Additive effects of norepinephrine and cyclic AMP on the activation of the protein kinase from adipose tissue. In this work the kinetics of activation of the cyclic AMP-dependent protein kinase by several catecholamines and ACTH, have been studied in rat epididymal fat pads and isolated fat cells. The method of Soderling and co-workers which permits the measurement of the state of activation of the protein kinase after hormonal stimulation in adipose tissue, has been used. Kinetics experiments where norepinephrine was used showed that the results obtained with isolated cells conform to the models of Sutherland and Brostrom and co-workers. Wtih intact tissue, norepinephrine not only stimulates the protein kinase activity measured without exogenous cyclic AMP but also the total activity measured in the presence of cyclic AMP (5 X 10(-6) M); thus the effect of norepinephrine, obtained during incubation of the tissue, and that of cyclic AMP, added to the soluble fraction after incubation, were additive. This effect seems to be of the beta type because it is blocked completely by propranolol. A weak, additive but significant effect was also obtained with epinephrine and isoproterenol but not with ACTH. Neither cyclic GMP nor cyclic IMP seems implicated in this effect. It was shown that stroma vascular cells which are present in the fat pads are not involved. These results suggest that the effects of norepinephrine on the protein kinase of the fat pads cannot be completely explained by the model of Brostrom and colleagues.


Current concepts of somatomedin and other biologically related growth factors. Since the discovery 20 years ago that the growth-promoting effects of somatotropin are mediated through a serum factor(s), research in this area has rapidly expanded. It is the purpose of this review to bring this area of scientific endeavor into perspective. The first part of this review will deal with aspects of total serum somatomedin activity and its biologic actions and measurements in health and disease. The last part of this review will summarize some of the physical and biologic characteristics of the recently purified "somatomedin-like" substances: somatomedin A, B, and C, NSILA-S (nonsuppressible insulin-like activity-soluble in acid ethanol) and MSA (multiplication-stimulating activity).


The metabolic fate of plasma lipoproteins in normal subjects and in patients with insulin resistance and endogenous hypertriglyceridaemia. Using I131 VLDL selectively labelled in the B-apoprotein and I125 LDL injected simultaneously into the patient we have derived some quantitative measures of VLDL and LDL metabolism in man. The effects of insulin resistance, associated with idiopathic hypertriglyceridaemia, adult onset diabetes and diabetic lipodystrophy on the metabolic behaviour of these molecules were also assessed. In the normal subjects 72-83% of the total daily plasma VLDL B-apoprotein flux was metabolised via a pathway which involved its ultimate conversion to plasma LDL, while 21-28% was degraded without such conversion. The amount of B-apoprotein metabolised by either of these routes was proportionate to the flux rate and the two pathways accounted for the total VLDL B-apoprotein removed from the plasma. In patients with idiopathic hypertriglyceridaemia and in the adult onset diabetics the total plasma VLDL B-apoprotein flux was higher than normal, indicating increased production of this apoprotein. On the other hand, the flux rate of plasma VLDL B-apoprotein in the patients with diabetic lipodystrophy was normal, suggesting that the increase in the circulating mass of these molecules was due to impaired clearance. In all the patients, however, the fractions of the total flux either converted to LDL or degraded were lower than normal, suggesting that insulin resistance limited the removal of this apoprotein by these pathways. The results also indicate that a fraction of the total VLDL removed from the plasma has been retained in an extravascular compartment, possibly representing VLDL molecules trapped in the vascular structures. In the control and the insulin resistant subjects the quantity of LDL apoprotein catabolised per day agreed closely with the amount derived from VLDL B-apoprotein conversion, suggesting that VLDL-B-apoprotein serves as the main source of LDL apoprotein. In patients with idiopathic hypertriglyceridaemia and in adult onset diabetics the absolute turnover rate of plasma LDL apoprotein was higher than normal, while in the lipodystrophic patients it was reduced. It is suggested that the increase in LDL turnover seen in the former groups could be an additive factor in the deposition of lipid rich material in arterial walls.


Clinical correlations of serum proinsulin-like material in islet cell tumours. To examine the possibility that the concentration of circulating proinsulin-like material (PLM) might be helpful in evaluating the therapeutic response of patients with islet cell tumours, serum levels of PLM in three patients with islet cell tumours were correlated with hypoglycaemic symptoms and plasma glucose concentrations before and after treatment. In two patients ranges of fasting PLM concentration were 0.21-0.29 and 0.91-0.93 ng/ml, respectively, before treatment. After surgical excision of their islet cell adenomas, PLM concentrations decreased to 0.06-0.09 and 0.03-0.05 ng/ml. Insulin concentrations were low preoperatively in both patients and were unchanged postoperatively. The resulting relief from hypoglycaemia was paralleled by a reduction of PLM, with no significant change in insulin. In a third patient, treatment with streptozotocin resulted in marked symptomatic improvement, a 65% reduction in PLM concentration, but no significant change in insulin levels. Relapse was associated with increasing frequency of hypoglycaemic symptoms and increasing PLM concentrations. These findings suggest that changes in the levels of serum PLM may prove to be a sensitive indicator of the response of islet cell tumours to therapy.


