Microassay with the NADH-induced light reaction, technique improved by means of purified enzymes from Achromobacter fischeri. Purification of a commercial preparation of Achromobacter fischeri was carried out by agarose-suspension electrophoresis and by molecular-sieve chromatography. Both the luciferase and an oxidoreductase, catalyzing reduction of FMN with NADH, were obtained in more than one form. Flavins, liable to interfere with the light production in analytical applications, were present in amounts worthy of consideration, but seem to be firmly bound to protein. The major quantity was found in the enzymatically inactive fractions. In free zone electrophoresis of the main luciferase component, the mobility of the zone containing enzyme activity was calculated to -4.0 X 10(-5) cm2 sec-1 V-1 at 12 degrees C. Fractions of the two enzymes were separated and mixed in different proportions to study how the intensity and time course of NADH-induced light emission can be modified. These experiments disclosed how reaction mixtures will have to be composed in appropriate photokinetic assays, using NADH as measurable product. A regenerating system based on the purified fractions is described. Instead of the light flash, following the consumption of NADH, the light is emitted on a well maintained level, permitting assays with a less elaborate equipment than the one required for the recording of fast reactions.


[Functional anatomy of low back pain and ischialgia (author's transl)]. The neuronal systems involved in low back pain are first described in a functional anatomical review and then an attempt is made to work out the special functional structure data which seem to be decisive for the origin and localization of low back pain and ischialgia. Low back pain may arise as a deep pain through stimulation of the nociceptive afferents in the musculature and supporting tissues and as a neural pain through irritation of nociceptive nerve fibers within the innervation area of the lumbosacral plexus. The nociceptive influx from the viscera only elicits pain in the rarest cases, however, it often has a conductive influence. Skeletal muscle plays a special role here, along with the sensory supply to the vertebral motor segment through the spinal nerve and the particular innervation scheme of the lumbosacral plexus caused by the limb budding.


[Neurological aspects of the differential diagnosis of low back pain (author's transl)]. The close relationships between the bony spinal column, spinal marrow and the emerging nerve roots explain the frequency of neurological symptoms of diseases in the region of the lumbar vertebrae, the sacrum and the pelvis. After a short description of the local diagnostic signs in lumbosacral pains, further important differential diagnostic aspects of the changes in the vertebral column besides those of the intervertebral disks, inflammatory changes in the vertebrae, intramedullary tumors as well as a few rare pain syndromes in peripheral nerve diseases are discussed.


[The role of cyclic AMP in the control of insulin secretion (author's transl)]. D-glucose stimulates the accumulation of cyclic AMP in isolated islets of Langerhans in a dose-dependent manner. This effect is present as long as the sugar remains elevated in the incubate. Under present experimental conditions the stimulation of cyclic AMP precedes insulin release. Islet or medium cyclic AMP, and insulin secretion are closely correlated at various incubation times. We conclude that the beta-cell cyclic AMP system is one of the major mediators of the glucose signal to insulin secretion. Tolbutamide and glibenclamide have similar effects in the absence or with low concentrations of glucose. These results may indicate that these sulfonylurea derivates act at sites where glucose exerts its effect.


Decline in mutation frequency in temperature-sensitive SV40 viruses before viral DNA synthesis. Lesions that promote reversion from a temperature-sensitive to a wild-type phenotype were induced in temperature-sensitive late mutants of SV40 virus by UV irradiation. When cultures infected with UV-irradiated temperature-sensitive mutants were grown for various times at permissive temperature (35 degrees C) and then at restrictive temperature (39 degrees C), the reversion frequency declined just before the onset of semiconservative DNA synthesis when DNA synthesis began at 32 degrees C. This can be explained by competition between reactions that lead to the onset of viral DNA synthesis and reactions that repair the lesions before the onset of viral DNA synthesis.


Paradoxical ACTH response to glucocorticoids in Cushing's disease. To define further the defect in the steroid feedback mechanism in Cushing's disease, we studied the acute effects of intravenous administration of glucorticoids on plasma ACTH levels in seven patients with this disease after total adrenalectomy. In seven other patients with hypoadrenocorticism ACTH was readily suppressed; a significant decrease (72.5+/-5 per cent, mean +/- S.E.M., P less than 0.002) occurred within 15 minutes of the start of an infusion of 50 mg per hour of cortisol. In contrast, in the seven adrenalectomized patients with Cushing's disease, cortisol induced a transient paradoxical rise in ACTH levels, with a maximum at 15 minutes (347+/-99 per cent,, P less than 0.05). A similar ACTH response was observed with dexamethasone. Cushing's disease is characterized by a paradoxical transient rise in ACTH after glucocorticoid administration. This effect was more pronounced in adrenalectomized than in nonadrenalectomized patients.


Factitious hypoglycemia. Diagnosis by measurement of serum C-peptide immunoreactivity and insulin-binding antibodies. In seven patients with factitious hypoglycemia due to the surreptitious injection of insulin, we made the diagnosis by measurements of plasma insulin and C-peptide immunoreactivity (in seven patients), facilitated by the finding of circulating insulin-binding antibodies (in two patients). The simultaneous demonstration of low plasma glucose, high immunoreactive insulin and suppressed C-peptide immunoreactivity represents a triad of results pathognomonic of exogenous insulin administration. Determination of plasma free C-peptide and free insulin permitted patients with high titers of insulin antibodies, including those with a history of insulin-treated diabetes, to be studied and diagnosed in a way similar to that in subjects who had no circulating insulin antibodies.


Variable phenotypic expression of an X-linked recessive lymphoproliferative syndrome. Investigation of a family with cancer in boys revealed that at least 20 males had the X-linked recessive lymphoproliferative syndrome. A variety of phenotypes occurred: aproliferative phenotypes consisted of aplastic anemia, agranulocytosis or acquired hypogammaglobulinemia; and proliferative phenotypes of B cells included disorders associated with the Epstein-Barr virus, American Burkitt's lymphoma, immunoblastic sarcoma of B cells, fatal infectious mononucleosis or plasmacytoma. The lymphoproliferative disorders observed in males could have resulted from an immunodeficiency to Epstein-Barr virus. The variable phenotypic expression could have resulted from individual differences in the viral dose, duration of exposure and age at which the boys were exposed to the virus. Aproliferative phenotypes such as acquired hypogammaglobulinemia could have ensued from excessive suppressor-cell activity on B cells, whereas proliferative phenotypes such as Burkitt's lymphoma or fatal infectious mononucleosis could have resulted from infection by Epstein-Barr virus and failure to stop proliferation of B cells.


Selective deficiency of 1,25-dihydroxycholecalciferol. A cause of isolated skeletal resistance to parathyroid hormone. To investigate the role of vitamin D metabolites in the pathogenesis of pseudohypoparathyroidism, we studied an elderly man with a unique variant of the disease, which was characterized by hypocalcemia, elevated serum parathyroid hormone (513 +/- 13 pg per milliliter, mean +/- S.E.M., normal, less than 450) but normal renal responses (phosphate and cyclic AMP) to exogenous parathyroid extract. Treatment with parathyroid extract did not produce a calcemic effect, suggesting an isolated skeletal hyporesponsiveness to parathyroid hormone. Although 25-hydroxyvitamin D levels were not reduced, levels of 1,25-dihydroxycholecalciferol were extremely low (0.52 ng per deciliter; normal 3.3 +/- 0.06, S.D.). Treatment with 1,25-dihydroxycholecalciferol (1 microgram by mouth per day for four days) increased circulating levels to normal (4.60 ng per deciliter) and restored to normal the calcemic response to parathyroid (change in calcium 3.0 mg per deciliter). These data suggest that 1,25-dihydroxycholecalciferol deficiency may explain the skeletal resistance, but not the renal resistance, often present in classic pseudohypoparathyroidism.


Iontrophoretic application of autonomic mediators and cyclic nucleotides in the sinus node cells. The effects of intracellular application of noradrenaline, acetylcholine, cAMP and cGMP were studied in the sinus-node of the rabbit. Noradrenaline and acetylcholine were without effect on electrical activity. In about one third of cases, cAMP increased, and cGMP decreased the slope of slow depolarization. These results suggest that adrenergic as well as cholinergic receptors which control the electrical activity of the sinus node cell are localized at the outer side of the cell membrane. It has also been shown that the cyclic nucleotides exert direct but opposite chronotropic effects.


alpha-Adrenoceptor-mediated inhibitory effect of the sympathetic nervous system on the isoprenaline-induced increase in plasma renin concentration. The importance of the sympatho-adrenal system for the isoprenaline-induced increase in plasma renin concentration was investigated in conscious rats Ganglionic blockade by trimethidinium (10 mg kg-1) increased the dose-dependent elevation of plasma renin concentration induced by isoprenaline (0.03-0.48 microgram kg-1 min-1). Also treatment of the rats with guanethidine (6 mg kg-1) or reserpine (2.5 mg kg-1, given 16 and 7 h prior to the experiments) further increased the effect of isoprenaline (0.5 microgram kg-1 min-1) on plasma renin concentration. Unilateral renal denervation combined with contralateral nephrectomy doubled the effect of the beta-sympathomimetic amine on renin release. The alpha-adrenoceptor antagonist phenoxy-benzamine (3 mg kg-1) also enhanced the effect of isoprenaline on this parameter. It is concluded that apart from a stimulation of renin release via beta-adrenoceptors the sympathetic nervous system may inhibit renin release via stimulation of alpha-adrenoceptors.


Effects of Sr2+ and Mg2+ on the phospholipase A and the presynaptic neuromuscular blocking actions of beta-bungarotoxin, crotoxin and taipoxin. 1.beta-Bungarotoxin, crotoxin and taipoxin, presynaptic neurotoxins of snake venom origin, have about the same phospholipid-splitting activities as a much less toxic cobra phospholipase A2 in the presence of Ca2+ and deoxycholate. 2. Sr2+ was a much less effective activator of the enzymes than is Ca2+, the activation by Sr2+ being only 3-6% for beta-bungarotoxin and crotoxin and 12% for taipoxin. 3. Sr2+ also inhibited the Ca2+ -activated enzymes by 80% in the cases of beta-bungarotoxin and crotoxin, but only 16% in the case of taipoxin. 4. Mg2" had no significant effect on beta-bungarotoxin or crotoxin, but activated taipoxin in the presence or absence of Ca2". 5. In Sr2+ -Tyrode lacking Ca2+ all three toxins exhibited the same immediate depression followed by facilitation in the rat and mouse diaphragms, but the final blocking activity was only 3-10% with beta-bungarotoxin and crotoxin and was 30% with taipoxin. 6. In Sr2+ -Tyrode, increasing in the rate of nerve stimulation had less accelerating effect on the development of neuromuscular block than in Ca2+ -Tyrode for any of the toxins. 7. Removal of Mg2+ from Sr2+ -Tyrode did not diminish the potency of taipoxin in blocking neuromuscular transmission, suggesting that enzyme activity at the outer surface of the axolemma does not contribute to the neuromuscular blocking action. 8. All of the results indicate that there are close correlations between the presynaptic activities of these toxins and their phospholipid-splitting activities in the cationic environment prevailing in the axoplasm. Apparently the final blocking effect of these toxins is due to phospholipase A action within the nerve terminal.


