Effect of K+ ions on kinetic properties of the (Na+, K+)-ATPase (EC 3.6.1.3) of bulk isolated glial cells, perikarya and synaptosomes from rabbit brain cortex.

Progress curves of the enzymatic reactions show that ATPases of bulk isolated glial cells, perikarya and synaptosomes exhibit hysteretic change. Initial velocities of enzyme activities were therefore obtained according to the equation valid for the hysteretic model. The (Na+, K+)-ATPase activities of the same brain fractions were measured before or after NaI treatment. Only glial and synaptosomal enzyme could be adequately extracted by using this procedure. Attempts to purify the (Na+, K+)-ATPase from brain perikarya by NaI extraction were unsuccessful. In order to determine the effect of the K+ ions on enzymic physiological efficiency (phys. eff.; i.e., the ratio Vmax/Kmapp) the variation of (Na+, K+)-ATPase activities from each brain fraction was measured as a function of Mg.ATP2- concentration in the presence of 5 and 20 mM K+ ions. High K+ ion concentrations (20 mM) increased the physiological efficiency of glial enzyme and decreased the same kinetic parameter in neuronal (perikaryal as well as synaptosomal) enzyme preparations. Results are discussed in relation to a possible distribution of distinct enzyme in different brain cell populations as well as a possible role of glial cells in an active regulation of K+ ion extracellular fluid in the CNS.


Maintained activity, excitation and inhibition of lateral geniculate neurons after monocular deafferentation in the adult cat.

Monocular deafferentation of the lateral geniculate nucleus (LGN) was accomplished by photocoagulation of the retinal vessels and the optic disc of one eye. Single cells with normal excitatory innervation from the intact eye (N-cells) and deafferented (D-) cells were recorded extracellularly with glass micropipettes from layers A and A1 of the LGN 10 days and 10 weeks after visual deafferentation. The maintained activity of D-cells was severely reduced after 10 days but had recovered significantly after 10 weeks. Different interspike interval distributions indicated a change in the main input source for the reactivated cells after 10 weeks. Stimulation of the intact retina with diffuse, high-intensity light flashes revealed missing or very small responses in the acutely deafferented cells. A still weak influence of the non-dominant eye on the deafferented cells after 10 days developed after 10 weeks into a biphasic response with an initial inhibitory period followed by a post-inhibitory exciation. Electrical stimulation of the optic chiasm revealed an increased inhibition during chronic deafferentation. Excitation following this inhibitory period was rare after 10 days but occurred frequently after 10 weeks. An enhanced excitability following stimulation of the visual cortex and the mesencephalic reticular formation was also observed after 10 weeks of deafferentation. An increased efficiency of all remaining inputs to the visually deafferented cells is concluded from the augmented inhibition and non-retinal excitation.


Effects of substance P on neurones in the dorsal horn of the spinal cord of the cat.

The activity of single, physiologically identified neurones has been recorded both extra- and intracellularly in the 6th and 7th lumbar segments of the pentobarbitone-anaesthetized cat. In the majority of dorsal horn neurones studies (laminae 4 and 5 of Rexed) microiontophoretically applied synthetic substance P and the active fragment, substance P (4--11), were found to cause a slow and prolonged increase of the spontaneous firing rate and/or an enhancement of L-glutamate induced activity. Intracellular studies revealed that substance P caused a reversible depolarization of both dorsal horn neurones and motoneurones without a detectable alteration of the membrane resistance, antidromic action potential or postsynaptic potentials. These results are compatible with a possible role of substance P in sensory transmission in the spinal cord of the cat.


Noradrenergic transmission and female sexual behavior of guinea pigs.

Treatment with the dopamine beta-hydroxylase (DBH) inhibitor U-14,624 (50, 100, or 150 mg/kg) blocked the induction of lordosis behavior be estradiol benzoate (EB) and progesterone (P) in ovariectomized guinea pigs. After treatment with U-14,624 (100 mg/kg), norepinephrine (NE) content of medial basal hypothalamus, preoptic area and cortex was reduced (by 55%) and dopamine (DA) content of medial basal hypothalamus was increased (by 155%) during the period when females treated with EB and P normally display lordosis. Treatment with the NE receptor stimulator clonidine (1.0 mg/kg) restored lordosis behavior in females treated with EB, P, and U-14,624 (100 mg/kg), but the putative DA and serotonin (5-HT) receptor blockers pimozide (1.0 mg/kg) and methysergide (20.0 mg/kg) were ineffective in this respect. Thus, inhibition of lordosis after treatment with U-14,624 appeared to be attributable primarily to a reduction in NE neurotransmission, rather than to increase in DA or 5-HT activity. Because clonidine induced lordosis in females treated with EB, P, and U-14,624, it seemed unlikely that the facilitatory effects of clonidine on lordosis were mediated by activation of presynaptic alpha-adrenergic receptors (i.e. inhibitory NE autoreceptors) rather than by postsynaptic alpha-receptors. In addition, pretreatment with the postsynaptic alpha-adrenergic antagonist phenoxybenzamine (20.0 mg/kg) blocked the facilitation of lordosis by clonidine (1.0 mg/kg) in females primed with EB alone and with EB plus P. Thus, the facilitatory effects of clonidine on lordosis appear to be mediated by activation of postsynaptic alpha-adrenergic (i.e. NE) receptors. The results of this study provide further evidence that NE neurotransmission facilitates the expression of female sexual behavior in guinea pigs.


[Effect of sodium n-dipropylacetate (sodium valproate) on GABA level of neuronal and glial cells in culture].

The effect of sodium n dipropylacetate (nDPA), a competitive GABA-T inhibitor with respect to GABA, has been investigated on glial and neuronal cellular GABA level. After 1 to 4 days incubation with nDPA in the culture medium, a decrease of GABA level in M5 neuroblastoma clonal cell lines and no modification of GABA level in C6 astrocytoma cells has been observed. The combined addition of nDPA 4 micrometer with dibutyryl cyclic AMP (1 mM) to the culture medium induces the same decrease in GABA level in C6 astrocytoma cells as the addition of DB-c-AMP alone. After shorter incubation time with nDPA (5-150 min), we observed a decreased GABA level in C6 astrocytoma glial cells.


[Effects of fasting and of ACTH on renal compensatory hypertrophy in the 48 hour uninephrectomized rat].

Renal compensatory hypertrophy (R.C.H.) is determined 48 h. after uninephrectomy in fed and fasting rats having free access to a NaCl solution. ACTH (18 gamma/100 g BW/d/2d) enhances R.C.H. in the fed animals. R.C.H. is severely impaired by food deprivation and the remaining kidney looses weight; a normal kidney weight can be maintained if the fasted, uninephrectomized rat is treated with ACTH. These experiments suggest that the stimulation of the adrenal cortex by ACTH has a renotrophic effect. This action may be related to the elevation of blood glucose or/and to the fall of the concentration of plasma K+.


[Proteasic polyseritis: effects of intraperitoneal injections of collagenase, alone or together with the administration of trypsin].

The authors showed previously that the effusions of the experimental polyseritis after intraperitoneal injections of trypsin and elastase, go from peritoneum to the pleural cavities and never from thorax to the peritoneum. The intraperitoneal injections of collagenase or of collagenase and trypsin can also cause polyseritis in the rat; but more often they provoke heavy hemorrhagic lesions of the wall of the abdomen and the diaphragm. Perforations of the diaphragm were observed in 8 rats of 32, with in 6, a intrathoracic hernia of the liver or the stomach. After the intrapleural injection of collagenase or of collagenase and trypsin, hemorrhagic lesions were seen in the thorax, but not in the abdomen. These facts are a new proof for the transdiaphragmatic propagation of the proteasic solutions injected in the peritoneum.


[Modification of the response of nude mice to mouse hepatitis virus by transfer of heterozygote splenic cells].

The course of infection with low-virulence mouse hepatitis virus in nude mice was modified by transfer of sensitized or non-sensitized heterozygous spleen cells after the infection had been established. Infected nude mice having received the transfer of heterozygous spleen cells seem to have more severe signs of illness at early stage of infection than those without the heterozygous cell transfer, while they resisted against the infection.


Epidemiology of equine upper respiratory tract disease on standardbred racetracks.

The outbreaks of upper respiratory tract infections in horses at Standardbred racetracks were investigated over a three year period. The most serious epidemics of respiratory disease occurred in the winter and spring seasons. Both influenza viruses and equine herpesvirus 1 were shown to be present in the horse population. The herpesvirus was associated with respiratory disease particularly in the winter but the equine influenza viruses apparently were responsible for the major epidemics of respiratory disease at these tracks. Younger horses, two or three years of age, were particularly susceptible to upper respiratory disease and showed the greatest rate of seroconversion to influenza viruses. Major outbreaks of respiratory disease occurred when the proportion of young horses which had not previously been exposed to epidemics of respiratory disease reached 30 to 40% of the population at the track. Most horses over four years of age appeared to develop resistance to the infections.


Experimental swine vesicular disease, pathology and immunofluorescence studies.

Two day old piglets were inoculated intravenously with 1 ml of swine vesicular disease virus UK-G 27-72 isolate. Using infectivity tests, immunofluorescent staining and gross and histopathological examination, pathogenesis of the infection was studied in tissue specimens collected daily from one through seven days postinoculation. Swine vesicular disease virus had a strong affinity for the epithelia of the tongue, snout, coronary band and lips, the myocardium and the lymphoid elements of the tonsil and the brain stem. The virus had the greatest affinity for the epithelium of the tongue. However, there was no evidence that the tongue was the initial replication site for swine vesicular disease virus. Prickle cells in the stratum spinosum appear to be the primary targets for the virus. The necrotic foci in the stratum spinosum appeared first, followed the next day by reticular degeneration and multilocular intraepidermal vesicular formation. In the digestive tract and most of the other visceral organs the short duration and sudden drop of the virus titres and the negative fluorescence and pathological findings suggest that these are not important sites for the replication of swine vesicular disease virus in this experiment. The virus was recovered from most of the central nervous tissue specimens. Although the piglets had significant central nervous system lesions, signs of impaired central nervous system function were not detected. However, subtle nervous signs could have been obscured by difficulties in locomotion resulting from severe lesions of the feet.


Alteration in testicular cyclic AMP system in the rat following vasectomy.