[Participation of the beta-receptor system in the genesis of the carotid aortic baroreceptor reflex in the dog (author's transl)]. The effects of propranolol, given by close arterial injection, on the reflex vasodilatation induced in the atropinized isolated gracilis muscle by rapid i.v. administration of norepinephrine were studied in dogs. The integrated areas of vasodilatation were significantly reduced after treatment with propranolol. Thus the conclusion was that adrenergic beta-receptors participate in the reflex vasodilatation, although how they act needs further elucidation.


Changes in lactic dehydrogenase isoenzymes after hepatic artery ligation in patients with hepatic carcinoma. Serum activities of LDH isoenzymes as well as total LDH, GOT and GPT were determined after hepatic artery ligation in five patients with primary or metastatic liver cancer. Transaminases and total LDH activities were raised after the operation showing their peaks on the first or third postoperative days. LDH2, LDH3 and LDH5 increased substantially during the first three postoperative days. These changes became nearly normalized within two weeks after hepatic artery ligation. As control the same enzymatic activities were measured in eight patients after usual laparotomies but no significant abnormalities were observed postoperatively. Thus, liberation of not only cathodic but also anodic migrating LDH isoenzymes seems to ensue possibly after acute liver damage induced by hepatic artery ligation. This study also suggests that serial determination of LDH isoenzymes as well as its total activity could be a valuable assessment for evaluating the anti-tumor effect of hepatic artery ligation.


Receptors for morphine and opioids.

Receptors for morphine and opioids. Two points concerning enzymatic systems acting on disposal of morphine are discussed, namely the multiplicity of glucuronyltransferase and the effect of nalorphine on N-demethylation of morphine. Evidences of the presence of at least two different glucuronyltransferases in microsomes of rat liver, kidney and intestine are presented. These evidences are given by the different distribution of the glucuronizing activity for morphine and rho-nitrophenol in microsomal preparations of these organs; by the different glucuronidation activity for both substrates induced by phenobarbital and 3-methylcholantrene; and finally by the chromatographic separation of fractions with enzymatic glucuronidating activity only for rho-nitrophenol or only for morphine. Nalorphine in concentrations of 1.3 X 10(-3) M and 1.3 X 10(-4) M inhibited in vitro de-N-demethylation of morphine 1.3 X 10(-3) M by the supernatant of rat liver homogenates, while in concentration of 1.3 X 10(-5) M it enhanced this reaction.


Tachyphylaxis and angiotensin receptors. Angiotensin tachyphylaxis was studied in the isolated guinea pig ileum under isometric conditions. 2. In tachyphylactic organs, the initial phasic response to angiotensin was abolished, while the tonic component of the response became faster in its onset and in its reversal (relaxation). 3. The relaxation of non-tachyphylactic organs after angiotensin washout was significantly slower than with bradykinin, des-amino-angiotensin and hexanoyl-angiotensin. This difference did not occur in tachyphylactic organs. 4. There was no correlation between production of tachyphylaxis or rate of relaxation after washout and the partition coefficient of the four peptides in an organic:aqueous solvent system. 5. It is suggested that tachyphylaxis depends on a slowly reversible alteration of a calcium translocation step in the stimulus-response coupling.


Relative responsiveness (rho): a critical analysis of a new method in receptor differentiation. The rho ratio, which measures the capability of a receptor system to induce a maximal contraction after saturation by a full agonist was analysed from the standpoint of its variability considering the values obtained either for a given receptor system in the same type of preparation or for different types of preparation. This variability was discussed on the grounds of receptor theory, as the basis for a new method in receptor differentiation.


Evidence of depolarization-induced cAMP increase in the superior cervical ganglion of several mammalian species. 1. The activation of cAMP synthesis resulting from exposure of superior cervical ganglion (SCG) tissue to high K+ solutions observed in calf has been found to occur in three other mammalian species, namely rat, cat, and rabbit. 2. The cAMP accumulation in the ganglion of calf is much less pronounced than that occurring in the other species. The enhancing effect oftheophylline on the increase of cAMP was found to be much greater in the SCG of calf than in that of other species. When veratridine was used as the depolarizing agent the species differences were found to be similar to those observed after potassium stimulation. 3. The effectiveness of several other depolarizing agents in causing an increase in the cAMP level was tested in rabbit SCG.


When drug therapy for lung disease affects the heart. The development of effective drugs for chronic lung disease, especially bronchodilator aerosols, has been a boon to patients and physicians alike, but these agents also may provoke arrhythmias. Fluorocarbon propellants, once regarded as harmless, are now known to disrupt cardiac function, sensitizing the heart to the arrhythmic effects of sympathomimetic amines. Catecholamine drugs as a group have a strong impact on heart rate and contractility. But the danger of rhythm disturbances often can be reduced by cutting the dosage or choosing a preparation with more beta 2 activity. Methylxanthines, generally safer than catecholamines, nevertheless must be used with caution and preferably alone in patients with heart and lung disease.


Differential utilization of manpower.