Adrenal cortex adenylate cyclase. In vitro modification of the enzyme by cholera toxin. Pretreatment of rat adrenal particulate fractions with cholera toxin in vitro markedly changed the properties of the membrane-bound adenylate cyclase. The basal activity of the enzyme was increased after cholera toxin treatment. The main action of the toxin was on the Vmax of the enzyme. In the absence of added GTP Lineweaver-Burk plots indicate a deviation from normal Michaelis-Menten kinetics with respect to substrate, the slopes being concave downward for control and toxin-treated membranes. Although hormonal stimulation of the enzyme was diminished in toxin-treated membranes, the hormone receptors were still functionally active as revealed after addition of Gpp(NH)p, GTP or GTPgammaS. The response to NaF was decreased in the toxin-treated membranes. Whereas GTP behaves as an antagonist (or a partial agonist with low intrinsic activity) compared to Gpp(NH)p in control membranes, it has similar intrinsic activity as Gpp(NH)p in the toxin-treated membranes. The results indicate that cholera toxin modification of the adenylate cyclase complex is located at the guanyl nucleotide sites or factors controlling the turnover of GTP at these sites. Cholera toxin modification may be a useful tool to investigate the role of guanyl nucleotide sites in the regulation of adenylate cyclase activity.


[Synaptic effects induced in lamprey motor neurons by direct stimulation of individual presynaptic fibers]. Parallel intracellular recording from giant reticulospinal axons and target motoneurons of the lamprey (Lampetra fluviatilis) revealed unitary synaptic actions produced in the post-synaptic membrane by direct stimulation of individual presynaptic fibres. It is shown that monosynaptic reticulomotoneuronal EPSPs contain both electrical and chemical components which have different synaptic delay, time course, amplitude, sensitivity to Cadeficit and operating characterisitcs. A single motoneuron may receive direct electrotonic inputs from several giant axons. Individual giant axons may affect segmental motoneurons not only via monosynaptic connections but also via additional synaptic relays.


Morphological and histochemical properties of human embryonic cells transformed by Rous and polyoma viruses. It is shown that human embryonic cells transformed by Rous sarcoma virus (stable cell line 23) and those transformed by polyoma virus (stable cell line P-2) are morphologically distinguished from the normal human embryonic cells. The mitotic activity of P-2 cells was 51% and the mitotic activity of 23 cells was 48%. While the mitosis activity of human embryo fibroblast was 28%. The duration of the mitosis of P-2 cells was 20 hours and that of 23 cells was 18 hr. The duration of the mitotic cycle of human embryo fibroblast was 18 hr. The G1 periods lasted 6 hours for both the cell lines; the S period of P-2 cells lasted 8 hr and the S period of 23 cells was 6 hr. Both the cell lines had a high content of RNA, DNA, protein bound SH-groups, and a high activity of acid phosphatase, acid RNAase and glucose-6-phosphatase. The content of glycogen, and acidic mucopolysaccharides, the activity of NADPH-tetrazolium reductase, succinic dehydrogenase of both the lines were the same as in normal human cells.


Notes on the mechanism of postvaccination immunity in Marek's disease. The investigated 16th and 45th in vitro passages of non-pathogenic variant 83 of the Kekava strain Marek's disease virus have led in chickens to resistance to Marek's disease by introduction of the above-mentioned virus 14 days before application of pathogenic variant 55 of the Kekava strain Marek's disease virus. Simultaneous administration of both variants of the Kekava strains Marek's disease virus did not protect chickens from the disease. Presence in those variants of the Kekava strain Marek's disease virus of genetic markers manifesting themselves on passaging the virus in chicken fibroblast cultures created the possibility to investigate interrelations between them in the organism of chickens, utilizing in isolation of the virus the method of infecting cultures with chicken fibroblasts. The results of isolation of the virus from the blood cells of vaccinated chickens have shown that in their organism there is interference between those virus variants since the frequency of isolation of the pathogenic virus variant was 3-times lower than that of the apathogenic Kekava strain Marek's disease virus, and both virus variants persisted in various cells. After simultaneous administration of both virus variants to chickens equal amounts of the pathogenic and of the apathogenic Kekava strain Marek's disease virus were isolated from their blood cells. In that case also persistance of both virus variants in one cell may occur.


Benzo(a)pyrene free-radicals formation in the presence of butylated hydroxyanisole and their possible importance in carcinogenesis. By the method of UV irradiation of benzopyrene in the presence of butylated hydroxyanisole, the benzopyrene EPR spectra was investigated and the possible mechanism by which the carcinogenic activity of benzopyrene in the presence of butylated hydroxyanisole is lowered, was studied.


Observations on the etiology of trigeminal neuralgia, hemifacial spasm, acoustic nerve dysfunction and glossopharyngeal neuralgia. Definitive microsurgical treatment and results in 117 patients. Microsurgical observations have been made of the cranial nerve root entry or exit zones 117 patients operated upon for the treatment of hyperactive-hypoactive dysfunction syndromes (trigeminal neuralgia, hemifacial spasm, acoustic nerve dysfunction, and glossopharyngeal neuralgia). Cross-compression or distortion of the appropriate nerve root at its entry or exit zone was noted in all patients. This compression or distortion was usually caused by normal or arteriosclerotic, elongated arterial loops, it was usually relieved by decompressive microsurgical techniques. A small percentage of patients were found to have compression of the nerve root at the entry-exit zone by a tumor, a vein, or some other structural abnormality; they were relieved by tumor excision or other measures as described. Relief was gradual postoperatively if the treated nerve was not stroked or manipulated at operation but it was immediate if the nerve was manipulated. Preoperative evidence of decreased nerve function improved postoperatively.


Effect of electrical stimulation of the canine brain stem on the secretion of ACTH and growth hormone (GH). The effects of electrical stimulation of the brain stem on ACTH and GH secretion were studied in pentobarbital-anesthetized dogs. Stimulations in or near the subcoeruleus area or ventral ascending noradrenergic pathway inhibited the stress-induced increase in ACTH secretion, while stimulations in other areas of the brain stem did not. No significant changes in GH secretion occurred following stimulation of the subcoeruleus area, ventral ascending noradrenergic pathway or adjacent regions of the canine brain stem. These results are consistent with the hypothesis that there is central noradrenergic input to the hypothalamus which can inhibit ACTH secretion. This input appears to be separate from the postulated noradrenergic input that may increase the secretion of GH.


Effect of electrical stimulation of the canine diencephalon on the secretion of ACTH, growth hormone (GH) and prolactin (P). Plasma levels of ACTH, growth hormone (GH), prolactin (Prl) and corticoids were simultaneously monitored before, during and after diencephalic stimulation in 18 pentobarbital-anesthetized dogs. Constant-current, monopolar biphasic stimulation of several regions of the canine ventral medial hypothalamus caused rapid increases in ACTH and corticoid concentrations. No increases in plasma GH were seen during stimulation, but plasma GH increased after the stimulus was turned off when the stimulating electrodes were in the arcuate-median eminence region. Plasma Prl concentration was not increased or decreased by stimulation of the ventral medial hypothalamus, but in 2 instances in 1 dog, it was increased by stimulation of the nucleus supraopticus difusus. The data indicate that different areas in the hypothalamus regulate the secretion of these 3 anterior pituitary (AP) hormones.


The use of coronal scans for computed tomography of the orbits. The use of coronal tomograms for CT scanning of the orbits is emphasized and the technic for patient positioning is described. Normal tomographic images are described and the contribution of coronal scans to a correct diagnosis is stressed in the light of 16 pathological cases. Coronal tomograms are especially valuable to show the regions of the orbital floor and roof and complement axial scanning.


Neuromuscular blockade with anti-axoplasmic antibodies. In the process of developing an immunopharmacologic method for identifying a "trophic" protein released from motor nerve terminals, a soluble fraction of peripheral nerve axoplasm was prepared. An attempt was made to eliminate contaminating myelin and basic protein. Antibodies were produced to soluble nerve proteins in all sheep immunized. On boosting, after a 6-week interval, the animals became weak, and some could not stand on the fifth day after injection. A distinct component of neuromuscular blockade was demonstrated electrically and in response to edrophonium. Because this syndrome was (1) in the broadest sense, an experimental allergic neuropathy but produced by a distinctly different antigen than has been utilized previously (the soluble nerve proteins represented the axoplasmic compartment) and (2) a clinical condition associated with a defect in neuromuscular transmission, this may represent a new and potentially important model in the study of neuromuscular and other neurologic diseases.


Conjugated estrogens and the overnight dexamethasone suppression test. Basal (8:00 AM) serum cortisol levels in peri- or postmenopausal women treated with 0.625 mg daily of conjugated equine estrogens were only slightly higher than pretreatment levels. A 1.25 mg/day dosage resulted in a significant increase in basal serum cortisol levels. In women treated with either dosage, 8:00 AM serum cortisol levels 8 to 10 hours after 1 mg of oral dexamethasone were suppressed to below 5 microgram/100 ml, although there was a significant rise in post-dexamethasone serum cortisol levels in those treated with the higher dosage. It is concluded that while replacement dosages of estrogens increase circulating total serum cortisol levels, they do not interfere with the interpretation of the overnight dexamethasone suppression test.


