fraction of anti-guinea pig gamma-globulin serum from rabbits. The fine structure of the stained cells was identical to that of the ACTH-secreting cell, as described by Siperstein and coworkers. Immunoreactive granules were mainly located at the periphery of the cell. Numerous granules of the inner cytoplasm and also the Golgi complex were nonreactive to the antiserum. The differential labeling for granules and Golgi apparatus peptide.


Parathyroid hormone stimulation of adenylate cyclase activity and lactic acid accumulation in calvaria of osteopetrotic (ia) rats. We have examined the effect of parathyroid hormone (PTH) on the adenylate cyclase activity of newborn osteopetrotic rat calvaria, to study a possible molecular basis for the reduced response of this mutant to PTH. Phenotypically normal littermates served as controls. We also measured the effect of PTH on kidney adenylate cyclase activity and on lactic acid accumulation in short term cultures of calvaria. PTH stimulated calvarial adenylate cyclase activity in a dose-dependent manner in both mutant rats and normal littermates. Lactic acid production was also enhanced by PTH, and no significant difference between mutants and normal littermates was observed. These findings indicate that the reduced response of the young osteopetrotic rats to PTH is not due to an absence of PTH receptors coupled to adenylate cyclase.


Triiodothyronine receptors in rat lung.

Triiodothyronine receptors in rat lung. Specific triiodothyronine receptor activity has been detected in rat lung nuclei isolated by Triton X-100, which was sensitive to pH and exhibited temperature-dependent association kinetics. This activity represented a single order of high affinity binding sites, with an apparent dissociation constant for 3,3',5-triiodo-L-thyronine (L-T3) of 0.254 +/- 0.027 nM and an apparent capacity of 249.4 +/- 15.5 fmol 3,3',5-triiodo-L-thyronine/mg DNA. Its specificity was such that triiodothyronine derivatives were bound at least 10 times more avidly than were the tetraiodothyronines. The presence of specific triiodothyronine receptors in lung nuclei suggests that the effect of thyroid hormones on pulmonary surfactant metabolism may be mediated through these binding sites.


Glucagon administration decreases hepatic nuclear triiodothyronine binding capacity. The maximal binding capacity (MBC) of nuclear triiodothyronine (T3) receptor sites in rat liver decreases markedly after glucagon administration. Administration of serial doses of glucagon (2.5 microgram/100 g BW) resulted in a 33% decrease in MBC in 3.5 h and MBC was reduced by 45% in 6.25 h. The individual doses used were in the same order of magnitude as those used in the treatment of hypoglycemic human subjects (1.5 microgram/100 g BW). This report presents the first evidence that a peptide hormone can change the number of nuclear T3 receptor sites. The physiological significance of these findings remains to be clarified.


In vivo effects of antisera to prolactin receptors in female rats. Antisera generated in guinea pigs against partially purified PRL receptors derived from rabbit mammary glands were tested for their effect on PRL binding and the biological effects of PRL in vitro and in vivo. Several dilutions of either normal guinea pig serum or anti-PRL receptor serum were incubated in vitro with homogenates of rat ovaries. Although specific binding of PRL was inhibited as much as 90% by the anti-serum, normal guinea pig serum had little effect. There was no inhibition of binding of LH and FSH to ovaries by these antisera. When these antisera were administered to normal cycling rats, the most pronounced effect was an increase in the number of corpora lutea, presumably due to the prevention of the luteolytic effect of PRL. When one of these antisera was given to adult rats immediately after parturition, weight gain of their pups decreased significantly, possibly reflecting a decrease of milk yield. Thus, passive immunization of animals with anti-PRL receptor sera modifies the actions of PRL on some of its target tissues.