Differential utilization of manpower. Employers often ignore the different levels of skill among social workers and regard the non-MSW as merely a less expensive, second choice after the MSW for the same job. The author discusses a more productive allocation of work among MSWs and BSWs and describes its application in a medical setting.


Social work services in a high-risk nursery. Why should a social worker be on the team of a neonatal intensive-care nursery? Helping parents cope with the crises that arise with high-risk births is only one important reason. Ameliorating staff stress is another. Also, by following up the high-risk babies, the social worker has an opportunity to play a preventive role.


[Effects of monoamine-related compounds on the sleep-awake cycle in rabbits]. The role of 5HT and NA in the regulation of the sleep-awake cycle, especially in the triggering mechanisms of paradoxical sleep (PT), was studied in rabbits using reserpine, parachlorophenylalanine (PCPA), alpha-methyl-p-tyrosine (alpha-MT) and alpha-methyl -m-tyrosine (alpha-MMT). 1) Reserpine (0.5 mg/kg, i.v.) caused a diphasic action on the brain electrical activity in rabbits: an initial activation pattern in the EEG which continued to 2approximately3 hr, was followed gradually by a slow wave. Forty to sixty min after reserpine administration, spike waves resembling the PGO waves in cats appeared from the medial nucleus Trapezoides (Trap. m.) in rabbits. From the electrophysiological point of view, these spike waves (reserpine spike waves) were in many respects similar to those recorded during PS (TR spike waves). Later reserpine spike waves appeared almost continuously for about 8approximately12 hr during arousal and slow wave sleep stages. When the amplitude and grouping of spike waves became prominent, severe myoclonic jerky movements resembling "pseudo hallucinatory" behavior were observed. Polygrams of these periods were similar to those of PS except for abrupt arousal and orienting reactions. The onset of the typical PS episode was delayed by more than 8approximately9 hr. 2). Repeated doses of PCPA (500 mg/kg/day for 3 consecutive days, i.p.) caused only slight suppressive effect on slow wave sleep (SS), but spike waves similar to TR spike and/or reserpine spike waves developed throughout SS. When the amplitude and grouping of these spike waves were progressively increased, SS was often followed by PS. Abrupt enhancement of these spike waves as associated with "pseudo hallucinatory" behavior and aroused the animal. Usually these spike waves were not seen during arousal phase. When reserpine (0.5 mg/kg) was given i.v. to PCPA-treated rabbits, the latent period of appearance of reserpine spike waves was markedly shortened. 3) alpha-MMT, at a dose which had no effect on SS, markedly suppressed PS for a long period. These findings suggest that 5HT and NA in the lower brainstem play inhibitory roles on the triggering mechanism of PS.


[Therapy of chronic non-specific respiratory tract diseases. 2. Therapy of infection, oxygen therapy, supplementary therapy]. Based on the pathological findings and functional analysis treatment of chronic non-specific lung disease (CNSLD) consists in improvement of secretion disorders and airways obstruction as well as in antiphlogistic treatment. The agents and drugs for therapy of pulmonary impairment are reviewed, the efficacy, way of administration, and side effects are outlined. Drug therapy is complemented by digitalis, oxygen administration, physical measures, and causative therapy. With these complex therapeutic efforts an effective therapy of chronic non-specific lung diseases is possible.


[Metabolism of nicotinic acid in plant cell suspension cultures, III: Formation and metabolism of trigonelline (author's transl)]. Cell suspension cultures of Phaseolus aureus, Glycinemax., Cicer arietinum and Chenopodium rubrum convert nicotinic acid and nicotinamide into N-methyl nicotinic acid (trigonelline). Application of [carboxyl-14C]- and [N-methyl-14C]nicotinic acid to cell cultures demonstrated that 1) the nicotinic acid moiety of trigonelline is funnelled into the pyridine nucleotide cycle, 2) trigonelline is demethylated partly oxidatively, but predominantly non-oxidatively, transferring the methyl carbon atom to still unknown acceptors, and 3) uptake of trigonelline by mung bean cell cultures is accompanied by demethylation and instantaneous remethylation reactions. Cell suspension cultures of parsley (Petroselinum hortense Hoffm.) show uptake but no metabolism of trigonelline. The data are compared with trigonelline metabolism in intact plants.


[Metabolism of nicotinic acid in plant cell suspension cultures, IV: Occurrence and metabolism of nicotinic acid N-alpha-arabinoside (author's transl)]. Application of nicotinic acid to cell suspension cultures of Petroselinum hortense Hoffm., Daucus carota, Nicotiana tabacum and Nicotiana glauca leads to the formation of the recently isolated[2] nicotinic acid N-alpha-L-arabinoside. In these cell cultures the arabinoside is a metabolically active compound; the nicotinic acid moiety is used for NAD synthesis and nicotinic acid degradation involving decarboxylation and ring fission. N-Methylnicotinic acid (trigonelline) and nicotinic acid N-alpha-L-arabinoside occur alternatively in plant cell suspension cultures, but seem to fulfil the same function as a reserve form for nicotinic acid. Catabolism of nicotinic acid in parsley cell suspension cultures does not involve 6-hydroxynicotinic acid as an intermediate.