Endogenous cyclic AMP levels and the activities of adenylate cyclase, cyclic AMP-dependent and independent protein kinases were examined in testes of mature rats bilaterally vasectomized for one, three and seven months. Although no significant alteration in testicular cyclic AMP was detected one month following vasectomy, marked decreases (by 55% and 32%, respectively) were seen three and seven months postvasectomy. Likewise, vasectomy also resulted in a significant decrease (by 25%) in the activity of testicular adenylate cyclase three and seven months after vasectomy. Although soluble cyclic AMP-dependent protein kinase activity remained unaffected three months postvasectomy, the activity of the cyclic nucleotide-dependent enzyme was significantly increased (by 21%) when compared to the sham-operated controls. Furthermore, while the protein kinase ratio (--cyclic AMP/+cyclic AMP) was decreased in animals vasectomized for three months, the ability of the enzyme to bind (3H) cyclic AMP in vitro was significantly enhanced (18%). Rats vasectomized for seven months showed similar biochemical alterations but the effects of this procedure were more pronounced. Moreover, while short-term vasectomy increased the responsiveness of seminiferous tubular adenylate cyclase to in vitro stimulation by follicle stimulating hormone, the activity of the enzyme was also increased (by 100%) in the presence of luteinizing hormone in vasectomized rats. These data raise the possibility that changes in testicular function seen following vasectomy may be related to the alterations in cyclic AMP metabolism as well as in the sensitivity of testicular adenylate cyclase to regulation by gonadotropins.


Comparison of results using electron microscope, immunodiffusion and fluorescent antibody analyses to detect rotavirus in diarrheic fecal samples of calves.

Seventy-nine diarrheic calf fecal samples were examined by electron microscopy, immunodiffusion and the fluorescent antibody technique for the presence of rotavirus (reovirus-like agent). Thirty-eight (48%) of the samples were positive by electron microscopy, 59% by immunodiffusion and 20% positive by fluorescent antibody technique analyses. Another 9% were suspect-positive by fluorescent antibody technique. Chymotrypsin treatment of the fecal samples increased the ease of observing the viral particles by electron microscopy and also intensified the immunodiffusion arcs obtained. Immunodiffusion analyses using specific antisera to the virus would appear to be a practical method of detecting rotavirus in diarrheic fecal samples.


The calf reo-like virus (rotavirus) vaccine: an ineffective immunization agent for rotaviral diarrhea of piglets.

Rotavirus, in a commercially available calf vaccine, did not replicate in newborn colostrum-free piglets inoculated orally with one half of a calf dose. Gross and microscopic examination of these vaccinated piglets revealed no lesions consistent with rotaviral infection and vaccinated piglets were susceptible to challenge by porcine rotavirus. Challenged piglets vomited, had diarrhea and became severely dehydrated. Rotavirus was visualized in their gut fluid. Villi in the small intestines were shortened, blunted and fused. Rotaviral antigens were seen in enterocytes.


A comparative study of bovine herpesvirus 1247 and equine herpesvirus 1 in ponies.

The clinical and immunological response of ponies exposed to a bovine herpesvirus isolate and equine herpesvirus 1 were compared. Each virus was inoculated into two ponies by the intranasal route. One uninoculated pony was used with each group as a contact control. The four inoculated ponies developed a mild rhinitis with an increase in rectal temperature. Virus was recovered from nasal secretions collected from the four inoculated and one contact pony. All ponies developed a serum neutralizing antibody to each virus. The data show that the two viruses are similar.


Identification of enterotoxigenic Clostridium perfringens type A in mixed cultures.

Three known enterotoxigenic Clostridium perfringens type A strains were mixed in various combinations with three nonenterotoxigenic strains and three lots of animal intestinal contents. They were grown as mixed cultures and tested for the presence of enterotoxin by the fluorescent antibody, reversed passive hemagglutination and immunodiffusion techniques. The fluorescent antibody and reversed passive hemagglutination tests detected enterotoxin in all 16 cultures prepared but the immunodiffusion test failed on two cultures. Attempts to reisolate the enterotoxigenic strains from the mixed cultures was successful in 12 cases when two of five isolated colonies were selected.


Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in Myxococcus xanthus.

Exogenous cyclic nucleotide phosphodiesterase (PD) accelerated fruiting body (FB) formation and increased territory size of aggregates in Myxococcus xanthus. Both guanosine 3'5'-monophosphate (cGMP) and guanosine 5'-monophosphate (GMP) were antagonistic to the PD effect. Adenosine 3'5'-monophosphate (cAMP) increases FB numbers twofold in the absence but not in the presence of PD. PD induction is not affected by methionine or isoleucine, which inhibit, or by threonine, which stimulates, FB formation. There is an increase and subsequent decrease in cAMP levels during early glycerol-induced microcyst development but 10 mM theophylline or caffeine not only inhibited microcyst development but induced germination in the presence of glycerol. On the basis of these results and the reports of other investigators a tentative model is proposed based on a dual role for cyclic nucleotides in the development in M. xanthus.


Effect of serum-antigen incubation times on the expression of non-specific inhibitors of rubella hemagglutination.

Under certain conditions, serum very low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) can inhibit rubella hemagglutination. The level of this non-specific hemagglutination-inhibition (HI) activity increases as the incubation period between serum and antigen is increased. Treatment of serum with heparin-MnCl2 does not precipitate HDL, and may not effect complete removal of all VLDL. This treatment method, therefore, should be considered a source of false-positive reactions, especially when extended serum-antigen incubation periods are used to enhance HI activity and to detect low levels of IgM.


Studies of the microaerophilic nature of Campylobacter fetus subsp. jejuni. II. Role of exogenous superoxide anions and hydrogen peroxide.

The addition of bovine superoxide dismutase to Brucella broth or Brucellar agar greatly echanced the oxygen tolerance of Campylobacter fetus subsp. jejuni strain H840 (ATCC 29428). Catalase also enhanced oxygen tolerance, but to a lesser extent. These enzymes must act externally to the bacteria. All of the diverse compounds which enhance oxygen tolerance of C. fetus, including nor-epinephrine and a combination of ferrous sulfate, sodium metabisulfite, and sodium pyruvate, share the ability to quench either superoxide anions or hydrogen peroxide. On the basis of these and other data, we propose that C. fetus is more sensitive to exogenous superoxide anions and hydrogen peroxide than are aerotolerant bacteria, despite the occurrence of superoxide dismutase and catalse activities in C. fetus. Compounds that enhance oxygen tolerance in C. fetus appear to act by quenching superoxide anions and hydrogen peroxide which occur spontaneously in the culture medium.


Surgical treatment of tendosynovial sarcoma.

Tendosynovial sarcoma is notorious for its high rates of local recurrence and metastases after surgical treatment. A retrospective study was made of 109 cases of monobloc wide soft part resection and amputation for primary tumors, and 29 patients who underwent resection of pulmonary metastases. Actuarial five-year survival rates after soft part resection was 70% for untreated, and 61% for previously treated, locally recurrent neoplasms. Corresponding rates after amputation were 47% and 64%. Local tumor recurrence developed in 18% of the primary soft part resections and 4% of the amputations, usually when some basic surgical principle had been violated. 35% five-year survival was achieved with judicious resection of solitary and multiple lung metastases in most cases without chemotherapy. Local control of a tendosynovial sarcoma can be achieved with properly executed surgical procedures which adhere to established tenets of cancer surgery. Resection of pulmonary metastases merits an important position in the management of these patients.


Cytotoxic drugs and the human adrenal cortex: a cell culture study.

Primary monolayer cultures of nonproliferating adult human adrenocortical cells have been used to screen 18 cytotoxic drugs used in cancer chemotherapy for direct effects on corticosteroidogenesis. None of the drugs tested, with the exception of 5-fluorouracil (5-FU) and its metabolite 5-fluorodeoxyuridine, showed significant activity at levels compatible with cortical cell viability and/or likely to be encountered during therapy. These two antimetabolites, however, resulted in a slow but long-lived reversible suppression of corticosteroidogenesis in both ACTH- and monobutyryl cyclic AMP-stimulated, as well as unstimulated cultures of human cells. Thus 10 micrograms/ml resulted in less than 80% inhibition after seven days treatment without any evidence of overt cytotoxicity. High-pressure liquid chromatography showed a suppression of all UV-absorbing steroids secreted. Examination of the ultrastructure of the treated cells showed significant changes in mitochondrial morphology, suggesting a possible site of action for the antisteroidogenic effects of 5-fluorouracil. These in vitro results suggest the possibility of adrenal suppression in vivo during long-term or high dose infusion 5-FU chemotherapy.


Enhancement of biochemical transformation of mammalian cells by herpes simplex virus following nitrosomethylurea treatment.

A quantitative assay for the biochemical transformation of thymidine kinaseless mouse cells (N cIA cI10 cells) to an enzyme-positive phenotype by herpes simplex virus has been used to examine the effect of the carcinogen nitrosomethylurea on cell transformation. Exposure of cells to the chemical carcinogen, followed by virus infection, resulted in enhancement of transformation when compared to that seen with virus or chemical alone. Enhancement occurred after doses of nitrosomethylurea that were nontoxic to treated cells. A strong time dependence after treatment was demonstrated for enhancement which was correlated with the presence and excision-repair of alkylated DNA in treated cells.


Humoral antibody response to bovine leukemia virus infection in cattle and sheep.

In this study, 345 cattle from 7 herds with a history of lymphosarcoma were tested for antibody to BLV antigens by three serological methods, namely immunodiffusion using a bovine leukemia virus glycoprotein with a molecular weight of 60,000 as antigen, and radioimmunoassay using a bovine leukemia virus glycoprotein with a molecular weight of 60,000 and a bovine leukemia virus protein with a molecular weight of 24,000 as antigen. The three tests under comparison agreed for 335 animals, 240 being negative in the three tests, and 95 being positive. Results were variable in ten cases only. Glycoprotein with a molecular weight of 60,000 antibody titers were systematically higher than were protein with a molecular weight of 24,000 antibody titers in bovine sera and milk, as well as in sera of experimentally infected sheep. In the latter case, antibodies to bovine leukemia virus antigens reached maximal values at the animal death in the tumor phase of the disease. Ratios of serum antiglycoprotein titer to milk titer varied between 4 and 117, showing that, if milk pools are to be used in surveys of bovine leukemia virus infection, use of very sensitive techniques of detection is mandatory.


Immunosuppressive properties of a virion polypeptide, a 15,000-dalton protein, from feline leukemia virus.