[Characteristics of the functional maturity of the insular apparatus of the human pancreas during intrauterine development]. The reactivity of human foetal pancreas was determined by the increase of insulin secretion in vitro in response to the effect of glucose, arginine, theophylline, cyclic AMP and somatotrophic hormone (STH). The results of the experiments have shown that the beta-cells of the islet system in the pancreas of 7-9 weeks old embryos are as yet not able to respond to the main physiological stimulus, glucose, but respond already to cAMP, STH, arginine with glucose. The glands of 10-14 weeks old foetuses are already able to react to glucose and respond to all other stimuli, except arginine. Taken for comparison, the glands of 19-22 weeks old foetuses respond to glucose by the increase of insulin secretion.


[Effect of thyroid hormones on cytochrome concentration on the liver and heart tissues of chickens during ontogenesis]. Changes in the concentration of cytochromes c, c1 and a in the liver and heart homogenates during chick pre- and postnatal development were detected by means of low temperature differential spectrometry. At the early stages of development the high content of respiratory enzymes comparable with that in the adult organism is noted. By the end of embryogenesis the content of cytochromes decreased and, subsequently, increased. The injection of thyroxine in the 16-17 days old embryos and 9-10 days old chickes resulted in more distinct changes in the content of respiratory enzymes in mitochondria of the heart, as compared with those of the liver. No changes in the content of cytochromes were recorded after the injection of 6-methyl thiouracyl in the 10 days old embryos, thus suggesting the inactive state of thyroid parenchyma during this period of development.


[Isoenzyme makeup of the malate dehydrogenase in 2 species of Acetabularia]. The isozymes of malate dehydrogenase (MDH) were studied by means of electrophoresis in polyacrilamide gel in Acetabularia crenulata and A. mediterranea. The isozyme profile of MDH was shown to be variable in different parts of the plant. Distinct differences in isozyme profiles of MDH between A. crenulata and A. mediteranea were found when studying the cell fractions which consisted mainly of chloroplasts. The chloroplast fraction of A. mediterranea contained 8 isozymes which form 2 groups with different electrophoretic mobility. The chloroplast fraction of A. crenulata contained 9 isozymes. All the isozymes of the first group were common for both the species under study.


[Interrelations of cyclic adenosine monophosphate and calcium in regulating glycogen synthetase in developing skeletal and cardiac muscles in chickens]. The decrease of glycogen synthetase activity under the effect of Ca++ and cAMP is observed in the skeletal muscles of chick embryos only at the later stages of embryogenesis (15-18 days). In the cardiac muscle the activity of glycogen synthetase is inhibited by Ca++ apparently prior to the 12th day of development, long before the effect of cAMP which appears, as in the skeletal muscles, on the 18th day. Under the combined effect of cAMP and Ca++ in vitro and that of Ca++ and epinephrine in vivo, no additivity was observed. The possibility of Ca++ participation in the realization of catecholamine effect on the glycogen synthetase system of muscle and their interrelations with cAMP are discussed.


Pleomorphic adenomas of the parotid gland resembling mesenchymal tumors. Two cases of unusual pleomorphic adenoma of the parotid gland are described. Each showed marked palisaded zones. Distinction from smooth muscle tumors, neurofibromas, and other spindle-cell neoplasms is described.


Ultrastructural, electron-probe, and microhardness studies of the controversial amorphous areas in the dentin of regional odontodysplasia. The amorphous areas, which are gray in sections stained with hematoxylin and eosin and which are a prominent feature in the coronal dentin of the more severely affected cases of regional odontodysplasia, were studied ultrastructurally and with an electron probe and a Vickers microhardnesss tester. The ultrastructural findings confirmed previous histochemical evidence that the amorphous material consists of glycosaminoglycans and represents collagen-free dentinal matrix, that is, ground substance. Phosphoproteins may also present. The ultrastructural examination of nondecalcified material and electron-probe and microhardness tests supported previous microradiographic evidence that the amorphous areas are more heavily mineralized than normal dentin. The boundaries of the amorphous areas were found on ultrastructural examination to be distinct but very irregular.


Fine structure of a bronchial oncocytoma. The fine structure of a bronchial oncocytoma is described. The tumour cells are characterized by abundant, normal sized mitochondria with lamellar cristae. Membrane bound protein granules, which measure 0,1 to 0.5 micron in diameter, are also regularly found. Furthermore, all cell organelles involved in protein synthesis occur in oncocytes. The tumour cells lack basement membranes. The protein storing granules of oncocytes are comparable to peptide hormones storing granules. Positive staining with lead hematoxylin and a faint argentaffin staining suggest that oncocytes in the case studied belong to an oncocytic carcinoid. This view is further substantiated by a high serotonin content of the tumour (50.6 microgram/g wet weight).


[Cerebral gigantism (Sotos-syndrome) (author's transl)]. Case report of a nine month old girl with Cerebral Gigantism. It is a gigantism syndrome characterized by advanced bone age, a typical craniofacial dysmorphia and a not progredient mental retardation. Etiology and pathogenesis of this syndrome are unknown.


Observations on the algogenic actions of adenosine compounds on the human blister base preparation. The action of the adenyl compounds adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine was studied on the human blister base preparation. All 4 adenyl compounds produced pain which was slow in onset and not maintained. The threshold concentration for pain was of the order of 1-3 micron. The slopes of log concentration:pain intensity plots were relatively shallow and for moderate to severe pain a 100-fold increase of the threshold concentration was required. The adenyl compounds resembled 5-hydroxytryptamine and bradykinin with respect to onset and duration of action but were less potent. On the other hand, for threshold effects they were more potent than acetylcholine or potassium. Evidence was found for an interaction of adenyl compounds with 5-hydroxytryptamine but not with potassium, acetylcholine or bradykinin. Cyclic adenosine 3',5'-monophosphate or the chelation of extracellular calcium or magnesium were shown not to be involved in the algogenic action of adenyl compounds and the action of adenyl compounds on the rabbit isolated jejunum too was found to be unrelated to their algogenic action on the human blister base preparation.


Longitudinal study of calcium and bone metabolism in paraplegic patients. A longitudinal study of bone and calcium metabolism in 28 patients with spinal chord lesion shows an enhancement of bone calcium accretion, generalized to the whole skeleton. The bone calcium turnover rate is more increased in the non-paralysed area during the first 2 months.


The activity of galactose-1-phosphate uridyltransferase and galactokinase in human fetal organs. The activity of galactose-1-phosphate uridyltransferase (transferase) and galactokinase in several organs from human fetuses 7-28 weeks old was measured by using radioactive substrates and column chromatography for product identification. The specific activity of transferase and galactokinase increased with gestational age and reached, at the 28th week, a maximal level of 30.0 and 7.9 nmol/min/mg protein (units) in liver, 4.7 and  2.5 units in kidney, 6.0 and 4.0 units in lung, 6.7 and 2.9 units in spleen, 5.2 and 2.6 units in cardiac muscle, and 4.0 and 1.4 units in skeletal muscle, respectively. The activity in brain, on the other hand, remained quite constant with 1.2 units in the case of transferase and 0.5 units in the case of galactokinase during this period. The activities of both enzymes in the liver of children were slightly lower than the highest fetal level during the period of pregnancy studied. Galactokinase activity in fetal erythrocytes was approximately 4 times higher and the transferase activity approximately 30% higher than in adults. The Km value of fetal liver transferase for galactose-1-phosphate was found to be 0.330-0.357 mM and that of galactokinase for galactose, 0.265-0.277 mM.


Recent advances in pediatric oncology.

Recent advances in pediatric oncology. There have been remarkable improvements in our knowledge of how to treat children with cancer and also in the results of treatment. Much of this improvement has come from careful and systematic clinical therapeutic studies. We have learned much about the natural history and some about the biology of childhood cancer. For the future it would seem that major advances are going to come not from clinical therapeutic trials but from combined laboratory and clinical research approaches. Chemotherapy will be the main stay of treatment for the foreseeable future. Immunotherapy may play some role as our knowledge of that modality improves. Basically, however, we need much more understanding of the biology of childhood tumors to effect significant answers in this disease area.


Rapid eye movement sleep, apnea, and cardiac slowing influenced by phenobarbital administration in the neonate. Polygraphic recordings were performed in seven preterm infants who had been given phenobarbital (phenobarbitone) to evaluate its effect on neonatal sleep behavior and on the incidence of neurogenic apnea and/or bradycardia. The amount of active sleep, as well as the incidence of apnea and/or cardiac slowing occurring predominantly in active sleep, were decreased at therapeutic serum levels of phenobarbital. With declining serum drug levels, active sleep showed a rebound effect; at the same time, apnea and/or cardiac slowing relapsed. Thus, our previously proposed neurophysiologic concept that neonatal apnea is facilitated by active sleep-inhibitory brain mechanisms seems to be confirmed.


The repeating nucleotide sequence in the repetitive mitochondrial DNA from a "low-density" petite mutant of yeast. The repeating nucleotide sequence of 68 base pairs in the mtDNA from an ethidium-induced cytoplasmic petite mutant of yeast has been determined. For sequence analysis specifically primed and terminated RNA copies, obtained by in vitro transcription of the separated strands, were use. The sequence consists of 66 consecutive AT base pairs flanked by two GC pairs and comprises nearly all of the mutant mitochondrial genome. The sequence, moreover, also represents the first part of wild-type mtDNA sequence so far.


Characterization of human enzymes specific for damaged DNA: resolution of endonuclease for irradiated DNA from an apparent N-glycosidase active on alkylated DNA. An endonuclease partially purified from human lymphoblasts, and active against ultraviolet-irradiated DNA, was found to act additionally on DNA damaged by either x-radiation or methylmethanesulfonate. To determine if these activities were truly endonucleolytic, the reaction products were analyzed under conditions that prevented conversion of apurinic or apyrimidinic sites to single-strand breaks. With either ultraviolet- or x-irradiated DNA, strand breakage remained maximal, hence confirming the endonucleolytic character of the enzyme. By contrast, with DNA alkylated with methylmethanesulfonate, strand breakage was sharply reduced. Additional experiments indicated that the activity for alkylated DNA induces strand breaks only in concert with a purified endonuclease specific for apurinic sites, suggesting that it is an N-glycosidase that depurinates alkylated bases. This enzyme was separated from the endonuclease specific for irradiated DNA, by chromatography on DNA-agarose.