[Studies on cytochrome c oxidase, I. Purification and characterization of bovine myocardial enzyme and identification of peptide chains in the complex]. As part of the preliminary work for the structural elucidation of cytochrome c oxidase, the enzyme complex was isolated from bovine heart muscle and characterised chemically. The enzyme contains 10-11 nmol haem a, and 12-13 nmol copper per mg protein. The solubilised active enzyme also contains 5% phospholipid, comprising about 2 mol each of cardiolipin and phosphatidylethanolamine per mol haem a. In addition, the preparation contains a small number of detergent molecules (Tween-80). Eight polypeptide components were isolated by preparative dodecylsulphate gel electrophoresis, gel filtration on Biogel P-60, and counter current distribution. The apparent molecular weights of these components were I - 36 000, II - 28 000 (21 000), III - 19 000, IV - 14 000, V - 12 500, VI - 11 000, VII - 10 000 and VIII - 6000. At least seven intact polypeptide chains contribute to the structure of the enzyme complex of the terminal oxidase. On the basis of amino acid analysis and end group determination, they can be divided into two groups. The high molecular weight peptides I -III are hydrophobic and their amino acid compositions differ markedly from those of known enzyme proteins, especially with respect to their contents of leucine and methionine. Components I and II have formyl methionine at their N-termini. They are therefore possibly mitochondrial membrane components from complex 4 of the respiratory chain. Polypeptides IV - VII resemble functional enzyme subunits in their amino acid composition. Some of them possess free N-termini (alanine). The low molecular weight component VIII is heterogeneous and contains the N-terminal amino acids isoleucine, serine and phenylelanine in non-stoichiometric amounts. Analysis gives a minimal protein molecular weight of 130 000 (65 000 per haem a) for the two haem and two copper-containing "monomers". The molecular weight of the moiety preliminarily defined as enzymatic is about 48 000. The chemical characterisation provides data for the strategy of the subsequent sequence analysis of the polypeptides.


Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli. The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.


Legal issues in the treatment of adolescent psychiatric inpatients. When planning a specialized adolescent treatment unit for a regional state mental hospital in North Carolina, staff of the child psychiatry training program explored several critical legal issues surrounding the admission, treatment, and discharge of minors, as well as issues of confidentiality. Through a question-and-answer format they conclude that, to help avoid conditions that could result in successful litigation against the hospital, parents, adolescents, and staff must have a documented, mutual agreement to and understanding of the techniques, procedures, and limitations of the treatment program. Such documentation could be provided through a signed treatment contract.


Primary hyperlipoproteinaemia in a Danish family. A Danish family consisting of 80 persons, 72 between the ages of 3 and 70 years, was examined for the presence of primary hyperlipoproteinaemia (HLP), resulting in the finding of types II A (8 cases), II B (4 cases), III (2 cases), IV (5 cases), and V (2 cases). The pedigree of the family is presented together with lipid data and HLP type. Five cases of the so-called 'sinking pre-beta midband', which can often be correlated to ischaemic heart disease, have been found using polyacrylamide gel electrophoresis (PGE); two of these subjects had arteriosclerotic disease, one had uratic arthritis, while the other two were normal. A comparative evaluation of the results of electrophoresis should be considered of greater importance when studying families, rather than individual persons, inasmuch as intra-familial lipid patterns appear to occur with a positive correlation to at least serum cholesterol. Continued observation will probably reveal a lipid abnormality characteristic for the family even though according to the reference material available it must be considered as normal.


C1 and human platelets. III. Role of C1 subcomponents in platelet aggregation induced by aggregated IgG. Studies have been performed with platelets using C1 haemolytic assays and platelet aggregation induced by anti-C1q, anti-C1s and aggregated IgG in the presence of C1 subcomponents C1q, C1r and C1s. C1q was removed by EDTA or modified by collagenase from human platelets while after the same treatment C1s remained bound to the platelets. EDTA treated platelets were no longer aggregated by aggregated IgG. The addition of C1q restored the reactivity of the platelets to aggregated IgG while the addition of C1r or C1s was without effect. Furthermore, the addition of C1r or C1s to C1q inhibited the action of C1q in platelet aggregation induced by IgG.The possible association between the different C1 subcomponents and human platelets is discussed.


Mechanism of fever induction in rabbits. Three exogenous pyrogens (Escherichia coli lipopolysaccharide, synthetic double-stranded ribonucleic acid. Newcastle disease virus) were compared with respect to their mechanisms of fever induction in rabbits. All inducers stimulated the production of an endogenous pyrogen demonstrated in the blood as well as prostaglandins of the E group, and of cyclic adenosine 3',5'-monophosphate in the cerebrospinal fluid. The concentrations of these compounds were elevated approximately twofold as compared to the controls. Independently of the mode of induction, the fever reaction could be prevented by pretreatment with 5 mg of cycloheximide per kg, although the three fever mediators were induced as in febrile animals. Consequently, at least one additional fever mediator that is sensitive to a 30 to 50% inhibition of protein synthesis by cycloheximide has to be postulated. The comparable reactions of the rabbits after administration of different pyrogens argues for a similar fever mechanism. In contrast to fever induction there was no stimulation of endogenous pyrogen, prostaglandins of the E group, and cyclic adenosine 3',5'-monophosphate in hyperthermia as a consequence of exposure of the animals to exogenous overheating. Furthermore, hyperthermia could not be prevented by cycloheximide.