The 15,000-molecular-weight polypeptide (p15) of feline leukemia virus (FeLV) was shown to impair normal lymphocyte function in vitro and to abrogate immunity to feline oncornavirus disease in vivo. FeLVp15 suppressed concanavalin A-induced blast transformation of normal feline lymphocytes by 68%, while other virion proteins had no effect. p15 suppression was not due to toxicity, nor was p15 a competitive inhibitor of concanavalin A binding. Capping of receptors for concanavalin A on normal feline lymphocytes also was inhibited by either inactivated FeLV or FeLV p15. Groups of cats were immunized with either killed feline oncornavirus-associated cell membrane antigen bearing tumor cells or tumor cells plus FeLV p15. After challenge with feline sarcoma virus, three of four p15-treated cats developed progressive fatal fibrosarcoma as compared to one of five non-p15-treated cats. The cats receiving p15 also had lower cytotoxic antibody titers against feline oncornavirus-associated cell membrane antigen (mean peak titer, 1:6) than did the non-p15 group (1:74). These data support the hypothesis that the immunosuppression in cats infected with FeLV is mediated by FeLV p15.


Mobility of lymphocyte surface membrane concanavalin A receptors of normal and feline leukemia virus-infected viremic felines.

The virus-associated depression of concanavalin A mitogenesis which accompanies feline leukemia virus-induced cat lymphoma was investigated by comparing lymphocyte surface receptor mobility of normal cats to that of viremic diseased animals. The mechanics of feline lymphocyte receptor mobility were studied using fluorescein-conjugated concanavalin A to quantitate lymphocyte capping. The results of a study of 21 disease-free animals showed that cat lymphocytes undergo appreciable concanavalin A capping, with a mean capping rate of 17% under conditions developed in this study. In contrast, morphologically normal peripheral blood lymphocytes of six feline leukemia virus-infected viremic cats, with or without lymphoma, exhibited a mean capping of only 7%, significantly less than that of the control animals (p less than 0.005). These findings suggest that a membrane-related lymphocyte deficiency accompanies the development of virus-induced lymphoma in the cat.


Insulin and glucagon receptors in Morris hepatomas of varying growth rates.

The binding of both insulin and glucagon to receptors in plasma membranes from five hepatomas of varying growth rates was diminished when compared to plasma membranes from normal liver. Scatchard analyses of the binding data suggested that the decrease in glucagon binding was due to a decrease in binding capacity, whereas the decrease in insulin binding was due either to a decrease in binding affinity or to site-site interactions. The decreased binding of insulin, but not of glucagon, showed a significant correlation with increasing growth rate of the tumors. These data suggest, therefore, that decreased binding of insulin to receptors could be a feature of increasing growth rate in hepatomas.


High incidence of mammary tumors in mice with inherited asplenia carriers for the nude gene.

A colony of mice suffering from dominant hemimelia associated with agenesis of the spleen has been developed and characterized during the past 7 years. The hereditarily asplenic (Dh/+) mice have a very low incidence (9%) of spontaneous mammary tumors (SMT). Asplenic (Dh/+) females were mated with mice homozygous (nu/nu) for hereditary athymia (nude) having a BALB/c background. BALB/c females heterozygous for the nu gene and with spleen (nu/+,+/+) have a moderate incidence (12%) of SMT, whereas nu/+,Dh/+ breeders have a drastic increase in the incidence of SMT to 46% when bred under identical conditions. Since all parent strains have a very low incidence of SMT, it appears that the spleen agenesis is a major factor accounting for an earlier and higher incidence of SMT in hereditarily asplenic (nu/+,Dh/+) mice than in normal (nu/+,+/+) siblings. The SMT express mammary tumor virus antigen(s) and possess estrogen, progesterone, and glucocorticoid receptors. The SMT rapidly metastasize and kill the host within 30 to 45 days. The BALB/c asplenic mice with SMT represent a unique model relevant to human breast cancer and for study of the function of the spleen in the development of solid tumors in general and of SMT in particular.


Correlation of the induction of transcription of the AKR mouse genome 5-lododeoxyuridine with the activation of an endogenous murine leukemia virus.

5-iododeoxyuridine (IdUrd) is highly effective in inducing the production of endogenous viruses (e.g., RNA-containing murine leukemia virus) from a variety of cell lines that normally do not release such viruses. For the activation of murine leukemia virus by IdUrd, its incorporation into cellular DNA is necessary. We have explored the possibility that incorporated IdUrd qualitatively alters the transcription of cell DNA. Nucleic acid hybridization between radioactive mouse unique DNA and RNA from the highly activatable mouse AKR-2B cell line indicates that the normal extent of transcription in AKR-2B cells is considerably lower than that observed in other lines of mouse cells studied. Treatment of AKR-2B cells with IdUrd increases the extent of transcription of unique DNA by 60%, which corresponds to an induction of approximately 2.5 X 10(4) gene equivalents. Included among this new set of RNA's are sequences that are transcribed from the DNA genome of the endogenous AKR-type murine leukemia virus present in AKR-2B cells. IdUrd treatment also markedly increases the synthesis and/or the accumulation of those RNA transcripts which are normally expressed in untreated cells. These results suggest that IdUrd stimulates the overall transcription activity of AKR-2B cells. It is possible that IdUrd-induced activation of endogenous murine leukemia virus is a consequence of this stimulation.


Modulation of plasminogen activator synthesis in chick embryo fibroblasts by cyclic nucleotides and phorobol myristate acetate.

To explore the interaction of tumor promoters and sarcoma virus transformation with cellular regulatory mechanisms, we have studied induction of plasminogen activator synthesis by these agents in a background of changing cyclic nucleotide concentrations. We have confirmed the original report of Wigler and Weinstein (Nature, 259: 232, 1976) that phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, induces high levels of plasminogen activator production by chick embryo fibroblasts. Sarcoma virus transformation sensitizes the fibroblasts by lowering the threshold concentration for response to the action of PMA, and the effects of transformation and PMA on plasminogen activator synthesis are synergistic rather than additive. The plasminogen activators produced in the PMA-, virus-induced, or synergistically stimulated cultures are indistinguishable. Enzyme production in all three conditions is strongly but reversibly inhibited when cyclic nucleotide levels are raised by exposure to cyclic adenosine-3':5'-monophosphate or cholera toxin. A substantial fraction of the morphological effect that accompanies transformation is not affected by concentrations of cyclic nucleotides that suppress plasminogen activator production, and the two phenomena are therefore at least partially independent expressions of transformation in this system.


Decrease in collagen production in normal and Rous sarcoma virus-transformed chick embryo fibroblasts induced by phorbol myristate acetate.

The effect of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on collagen synthesis, a differentiated property of chick embryo fibroblasts, was examined. Collagen synthesis, as measured by the rate of formation of [3H]hydroxyproline from [3H]proline, was found to be decreased in cells treated with PMA but not in cells treated with the parent alcohol phorbol. The decrease in collagenase-sensitive proteins was confirmed by polyacrylamide gel electrophoresis of cell lysates, indicating that the decrease could not be ascribed simply to an effect on prolyl hydroxylase. Although a decrease in collagen synthesis was observed after one day, five days were required for a maximal reduction to 20% of that of dimethyl sulfoxide-treated controls. The effect of PMA on collagen synthesis was reversible. It was therefore not the result of a permanent transformation of the cells or of the selection of a population of cells with a reduced capacity for collagen synthesis. Collagen synthesis was decreased in chick embryo fibroblasts transformed by Rous sarcoma virus. Treatment of these cells with PMA for 5 days brought about a further decrease to 50% of the level in dimethyl sulfoxide-treated transformed controls.


Virus particles in the basal plate of rhesus monkey and baboon placenta.

C-type virus particles and particles, approximately 35 nm in diameter, were present in the region of the basal plate from the placenta of a rhesus monkey and two baboons. Both particles appeared to bud from the plasma membrane of the cytotrophoblast: large, pleomorphic cells with cytoplasmic extensions, indented nuclei, well-developed endoplasmic reticulum, and glycogen deposits. Extracellular particles were enmeshed within a fibrous matrix. Particles were also observed in the junctional zone, but not in the decidua. C-type virus particles from the rhesus monkey and baboons differed in ultrastructure from each other and from C-type mouse leukemia virus particles. The 35-nm-type particle was spherical with a dense central core.


Thymidine and hypoxanthine requirements for the proliferation of normal and Rous sarcoma virus-infected chicken fibroblasts in the presence of methotrexate.

Cultured normal and Rous sarcoma virus-infected chicken fibroblasts do not differ in the concentrations of thymidine or of hypoxanthine that they require to proliferate in the presence of a methotrexate block. For maximal proliferation, thymidine is required at 10(-6) M, while hypoxanthine is required at 10(-5) M. The normal and Rous-infected fibroblasts show very similar, if not identical, decreases in proliferation rates at suboptimal concentrations of thymidine or hypoxanthine. These results suggest that conversion of fibroblasts to the neoplastic state does not alter their capacity to salvage thymidine or purines from the extracellular fluid or to metabolize these compounds.


Patterns of relapse in patients with small cell carcinoma of the lung treated with adriamycin-cyclophosphamide chemotherapy and radiation therapy.

Twenty-seven patients with small cell carcinoma of the lung were treated sequentially with induction chemotherapy (adriamycin and cyclophosphamide), radiation therapy (chest and whole-brain), and then maintenance chemotherapy for 2 years. Twenty responding patients were followed to relapse and patterns of recurrence were observed. This combined treatment resulted in a complete remission rate of 80% and a median survival of 565 days in limited-disease patients. Relapse overwhelmingly occurred in the chest, but patients receiving higher-dose radiation (4000-4500 rads in split-course) had a significant prolongation of time to recurrence compared to patients receiving 3000 rads in a single course of radiation (540 versus 270 days). Despite a long mean survival, only one limited-disease patient relapsed outside of the brain or chest, suggesting that chemotherapy had a good protective effect against micrometastatic disease. Three patients relapsed in the brain at 330, 450, and 520 days, suggesting that in future studies the prophylactic whole-brain radiation (3000 rads) should be intensified.


Action of 2-acetamido-2-deoxy-beta-D-hexosidase from Turbo cornutus on periodate-oxidized and Smith-degraded, blood-group HLeb and Lea substances from human, ovarian-cyst fluids.

Immunochemical and chemical studies were used to monitor and evaluate the structural changes produced by an enzyme from Turbo cornutus in periodate-oxidized and Smith-degraded, human blood-group substances from ovarian cysts. After the first step of periodate oxidation and Smith degradation, two blood-group substances, JS (HLeb and N-1 (Lea), were precipitated by mouse-myeloma S117 serum, specific for terminal, nonreducing, beta-D-linked 2-acetamido-2-deoxy-D-glucosyl groups, but not by type XIV antipneumococcal horse serum specific for terminal, nonreducing, beta-D-linked D-galactosyl groups. An exoglycosidase, 2-acetamido-2-deoxy-beta-D-hexosidase (beta-N-acetylhexosaminidase) from Turbo cornutus, split off 2-acetamido-2-deoxy-D-glucose amounting to 22.5 and 20.4% of the total weight of JS and N-1 blood-group substances, respectively. After enzymic digestion, both blood-group substances precipitated with type XIV serum, and did not precipitate with S117 serum. The findings are in agreement with the structure propsed for the water-soluble, blood-group substances [Lloyd and Kabat, Proc. Natl. Acad. Sci. U.S.A., 61 (1968) 1477]. Specific enzymes can be of value in structural studies when used in conjunction with sequential periodate oxidation and Smith degradation.