Influences of amino acid, ATP, pyrophosphate and tRNA on binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600. Aminoalcohol-AMP esters, structurally related to the assumed intermediates of the amino acid activation reaction, behave as competitive inhibitors both with respect to the amino acid and ATP, when tested in the ATP-(32P) PPi-exchange or the tRNA-charging reaction. However, closer investigation of the binding of norvalinyl adenylate to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 by an equilibrium method shows that only the amino acid is a true competitor, while ATP cannot displace the ester from binding. Pyrophosphate enhances the stability of the ester-enzyme complex whereas tRNA is without detectable influence.


[Respiratory diseases in poultry caused by viruses--occurrence and diagnosis (author's transl)]. The highly intensive conditions, that economic necessity has forced upon the poultry industry, have resulted in strongly changed environmental conditions and management which combined with the use of a constantly increasing number of live vaccines has highly complicated clearing up the etiology in diseased flocks. This is true not least as far as respiratory diseases concerns, which thereby often run an atypical course. A review, however, not complete, is given of the occurrence and diagnostic procedures in respiratory diseases caused by viruses, the greatest importance attached to infections caused by adeno- and infectious bronchitis virus.


Restoration by T4 ligase of DNA sequences sensitive to "flush" cleaving restriction enzyme. Fouteen "flush"-ended segments originate from the action of the restriction endonuclease Hae III of Haemophilus aegiptius on the DNA of the colicinogenic factor ColE 1 (A. Oka and M. Takanami, Nature, 264, 191, 1976). They are joined by the T4 polynucleotide ligase. The reaction can be monitored by gel electrophoresis, electron microscopy and resistance to phosphatase of the 5'-32P labelled ends. The joined products are a random recombination of the original segments, and can be cleaved by the same Hae III endonuclease to restore the exact electrophoretic pattern of the Hae III-cut ColE 1 DNA. In a properly diluted mixture of 5'-32P segments treated with T4 ligase, the level of phosphatase resistance is very close to the frequency of circle-formation as determined by electron microscopy: thus, the joining of the "flush"-ends involves the formation of circular structures covalently closed in both strands.


Natural occurence of a biopolymer, poly (adenosine diphosphate ribose). Evidence for the natural occurrence of poly(adenosine diphosphate ribose) in vivo was obtained using a sensitive radioimmunoassay and poly(adenosine diphosphate ribose) glycohydrolase, which specifically hydrolyzes poly(adenosine diphosphate ribose). Calf thymus, liver, kidney, brain, pancreas and spleen contained poly(adenosine diphosphate ribose). Naturally occurring poly(adenosine diphosphate ribose) in calf thymus is composed of molecules of various chain lengths, like that synthesized by an in vitro system. Calf thymus was estimated to contain about 0.02 microgram/mg DNA of poly(adenosine diphosphate ribose).


Nucleotide sequence of the gene for the major structural protein of SV40 virus. We have determined the sequence of the portion of Simian Virus 40 (SV40) that codes for the major structural protein of the virus. The gene contains 361 codons. Synonym codons for an amino acid are not used randomly. The dinucleotide CG occurs only once and there is 2 to 1 preference for uridylic acid in the third position of codons.


Comparison of the nucleotide sequence of the messenger RNA for the major structural protein of SV40 with the DNA sequence encoding the amino acids of the protein. The 16S late mRNA from SV40 directs the synthesis of the major viral structural protein, VP1. We have compared the oligonucleotides in the 16S mRNA with those that would be present in a transcript of the portion of SV40 DNA coding for VP1. The results indicate that a segment of about 200 nucleotides of RNA transcribed from a distant part of SV40 DNA have become linked to the transcript of VP1 codons by a bond resistant to phenol extraction and denaturation in formamide.


Nonuniform distribution of DNA repair in chromatin after treatment with methyl methanesulfonate. The distribution of methyl methanesulfonate induced DNA repair was measured in mouse mammary cell chromatin by digestion of "repair labeled" nuclei with micrococcal nuclease. The results indicate that there is a nonuniform distribution of DNA repair in chromatin. The chromatin fraction digested during the first 5 minutes of incubation with micrococcal nuclease appears to be a primary site of DNA repair after methyl methanesulfoante treatment. The observed nonuniform distribution of DNA repair in chromatin may be due to 1)a nonrandom alkylation of DNA in chromatin by methyl methanesulfonate or 2)areas in chromatin of increased accessibility for the repair enzymes to the DNA lesions.


Methylated constituents of Aedes albopictus poly (A)-containing messenger RNA. Poly (A)-containing mRNA prepared from cultured mosquito (Aedes albopictus) cells was found to contain methylated 5'-terminal "caps" as well as internal m6A residues. Both type I [m7G(5')ppp(5')Xmp] and type II [m7G(5')ppp(5')XmpYmp] caps were present, at molar ratio of ca five to one. All four common RNA bases were represented in the second position (Xm) of the caps, adenine being the most abundant and N6-methyladenine being absent. The four bases were also represented in the third position (Ym), but here uracil was the predominant base. There was approximately one internal m6A residue for every three caps. These studies demonstrate that mRNA from an invertebrate source can have a methylation pattern comparable with that of mammalian cells in it complexity.


Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences. A method of enriching for long double-stranded segments of eukaryotic DNA carrying particular genes is described. A purified RNA coded for by the gene is covalently attached to biotin via the protein, cytochrome c. This modified RNA is hybridized to total nuclear, double-stranded DNA under conditions that allow the formation of R-loops. Avidin, which has a high affinity for biotin, is covalently attached to polymer spheres. The complexes of avidin-spheres with DNA:RNA-biotin R-loop hybrids band in CsCl at a much lower bouyant density than does free DNA. This density is a function of the length of DNA coupled per avidin-sphere. This method was used to prepare very long double-strands of DNA highly enriched in the coding sequences for the large rRNAs of D. melanogaster and L. donovani and the histone mRNAs of S. purpuratus.


DNA synthesis by partially purified replicating simian virus 40 chromosomes. We have partially purified replicating simian virus 40 (SV40) chromosomes in a form which allows continued DNA synthesis in vitro. We first prepare a soluble DNA-synthesizing system from SV40-infected monkey cells and then sediment the components through a neutral sucrose gradient of extremely low ionic strength. Replicating SV40 chromosomes isolated from such gradients are capable of continuing DNA synthesis in vitro in the same manner as two crude subnuclear systems we have previously described (4). This indicates that the enzymes and other proteins required for in vitro DNA synthesis are bound to the replicating chromosomes.


The preparation of silica gel thin layers with a new apparatus. An apparatus for the preparation of uniform thin layer chromatography gel layers is described. The apparatus employs rubber cushion runners to compensate for differences in plate thickness and a gel applicator which functions independently of plate edge variability. Consistently uniform layers are prepared by careful establishment of the plate-to-applicator distance. Silica gel layers averaged 87% of the applied thickness with a variability of +/-2% within a single run and +/-8% between independent runs.


[Hormonal regulation of lipoate-35S transport into rat liver mitochondria]. The effect of adrenalectomy, and also of adrenaline, hydrocortisone and ACTH on the rate of lipoate-35S penetration into the mitochondria of the liver was studied in rats. Bilateral adrenalectomy proved to decrease the rate of the lipoate penetration into the organellae significantly; this was more pronounced in female than in the male rats. The capacity of hepatic mitochondria to absorp the lipoic acid was restored under the effect of single adrenaline, hydrocortisone and ACTH injections. Actinomycin D administered to the animals for a period of 3 days in combination with hydrocortisone eliminated the stimulating effect of the hormone on the lipoate transport into the mitochondria of the liver of the intact and adrenalectomized rats completely. The data obtained pointed to the regulating effect of some hormones on the intensity of penetration of lipoate into the biological structures.


[Mechanism of hypoglycemia in insulinoma]. A study was made of the capacity of insulinoma to catalyze the splitting of hippuryl-L-arginine (HA) and the contents of proinsulin-like component in the tissues of the tumours and in the blood serum of the patients. As revealed, in the absence of HA splitting by the tumour cytoplasmic fraction in the neutral pH zone there was noted a higher proinsulin-like component both in the tumour tissue and in the blood serum. An increase amount of proinsulin-like component in the blood serum stipulates possibly a more prolonged period of starvation before the occurrence of hypoglycemia, and a less pronounced picture of hypoglycemia in such patients in comparison with the patients whose tumours were capable of splitting HA similarly to the normal islands of Langerhans.


[Disorder of the activity of the cardiovascular system in obesity in children and adolescents]. The author describes the results of study carried out in 171 children aged from 6 to 14 years suffering from adiposity of the constitutional-exogenous and neuro-endocrine form. A complicated character of hemodynamic dicturbances in adiposity (intensified cardiac activity, hyperdynamia replaced by the hypodynamia of the myocardium syndrome in marked degrees of adiposity, incoordination between the central and the peripheral circulation links is noted. All this points to the early involvement of the heart and the vessels in the pathological process.


[Mineralocorticoid and glucocorticoid functions of the adrenal cortex and some mechanisms of their regulation in obesity]. A study was made of 77 patients with adiposity and a group of healthy persons with normal weight. Patients with adiposity had a significant increase of blood plasma aldosterone level; of decisive role in the formation of secondary hyperaldosteronism was played by the elevation of blood renin level. Apparently activation of the renin-angiotensin system and the associated intensification of mineralocorticoid function of the adrenal glands served as one of the causes of development of arterial hypertension in persons with excessive weight. In adiposity there proved to be an increased rate of cortizol secretion and of 17-OCS excretion with the urine; as to the blood plasma cortizol concentration, it failed to show any significant difference from the normal. Apparently stable blood cortizol level in the patients with adiposity was maintained as a result of intensified ACTH secretion; in these patients reserve possibilities of the adrenocorticosteroid function of the hypophysis were diminished.


[Correlation of hydrocortisone and corticosterone content in assessing the functional state of the adrenal cortex in varying forms of hypercorticism]. Separate determination of hydrocortisone and corticosterone in the blood of patients with the hypercorticism symptom-complex showed that elevation of the corticosteroid concentration could occur on account of similar elevation of the concentration of both hormones, or one of the hormones alone. Analogous data were observed in patients after ACTH administration. Dexamethasone administration caused a reduction of the hydrocortisone and corticosterone concentration in patients with Itsenko-Cushing's disease, but to a lesser degree than in healthy individuals. Administration of the preparation to the patients with juvenile dyspituitrism decreased the cortizol level, but corticosterone chiefly; as to the patients with the hypothalamic syndrome--only the cortizol level was reduced, the level of corticosterone remaining unchanged. Thus, the evidence obtained before and after the administration of the ACTH preparations and dexamethasone permitted to assess the adrenal cortex function more fully and to defect disturbances of the corticosteroid secretion of interest of the understanding of the clinical symptoms of the disease.