The Syrian hamster: a reproducible model for studying changes in intestinal fluid secretion in response to enterotoxin challenge. Syrian hamsters respond in a predictable and reproducible manner to intragastric administration of purified cholera enterotoxin by intraluminal accumulation of fluid in the small bowel, cecum, and proximal colon. In the majority of animals this process is self limiting, and recovery occurs with full reabsorption of intestinal fluid by 30 to 35 h. The secretory response to 75 mug of cholera toxin has been defined, and the model was utilized to study the inhibitory effects of indomethacin, polymyxin B sulfate, glucose electrolyte solutions, and colchicine. These studies demonstrate its potential usefulness as a convenient and inexpensive technique for evaluation of pharmacological agents that might inhibit intestinal fluid secretion.


Viral spread in the presence of neutralizing antibody: mechanisms of persistence in foamy virus infection. Several viruses were categorized on the basis of their ability to spread from cell to contiguous cell and form plaques in the presence of antiviral antibody. Herpes simplex virus, cytomegalovirus, and vaccinia, measles, and foamy viruses were able to spread in the presence of neutralizing antibody, whereas coxsackievirus, encephalomyocarditis virus, vesicular stomatitis virus, mumps virus, and simian virus 5 failed to spread. A detailed study of one of these virus groups (simian foamy viruses) suggested that the ability of these viruses to spread from cell to cell in the presence of antiviral antibody, the failure of antiviral antibody and complement to lyse infected cells, and the poor induction and relative resistance of these viruses to the antiviral action of interferon contribute to the persistent nature of this infection.


Antisera to human cytomegalovirus produced in hamsters: reactivity in radioimmunoassay and other antibody assay systems. Hamsters immunized with human cytomegalovirus (CMV) concentrated and purified by polyethylene glycol precipitation and density gradient centrifugation produced antisera with high titers of specific viral antibody, and which showed no significant reactivity with human host cell components. The antisera had high titers of CMV antibody in complement fixation, indirect fluorescent-antibody (FA), and neutralization tests, but titers obtained by indirect radioimmunoassay (RIA) were markedly higher. The antisera were used to follow the development of CMV antigen in infected host cells by indirect RIA and indirect FA staining. Virus-specific antigen was first detectable by RIA at 8 h after infection, and by FA staining at 16 h; cells contained optimal amounts of antigen for RIA and FA assays at 72 to 100 h postinfection. Immune globulins from the antisera were labeled with 125I for use in direct RIA. The labeled globulins gave highly specific reactions with CMV-infected cells, including those infected with low-passage isolates, and showed no reactivity with cells infected with other human herpesviruses or certain other human viruses.


Intrauterine transmission of Sendai virus in inbred mouse strains. A study of Sendai virus infection in adult mice (2 to 3 months of age) showed that the inbred strains were more susceptible to infection than randomly bred Swiss white mice and that virus could be isolated from inbred strains for as long as 21 days postinfection. For this reason, these mouse strains (C57B1/6J [black] and C57Br [brown]) were selected for the study of intrauterine transmission of virus. The major effect of infection was a decreased weight of both embryos at 16 days of gestation and newborn mice. Virus was isolated from 17 to 20% of the embryos and at least 20 to 30% of the newborns from intravenously infected mothers. Fluorescent-antibody studies showed that the virus was widely distributed in the tissues of both embryos and newborns, including the central nervous system.


Central nervous system involvement in infectious mononucleosis with studies for Epstein-Barr virus. Neurologic complications in three girls, aged four, fourteen and fifteen years, with infectious mononucleosis are reported. All three patients had meningoencephalitis, in two of them cerebellar involvement predominated, while the third patient had cerebral involvement with paresis of cerebral nerves. The diagnosis of an Epstein-Barr virus infection was established serologiccally and in the first patient also by the detection of the Epstein-Barr virus.


[Mouse cytomegalovirus infection under cytosine arabinoside treatment]. The antiviral activity of cytosine arabinoside (ARA-C) against the mouse cytomegalovirus was investigated in animal experiments using various dosages. The substance did not exhibit any virostatic effect, but on the contrary fostered the viral infection. The virus concentration in the liver after administration of five dosages of 30 mg/kg of cytosine arabinoside was 35 times higher than that in untreated animals, and after five dosages of 45 mg/kg was 70 times higher. In addition the mortality rate for infected animals treated with cytosine arabinoside was significantly higher in relation to dosage. Cytosine arabinoside also acts as an immunosuppressor in humans. Adverse effects in cytomegalo-infection can therefore not be excluded. The use of cytosine arabinoside in therapy would thus appear to be highly questionable.