The effect of DDT on vitamin D metabolism and calcium binding activity in the chick.

The mechanism of DDT impaired calcium absorption was studied in control, DDT fed and starved chicks. The metabolism of [3H]cholecalciferol was the same in the 3 groups, but the DDT fed and starved chicks had less intestinal calcium binding activity than the control chicks. These results suggest that DDT impaired calcium absorption and intestinal calcium binding activity may be a result of DDT induced anorexia.


Studies on the nephrotoxicity of p-nitrophenylarsonic acid: changes in rat kidney and urinary enzyme activities following the administration of p-nitrophenylarsonic acid.

Histological studies showed that the administration of p-nitrophenylarsonic acid to rats resulted in renal tubular necrosis. The nephrotoxin was administered intraperitoneally and doses greater than 30 mg/kg were found to be fatal. The severity of the renal lesion depended on the amount of the nephrotoxin used. Elevated serum urea levels, urinary protein and volume were recorded over an 8-day period following the injection of the nephrotoxin. These changes were paralleled by an increase in the activity of lactate dehydrogenase, acid and alkaline phosphatase, N-acetyl-beta-glucosaminidase and beta-glucosidase in the urine. beta-Glycosidase activities increased in kidney homogenates, immediately after the injection of the nephrotoxin, but this eventually fell to well below the normal range. Subcellular fractions were prepared from sucrose homogenates by differential centrifugation and beta-glycosidases and cytochrome oxidase were used as enzyme markers. Only minor changes in the activity of cytochrome oxidase activity resulted from the administration of p-nitrophenylarsonic acid. One of the earliest indications of renal damage was a decrease in lysosomal latency. The activities of the lysosomal and soluble enzymes were elevated above normal during the first two days after the injection of p-nitrophenylarsonic acid, but they fell to values, significantly lower than normal, on the third day. The isoenzymic forms of beta-galactosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase in normal and damaged kidneys were studied, using starch gel electrophoresis. The activities of both the lysosomal and the soluble forms of these enzymes decreased following the injection of the nephrotoxin, confirming the results obtained with whole homogenates. The relationship between the changes in renal enzyme activity and urinary enzyme excretion during the nephrotoxic process is discussed.


Evaluation of smallpox vaccination policy.

During 1978 and 1979 the Global Commission for the Certification of Smallpox Eradication will proceed with verification of smallpox eradication in 31 countries. If current surveillance activities do not discover any further cases before the end of 1979, the world will be declared smallpox free. However, the recent occurrence of two laboratory associated smallpox cases in Birmingham, England, revealed that the stocks of variola virus held in at least 12 laboratories in the world pose a real danger to the achievement of smallpox eradication and efforts are being made to reduce the number of laboratories retaining the virus to not more than 4-all WHO collaborating centres-by 1980. Scientific data indicate the unlikelihood of smallpox recurring once it has been eradicated from the human population, although further confirmatory studies are continuing. If the Global Commission verifies the eradication of smallpox, continuation of smallpox vaccination will be unjustifiable in view of its severe complications, however infrequent, and the expected global savings that would result from the termination of vaccination programmes.


Epidemiological events surrounding a paralytic case of poliomyelitis in Sweden.

A case of clinical poliomyelitis occurred in Sweden in January 1977; it was the first indigenous case in Sweden since 1962. This incident was of particular interest as it provided an opportunity to study the spread of virus in a cluster of unvaccinated persons and in individuals more or less fully vaccinated with inactivated vaccine. The patient excreted wild type 2 virus and the same type was isolated from 25 other individuals, all of whom were close contacts of virus excretors. Among the close contacts, 14 of 20 unvaccinated preschool children were found to excrete virus, but excretion was not found in any of the 7 vaccinated children examined.


Direct evidence for the presence of a different converting enzyme in the hamster cheek pouch.

Kininase II (angiotensin I-converting enzyme) is generally accepted to be the enzyme responsible for the conversion of angiotensin I (A I) to angiotensin II (A II). This study examined the response of the microvasculature of the hamster cheek pouch to the local application of A I, A II, and the renin substrate, tetradecapeptide (TDP). A I and TDP caused a localized vasoconstriction that was not blocked by converting enzyme inhibitors (CEI: BPF5a for A I and BPF5a and the nonapeptide inhibitor for TDP). However, both the A II antagonist [Sar1, Ala8]angiotensin II and the antiserum to A II blocked completely the A I- and TDP-induced vasoconstriction. Sixty-eight percent of the applied A I was converted to A II in the presence of CEI as well as in its absence. It is concluded that the vasculature of the hamster cheek pouch converts significant amounts of A I to A II by a route that does not involve kininase II.


Reduction of plasma renin activity by inhibition of the fatty acid cyclooxygenase in human subjects: independence of sodium retention.

We carried out the present studies to determine whether the suppression of plasma renin activity (PRA) that follows inhibition of prostaglandin (PG) synthesis can be dissociated from the sodium-retaining effects of these drugs. In an initial investigation we studied the effect of indomethacin on PRA in normal subjects in balance on a 10 mM Na+ diet to prevent Na+ retention. Under these experimental conditions indomethacin did not lower PRA even though the fatty acid cyclooxygenase was inhibited, as indicated by a greater than 70% reduction in the major urinary metabolite of prostaglandin E (PGE-M). Sodium depletion leads to enhanced sympathetic activity. We therefore studied the effect of indomethacin on a group of subjects in 10 mM Na+ balance in whom the effect of increased beta-sympathetic activity was blocked by the administration of propranolol. In this group, indomethacin caused 65% suppression of PGE-M and had no effect on Na+ balance, but reversibly reduced PRA in the supine and upright positions by 84% and 70%, respectively. In normal subjects in 10 mM Na+ balance, the isoproterenol-induced increase in PRA also was unaffected by indomethacin. These data establish that inhibition of the cyclooxygenase can result in a reduction of PRA that is independent of changes in Na+ balance or beta-sympathetic tone.


Suppression of plasma ACTH concentration by angiotensin II infusion in normal humans and in a subject with a steroid 17 alpha-hydroxylase defect.

Six healthy subjects were infused with angiotensin II and plasma concentrations of angiotensin, ACTH and cortisol were measured before, during and after the infusion. In all cases the plasma ACTH concentration fell as plasma angiotensin increased and rose again, sometimes to higher than basal levels, when the angiotensin infusion was terminated. These effects were most marked at the highest rate of infusion (8 pmol/kg/min) and, at the lower rates (2 and 4 pmol/kg/min), there was some recovery of ACTH levels during the infusion period in some subjects. Plasma ACTH concentrations also fell when angiotensin was infused into a patient with high ACTH levels due to a steroid 17 alpha-hydroxylation defect. The inhibition of ACTH secretion is not due to a rise in plasma cortisol operating a negative feedback inhibition. It could be a direct effect of the infused angiotensin on the brain-hypothalamus-pituitary complex or an effect on the metabolism of ACTH.


Lipodystrophy with hyperlipidaemia: the role of insulin in very low density lipoprotein over-synthesis.

A patient with partial lipodystrophy is described in whom hypertriglyceridaemia was accompanied by marked hyperinsulinaemia. The hyperlipidaemia was due to increased plasma levels of very low density lipoprotein (VLDL). Kinetic studies, performed after injection of autologous radioiodinated VLDL, indicated that the raised VLDL levels were associated with over-production of this lipoprotein. Administration of diazoxide led to a substantial fall in serum insulin levels, accompanied by reduction in VLDL production and in serum triglyceride concentration. The possible role of insulin in inducing hyperlipidaemia by causing over-production of VLDL is discussed.


Testicular function following irradiation of the human prepubertal testis.

Testicular function was studied in ten men, aged between 17 and 36 years, who had received irradiation for a nephroblastoma during childhood. The dose of scattered irradiation to the testes ranged from 268 to 983 rad. Eight subjects had either oligo- or azoospermia (0 to 5.6 million/ml), seven of whom had an elevated serum follicle-stimulating hormone (FSH) level. One subject showed evidence of Leydig cell dysfunction with a raised serum luteinizing hormone level (LH) and a low plasma testosterone concentration. A second group of eight prepubertal males, aged between 8 and 14 years, were studied. These had also been irradiated for abdominal malignancies during childhood and received a similar dose of irradiation to the testis as the first group studied. The plasma testosterone levels were within the normal range for prepubertal boys in all eight. The mean gonadotrophin levels were not significantly different from the mean levels of normal prepubertal males. Thus irradiation-induced damage to the germinal epithelium in prepubertal boys produces raised FSH levels after puberty but not before it. We conclude, therefore, that inhibition has a minor role in the control of the prepubertal hypothalamic-pituitary testicular axis and its contribution to gonadal control of gonadotrophin secretion changes with sexual maturation.


Plasma ACTH and cortisol profiles in Addisonian patients receiving conventional substitution therapy.

Plasma ACTH and cortisol profiles were studied over 24 h in five Addisonian patients maintained in good health by conventional gluco- and mineralocorticoid treatment. Patients received their usual treatment (15--30 mg cortisol daily) in divided doses at 08.00 and 16.00 hours. Plasma cortisol concentrations differed from those of control subjects in being higher from 08.00 hours-noon and 17.00-18.00 hours and were unmeasurable (less than 20 nmol/l) during the period of active cortisol secretion (03.00-08.00 hours) observed in normal subjects. Peak plasma ACTH levels (618- greater than 1600 ng/l) occurred at 08.00-09.00 hours in the Addisonian patients and were much greater than those seen in control subjects (60-220 ng/l). Despite this, plasma ACTH fell promptly within 3 h of the morning dose to less than 100 ng/l and levels were indistinguishable from those seen in normal subjects over the period 12.00-02.00 hours. These results show that there are substantial differences between treated Addisonian and control subjects in respect of plasma cortisol and ACTH concentrations. The findings may lead to a better understanding of ACTH-cortisol relationships in disease states, including congenital adrenal hyperplasia where control of excessive ACTH secretion is clinically desirable.


Cytomegalovirus infection and chronic hemodialysis.