[Effect of hypophyseal somatotropic, adrenocorticotropic and thyrotropic hormones on the protein component level in the aortal tissue of hypophysectomized rats]. The authors investigated changes in the content of collagen, noncollagen proteins, total glycoproteins, glycosaminoglycuronans in the aortic tissue of hypophysectomized rats with due regard for the regulatory effects of STH, ACTH and TTH. Replacement STH therapy led to an increase of oxyproline, hexuronic acids, hexoses in the aortic tissue of the hypophysectomized rats. With the action of ACTH under conditions of hypophysectomy there was a marked increase in the content of glyco- and mucoproteins and a reduction of glycosamines in the aortic tissue. The amount of glycosaminoglycuronans, collagen increased considerably under the effect of TTH in the aortic tissue of the hypophysectomized rats; as to the glyco- and mucoprotein content--in remained unchanged. ACTH, TTH and STH were administered from the 15th to the 21st day after hypophysectomy.


[Diabetic peripheral polyneuropathy].

[Diabetic peripheral polyneuropathy]. Pain, tactile, vibration and temperature sensitivity, and also electroexcitability of individual nerves were determined. Neuropathy incidence was almost the same among males and females. There was found no association between neuropathy and excessive weight of the patients and the type of treatment. The frequency of affection increased with the advance of age and was maximum (40.4%) in patients aged from 55 to 64 years. The frequency of neuropathy proved to depend on the duration of diabetes. Sensitivity was chiefly distributed by distal and polyneuritic type. Affection of the lower limbs occurred in all the cases. Improvement after the treatment was noted in 69.3% of patients; however, in 13.5% of cases only it was objective in character. The irritative-pain syndrome was the one which yielded to the treatment best. Analysis of the results demonstrated the treatment to be more affective in younger persons with a lesser duration and severity of diabetes. The duration of partial remission constituted from 3 to 13 months. This pointed to the necessity of prophylactic therapeutic courses.


[Diagnosis of virilizing ovarian tumors and surgical tactics for their treatment]. The authors present the results of study of the basic blood testosterone level and of the 17-ketosteroid urinary excretion under conditions of a test with dexamethasone and choriongonin. The results obtained were compared with the results of analogous examination of a group of patients with the virile form of the Stein-Loewenthal's syndrome. Blood testosterone level served as a significant criterion in the diagnosis of virilizing tumours of the testes. Besides, a study was made of the remote results of sparing surgical tactics in the treatment of virilizing tumours of the testes lasting up to 13 years in 37 patients. Favourable results pointed to the benign course of these tumours and justified conservative therapy.


Oxygen "pulsed" cytochrome c oxidase: functional properties and catalytic relevance. The kinetics of the reaction of cytochrome c with solubilized mammalian cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) has been studied by a stopped-flow technique under two different experimental situations: (i) the completely oxidized enzyme (resting oxidase as obtained from the preparation) was mixed with reduced cytochrome c, and (ii) the completely reduced enzyme in the presence of reduced cytochrome c was exposed to a "pulse" of O2 (pulsed oxidase). Both sets of experiments were performed with either "limiting" or "excess" O2 (relative to oxidase), in the presence or absence of CO. Both the pre-steady-state events and the steady-state kinetics of cytochrome oxidase are found to be different in the two cases. This shows that the product of the reaction of fully reduced oxidase with O2 (pulsed oxidase) is functionally different from the oxidase as prepared (resting oxidase). These differences are interpreted with the assumption of a different rate of intramolecular electron transfer in the pulsed and resting oxidases. Implications of these experimental findings are discussed in the general framework of a tentative model for the catalytic cycle of the oxidase.


Analysis of polyoma virus nuclear RNA by mini-blot hybridization. The size and sequence composition of virus-specific RNA extracted from the nuclei of mouse cells late during polyoma virus productive infection were studied by blot-hybridization analysis of 32P-labeled RNA fractionated on CH3HgOH/agarose gels. Viral RNA molecules between approximately 0.4 and 4 times the length of a complete transcript of the 5.4-kilobase circular viral DNA were found. Less than 20% of such molecules were polyadenylylated. Although viral RNA of all sizes contained species that together hybridized to the entire polyoma genome, sequences complementary to the late region were more abundant than sequences complementary to the early region in transcripts less than 10-12 kilobases long.


[Effect of 5alpha-androstan-3beta, 17beta-diol on serum lipids and lipoproteins and the development of experimental atherosclerosis]. The author studied the influence of testosterone metabolite--5alpha-androstan-3beta,17beta-diol--on the development of hyperlipidemia and atherosclerosis in chinchilla rabbits given cholesterol-free semisynthetic atherogenic diet. The metabolite under study inhibited the development of hypercholesterolemia and hyperbetalipoproteinemia and decreased blood phospholipid content in the blood serum of experimental animals below the initial level. Lipid content in the sum total fraction of pre-beta and beta-lipoproteins decreased under the effect of the mentioned metabolite; there was also a fall in the amount of lipoproteins of low density and their greater saturation with cholesterol. Development of experimental atherosclerosis was intensified.


Enzyme associations in T4 phage DNA precursor synthesis. A DIRECT APPROACH IS DESCRIBED TO THE QUESTION: Are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation analysis of several T4 phage-coded early enzyme activities in crude lysates of infected Escherichia coli. One-third to one-half of several activities tested-dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5'-monophosphate kinase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase-sedimented much more rapidly than expected from molecular weight. About 5% of the host cell nucleoside diphosphate kinase, known to participate in T4 DNA precursor synthesis, cosedimented with these activities. To show that this rapidly sedimenting material represents an organized enzyme complex rather than a nonspecific aggregate, we studied the kinetics of formation of dTTP with dUMP as the initial substrate. This three-step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, whereas an equivalent mixture of uncomplexed enzymes required nearly 20 min before dTTP synthesis reached its maximal rate. The effect of aggregation is evidently to decrease the volume into which intermediates are free to diffuse. Because there is reason to believe that intracellular concentration gradients of DNA precursors exist, the properties of this enzyme aggregate in vitro may help to explain how such gradients are maintained.


Stimulation of RNA synthesis in isolated nuclei by partially purified preparations of simian virus 40 T-antigen. T-Antigen was partially purified from nuclei of cells transformed by simian virus 40 (SV 40). When nuclei isolated from either rat liver or quiescent hamster cells were preincubated with T-antigen preparations, there was a marked stimulation of RNA synthesis in an in vitro assay, up to 150% above control levels. The stimulation of RNA synthesis was inhibited by hamster antiserum against T-antigen but not by normal hamster serum. When the T-antigen preparations were fractionated on glycerol gradients, the fractions containing complement-fixing activity with antiserum to T-antigen also had the highest stimulatory activity on nuclear RNA synthesis. T-Antigen was also partially purified from nuclei of cells transformed by a temperature-sensitive A mutant of SV40. When preincubated up to 2 hr at 50 degrees, the T-antigen preparation from these temperature-sensitive A mutants was rapidly inactivated, in terms of both complement-fixing activity and ability to stimulate RNA synthesis in isolated rat liver nuclei. Under the same conditions of preincubation, T-antigen preparations from cells transformed by wild-type SV40 maintained their complement-fixing activity and ability to stimulate RNA synthesis. These results suggest that the biological action of T-antigen may be exerted at the level of transcription.


Nucleotide phosphotransferase of Escherichia coli: purification by affinity chromatography. Improved extraction and purification procedures permit the isolation from Escherichia coli W cells of much larger quantities and of more highly purified preparations of nucleotide phosphotransferase. Of various affinity resins tested for efficiency of purification, columns of agarose/5'-AMP (AGAMP), type 3, proved the best. In this way a 300- to 450-fold purification of the enzyme was achieved in a few steps. The enzyme, which, as reported before, transfers organically bound phosphate to the 2' or 3' hydroxyls of nucleosides and nucleotides, was tested in its behavior toward a series of ribonucleosidonucleotides, namely, CpC, ApA, CpA, and ApC. All were phosphate acceptors, but a detailed comparative study of adenosine and cytidine, 5'-AMP and 5'-CMP, and ApA and ApC revealed peculiar specificities in the relative distribution of the phosphorylated products.


[Functional activity of the adrenal cortex and thymus of mice of strains AKR, C57BL/6, C3H/He and C3Hf]. The authors studied the relationship between the functional activity of the adrenal cortex before and after the ACTH administration and the thymus mass in AKR, C57BL/6, C3H/He, and C3Hf mice. Blood 11-OCS level of these mice proved to differ in accordance with the differences in the 11-OCS production by the adrenal glands. There was revealed no expected inverse correlative dependence between the basal blood glucocorticoid content and the thymus mass. After ACTH administration the mouse species could be placed thus in order of increasing reaction of the thymus: AKR less than C3Hf less than C3H/He less than C57BL/6; this correlated positively with the level of 11-OCS production by the adrenal glands and basal 11-OCS concentration in the peripheral blood.


Frog oocytes synthesize and completely process the precursor polypeptide to virion structural proteins after microinjection of avian myeloblastosis virus RNA. After microinjection of Xenopus laevis oocytes with RNA from avian myeloblastosis virus, viral structural proteins p27, p19, p15, and p12 are formed by a sequence of posttranslational cleavages of a high-molecular-weight precursor polypeptide. The 60-70S RNA aggregate or its 30-40S RNA subunits obtained by heat or formamide treatment possess the same ability to serve as template in X. laevis oocytes. The processing pattern of virus-specific precursor polypeptides is the same in X. laevis oocytes as in chick embryo fibroblasts infected with avian myeloblastosis virus, but the processing takes place at a much slower rate.