Immunochemical measurement of the vitamin D-binding protein in rat serum. The vitamin D-binding protein (DBP) was measured in rat serum using a single radial immunodiffusion technique. Normal serum levels at birth (74 +/- 11 mg/liter, mean +/- SD) were lower than during the last day of fetal life (130 +/- 14 mg/liter) and much lower than in adult rats. A marked sex difference in DBP occurred after puberty: male values (656 +/- 52 mg/liter) were significantly higher than female values (472 +/- 46 mg/liter). The sex difference could be abolished by either adult gonadectomy or transpharyngeal hypophysectomy. Implantation of a pituitary gland under the renal capsule in hypophysectomized male rats further decreased the DBP concentration, suggesting that PRL suppresses the DBP level. A similar decrease was also observed at the end of pregnancy and during lactation. Administration of androgens to either normal female or gonadectomized male rats increased their DBP concentration to the normal adult male level. The serum levels of total 25-hydroxyvitamin D did not fluctuate according to the concentration of DBP, indicating that the concentration of "free 25-hydroxyvitamin D" is not regulated at a constant level.


Binding of bovine thyrotropin to receptors in rat testis and its interaction with gonadotropins. Previously, we have shown that preparations of hCG bind to bovine thyroid membranes, as judged from their ability both to inhibit the binding of 125I-labeled bovine TSH (bTSH) and to activate adenylate cyclase (Amir, S.M., H. Uchimura, and S.H. Ingbar, J Clin Endocrinol Metab 45: 280, 1977). In the present studies, 125I-labeled, highly purified bTSH ([125I]bTSH) has been shown to bind specifically and saturably to receptors in a particulate fraction from rat testis. At 37 C, binding was rapid, reaching a maximum level in less than 15 min, but then declining markedly during the next several hours. At 22 C, binding reached a steady state after 2 h and remained unchanged for another 22 h. Binding of [125I]bTSH was greatest at pH 5.5, at which pH more than 50% of [125I]bTSH was bound in the presence of 330 microgram/ml particulate protein, the concentration of protein that yielded maximum binding. Nevertheless, the majority of experiments were conducted at lesser protein concentrations and at physiological pH (7.45), under which conditions total binding was only 25% of that measured at pH 5.5. Scatchard plots indicated the presence of a single binding site with a dissociation constant of 5.8 X 10(-8) M and a binding capacity of 0.22 nmol/mg protein on the basis of data obtained at 22 C and pH 7.45. Both crude and highly purified preparations of hCG inhibited the binding of [125I]bTSH to testis particulate fraction; crude hCG had 46 times the activity, and purified hCG had only one-tenth the activity of bTSH itself in this respect. This was true despite the fact that with respect to the displacement of [125I]hCG, crude and purified hCG were almost equally active. Bovine LH had one-third the activity of bTSH in displacing [125I]bTSH. Human FSH inhibited [125I]bTSH binding only slightly at the highest concentration tested, while glucagon, insulin, PRL, and GH were inactive. Purified bTSH inhibited the binding of [125I]hCG to testis particulate fraction but contained only about 2% of the activity of purified hCG. Lineweaver-Burk analysis suggested that inhibition of [125I]hCG binding by bTSH was competitive in nature. Purified bTSH stimulated cAMP production in Leydig cells, but with only about 0.1% of the activity of purified hCG. It is concluded that bTSH binds reversibly, saturably, and with relatively high affinity to receptors in rat testis that are either the same as receptors for hCG and LH or that interact therewith. bTSH, like hCG, is capable of stimulating the production of cAMP in rat Leydig cells, but is much less potent than hCG in this regard. Preparations of crude hCG contain a factor lacking hCG activity in bioassay, immunoassay, and receptor assay that is especially potent in displacing [125I]bTSH from receptors in testis, as has earlier been described for bTSH receptors in bovine thyroid membranes.