Serologic and virologic studies of cytomegalovirus (CMV), a virus infection often disseminated in immunosuppressed patients, were initiated among hemodialysis patients, home dialysis partners, hemodialysis center personnel, and several groups of patients. No evidence was found of activation or persistence of CMV infections in connection with chronic renal disease or in association with hemodialysis. Evidence of increased CMV activation and/or infection was found among individuals who had re-entered the dialysis program following renal allograft rejection. The data indicate that dialysis personnel and home dialysis partners are not at increased risk for CMV infection. The findings of this study, which contrast with those pertaining to hepatitis B infection, suggest that different mechanisms are responsible for establishing both infection and persistence of CMV and hepatitis B.


Biparietal osteodystrophy.

Following an exhaustive literature review of 126 cases, a personal series of 8 instances is presented. The following inferences are drawn: 1) the condition is not an anatomical variant, but a pathological lesion. 2) the lesion occurs in all races, and at all latitudes; its prevalence can be estimated to range between 0.4 and 0.5%. 3) the lesion occurs predominantly in females (sex ratio male: female = 1 : 2.5) and after the age of 60. 4) histological evidence indicated erosion of the external table, compensated by remodeling of the diploe, with resultant loss of the diploe. 5) Neither senility nor senescence, nor chronic trauma, nor muscular traction, nor inflammation, can explain the lesion, its symmetry, its site or its age/sex predilection.


Computed tomography of supratentorial astrocytoma.

In a group of 40 astrocytomas the C.T. appearance was correlated with the grade of malignancy. On the plain scan the majority presented an irregular, nonhomogeneous low density lesion, better demarcatable in high than in low grade astrocytomas. Contrast enhancement was seen in 28 of the 32 high grade astrocytomas, 50% of the annular, 25% of the nodular and 25% of the mixed type. Six of the 8 low grade astrocytomas showed no contrast enhancement and 1 showed an annular type lesion. A relationship was found between the degree of contrast enhancement and the vascularity. Mass effect was observed in all but 1 patient, but more pronounced in high grade astrocytomas. At the first interpretation 98% were recognized as a lesion, 93% as a tumor and 68% as an astrocytoma. One false negative and no false postive C.T.scans were obtained. In review, high and low grade malignancies were correctly differentiated in 90%. As a screening method, the combination of EEG and echo equalled C.T.; in establishing a definite diagnosis angiography was inferior to C.T. Solely on the basis of the C.T. appearance, astrocytomas can hardly be differentiated from the other malignant brain tumors, but the grade of malignancy can be predicted reliably.


A study of the anatomy of the anterior communicating artery with the aid of the operating microscope.

The anatomy of the Anterior Communicating Artery complex is studied with the aid of the Zeiss operating microscope in 75 human brains removed at autopsy. The variations in the structure of the Anterior Communicating Artery and the A1 and A2 segments of the Anterior Cerebral Artery are reported. Special attention is given to the site of origin and number of branches from the Anterior Communicating Artery and the site of origin of Heubner's arteries. The necessity of using the operating microscope during surgical treatment of aneurysms of the Anterior Communicating Artery complex is stressed.


A tumour in the cerebellopontine angle region: an unusual case.

This paper describes a nearly 60-year old patient with unilateral hearing loss, developed in one year, and complaints of tinnitus and dizziness. Additional findings were unilateral peripheral facial paresis and a diminished corneal reflex. The cause was found to be a metastasis of an anaplastic carcinoma, localized at the site of the internal acoustic porus.


Cerebral metastasis without known primary tumour: a retrospective study.

A retrospective study was made of 19 patients who underwent operation for an intracerebral metastasis of an unknown primary tumour. Most patients survived for a few months only, with 2 remarkable ecceptions. In only 8 of them, the primary tumour became manifest in the further course of the illness.


Atlas of 99mTc-methylene diphosphonate renal images in pediatric oncology.

We describe the urinary tract abnormalities observed with bone scintigraphy during the evaluation of malignant disease. The possible abnormal renal patterns are discussed.


Myocardial infarct imaging with 99mTc-pyrophosphate and 99mTc-methylene diphosphonate: lack of correlation.

A prospective study of eight patients with recent transmural myocardial infarction was performed using 99mTc-Sn-pyrophosphate and 99mTc-Sn-methylene diphosphonate in each patient. All pyrophosphate scans were strongly positive whereas the diphosphonate scan was strongly positive in only one case. We conclude that 99mTc-Sn-pyrophosphate is preferable to 99mTc-Sn-methylene diphosphonate for myocardial imaging.


Circadian rhythms of the functions of thrombocytes and basophils in the blood of healthy volunteers and of patients with ischaemic heart disease.

The circadian rhythms of the functions of the cellular components of haemostasis, and their response to administration of ACTH, were studied in 30 healthy volunteers and in 17 patients with cardiosclerosis secondary to past myocardial infarction. A biphasic periodicity was found of the counts and functional activities of thrombocytes and basophilic leukocytes during 24-h periods. The phase of peak activities of both formed blood elements was established between 11.00 and 14.00 hours; in the night their functional capacities were lower than in the morning. Administration of ACTH elicited analogous shifts in the above mentioned indicators, but with a faster development of the first phase. In ischaemic heart disease the normal circadian rhythm of functions of the specified indicators was disturbed, and they exhibited signs of inertia, which phenomenon, in the author's opinion, reflects a lowered reliability of function of the haemostatic system.


[Macro-electrode recording in man's brain-stem 1. Electrical activity during wakefulness and nightsleep (author's transl)].

In the context of a functional treatment for pain five patients had electrodes chronically implanted in the periaqueductal gray matter (PGM) and at the same time in thalamic nuclei. The spontaneous activity of these structures was analyzed in comparison with the simultaneously recorded scalp-EEG. The structure-specific differences between the activity of the PGM and the thalamic nuclei were pointed out. In waking state a close functional relation between the alpha-activity of the PGM and the scalp-EEG was found. Physiological sleep patterns were recorded in the mesencephalon earleir than at the surface. REM-phases were initiated by 4/sec rhythms in the PGM. During rapid eye movements sharp waves in the PGM appeared which are evidently related to the eye movements themselves. The findings are discussed in reference to the literature. Special emphasis is placed on the anatomical connexions between the explored mesencephalic and thalamic structures and the temporo-occipital cortex and on the role of ponto-mesencephalic structures in slow-wave-sleep and REM-sleep.


Increase of ceruloplasmin level in blood of rats after ACTH.

The level of ceruloplasmin in plasma was measured as polyphenol oxidase activity in following groups of rats: 1. controls; 2. adrenalectomized; 3. shamadrenalectomized; 4. formalin arthritis; 5. ACTH treated (25 micrograms ACTH daily for 5 days); 6. adrenalectomized and ACTH treated (as in a previous group). The level of ceruloplasmin was markedly increased after formalin arthritis, after ACTH treatment in controls and in adrenalectomized animals.


Evidence for the involvement of guanosine 3',5'-cyclic monophosphate in the regulation of gonadotropin release.

Addition of dibutyryl adenosine 3'-5'-cyclic monophosphate (DBcAMP) or dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP; 1--6mM) to to enzymatically dispersed, overnight-cultured rat anterior pituitary cell preparations stimulated the release of gonadotropins (LH and FSH) from the cells into the incubation medium. Stimulation of gonadotropin release by DBcGMP was observed after only 10 min of incubation, whereas that caused by DBcAMP appeared at 180 min. Synthetic LHRH (2.5 X 10(-9) M) induced a small, transient increase in intracellular cAMP content (+16%, P less than 0.05, after 5 min coincubation), while levels of cGMP in the same cells were rapidly and markedly low for 2 h. The decrease in cGMP content was accompanied by a discharge of gonadotropins lasting for 2 h, which was detectable after 5 min in the case of LH and after 10 min in the case of FSH. These results strongly suggest that cGMP might be an intracellular mediator in the process of LHRH-stimulated release of gonadotropins.


Phosphodiesterase and its Ca2+-dependent activating factor in bovine thyroid.

Properties of cyclic 3',5'-nucleotide phosphodiesterase in the 100,500 X g supernatant of the bovine thyroid were investigated. The enzyme activity was measured by a radioisotopic method using an anionic-exchange resin, and it was found that the activity was stimulated by Mg2+. Sephadex G-200 gel filtration separated the supernatant into an activating factor, which required the presence of Ca2+, and an enzyme form dependent on the factor. The molecular weights were estimated to be 25,000 and 130,000, respectively. There appeared to be another enzyme form of cAMP phosphodiesterase with different dependence on the activating factor as suggested by gel filtration, but this enzyme form could not be clearly separated. cGMP phosphodiesterase purified by gel filtration showed biphasic kinetic behavior in the absence of Ca2+ and the activating factor, whereas, in their presence, the Lineweaver-Burk plot gave a single Km. The activating mechanism of phosphodiesterase may play a role in the control of concentrations of intracellular cyclic 3',5'-nucleotides in the bovine thyroid.


Biochemical studies on rats with insulin-secreting islet cell tumors induced by streptozotocin: with special reference to physiological response to oral glucose load in the course of and after tumor induction.

Pancreatic islet cell tumors were induced in 32 of 49 male Wistar rats (73%) surviving 9 months or longer following treatment with streptozotocin alone, with streptozotocin and nicotinamide, or with streptozotocin and picolinamide. Serial oral glucose tolerance tests in rats treated with streptozotocin and nicotinamide showed that the elevation of blood glucose levels after oral glucose load was depressed significantly 7 months after treatment. Plasma insulin responses were distinctly elevated 9 months after treatment. Blood glucose levels remained lower and plasma insulin levels rose markedly after a glucose load in tumor-bearing rats as compared to the response of tumor-free rats. These findings suggest that pancreatic islet cell tumors induced by streptozotocin with and without combined treatment are insulin-secreting, and that streptozotocin itself has oncogenic effects on the rat pancreas. Mean insulin concentration in islet cell tumors amounted to 401 U/g wet wt, whereas the concentration was 14 U/g wet wt in the pancreatic tissue from tumor-free rats.


Growth hormone causes rapid induction of lactogenic receptor activity in the Snell dwarf mouse liver.

The Snell dwarf mouse (dw/dw) has no detectable PRL-binding sites in the microsomal fractions of its liver. Both bGH and bPRL, purified by preparative gel electrophoresis, induce PRL-binding sites when injected into dw/dw. Intraperitoneal injection of 100 micrograms bGH every 8 h results in the appearance of a high affinity PRL receptor 8--16 h after initiation of treatment. This induced binding capacity plateaus after 32 h of treatment and subsequently decreases to nondetectable levels 48 h after the last injection. [125I]Iodo-ovine PRL is displaced from the induced receptor equally well by similar concentrations of ovine PRL, human GH, and bovine PRL, but is displaced by bovine GH (bGH) only at approximately 100-fold higher concentrations. While untreated dw/dw livers do possess high affinity bGH-binding sites, treatment with bGH did not alter the bGH-binding activity. Treatment of dw/dw with cycloheximide does not prevent induction of PRL receptors by bGH injections. These observations indicate that bGH induces the PRL receptor by interacting with the cell at some point distal to the induced receptor site and does not require the synthesis of new proteins.