Adenosine 3':5'-cyclic monophosphate- and guanosine 3':5'-cyclic monophosphate-dependent protein kinases: possible homologous proteins. The properties of purified mammalian adenosine 3':5'-cyclic monophosphate (cAMP)- and guanosine 3':5'-cyclic monophosphate (cGMP)-dependent protein kinases were compared. Several physical characteristics of the two enzymes were similar, including size, shape, affinity for cyclic nucleotide binding, and K(m) for ATP. In addition, the amino acid composition of the two proteins indicated a close composition homology (70-90%). Both cyclic nucleotide-dependent protein kinases catalyzed phosphorylation of rat liver pyruvate kinase (EC 2.7.1.40) and fructose 1,6-diphosphatase (EC 3.1.3.11), rabbit skeletal muscle glycogen synthase (EC 2.4.1.11) and phosphorylase b kinase (EC 2.7.1.38), and calf thymus histone H(2)b. The phosphorylation of several synthetic peptides and of trypsin-sensitive and trypsin-insensitive sites in glycogen synthase suggested similar recognition sites on the protein substrates for the two kinases. The cAMP-dependent protein kinase was the better catalyst with each protein or peptides substrate. The results suggest that the two enzymes evolved from a common ancestral protein.


Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Membrane assembly was observed to proceed in cell-free extracts. Specifically, the membrane glycoprotein of vesicular stomatitis virus was synthesized in crude extracts of wheat germ in the presence of membrane vesicles derived from pancreatic endoplasmic reticulum. The resulting glycoprotein spans the lipid bilayer asymmetrically, is glycosylated, and is indistinguishable in these respects from the form of the glycoprotein found in the rough endoplasmic reticulum of virus-infected cells. Both glycosylation and asymmetric transmembrane insertion of the glycoprotein into membranes in vitro require protein synthesis in the presence of membranes. The carboxyl-terminal 5% of the polypeptide chain is located on the external surface of vesicles, corresponding to the cytoplasmic surface of the endoplasmic reticulum in cells. Most, or all, of the amino-terminal portion of the glycoprotein, as well as the protein-bound carbohydrate, appears to be located within the lumen of the membrane vesicles. These findings demonstrate that insertion of this membrane protein occurs during or immediately after protein synthesis. The results are consistent with the concepts that the growing membrane protein is extruded across the endoplasmic reticulum membrane amino terminus first and that glycosylation is restricted to the lumenal surface of the membrane. The cell-free system reported here should prove valuable for studying these processes.


Glucocorticoid effect on the level of corticotropin messenger RNA activity in rat pituitary. In an attempt to understand the molecular mechanism underlying the depressive effect of glucocorticoids on corticotropin production, the level of corticotropin messenger RNA activity in rat pituitaries was measured with the use of the cell-free protein-synthesizing system derived from wheat germ. The large translation product of corticotropin messenger RNA was identified and quantitated by indirect immunoprecipitation with antibody against corticotropin. The level of corticotropin messenger RNA activity was increased 3- to 6-fold by adrenalectomy. Dexamethasone administration to adrenalectomized rats resulted in a marked suppression of corticotropin messenger RNA activity. Cortisol and corticosterone also exhibited a suppressive effect but were less effective than dexamethasone. In contrast, nonglucocorticoids such as progesterone and aldosterone had no suppressive effect. These results indicate that at least part of the glucocorticoid effect on corticotropin production in the pituitary is exerted at the pretranslational level.


In vitro translation yields a possible Rous sarcoma virus src gene product. In vitro translation of Rous sarcoma virus (RSV) virion RNA in the messenger-dependent reticulocyte lysate system yielded polypeptides that were not synthesized by translation of RNA from a transformation-defective deletion mutant of RSV. These RSV-specific products migrated on sodium dodecyl sulfate/polyacrylamide gels as two doublets of approximately 25,000 and 17,000 daltons. Synthesis of these proteins was not sensitive to inhibition by m7GTP; however, synthesis of the 76,000-dalton precursor of the internal structural proteins was sensitive to inhibition by m7GTP. Tryptic peptide maps showed the 25,000- and 17,000-dalton proteins to be related to one another but to be distinct from the 76,000-dalton protein. The 25,000-dalton protein was translated only from a polyadenylylated RNA of approximately 2500 nucleotides, whereas the 76,000-dalton protein was translated from 38S RNA, corresponding to the entire viral genome. A 180,000-dalton protein was also synthesized from 38S RSV virion RNA. From the absence of the 25,000- and 17,000-dalton proteins in the translation products of transformation-defective RSV RNA and the size of their RNA templates, we conclude that these proteins may be derived from coding sequences within the RSV src gene.


Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Treatment of turkey erthrocyte membranes with cholera toxin caused an enhancement of the basal and catecholamine-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activities. Both of these activities required the presence of GTP. The toxin effect on the adenylate cyclase activity concided with an inhibition of the catecholamine-stimulated guanosinetriphosphatase activity. Inhibition of the guanosinetriphosphatase, as well as enhancement of the adenylate cyclase activity, showed the same dependence on cholera toxin concentrations, and the effect of the toxin on both activities was dependent on the presence of NAD. It is proposed that continuous GTP hydrolysis at the regulatory guanyl nucleotide site is an essential turn-off mechanism, terminating activation of the adenylate cyclase. Cholera toxin inhibits the turn-off guanosinetriphosphatase reaction and thereby causes activation of the adenylate cyclase. According to this mechanism GTP should activate the toxin-treated preparation of adenylate cyclase, as does the hydrolysis-resistant analog guanosine 5'-(beta,gamma-immino)triphosphate [Gpp(NH)p]. Indeed, the toxin-treated adenylate cyclase was maximally activated, in the presence of isoproternol, by either GTP or Gpp(NH)p, while adenylate cyclase not treated with toxin was stimulated by hormone plus GTP to only one-fifth of the activity achieved with hormone plus Gpp(NH)p. Furthermore, the toxin-treated adenylate cyclase activated by isoproterenol plus GTP remained active for and extended period (half-time of 3 min) upon subsequent addition of the beta-adrenergic blocker, propranolol. The native enzyme, however, was refractory to propranolol only if activated by Gpp(NH)p but not by GTP.


Mechanism of translational control by hemin in reticulocyte lysates. The formation of translational inhibitor (active eIF-2 kinase) from proinhibitor (inactive eIF-2 kinase) in reticulocyte lysates, known to be controlled by hemin, can, as we recently reported, be induced by 3':5'-cyclic AMP(cAMP)-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) or its catalytic subunit. We find that in crude preparations from rabbit reticulocyte lysates, hemin inhibits the conversion of proinhibitor to inhibitor catalyzed by endogenous cAMP-dependent protein kinase upon addition of cAMP, but not that caused by the addition of free protein kinase catalytic subunit. Hemin prevents the binding of cAMP to the regulatory subunit of cAMP-dependent protein kinase and blocks the cAMP-induced dissociation of regulatory and catalytic subunits of the enzyme whereby the enzyme is inactivated. The mechanism by which hemin prevents the formation of the inhibitor and maintains protein synthesis in reticulocyte lysates is thus explained.


Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts. Transformation of chick embryo fibroblasts by Rous sarcoma virus results in decreased amounts of a major cell surface protein and of collagen. To determine the mechanism accounting for the decreased production of these proteins, we have measured the relative amounts of functional mRNAs for these and other transformation-sensitive proteins. Total cellular RNAs extracted from normal cells and from cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus were translated in a cell-free system derived from wheat germ. Analysis of the in vitro translation products of RNAs from normal and transformed chick embryo fibroblasts shows a 5-fold reduction in the translatable mRNA for cell surface protein and a 10-fold reduction in translatable mRNA for two collagen precursors. In addition, increases in functional mRNA are observed for myosin and for two membrane polypeptides with molecular weights of 95,000 and 78,000; the latter two proteins increase on transformation, but the increases are in large part secondary to the depletion of glucose from the medium of transformed cells. Our data suggest that some of the major cellular changes induced by oncogenic viruses are due to changes in the activity of specific cellular genes.


Effects of colchicine on cyclic AMP levels in human leukocytes. The increase in human leukocyte adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels seen in response to various substances was markedly potentiated by colchicine and other agents that affect microtubule assembly. Addition of dl-isoproterenol (2 muM) or prostaglandin E(1) (10 muM), together with the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM), caused a much greater increase in cyclic AMP in colchicine-pretreated cells than in control cells. With isoproterenol (2 muM) plus isobutylmethylaxanthine (1 mM), cyclic AMP levels rose about 3-fold but, in combination with colchicine, these drugs caused a more than 15-fold increase in cyclic AMP. The effects of colchicine were both time- and dose-dependent; they could be seen within 1 min after addition of colchicine or at concentrations as low as 10 nM. In addition to its potentiation of hormonally induced increases in cyclic AMP levels, colchicine also potentiated the effect of isobutylmethylxanthine alone on leukocyte cyclic AMP levels. Vinblastine, vincristine, podophyllotoxin, and oncodazole all had effects similar to those of colchicine but lumicolchicine did not. The data suggest that cytoplasmic microtubules interact with the leukocyte plasma membrane to impose constraints on the expression of hormone-sensitive adenylate cyclase; the therapeutic effects of colchicine may depend in part upon the relaxation of such constraints. Moreover, the synergism described here between colchicine-like agents and hormones is of potential therapeutic importance in clinical conditions in which either alkaloid or hormone has been useful separately.


Intermediate role of adenosine 3':5'-cyclic monophosphate and protein kinase during gonadotropin-induced steroidogenesis in testicular interstitial cells. Discrepancies between adenosine 3':5'-cyclic monophosphate (cAMP) and steroid production have been frequently observed in isolated target cells stimulated by low concentrations of trophic hormone. This dissociation is particularly marked in the interstitial cells of the testis, where testosterone production is elicited by gonadotropin concentrations in the picomolar range. Because of these observations, and a disparity between steroidogenesis and protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activation in Leydig cells, the role of cAMP as a mediator of the acute steroidogenic response has been questioned. This problem has been further analyzed by assay of free and occupied cAMP-binding sites of the regulatory subunit of protein kinase in basal and hormone-stimulated cells. Free sites were measured by a [(3)H]-cAMP-binding assay, and occupied sites were measured by radioimmunoassay of endogenous cAMP eluted from receptor protein. After stimulation of purified Leydig cells with 0.1-10 pM human chorionic gonadotropin, a dose-dependent decrease in available [(3)H]cAMP-binding sites was observed, with no change in binding affinity. The reduction in cAMP-binding sites was equivalent to the increase in occupancy of cAMP receptors by endogenous nucleotide formed during gonadotropin action. Fractional occupancy of cAMP receptors rose progressively from basal values of 0.2-0.40 to full saturation as intracellular cAMP rose 10- to 30-fold during hormone stimulation. The testosterone dose-response curve was coincident with the initial part of the cAMP-receptor occupancy curve. These changes in endogenous cAMP binding to the regulatory subunit were accompanied by a significant increase in protein kinase activity in gonadotropin-stimulated Leydig cells. These observations provide direct evidence for the role of cAMP and protein kinase during hormonal activation of steroidogenesis in the Leydig cell by low concentrations of gonadotropin.