Surface glycoproteins and concanavalin-A-mediated agglutinability of clonal variants and tumour cells derived from SV40-virus-transformed mouse 3T3 cells. Cell strains isolated from an established line of SV40-transformed mouse 3T3 cells (SV3T3) showed large variations in the various parameters of transformation, viz. saturation density, serum requirement, contact inhibition of movement and of growth and Concanavalin-A-mediated agglutinability. These cell strains were studied for changes in elution profiles of fucose-labelled surface glycoproteins, using actively growing, untransformed 3T3 cells as controls. A cell strain established from a tumour arising after injection of wild-type SV3T3 cells and SV3T3 cells grown in vivo in diffusion chambers, was similarly studied. Changes in surface glycoproteins were observed only in the tumour-derived cell strain. The results suggest that alterations in surface glycoproteins are associated with the tumorigenic potential of the cells rather than with the transformed phenotype per se.


Neoplastic transformation of rat embryo cells with herpes simplex virus. Sprague Dawley rat embryo cells (REF) were transformed by inoculation with herpes simplex virus (HSV) and incubation at 42 degrees C for 8 days. The infected cultures were subsequently returned to 37 degrees C and two types of cell clone were isolated from foci of growing cells after 4 weeks. One of the clones consisted of epithelial-like cells and did not produce HSV (REF-Tep-NP). The second consisted of spindle-shaped cells and cultures of these cells persistently developed small areas of degeneration where production of infectious HSV (REF-Tsp-P) took place. An additional clone which did not produce any more HSV (REF-Tsp-NP) was isolated from REF-Tsp-P in the presence of HSV-antiserum. REF-Tsp-P and REF-Tsp-NP grew more rapidly than REF and also formed foci in soft agar. REF-Tep-NP had a growth rate between that of normal rat embryo cells and that of both REF-Tsp-NP and REF-Tsp-P and did not form foci in soft agar. REF-Tsp-NP cells, in contrast to REF-Tep-NP cells, were resistant to superinfection with HSV types 1 and 2. REF-Tsp-P and REF-Tsp-NP produced metastasizing sarcomas in rats. After inoculation of 10(3) REF-Tsp-NP cells into 1-day-old rats tumours developed rapidly. REF-Tep-NP cells did not induce tumours in rats. The parental REF cells produced no tumours, even when 10(8) cells were inoculated into the rats. Positive immunofluorescence was observed in all three transformed cells only with the hyperimmune rabbit sera but not with human anti-HSV reconvalescence immune sera.


Markers to distinguish normal and neoplastic mammary epithelial cells in vitro: comparison of saturation density, morphology and concanavalin A reactivity. Normal and premalignant mouse mammary epithelial cells can be prepared in high yields by collagenase dissociation of minced glands followed by a brief, differential centrifugation to remove contaminating fibroblasts and fat cells. The major difficulties in preparing pure cultures in quantity are 1) incomplete dissociation of gland material, and 2) cell death during enzymatic digestion. These problems are eliminated by careful selection of collagenases for dissociation. Normal and premalignant mammary epithelial cells are morphologically indistinguishable from malignant mouse mammary epithelial cells in primary monolayer cultures. In addition, the growth rates and saturation densities achieved by normal mammary epithelial cells are indistinguishable from those of malignant mammary epithelial cells in primary culture. In both cases, a monolayer of cells is preserved with no evidence of focal overgrowth. Malignant adenocarcinoma mammary cells can however be distinguished from normal mammary epithelial cells by virtue of differences in their surface interactions with concanavalin A. A hemadsorption assay using Con-A-coated erythrocytes was the most sensitive indicator for these differences. In hemadsorption assays malignant mammary epithelial cells were half-maximally reactive with 2.5 mug/ml concanavalin A, while normal cells were completely unreactive even at concanavalin A concentrations five-times higher. Premalignant mammary epithelial cells were as reactive as malignant mammary epithelial cells in the hemadsorption assays. Hemadsorption of malignant cells was observed in primary and secondary cultures of epithelium as well as in cell lines. Malignant cells forming mammary adenocarcinomas were as highly reactive as malignant cells forming scirrhous carcinomas. Malignant cells not releasing mammary tumor virus (MuMTV) were as reactive as cells releasing that virus. Adsorption of concanavalin-A-coated erythrocytes to normal mammary epithelial cells could be induced by brief treatment of cell monolayers with hyaluronidase. Exposure of active sites was not affected with either trypsin or collagenase. Our results show that while the growth of malignant cells does not serve to distinguish them from normal cells in monolayer culture, surface changes do exist which can be identified by differences in concanavalin A reactivity. Since the earliest transformants identifiable in vivo (premalignant) have undergone conversion of the surface marker, concanavalin-A-mediated hemadsorption provides a sensitive measure for mammary epithelial cell transformants in vitro.


Cell surface changes observed in MC-29 virus-infected chicken-embryo fibroblast (CEC) cultures. By transmission and scanning electron microscopic studies of MC-29 virus-infected CEC cultures, C-type virus particles were detected in the medium of infected cultures, in infected cells and in intracellular vacuoles. Virus budding was also observed. In the scanning electron microscope (SEM) so-called blebs appeared on the surfaces of the infected cells, in a number proportional to the concentration of the virus preparation. On the basis of the size of the blebs, it could be considered that virus formation was taking place in their center. In control cells, neither viruses nor blebs were observed. This method will presumably be suitable for the titration of virus preparations.