Developmental changes in testicular gonadotropin receptors: plasma gonadotropins and plasma testosterone in the rat. The relationships between plasma gonadotropins, testicular gonadotropin receptors, and plasma testosterone were examined during neonatal life and throughout sexual maturation in the rat. The binding affinity of testicular LH receptors (2.4 X 10(10) M-1) was significantly higher than that of FSH receptors (2.1 X 10(9) M-1) at all stages of development. The concentration of FSH receptors in the testis reached a peak between 10-15 days of age, then fell to a constant level from 25-90 days. However, the testis content of FSH receptors increased continually with age and reached a plateau at day 60. Plasma FSH declined after birth to a nadir at 15 days, then rose rapidly to a peak at day 38, and fell to a plateau from day 50 through adult life. In contrast to the rapidly changing profile of plasma FSH during early maturation, alterations in plasma LH were less marked throughout development. Although a progressive rise in plasma LH concentration was observed between days 36-51, the simultaneous changes in testicular LH receptors and plasma testosterone were much more prominent. Testicular LH receptors showed a continuous increase in concentration and total number with advancing age and testis growth. The major rise in LH receptor concentration occurred between 15-38 days age, at the same time as the rise in plasma FSH concentration and the phase of rapid testicular growth. Plasma testosterone fell during the 8th-24th days after birth, then rose rapidly between days 35-55. The pubertal rise in plasma testosterone occurred about 15 days after testicular LH receptors began to increase and was coincident with the continuing rise in LH receptor content from day 35 until day 55 and with the progressive increase in plasma LH during this period. These observations have demonstrated that the early development of testicular FSH receptors in followed by a prominent rise in plasma FSH, with concomitant increases in testicular growth and LH receptor concentration. The resulting increase in gonadal sensitivity to LH could be responsible for the marked increase in secretion of testosterone which occurs during puberty in the presence of a relatively small change in the circulating LH concentration. The sequence of changes observed in gonadotropins and their testicular receptors is consistent with the view that FSH-induced testicular sensitivity to LH is an important factor in sexual maturation in the male rat.


Binding of thyroid stimulators to guinea pig testis and thyroid. We have demonstrated TSH binding sites in membranes prepared from guinea pig testis. The binding of TSH to testicular homogenate was localized to the interstitial tissue and showed similar characteristics to the binding of TSH to thyroid membranes with high affinity (Ka = 2.5 X 10(10) M-1) but limited capacity (approximately 0.9-1.7 pg/mg equivalent testis homogenate). In addition, thyroid-stimulating antibodies inhibited the binding of TSH to both testicular and thyroid homogenate but failed to inhibit the binding of hCG to the testis preparation. These data suggested that the TSH-binding site was distinct from the hCG/LH binding site. Testicular and thyroid TSH-binding sites also appeared to demonstrate a similar degree of cross-reactivity with a crude preparation of hCG. In conclusion, our findings suggest that thyroid-stimulators may influence gonadal function. Furthermore, the ability of hCG to inhibit the binding of TSH to thyroid membranes provides a possible mechanism for the known thyroid-stimulating properties of hCG.


Role of the carbohydrate residues of human chorionic gonadotropin in binding and stimulation of adenosine 3',5'-monophosphate accumulation by porcine granulosa cells. Removal of the sugar residues internal to sialic acid from the hCG molecule markedly diminished the ability of hCG to stimulate cAMP accumulation by porcine granulosa cells during a 30-min incubation period. At the same time, removal of sugars internal to sialic acid enabled the resulting hCG derivatives to become competitive inhibitors of hCG stimulation of cAMP accumulation. This contrasts with the finding that removal of sugars internal to sialic acid residues has a lesser effect on the ability of hCG to inhibit the binding of human [125I]iodo-CG to granulosa cells, provided the hCG derivatives were added before the tracer. It would, therefore, seem that hCG with sugars internal to sialic acid removed is able to bind to the receptor, but that, once bound, it is unable to activate adenylate cyclase. Under these conditions, it can also act as a competitive inhibitor of hCG stimulation of cAMP accumulation.