Changes in surface properties of normal and transformed cells caused by tunicamycin, an inhibitor of protein glycosylation. Normal and virally transformed mouse (3T3) and human (WI-38) cells were treated with tunicamycin, an inhibitor of lipid-carrier-dependent glycosylation of proteins. Incubation of cells with tunicamycin (1 microgram/ml) caused detachment and death of simian virus 40- and polyoma-transformed cells within 24 hr; these effects were not seen with nontransformed cell lines. However, the proliferation of 3T3 cells was inhibited by tunicamycin and, after a few days, a distinct change from an epithelioid to an abnormally elongated shape was observed. Both inhibition of growth and the morphological changes were reversible. A marked decrease in concanavalin A agglutinability was observed in virally transformed cells treated with tunicamycin (0.5 microgram/ml), but agglutination by wheat germ agglutinin or soybean agglutinin was unaffected. Analysis of biosynthetically labeled proteins showed that a high-molecular-weight protein, presumed to be related to fibronectin, is markedly reduced in the medium of cells cultured in the presence of tunicamycin. These results suggest that tunicamycin interferes with the insertion or function of one or more cell-surface glycoproteins. Such cell-surface changes could affect a number of cellular properties, including attachment, cell shape, and agglutinability by some lectins.


Biological properties of poliovirus encapsulated in lipid vesicles: antibody resistance and infectivity in virus-resistant cells. We present evidence that poliovirus can be encapsulated in synthetic large phospholipid vesicles. The virus associated with the vesicles is found to be (i) resistant to antiserum against poliovirus and (ii) infectious for cells that are normally resistant to virus infection because of a membrane restriction. Our interpretation of these results is that the virus is entrapped in the interior aqueous space of the vesicles and that this vesicle-associated virus is introduced directly into the cytoplasm of the cells via fusion of the vesicles with the cellular plasma membrane, bypassing the surface receptor-mediated restriction.


Genetic analysis of host range mutant viruses suggests an uncoating defect in simian virus 40-resistant monkey cells. Host range mutations that permit simian virus 40 (SV40) to grow with increased efficiency on SV40-resistant monkey cells have been positioned within the viral B/C gene by a mapping method that relies on the coupling of specific DNA fragments. Pairs of restriction endonucleases that each cleave SV40 DNA at only one site were used to generate pairs of specific DNA fragments. Corresponding pairs of fragments were purified from host range mutant and wild-type DNA and joined in known combinations to determine the location of the host range mutations. The map position of the host range mutations was confirmed by using the same technique to generate and couple genetically marked viral DNA fragments to produce the predicted double mutants. Three different double mutants were constructed that carry both host range and temperature-sensitive A mutations. The mutations in three independently isolated host range mutant viruses are located at very close, perhaps identical, sites, because no wild type viruses were produced from the cell-mediated repair of pairwise heteroduplexes between them. The location of these host range mutations suggests that their phenotype results from mutational alteration of the major capsid protein, the product of the B/C gene. In addition it was demonstrated that monkey cells can efficiently join appropriate pairs of restriction endonuclease fragments intracellularly to produce infectious genomes. That reaction has been partially characterized. The general utility of fragment coupling (in vitro and in vivo) and heteroduplex repair for constructing and analyzing multiple mutants of SV40 is discussed.


Membrane-controlled depletion of complement activity by spin-label-specific IgM. Complement depletion mediated by high molecular weight (IgM) rabbit antibodies specifically bound to spin-label lipid haptens dispersed in model membranes is controlled by various physical attributes of those membranes other than the total number of exposed determinants that they provide. Carrier lipids used at 32 degrees were (i) a "fluid" phosphatidylcholine (PC), (ii) a "solid" PC, and (iii) a cholesterol/PC mixture. The concentration of hapten in the plane of the membranes (two-dimensional concentration) was varied while the overall hapten molarity (three-dimensional concentration) was kept constant. Both specific binding and the efficiency of depletion by IgM are markedly enhanced by systematically decreasing the average distance between haptens (infinity --> 26 A). Heterogeneous distribution was found to be more favorable than a random homogeneous distribution of the same number of haptens in the same total quantity of lipids. IgM efficiency is also markedly increased by the inclusion of cholesterol in PC membranes, an effect thought to result from enhanced projection of the determinant from the surface of the membrane and hence increased accessibility to the antibody-binding site. Furthermore, the efficiency of IgM was increased by using haptens dispersed in fluid rather than in solid PC membranes. The results are consistent with the hypothesis that IgM molecules must be bound to a critical multiple of antigenic determinants at a membrane surface in order to induce complement-mediated attack and that subtle variation of the physical state of membrane antigens can be the crucial factor in determining the outcome of this type of efferent immune response.


Immunohistochemical localization of 3':5'-cyclic AMP and 3':5'-cyclic GMP in rat renal cortex: effect of parathyroid hormone. Adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) were localized in cells of rat kidney cortex by an immunocytochemical technique before and after perfusion with parathyroid hormone (PTH). In control tissues the cAMP antiserum detected approximately the same intensity of fluorescence in cytoplasmic epithelial cell elements of cortical tubules and glomeruli (cells of Bowman's capsule and podocytes). PTH increased fluorescence in these glomerular cells and increased cAMP fluorescence in cytoplasmic granules in proximal tubular cells. These granules, whose structure has not been identified, were located predominantly on the luminal side of the tubular cells. In control rats, the renal cortical fluorescence detected with the cGMP antiserum was more pronounced in glomeruli (predominantly in the mesangial areas) and lesser amounts of fluorescence were observed in tubules. After PTH treatment, cGMP fluorescence increased in glomeruli and in renal tubular cells. A bright linear pattern of fluorescence was found in the area of the tubular luminal membrane. Perfusion with PTH caused relatively small increases in total tissue cAMP and no consistent increases in total tissue cGMP. Our observations suggest that both cAMP and cGMP are involved in the glomerular and tubular responses to PTH and point out the added dimension that this immunocytochemical technique brings to studies of cyclic nucleotide dynamics in heterogeneous tissues.


Supercoiled circular DNA of an insect granulosis virus. The DNA of the granulosis virus of the Indian meal moth, Plodia interpunctella, was characterized by physical chemical and electron microscopic techniques. Twenty-five percent of the DNA extracted from purified virus was isolated as supercoiled circular molecules. The remaining 75% consisted of relaxed circular molecules. These molecular forms were indicated by the production of two radioactive bands during sedimentation of (3)H-labeled granulosis virus DNA in alkaline sucrose gradients or in equilibrium density gradients of neutral cesium chloride/propidium iodide. Electron microscopic visualization of the DNA that banded at the higher density in the latter gradients revealed supercoiled structures whereas that of DNA that banded at the lower density demonstrated relaxed circular molecules. The superhelical molecules were converted to relaxed circles by treatment with pancreatic DNase. The molecular weight of the viral DNA was calculated to be 81 x 10(6) by sedimentation in neutral sucrose and 78 x 10(6) by sedimentation in alkaline sucrose. The molecular weight estimated from length measurements in electron micrographs was 76 x 10(6). The buoyant density of the granulosis virus DNA was 1.703 g/cm(3) and that of its insect host DNA was 1.697 g/cm(3). Equilibrium sedimentation in cesium chloride and thermal denaturation indicated G + C contents of 44% and 39% for the viral and host DNA, respectively.


Mechanisms by which diazepam, muscimol, and other drugs change the content of cGMP in cerebellar cortex. THE CEREBELLUM CONSISTS OF TWO PARTS: the cerebellar nuclei whose connections to the various parts of the central nervous system coordinate muscle movements, and the cerebellar cortex which exerts an inhibitory influence on the cerebellar nuclei through the release of gamma-aminobutyric acid (gammaAbu) from Purkinje cells. The activity of Purkinje cells is regulated by two excitatory inputs to the cerebellar cortex-the climbing and mossy fibers-and by a neuronal network within the cortex which inhibits the activity of Purkinje cells through the release of gammaAbu from interneurons. The net activity of Purkinje cells is related to their content of guanosine 3':5'-cyclic monophosphate (cGMP) which increases or decreases according to changes in the activity of climbing and mossy fibers as well as to changes in the activation of gammaAbu receptors. When these receptors are activated, the cGMP of Purkinje cells decreases; when they are inhibited, the cGMP increases.The cGMP content of the cerebellar cortex is altered by drugs that change either the excitatory input of climbing or mossy fibers or the inhibitory input mediated by the activation of gammaAbu receptors. Mechanisms by which various drugs alter the cerebellar content of cGMP were investigated. By using various experimental designs, it was shown that diazepam and muscimol lowered the cGMP content by activating gammaAbu receptors. In contrast, morphine and haloperidol lowered the cerebellar cortex cGMP by decreasing the excitation of mossy fibers whereas harmaline increased the cGMP by increasing the excitation of the climbing fibers.


31P nuclear magnetic resonance kinetic measurements on adenylatekinase. By using 31P double-resonance nucelar magnetic resonance, preliminary kinetic measurements were made on the equilibrium reaction 2ADP in equilibrium ATP +AMP catalyzed by adenylate kinase. The double-resonance method used consisted of selectively inverting the spin population in a particular chemical environment and observing the transfer of the inverted spin to another chemical environment. The chemical transfer time between AMP and ADP, free in solution, was proportional to the substrate-to-enzyme ratio, and a transfer rate of 95 mol of AMP/see per mol of enzyme was obtained. In the same series of experiments, the life-time of AMP in solution was determined from the extra broadening of its resonance line due to the active enzyme. This gave a rate 3.2 times faster than the overall transfer rate given above. From these rates and other nuclear magnetic resonance  measurements wehave calculated the individual rate constants between the ternary complexes, AMP-enzyme-ATP and ADP-enzyme-ADP. In addition, we obtained one of the ADP off-rates from this latter complex. The rates on the enzyme are approximately 1250 and 500mol/sec per mol of enzyme for the forward and reverse directions, respectively. The ADP has an off-rate of 450 sec (-1).


Coupling in cytochrome c oxidase.