Kinetics of dog thyroid secretion in vitro.

The time sequence of radioiodine sequestration and secretion (BE131I) have been compared in dog thyroid slices prelabeled with 131I in vivo and incubated in vitro with or without TSH. Sequestration has been taken to be the amount of radioiodine present in phagocytic vacuoles or colloid droplets; the TSH or (Bu)2cAMP stimulation of the basal values was suppressed by endocytosis blocking drugs. TSH induced a sequestrated radioactivity (S) after 5 min and a stimulated secretion after 20 min. The secretion rate was constant: 1%/h (mean +/- SD = 1.0 +/- 0.4; n = 7) of the total radioactivity of the slices. At equilibrium, S was constant and equal to less than 1% of the total radioactivity. The half-life of S, assuming a disappearance rate proportional to S, was 26 min (26 +/- 4; n = 5); assuming a disappearance rate independent of S, the lifetime was 44 min (44 +/- 7; n = 6). At the steady state, the limiting step of maximally stimulated secretion was the hydrolysis of the sequestrated radioactivity and endocytosis rate was equal to secretion rate. Without TSH, a constant BEI release (0.23% +/- 0.07%/h; n = 7), insensitive to cytochalasin B, was observed, which corresponded to basal secretion.


Estradiol inhibition of luteinizing hormone-stimulated progesterone synthesis in isolated bovine luteal cells.

The effects of several steroid hormones on progesterone synthesis and cAMP accumulation in isolated bovine corpora luteal cells were investigated in an attempt to determine if any of the steroids would affect the basal level of these processes or their response to gonadotropin. Isolated bovine corpora luteal cells responded to LH with a significant (P less than 0.05) increase in progesterone synthesis and cAMP accumulation when incubated at 37 C for up to 1 h. Exogenous cAMP and analogs of cAMP also significantly stimulated steroidogenesis in these incubated cells. Stimulation of progesterone synthesis by 1 microgram/ml LH was significantly suppressed (P less than 0.05) in the presence of 5--10 microgram/ml estradiol. This inhibition appeared to be largely specific for 17beta-estradiol, in that other steroids such as estrone, estriol, 17alpha-estradiol, cortisol, and dihydrotestosterone were not inhibitory. Testosterone was found to be inhibitory, but it is uncertain if this effect was due to the androgen itself or to its conversion to estradiol. Estradiol did not affect the increase in endogenous cAMP caused by LH in these cells, but did inhibit the effect of exogenous dibutyryl cAMP on progesterone synthesis. The magnitude of this inhibition of the effect of dibutyryl cAMP was not, however, equal to the estradiol inhibition of the stimulation of progesterone synthesis by LH. These data indicate that estradiol, a possible physiological luteolytic agent, has a direct inhibitory action on the corpus luteum and produces its suppression by blocking the stimulatory effect of LH at a step after cAMP.


Triiodothyronine nuclear binding in fetal and adult rabbit lung and cultured lung cells.

To investigate the possible mechanism of thyroid hormone action in the lung, we examined fetal and adult rabbit lung, and cell lines derived from lung, for specific nuclear binding sites for T3. Using incubation of isolated nuclei with L-[125I]T3 at 37 C, we found approximately 2400 specific binding sites/cell in fetal lung and 1120 sites/cell in adult lung, with a similar dissociation constant (approximately 500 pm) for both tissues. The L-2 and A549 cell lines, which may have originated from pulmonary type II alveolar cells, contained 2280 and 1580 nuclear sites/cell, and the dissociation constants were 280 and 200 pm, respectively. In fetal lung, the ability of various analogs to compete for L-T3 (100%) binding was: 3,5-diiodo-3'-isopropylthyronine, 81%; D-T3, 73%; L-T4, 6.7%; 3,3'-diiodothyronine, 0.19%; 3,5-dimethyl-3'-isopropyl-L-thyronine, 0.15%; and rT3, 0.08%. These findings indicate that both fetal and adult lung, and cultured lung cells, contain specific nuclear binding sites for T3, suggesting that these tissues and their type II alveolar cells may be directly influenced by thyroid hormones.


Thyroid plasma membrane-associated protein kinases: properties and substrates of solubilized and insoluble enzymes.

The protein kinase activities of thyroid plasma membranes were characterized after treatment by the nonionic detergent, Triton X-100. With endogenous substrate the protein kinase activity of intact plasma membranes appeared to be cAMP independent, whereas the solubilized plasma membranes contained a cAMP-dependent protein kinase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of intact plasma membranes demonstrated approximately 30 protein bands, of which several were substrates for endogenous protein kinase, cAMP had a slight, but reproducible, stimulatory effect on some of these. In solubilized plasma membranes cAMP significantly augmented phosphorylation of at least seven of these proteins. Solubilized plasma membranes bound significantly more cAMP per mg protein than intact plasma membranes. The inability to unequivocally detect cAMP-dependent protein kinase in intact membranes using endogenous substrate probably reflects the much greater activity of the cAMP-independent enzyme activity. The protein kinase activity of intact plasma membranes which was stimulated by cAMP when histone was the substrate was primarily recovered in the solubilized plasma membranes. Most of the protein kinase activity of the intact plasma membranes was insoluble and was not augmented by cAMP. The solubilized protein kinase demonstrated the same Km values for ATP, cAMP, and MgCl2 as did the cytosolic protein kinase of the thyroid. Cytosolic and solubilized protein kinase activities were more sensitive to cAMP and cGMP stimulation when histone and protamine were used as substrates. Both enzyme activities were depressed by protein kinase modulator when histone, but not protamine and casein, were used as substrates. The protein kinase activity of insoluble plasma membranes was not inhibited by the protein kinase modulator.


Steroid induction of gonadotropin surges in the immature rat. I. Priming effects of androgens.

Sexually immature female rats were either primed with estradiol benzoate on day 23 or given daily injections of various androgens on days 23--25. Plasma for LH and FSH determinations was collected on day 26, 5 h after an injection of progesterone. Massive gonadotropin surges were found after priming with estradiol benzoate or treatment with dehydroepiandrosterone (DHEA), delta 4-androstenedione, and testosterone, but not with ring A-reduced androgens (5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol, its 3 beta-epimer, and androsterone) or the nonaromatizable 11 beta-hydroxy- and 11-ketoderivatives of delta 4-androstenedione. Rats bearing DHEA-containing Silastic implants also produced LH surges in response to progesterone. A single injection of an antiestrogen antiserum abolished gonadotropin surges in rats primed with estradiol benzoate or DHEA and greatly reduced the accompanying uterine hypertrophy. DHEA and delta 4-androstenedione were barely uterotrophic in ovariectomized rats but sustained progesterone-induced gonadotropin surges. The results indicate that certain (adrenal?) androgens are able to induce maturation of the steroid-sensitive surge system via extragonadal aromatization, whereas their uterotrophic effect is largely mediated by the ovaries. Coordinated increased conversion of androgens at central and peripheral sites may be of physiological importance for the triggering of puberty.


Steroid induction of gonadotropin surges in the immature rat. II. Triggering ability of progesterone metabolites, adrenocortical hormones, and adrenocorticotropin.

Several adrenocortical steroids were tested for their ability to trigger LH release in estrogen-primed sexually immature female rats. Massive LH surges, approaching these known to be triggered by progesterone (P), followed the injection of depot ACTH1-24 and also of the adrenal steroids deoxycorticosterone (DOC) and 4-pregnen-21-ol-3,20-dione 21-acetate, but not of 5 alpha- and 5 beta-pregnan-21-of-3,20-dione, corticosterone, or aldosterone. Estrogen-primed adrenalectomized/ovariectomized rats also responded to DOC, albeit to a lesser extent. The P metabolites 5 alpha-pregnana-3,20-dione, 4-pregnen-20 alpha-ol-3-one, and 3 alpha-hydroxy-5 alpha-pregnan-20-one proved ineffective, although their triggering ability in adults was confirmed. It is concluded that adrenal P and DOC are potent activators of the gonadotropin surge system underlying pubertal ovulation and that P metabolites may acquire biological properties during sexual maturation.


Extrapituitary regulation of the circadian rhythm of plasma corticosteroid concentration in rats.

Intravascular injections of adrenocorticotropic hormone in hypophysectomized rats were approximately 3 times more effective in stimulating increases in plasma corticosteroid concentration when given at the offset rather than the onset of the daily photoperiod (12 h:12 h light-dark cycle). Reversal of the photoperiodic schedule produced a reversal in the corticosteroid response after 5 days. Similar temporal variations in androgen and T4 responses to LH and TSH were not obtained. Although plasma corticosteroid concentrations were maintained at normal intermediate levels in rats with adrenal autotransplants, daily variations were not demonstrable. These results indicate that the circadian rhythm of plasma corticosteroid concentration is regulated by way of neural pathways to the adrenal.


Mitochondrial binding sites for triiodothyronine.

Metabolic effects upon rat liver mitochondria have been observed by others within 2 min of injection of massive amounts of L-T4. Reported here is the subcellular distribution of 2--5 ng high specific activity [125I]L-T3 2 min after ip injection with and without a loading dose of 30 micrograms unlabeled T3. The loading dose did not change the hormone concentration in the livers of normal or thyroidectomized rats, but it decreased the amount of hormone found in the nuclei in both groups and it increased the mitochondrial hormone only in the normals. The amounts of hormone in the nuclear and mitochondrial fractions were considerably higher in hypothyroid animals than in normals. By chromatography, over 90% of mitochondrial 125I was T3. The 125I content of Triton X-100 extracts of mitochondrial membranes was not affected by stable T3 in vivo or in vitro. It was concluded that specific high affinity, low capacity mitochondrial binding sites could not be found 2 min after hormone injection when specific nuclear binding sites are already evident. Specific binding may not be essential for the rapid effects of thyroid hormones upon rat liver mitochondria.


Some effects of adrenalectomy in the fatty rat.

Function of the pituitary-adrenal axis was examined in lean and genetically obese fatty (Zucker) rats. The diurnal rhythms of plasma corticosterone and plasma ACTH were similar in both groups. The secretion of corticosterone by adrenal glands incubated in vitro with graded doses of ACTH was also comparable in lean and fatty rats. Adrenalectomy reduced food intake and weight gain in the fatty rats to levels similar to those in the lean animals and raised plasma ACTH to the same level. The injection of corticosterone (2 and 10 mg/day) increased food intake more in adrenalectomized fatty rats than in the corresponding lean ones. Progesterone increased food intake similarly in both groups. The enhanced responsiveness to corticosterone could account for many of the metabolical defects in the fatty rat.