Thyroid hormone receptors from liver nuclei: characteristics of receptor from normal, thyroidectomized, and triiodothyronine-treated rats; measurement of occupied and unoccupied receptors, and chromatin binding of receptors. The capacity and characteristics of liver nuclear receptors for T3 have been compared in normal rats, thyroidectomized rats, and thyroidectomized rats treated with different doses of T3. Receptor capacity was unchanged, with the exception of a decrease of total nuclear receptors in rats treated with 15 microgram T3/day, which is believed due to lack of complete dissociation of the endogenously bound hormone during incubation in vitro. During incubation of the isolated nuclei in 0.32 M sucrose plus 1 mM MgCl2 plus 20 mM Tris-Cl buffer, pH 7.85, about 50% of the nuclear receptors were released to the medium and no effect of T3 treatment on the amount released was found. The affinity of the receptors free in solution was 50% higher than that of receptors retained in the nuclei. Receptors from normal and thyroidectomized rats were studied by ultracentrifugation through sucrose gradients. No difference in sedimentation coefficient was found between the groups of rats, whether the receptors were filled with the labeled hormone before or after centrifugation and separation of the gradient fractions. Occupied and unoccupied receptor capacity was estimated by incubation of receptor preparations at 20 and 0 C for 2 h. Observed capacity at 20 C represents unoccupied plus approximately 80% of occupied sites, while observed capacity at 0 C represents unoccupied plus 10% of occupied site. Binding of receptors to the chromatin was studied by incubating nuclear extract from normal rats with chromatin and measuring the amount of receptor that was not bound and remained free in the supernatant. Receptor not bound to chromatin was incubated at 0 and 20 C, with a saturating concentration of [125I]T3, to measure occupied and unoccupied sites. Both occupied and unoccupied receptor decrease in the supernatant after incubation with the chromatin when the concentration of chromatin in the incubation medium is increased. No saturation of the chromatin-binding sites was achieved by increasing the concentration of receptor several-fold, and no difference in the binding of occupied and unoccupied receptors to chromatin was observed. These data rule out the possibility that the concentration of receptors in the nucleus is under control of the hormone. Major molecular changes of the receptor that could be induced by T3, such as dissociation into subunits, are also ruled out. Binding studies to the chromatin are not necessarily conclusive, as nonspecific binding may be masking the physiological binding of the receptors to a limited number of acceptor sites.


Rabbit ovarian protein kinases. I. Effect of an ovulatory dose of human chorionic gonadotropin or luteinizing hormone on the subcellular distribution of follicular and luteal protein kinases. Studies on the subcellular distribution of protein kinase activity in popped estrous follicles from rabbit ovaries revealed that 15% of the total cellular protein kinase activity was compartmentalized in the nuclear, mitochondrial, and microsomal fractions. About 50% of the particulate protein kinase activity was unaffected by the heat-stable protein kinase inhibitor and was thus cAMP-independent. The majority of cellular protein kinase activity was identified in the 105,000 X g supernatant fraction as cAMP-dependent. hCG- or coital-induced ovulation and subsequent corpus luteum (CL) formation, and hCG-induced luteal regression promoted changes and a redistribution of protein kinase activity among the subcellular fractions. In follicles, hCG promoted a transient decline of nuclear protein kinase activity as well as transient increases of the relative amount of protein kinases localized in the microsomal fractions before ovulation. In CL induced by a fertile mating, the specific activity as well as the total amount of protein kinases in the nuclear fraction were reduced 2-fold. Mitochondrial protein kinase activity from CL of pseudopregnancy and pregnancy was reduced 2-fold. The relative amount of protein kinase activity in microsomes of CL was increased 2-fold, but the specific activity was not affected. hCG-induced luteal regression resulted in a transient decline of the nuclear protein kinase activity in CL of 4-day pseudopregnant rabbits. In interstitial tissue, the specific activity of the nuclear protein kinase was increased over luteal levels, the mitochondrial-specific protein kinase remained at the reduced luteal levels, and the microsomal and cytosol protein kinase specific activities increased 2-fold. Studies with the heat-stable protein kinase inhibitor revealed that the hCG- or coital-induced redistribution of intracellular protein kinase affected both the cAMP-dependent and cAMP-independent activity to a similar degree and no changes of the relative distribution of cAMP-dependent vs. cAMP-independent activity were observed. These results indicate that the intracellular distribution and enzymatic activity of cAMP-dependent protein kinases in ovarian structures are subject to regulation by LH (hCG) and depend upon the various reproductive stages of the rabbit.