Changes in cellularity induced by radiation in a solid tumour. The growth, and cellular responses of Morris hepatoma 3924 A to a locally-administered dose of 3750 R X-rays were studied using the following parameters; (1) relative tumour volume changes; (2) tritiated thymidine (3H-TdR) incorporation into DNA; (3) tumour DNA content and (4) cellular analysis, including 3H-TdR labelling index, mitotic index, aberrant mitotic frequency and relative cell density. Before depression of tumour growth, cell proliferation is temporarily interuppted. As proliferation is reinitiated, a short-lived synhcrony and prolongation of cell-cycle traverse are reflected in (a) the labelling index and mitotic index, (b) the relative cell density, and (c) the rate of incorporation of 3H-TdR into DNA. Within 4 days after radiation, cell proliferation and 3H-TdR incorporation are significantly depressed. Simultaneously there are reductions in both the relative cell density and tumour DNA contents, and these remain depressed as the tumours initiate regression. From these studies, it is apparent that the cellular responses to radiation insult occur well in advance of measurable volume changes and are observed both in tumours that continue to regress and in those that initiate regrowth.


Free radical inactivation of lactate dehydrogenase. The enzyme lactate dehydrogenase (LDH) has been irradiated under various conditions to assess the relative contributions of -H, -OH, H2O2 and -O2- to LDH inactivation, and it is concluded that -OH is the only important inactivating species. Further the effect of the selective free radicals, -(SCN)2-, -Br2- and -I2- on the activity has been studied. In neutral solution, the order of inactivating effectiveness is -I2- greater than -OH greater than -Br2- greater than -(SCN)2-. At pH 8-6, -OH and -Br2- are approximately equal in effectiveness, whereas -(SCN)2- is the least efficient. The radiation inactivation of LDH is accompanied by a loss of sulphydryl groups, and it is suggested that the primary target for radiation damage in LDH is the active site cysteine-165. Subsequent conformational changes are suggested to account for the apparent loss of coenzyme-binding ability and changes in the enzyme's kinetic parameters. The effect of bound coenzyme (NAD) on radiation-induced inactivation of N2O and air-saturated solutions was also investigated, and it is shown that NAD binding protects LDH.


Effects of X-irradiation on the hybridization of rat thymus nuclear RNA with repeated and unique DNA sequences. The kinetics of DNA hybridization with heterogeneous nuclear RNA (hnRNA) from normal and phytohaemagglutinin (PHA)-stimulated thymocytes has been studied in control rats and in animals 30 min after exposure to whole-body X-radiation with 400 rad. Irradiation results in a diminished ability of hnRNA to form hybrids with DNA at all C0t values ranging from 10(-3) to 10(4). Since this effect is most pronounced in the regions of low repetitive and unique DNA sequences, it is concluded that whole-body X-irradiation of animals may also suppress the transcription, particularly, in these regions.


E.s.r. of spin-trapped radicals in gamma-irradiated aqueous solutions of nucleic acids and their constituents. The gamma-radiolysis of de-aerated neutral aqueous solutions of uracil, thymine, cytosine and of the corresponding nucleosides and nucleotides and of calf-thymus DNA was investigated. For uracil and thymine, the U.V. photolysis of aqueous solutions containing H2O2 was also studied. The short-lived radicals were spin-trapped by tert-nitrosobutane and identified by electron-spin-resonance spectroscopy. For all compounds two or more radicals were observed, and these could be distinguished by following the thermal decay of the spin adducts. Radicals formed by the addition of H or OH at the C(5) or C(6) positions of the pyrimidine derivatives were observed in all cases. Sodium formate was used as a scavenger for H and OH to identify the radicals formed by eaq-. Spin-trapped radicals in gamma-irradiated aqueous solutions of polynucleotides exhibited broad e.s.r. lines. For DNA gel, additional narrow lines due to scission products were also found.


Acoustic reflex latency as a function of frequency and intensity of eliciting stimulus. The purpose of the present study was to examine the functional between acoustic reflex (AR) latency and the intensity and frequency of the eliciting stimulus. AR latency was determined at various intensities for 500 Hz and 300 Hz pure tones, and for wide-band noise. The results of the study indicated that AR latency decreased as stimulus intensity increased for all stimuli; the exact nature of the relationship depended on the stimulus, however. Latency differences between the stimuli at a given sound pressure level tended to disappear at higher presentation levels. Of particular interest was the dependency of inter- and intrasubject variability on parameters of the eliciting stimulus. The data do no indicate that AR latency is mediated solely by the loudness of the eliciting stimulus.


An epornitic of duck viral enteritis in a zoological park. An epornitic of duck viral enteritis occurred at the National Zoological Park in the spring of 1975. The disease affected 8 different species of ducks; geese and swan were spared. Diagnosis was based on characteristic pathologic changes, with herpetic inclusion bodies in the epithelium of the digestive tract and liver, and by viral isolation from 4 ducks. An experimental attenuated live-virus vaccine and sanitizing procedures were used to control the epornitic.