Effect of gastrointestinal hormones on isolated bovine parathyroid cells.

The effects of gastrointestinal hormones on cAMP accumulation and parathyroid hormone (PTH) release were investigated in dispersed bovine parathyroid cells. Secretin (10 (-7) M) caused a 4- to 6-fold increase in cAMP accumulation, while glucagon, vasoactive intestinal peptide, and gastrin caused little if any stimulation. Cholecystokinin caused a 2- to 3-fold increase in cAMP accumulation at 10(-6) M, but this effect may be related to contamination with endogenous secretin since synthetic cholecystokinin octapeptide had no effect. Maximal intracellular cAMP accumulation due to 10(-7) M secretin was reached within 5 min and returned to control over the next 30-60 min, concomitant with a progressive rise in extracellular cyclic nucleotide. cAMP accumulation was half-maximally stimulated by 5 x 10(-9) to 1 x 10(-8) M secretin and was unaffected by alpha- or beta-adrenergic or dopaminergic blockers. Parallel effects were noted on PTH release : 10(-8) M secretin caused a 20-50% increment in PTH release at 15 min which persisted for up to 2 h; PTH release was stimulated half-maximally by approximately 6--8 x 10(-9) m secretin. The specificity of the observed results for secretin and the lack of effect of adrenergic antagonists suggest the presence of a receptor for secretin on dispersed bovine parathyroid cells. These results also suggest the possibility that secretin may modulate parathyroid function in vivo in the cow.


Purification and properties of 17 beta-hydroxy-C19-steroid dehydrogenases of adult male guinea pig kidney.

The 17 beta-hydroxy-C19-steroid dehydrogenase activity of adult male guinea pig kidney was separated into one isolated and two contiguous enzymatically pure fractions by submitting the cytosol to (NH4)2SO4 (40-80% saturation) precipitation, Sephadex G-75 filtration, and DEAE-Sephadex A-50 and CM-Sephadex C-50 chromatography. Further DEAE-Sephadex A-50 and CM-Sephadex C-50 chromatography separated eight isozymes, which were divided into four groups in accordance with their behavior on chromatography and mobility on gel electrophoresis. The molecular weights of the purified enzymes were identical by Sephadex filtration (33,000) and SDS gel electrophoresis (34,000). Two of the enzymes were separated by SDS gel electrophoresis into two subcomponents of 23,000 and 11,000 daltons. The amino acid composition, Km values, and coenzyme and substrate specificities of the enzymes (except one) were very similar. The pI values varied from 5.1-6.4 HgCl2 and PCMB inhibited enzyme activity, but prior addition of cysteine prevented the inhibition.  The presence of phosphate or pyrophosphate greatly enhanced the trace of DPN+ -linked activity. The heterogeneity was due to at least two factors. Rapid (within 3 h) preparation of the cytosol in 7 mM 2-mercaptoethanol yielded one intense and one minor enzyme band on gel electrophoresis. Omission of the mercaptoethanol resulted in the appearance of three enzyme bands, which were reversible on the addition of 2-mercaptoethanol. Storage of the cytosol at 4 or -20 C in the absence or presence of 7 mM 2-mercaptoethanol and of the kidney before extraction also resulted in the exhibition of enzyme bands which were not reversible on addition of 2-mercaptoethanol. The purified enzymes exhibited only a single form on storage at -20 C with 2-mercaptoethanol, but multiple forms appeared when the mercaptoethanol was removed by dialysis. The multiple forms were reverted to a single form on addition of mercaptoethanol.


Hormone receptors in livers of GH3 tumor-bearing rats: the predominant effect of growth hormone and testosterone.

The relationship between serum levels of rat GH (rGH), rat PRL (rPRL), corticosterone, estrogen, and testosterone and the liver-binding sites specific to [125I]iodo-human GH (hGH), -human PRL (hPRL), and -rPRL were investigated in normal rats, in rats bearing the GH- and PRL-secreting tumor (GH3), and in rats 14 days after tumor removal. The GH3 tumor elevated serum levels of rGH and rPRL and concomitantly increased the hepatic binding of radioiodinated hGH, hPRL, and rPRL; male rats had a greater increase than female rats. The increased binding was due to an increase in the specific membrane-binding sites, the receptors, whereas the affinity constant of binding (Ka) was not altered. In both male and female rats, the specific binding of receptors and the total binding capacities of livers correlated positively with serum levels of rGH (P less than 0.02) and inversely with serum levels of testosterone (P less than 0.05). There was insignificant or no correlation between rPRL or other steroids and the total liver-binding capacities. These results suggested that GH was the primary inducer and testosterone was the predominant regulator of the hepatic receptors in rats. The observed sex difference in the increase of receptors by GH3 tumor indicated that in normal and tumor-removed male rats, testosterone had suppressed the induction of hepatic receptors.


Suppression of aldosterone biosynthesis by treatment of rats with adrenocorticotropin: comparison with glucocorticoid effects.

The treatment of rats with a high dose of ACTH resulted within 4 days in a marked suppression of aldosterone and deoxycorticosterone outputs by capsular adrenal tissue ("zona glomerulosa") incubated with and without serotonin and in decreased conversions of tritiated corticosterone and deoxycorticosterone to aldosterone and 18-hydroxycorticosterone, but in strikingly increased capsular adrenal conversions of tritiated deoxycorticosterone to corticosterone and 18-hydroxy deoxycorticosterone. ACTH also suppressed aldosterone biosynthesis in rats kept on a sodium-deficient diet. Corticosterone or dexamethasone, when added to the drinking fluid for 2 weeks, induced only small decreases in aldosterone biosynthesis from endogenous or exogenous precursors. Moreover, they significantly decreased the capsular adrenal conversion of added deoxycorticosterone to corticosterone and 18-hydroxydeoxycorticosterone. These results indicate that during prolonged ACTH treatment, the zona glomerulosa cell may be converted to a functional zona fasciculata type of cell. High levels of circulating mineralocorticoids and glucocorticoids seem to be minor contributory factors in the long term suppression of aldosterone biosynthesis by ACTH.


Prolactin, growth hormone, luteinizing hormone receptors, and seasonal changes in testicular activity in the golden hamster.

In adult male hamsters, 2 months of exposure to a short photoperiod (5 h of light:19 h of darkness) caused testicular regression and a precipitous decline in plasma PRL, in agreement with earlier reports from other laboratories. Depressed release of PRL cannot be explained by a reduction in testicular steroidogenesis, because castration of males kept in a long photoperiod did not reduce PRL levels and administration of testosterone to males kept in a short photoperiod failed to reverse the decline in plasma PRL concentration. Treatment of such "regressed" animals with PRL, GH, or ectopic pituitary transplants stimulated growth of the testes and the accessory reproductive glands, increased the concentration of LH receptors in the testes, and elevated plasma testosterone levels. A single injection of 250 microgram PRL was sufficient to increase testicular LH binding, and chronic treatment with pituitary grafts completely reversed testicular regression. The effectiveness of exogenous PRL in stimulating testicular growth and LH receptors was significantly influenced by the timing of the injection. In some experiments, gonadotropin levels appeared elevated in animals injected with PRL, but these differences were not statistically significant. In hamsters with gonadal regression induced by exposure to a short photoperiod, daily administration of 20 microgram H and/or 150 microgram FSH had no apparent effect on testicular function. However, treatment with large doses of hCG and/or PMS gonadotropin resulted in significant stimulation of testicular growth and steroidogenesis. Chronic treatment of males maintained in a long photoperiod (14 h of light:10 h of darkness) with an inhibitor of PRL release, 2-Br-alpha-ergocryptine, resulted in a decreased weight of the testes and seminal vesicles. Administration of this inhibitor for a longer period (2 months) produced a significant increase in body weight but had little effect on testicular function. These results indicate that changes in the release of PRL (and possibly also GH) may plan an important role in mediating the effects of the photoperiod on testicular function in the golden hamster.


Prolactin induces its own receptors in rat liver.

Attempts to restore PRL receptors (PRL-R) in liver membranes of hypophysectomized rats with injections of PRL have so far been only partly successful. In the present study, bovine PRL (bPRL) was mixed with polyvinylpyrrolidone (PVP) in order to sustain PRL blood levels with a single injection a day. PRL-R were measured by displacement of the binding of [125I]ovine PRL (lactoperoxidase oxidation) to a 5000 x g particulate fraction of liver by unlabeled ovine PRL. The number of binding sites was calculated by Scatchard analysis and expressed as femtomoles per mg protein. PRL-R were 85 +/- 12 fmol/mg in normal intact female rats. Seven days posthypophysectomy, PRL-R were undetectable. Daily injections of bPRL with PVP for 10 days fully restored  PRL-R (117 +/- 32 fmol/mg). No significant change in PRL-R was noted when bPRL was injected with bovine GH (bGH; 120 +/- 23 fmol/mg), bovine LH (bLH; 84 +/- 14 fmol/mg), bGH plus bLH (90 +/- 12 fmol/mg), or estrogens (79 +/- 12 fmol/mg). Daily injections of bGH or bLH, alone or in combination, or estrogens with PVP failed to restore PRL-R. These results demonstrate a direct role of PRL in stimulating the production of its own receptors when a sustained blood level of the hormone is achieved.


The effect of adrenocorticotropin and nucleotides on Ca2+ uptake in adrenal cortical microsomal vesicles.

Rat and bovine adrenal cortical microsomal fractions isolated at 27,000 x g and 105,000 x g accumulated Ca2+ by a nonmitochondrial, ATP-dependent uptake system that was stimulated by ammonium oxalate. ACTH (2 mU/ml) significantly increased Ca2+ uptake in bovine adrenal cortical microsomes and in adrenal microsomes from acutely hypophysectomized rats, but only when the hormone was preincubated with intact tissue and not when it was added after homogenization. ACTH did not stimulate C2+ uptake in adrenal microsomes isolated from nonhypophysectomized, ether-stressed rats, in which basal Ca2+ uptake was higher than that observed in microsomes from hypophysectomized animals. The peptides oxytocin, insulin, and TSH did not stimulate Ca2+ uptake by adrenal cortical microsomes. ACTH preincubated with intact tissue had no effect on Ca2+ uptake in microsomes from liver, kidney, esophagus, or aorta. cAMP, 5'-AMP, and several other nucleotides, nucleosides, and related compounds stimulated adrenal cortical microsomal Ca2+ uptake by as much as 540% of control. The stimulatory effects of nucleotides, unlike those of ACTH, were apparent even when the agents were added after homogenization. However, like ACTH, the nucleotides were unable to stimulate Ca2+ uptake when they were added to isolated membrane vesicles during Ca2+ uptake measurements. It is suggested that the microsomal Ca2+ uptake system may respond to physiological stimulants and regulate Ca2+ availability in the intact cell.