Coupling in cytochrome c oxidase. Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K(+) when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome c and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid.


Nucleotide sequences of ribosome recgonition sites in messenger RNAs of vesicular stomatitis virus. The ribosome recognition sites from vesicular stomatitis virus mRNAs have been isolated by ribosome protection of the mixture of viral mRNAs followed by complete separation of the protected sites by two-dimensional gel electrophoresis. The sites from the four major mRNAs each contain a single AUG initiation codon, indicating that they are true initiation sites, and the complete sequences of the N, NS, and G mRNA sites are reported. The sites do not all share common features nor do they have significant regions complementary to the known sequence at the 3'-terminus of 18S rRNA. The N and NS mRNAs show anine-nucleotide homology in the first eleven nucleotides extending from the capped 5'-end.


Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A). A template-dependent RNA polymerase has been isolated from poliovirus-infected cells by assaying for the ability of the enzyme to copy poly(A) complexed to an oligo(U) primer. The polymerase was solubilized with detergent, and RNA was removed by precipitation with 2 M LiCl. The solubilized polymerase required both poly(A) and oligo(U) for activity and was stimulated by Mg2+ but was inhibited by Mn2+. Poly(A)-oligo(U)-dependent poly(U) polymerase was not found in extracts of HeLa cells until about 2 hr after poliovirus infection, and then there was a linear increase in activity until about 5 hr. Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes. This poly(A)-oligo(U)-dependent polymerase activity could represent an important component of the poliovirus RNA-dependent RNA polymerase.


Novel mechanism for RNA maturation: the leader sequences of simian virus 40 mRNA are not transcribed adjacent to the coding sequences. The 5'-terminal 100-200 ribonucleotides of late simian virus 40 (SV40) mRNAs are not transcribed immediately adjacent to their coding sequences. This conclusion is based on the following observations. The major late SV40 cytoplasmic RNA species, 16S and 19S, were purified from poly(A)-containing cytoplasmic RNA by hybridization to and elution from an SV40 DNA fragment that maps between 0.67 and 0.76. This fragment is remote from the DNA fragments that include the coding sequences. The RNA transcripts from the fragment located between 0.67 and 0.76 were found in abundance. Even though selected on oligo(dT)-cellulose columns, the 5'-terminal sequences did not contain poly(A) tails directly adjacent to their 3' termini. The 5' terminus of the 16S mRNA, as monitored by hybridization of the sequences adjacent to the "cap" structure, was found adjacent to the coding sequences when intact [3H]methyl-labeled RNA was hybridized with restriction fragments. However, after fragmentation, the methyl label of this same RNA hybridized with a fragment that is remote from the coding sequences and maps between 0.67 and 0.73. These results imply a novel mechanism for biosynthesis of SV40 mRNA.


Isolation of adenylate cyclase-free, beta-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography. The adenylate cyclase [ATP pyrophosphatelyase (cyclizing); EC 4.6.1.1] and beta-adrenergic receptor of plasma membranes of turkey erythrocytes were solubilized in an active form by treatment with either NaF or guanylylimidodiphosphate and digitonin. The solubilized enzyme was no longer stimulated by catecholamines, NaF, or guanine nucleotides. The digitonin extract was chromatographed on an alprenolol-agarose derivative. While the bulk of protein and all the adenylate cyclase activity passed unretarded through the column, the receptor was retained. It eluted free of enzyme activity with an alprenolol solution containing 1 M NaCl; the yield was 25-30%. The protein content of the alprenolol eluates was too low to be estimated by the Lowry technique and was assessed by a more sensitive fluorometric method. Under these conditions, the beta-adrenergic receptor was purified approximately 2000-fold in a single step with retention of all its pharmacological properties. These experiments establish that the beta-adrenergic receptor and the adenylate cyclase are independent entities which may be separated on a functional basis.


Reconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes. Membranes of mouse L cells that contain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but lack beta-adrenergic receptors have been solubilized with Lubrol 12A9. Addition of such adenylate cyclase-containing extracts to beta-adrenergic receptor-replete membranes from adenylate cyclase-deficient S49 lymphoma cells results in the production of a catecholamine-sensitive adenylate cyclase system. The effects of beta-adrenergic agonists and antagonists on the reconstituted system reproduce those that are characteristic of the wild-type S49 lymphoma cell. The uncoupled variant of the S49lymphoma contains adenylate cyclase, but donor extracts from this clone fail to reconstitute the hormone-sensitive enzyme activity when added to adenylate cyclase-deficient membranes. Thus, the uncoupled and adenylate cyclase-deficient variants of the S49 cell are not complementary.


Single-stranded DNA from oncornavirus-infected cells enriched in virus-specific DNA sequences. We previously found that a minor fraction of single-stranded DNA (ss-DNA) isolated from native nuclear DNA of normal chicken embryonic cells and cells of other species hybridized with bulk nuclear DNA or cellular RNA in great excess. At least one-third of ss-DNA belonging to the nonrepetitious part of the cell genome could be hybridized to homologous RNAs. In the present work, similar results were obtained with ss-DNA from cells of chickens infected by avian myeloblastosis virus (AMV). To investigate whether this enrichment of ss-DNA in transcribed DNA sequences involves provirus DNA, radioactive AMV RNA and cDNA copies of AMV RNA were used. Most of the 70S AMV RNA hybridized much faster to ss-DNA from productively infected leukemic cells than to bulk DNA. cDNA, either double-stranded or single-stranded, made in the presence of actinomycin D hybridized to total nuclear DNA with similar kinetics. In contrast, about half of the double-stranded cDNA molecules hybridized 40-50 times faster to ss-DNA than to total DNA, indicating that only one of the provirus DNA strands seems to be present in ss-DNA. This was confirmed by the fact that relatively insignificant amounts of the ss-cDNA molecules made in the presence of actinomycin D could be annealed to ss-DNA as compared with bulk DNA. These results indicate that actively transcribed DNA sequences can be selectively distributed in the ss-DNA fraction, probably because of single strand breaks in the vicinity of transcription sites.


High aerobic glycolysis of rat hepatoma cells in culture: role of mitochondrial hexokinase. A tumorigenic anchorage-dependent cell line (H-91) was established in culture from an azo-dye-induced rat ascites hepatoma. When grown in a glucose-containing medium the cells exhibit high rates of lactic acid production characteristic of rapidly growing tumor cells. However, when glucose is replaced with galactose the cells grow equally well but exhibit only moderately elevated rates of lactic acid production. The molecular basis for this observation cannot be attributed to differences in permeability because initial rates of glucose and galactose entry into hepatoma cells are identical. Rather, the activity of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is found to be high in hepatoma cells, about 20-fold higher than that of control and regenerating rat liver. Moreover, tumor hexokinase activity is not inhibited by low concentrations (<0.6 mM) of the reaction product glucose 6-phosphate. Additionally, 50% of the hexokinase activity of hepatoma cells is found associated with the mitochondrial fraction. This fraction is 3-fold enriched in hexokinase activity relative to the homogenate and 4-fold enriched relative to the nuclear and postmitochondrial fractions. Tumor mitochondrial hexokinase appears to be coupled directly to oxidative phosphorylation, because addition of glucose to respiring hepatoma mitochondria (after a burst of ATP synthesis) results in stimulation of respiration. In contrast, glucose has no effect on the respiration of mitochondria from control and regenerating liver. These results suggest that the high glycolytic capacity of H-91 hepatoma cells is due, at least in part, to an elevated form of hexokinase concentrated in the mitochondrial fraction of the cell.


Purification of simian virus 40 tumor antigen from a line of simian virus 40-transformed human cells. Simian virus 40 tumor antigen can be isolated in a highly purified state from the nuclei ofSV80 cells, a continuous line of simian virus 40-transformed human fibroblasts. A five-step purification method was used. Its apparent molecular weight (in sodium dodecyl sulfate/polyacrylamide gels) is approximately 90,000-94,000. It contains a detectable amino-terminal residue.


Enzyme regulation in neuroblastoma cells in a salts/glucose medium: induction of ornithine decarboxylase by asparagine and glutamine. L-Asparagine is necessary and sufficient for the maximal induction of ornithine decarboxylase (ODC) (L-ornithine carboxy-lyase, EC 4.1.1.17) activity in confluent N18 mouse neuroblastoma cells in a salts/glucose medium; L-asparagine also induces maximal ODC activity when added to a tissue culture medium. L-Glutamine is about one-half as effective as asparagine. Cholera toxin and agents that are known to raise intracellular cyclic AMP concentrations have no effect on the induction of ODC activity unless suboptimal concentrations of asparagine are present in the salts/glucose medium. Whereas actinomycin D does not inhibit induction of ODC activity by asparagine, it inhibits the induction of ODC activity in association with cyclic AMP. In the salts/glucose medium, the rate of loss of ODC activity following the inhibition of protein synthesis by cycloheximide or puromycin depends upon the presence or absence of asparagine; loss is rapid only in the absence of asparagine and does not appear to be related to the inhibition of protein synthesis. These results are discussed in the context that the overlay of the growth medium tends to mask the minimal requirements for enzyme induction, because the composition of the medium defines: (a) the requirements for the induction of ODC activity; (b) the effect, or lack of effect, of cyclic AMP (and of inducers of intracellular cyclic AMP) on the induction of ODC activity; (c) the effect, or lack of effect, of actinomycin D on the induction of ODC activity; and (d) the action of puromycin and of cycloheximide on the rate of loss of ODC activity. It will be interesting to determine whether these results are uniquely applicable to ODC, whether many of the reactions attributed to cyclic AMP in the literature may be mediated by asparagine and glutamine, and whether actinomycin D, cycloheximide, and puromycin can be relied upon to differentiate between transcriptional and post-transcriptional control.


Interaction of L-alpha-didecanoyl phosphatidylcholine with the AI polypeptide of high density lipoprotein. Cooperative interaction of L-alpha-didecanoyl phosphatidylcholine with the AI polypeptide from human serum high density lipoprotein results in a complex particle containing 2 molecules of AI and 190 molecules of the lipid. Binding occurs two orders of magnitude above the critical micelle concentration of the lipid and requires the simultaneous presence of low levels of single-chain amphiphiles. The present investigation and previous studies from this laboratory suggest that AI contains "pockets" capable of interacting with a fixed hydrophobic volume and that amphiphilic head groups do not affect the binding capacity.