Thyroid hormone modulation of epinephrine-induced lipolysis in rat adipocytes: a possible role of calcium.

Adipocytes isolated from normal, hypothyroid, and hyperthyroid rats were characterized with respect to their lipolytic activity (assessed by glycerol release) and beta-adrenergic receptors (assessed by binding of (--) [3H]alprenolol). Fat cells from hypo- and hyperthyroid rats showed the same affinity (K = 1.4 X 10(10) M(-1) and binding capacity (N = 1.21 X 10(-13) mol/microgram DNA) toward alprenolol as those from normal animals. Adipocytes from hypothyroid rats were unresponsive to epinephrine in a concentration range of 0.1-10 micron, with moderate responses at higher concentrations; injection of T3 in hypothyroid rats restored lipolytic responsiveness of the adipocytes to normal levels. Quabain (1 mM) inhibited lipolytic responses to epinephrine by 40--45% in normal and hyperthyroid rats; the lipolytic increment due to the hyperthyroid state was uninfluenced by ouabain. The lipolytic refractoriness to epinephrine of hypothyroid adipocytes was restored to normal levels by theophylline (1 mM) or EGTA  (1 mM); the theophylline and EGTA effects were not additive, suggesting that they stimulated lipolysis via a common mechanism. Epinephrine-induced lipolysis in all groups was progressively inhibited by increasing concentrations of Ca2+ in the medium. The Ca ionophore, A23187, showed a concentration-dependent inhibitory action. Theophylline (1mM) almost completely overcame the inhibitory action of the ionophore; in the presence of lower concentrations of theophylline, the inhibitory effect of the ionophore was least in hypothyroid and greatest in hyperthyroid fat cells. The findings suggest that the differences in the lipolytic response to epinephrine observed in hyperthyroid, euthyroid, and hypothyroid adipocytes are not due to alterations in the number or affinity of beta-adrenergic receptors nor to a membrane mechanism that might show differential ouabain sensitivity, but may be related to altered cellular Ca2+ concentrations which may indirectly alter cellular phosphodiesterase activity.


Decreased hepatic nuclear L-triiodothyronine receptors in rats and mice bearing transplantable neoplasms.

The concentration of hepatic nuclear T3 receptors was measured in isolated nuclei from athyreotic mice bearing thyrotrophic tumors and intact rats with Walker 256 carcinoma. Receptor concentration was reduced in all tumor-bearing animals. The mean receptor capacity of the Walker tumor-bearing rats [0.31 +/- 0.05 (SEM) ng/mg DNA] was significantly decreased from simultaneously assayed controls (0.47 +/- 0.04 ng/mg DNA; P less than 0.01). No change in the apparent equilibrium association constant was observed. In individual rats, the magnitude of the decrease in nuclear T3 receptor concentration was highly correlated with the decrease in tumor-free body weight. Additional studies showed that the decrease in nuclear receptors was not due to delayed equilibration of added T3 with nuclear sites in vitro or to an increase in endogenous hepatic T3 concentration. The plasma concentration of total and free T4 and T3 was decreased in tumor-bearing rats. Plasma TSH concentration, however, remained unchanged. Thus, these transplantable neoplasms seem to be associated with decreased hepatic nuclear receptors and low concentrations of plasma thyroid hormones. The unchanged plasma TSH suggests that the animals remained euthyroid.


Time course of thyrotropin-dependent protein phosphorylation in thyroid slices.

The time course of TSH-dependent protein phosphorylation was studied in calf thyroid slices labeled in vitro with 32Pi. Several of the proteins identified by two-dimensional electrophoresis displayed striking increases in 32P labeling in the presence of TSH. Phosphorylation of histones H3 and H1 (two subgroups) was enhanced about 3.7-and 10-fold, respectively, after incubation with TSH (15 mu/ml) for 70 min. Histone phosphorylation showed a lag after exposure to TSH; a major increase occurred only after 30-min incubation but then increased progressively up to 2 h. The lag in histone phosphorylation was also observed with slices prelabeled with 32P before the addition of TSH. In contrast, phosphorylation of a minor basic protein, A5 was also increased by 15 mU/ml TSH but displayed different kinetics, increasing substantially at 10 min. This time course correlates well with the rise in intracellular cAMP levels and protein kinase activity in thyroid slices after TSH.


Purification and characterization of the messenger RNA coding for bovine corticotropin/beta-lipotropin precursor.

The mRNA coding for the common precursor of corticotropin and beta-lipotropin has been purified to homogeneity from neurointermediate lobes of bovine pituitaries. The homogeneity of the mRNA preparation is evidenced by analysis of its translation product, electrophoresis on polyacrylamide gel in the presence of formamide and analysis of the kinetics of hybridization with its cDNA. The purification procedure involves the isolation of RNA from membrane-bound polysomes, chromatography on oligo(dT)-cellulose and on poly(U)-Sepharose and sucrose density gradient centrifugation. The mRNA has a molecular weight of approximately 450000, equivalent to approximately 1360 nucleotides in length, and contains a polyadenylate sequence with an average length of 68 nucleotides. The size of the mRNA is sufficiently large to encode the corticotropin/beta-lipotropin precursor.


Brain pyridoxal kinase. Mobility of the substrate pyridoxal and binding of inhibitors to the nucleotide site.

Pyridoxal kinase has been purified 2000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N-dansyl-2-oxopyrrolidine (dansyl = 5-dimethylaminonaphthalene-1-sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd = 11 microns at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix. N-Dansyl-2-oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 microns. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N-dansyl-2-oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.


Mechanism of carbamoyl-phosphate synthetase. Binding of ATP by the rat-liver mitochondrial enzyme.

This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.


Mitochondrial GTP-AMP phosphotransferase. 1. Purification and properties.

GTP-AMP phosphotransferase has been purified 116-fold with a yield of 24% from beef heart mitochondria using freeze-thawing, alkali and acid treatment and successive column chromatography on phosphocellulose, Sephadex G-100 and blue-dextran--Sepharose. It has crystallized from poly-(ethylene glycol) and is essential homogeneous by sodium dodecylsulfate electrophoresis and isoelectrofocusing. The specific activity of the crystalline preparation was 290 U/mg. The molecular weight was found to be 26000 and the isoelectric point to be 9.8. Amino acid analysis showed 21 aspartic acid or asparagine, 19 threonine, 12 serine, 26 glutamic acid or glutamine, 15 proline, 16 glycine, 14 alanine, 15 valine, 4 methionine, 12 isoleucine, 28 leucine, 7 tyrosine, 7 phenylalanine, 5 histidine, 14 lysine, 16 arginine, 2 tryptophan, no --SS-- bonds or free --SH. Guanosine(5')pentaphospho(5')adenosine is a very strong inhibitor similar to adenosine(5')pentaphospho(5')adenosine as an inhibitor of cytosolic adenylate kinase.


Mitochondrial GTP-AMP phosphotransferase. 2. Kinetic and equilibrium dialysis studies.

Kinetic and equilibrium dialysis substrate binding studies have been done to investigate the properties of mitochondrial GTP-AMP phosphotransferase. The results show that the enzyme has a specific requirement for divalent metal ions, namely Mg2+, Mn2+ or Ca2+ (Ca2+ is active only in the forward direction, the direction of formation of ADP). The reaction rate depends upon the ratio [Mg2+]:[substrate] rather than on the metal ion concentration alone. The enzymatic activity is influenced by NaCl (or KCl) and optimum pH occurs at 11.5 and 9.5 for guanosine and inosine nucleotides respectively. Examination of binding of substrates to the enzyme showed that there is one binding site (GTP site) for MgGTP, GTP, MgGDP or GDP per molecule of enzyme, with dissociation constants of 4.5, 4.4, 3.0, 2.2 micron respectively and one binding site (AMP site) for AMP, ADP or ATP per molecule of enzyme with dissociation constants of 20.9, 33.4 and 33.4 microns respectively. Since, within the limitations of equilibrium dialysis used in the present studies, AMP binding to one site of the enzyme could be detected only when GDP or GTP is present, the mechanism of the forward reaction may be assumed to be nearly ordered. For the reverse reaction there is no requirement of order of binding of the two nucleotides and so the mechanism of reaction may be assumed to be random.


A DNA-unwinding enzyme induced in bacteriophage-T4-infected Escherichia coli cells.

A single-stranded DNA-dependent ATP gamma-phosphohydrolase of Mr 56000 induced after infection of Escherichia coli cells with bacteriophage T4, probably the ATPase dependent on gene dda of the phage, was isolated. Studies on the enzyme show that in the presence of ATP and M2+ ions it is capable of dissociating partially double-stranded fd bacteriophage DNA into the single strands and that some 3000 enzyme copies are required to unwind the 6400-nucleotides-long DNA. Unwinding is inhibited by reducing the length of the single-stranded portion of DNA to two nucleotides. In addition it can be inhibited by sulfhydryl reagents which block the ATPase or by trapping free enzyme molecules in the assay system. The results suggest that unwinding is initiated near the single-stranded portion of the DNA and is driven by the ATPase. It further appears that the enzyme unwinds by adsorbing to the DNA. Affinity of the enzyme for double-standed DNA is not detectable by DNA binding assay.


Diglyceride kinase from Escherichia coli. Purification in organic solvent and some properties of the enzyme.

The diglyceride kinase activity of membranes from Escherichia coli was extracted into acidic butan-1-ol. The enzyme was purified in organic solvent by precipitation at -20 degrees C, chromatography on DEAE-cellulose and repeated chromatography on Sephadex LH-60. The final 1460-fold purified enzyme preparation gave a single protein band upon isoelectric focusing in the presence of Triton X-100 (pI, 4.0) and upon polyacrylamide-gel electrophoresis in the presence of sodium dodecylsulphate. The latter method as well as gel chromatography on Sephadex LH-60 indicated a molecular weight of about 15400. The purified enzyme was devoid of lipid, and it required re-addition of lipid for activity. sn-1,2-Dipalmitate and ceramide were phosphorylated, whereas the C55-isoprenoid alcohol, ficaprenol, did not serve as a substrate under the same conditions. Conversely, the butanol-soluble C55-isoprenoid-alcohol kinase from Staphylococcus aureus did not phosphorylate sn-1,2-dipalmitate.