Necropsy and laboratory findings in free-living deer in South Dakota. In a diagnostic survey of diseases in wild white-tailed deer (62 cases) and mule deer (12 cases) the most common findings were traumatic injury (20%), nontraumatic hemorrhage (13%), polioencephalomalacia (11%), and bacterial infections (9%). Although epizootic hemorrhagic disease was suspected in several cases, the virus was isolated from only 1 white-tailed deer.


Parameters of fixation of the putative pain afferents in the ureter: preservation of the dense cores of the large vesicles in the axonal terminals. A study has been undertaken of the effects of a number of fixation procedures on the appearance of axons in the ureteric plexuses of the rat, and especially on the appearance of the dense-cored vesicles in those axons which have recently been characterized as pain afferents. Terminals were readily identified in material fixed by perfusion with glutaraldehyde, and many of them contained few if any vesicles. After fixation by immersion in cold glutaraldehyde terminals were not easy to identify because of the loss of microtubules. Immersion fixation in glutaraldehyde produced changes in the number and distribution of the vesicles in the axons which accentuated the similarity of the 'vesicle-containing regions' to the terminals of so-called 'purinergic' axons. Few of the dense-cored vesicles seen in glutaraldehyde-fixed material were preserved by fixation in permanganate, osmium or paraformaldehyde. The way dense-cored vesicles were or were not preserved by the various fixatives used indicated major differences between such cores and those of the vesicles in adrenergic axons. There was also evidence from the study of osmium-fixed material that both the number and appearance of the vesicles can be affected by the type of buffer used: the presence of more vesicles after fixation in osmium buffered with veronal than after osmium buffered in other ways was attributed to the membrane-stabilizing properties of veronal.


Naturally-occurring degeneration in chick muscle development: ultrastructure of the M. complexus. Ultrastructural study of the M. complexus muscle of the chick embryo has demonstrated two populations of fibres: the more common is the normal myotube or muscle fibre, which was observed in various stages of development; the other shows myofibrillar contractions which, at their greatest degree, produce the appearance of an amorphous mass of myofilaments. The contracted fibre had rounded and swollen mitochondria, and vacuoles (autophagic) containing glycogen, and it exhibited a cleavage of the cell which isolated the nuclear region from the main body of the fibre. When the fibres were less contracted they resembled degenerative mammalian fast-twitch-oxidative-glycolytic fibres after immobilization. The more normal fibre population was identical with that of the pectoralis muscle, which was used as a control. These results suggest that the contracted fibres are degenerating, which agrees with conclusions by earlier investigators using light miscroscopy.


Inclusions in the human thyroid.

Inclusions in the human thyroid. A survey of the thyroids of 350 infants and children suggests that the presence of thymus and parathyroid tissue within the thyroid is so common as to be classified as normal. One in a hundred thyroids contains masses of cartilage, and one in a hundred shows small foci of ciliated epithelium. Compact masses of dark-staining cells that bear a resemblance to primitive follicular plate cells are also so common as to raise the possibility of their being a normal component with, possibly, an endocrine function. It has been suggested that they produce calcitonin, but this could not be confirmed. Peculiar granular cells replacing follicle cells were also noticed.


The relationship of the house-dust mite to respiratory allergy. 1. The house-dust mite is a potent and important allergen. 2. 11.2% of patients with perennial respiratory allergy are allergic to the mite allergen and not to the various dust allergens. 3. 39.6% of the patients allergic to the allergens of house-dust are allergic to the allergen of the house-dust mites. 4. 23.7% of patients allergic to the allergens of house-dust are not allergic to the allergen of the house-dust mite. 5. 25.2% of patients with perennial respiratory allergy are not allergic to either the house-dust or mite allergens. 6. Endo, Center, or both dusts were negative in 222 cases, or 36.4%. 7.house-dust mites were negative in 297 cases, or 48.9%. 8. Positive intracutaneous skin tests with house-dust mite extract correlated with only 60.5% of the patients. Negative skin tests correlated with 67.3% of the patients.


Arginine-sensitive phenotype of mutations in pyrA of Salmonella typhimurium: role of ornithine carbamyltransferase in the assembly of mutant carbamylphosphate synthetase. The phenotype of certain mutations in pyrA, the gene encoding carbamylphosphate synthetase (CPSase), is expressed only in the presence od exogenous arginine. In unsupplemented media, synthesis of carbamylphosphate and growth was almost normal; in arginine-containing media, synthesis of carbamylphosphate stopped, as did growth, as a consequence of starvation for pyrimidine. Genetic and biochemical evidence suggests that arginine exerts this inhibition by repressing the synthesis of ornithine carbamyltransferase (OTCase), the intracellular presence of which is required for assembly of the unequal subunits and proper functioning of the mutant CPSase. After the addition of arginine to a culture of the mutant, CPSase activity (glutamine dependent) characteristic of the intact holoenzyme progressively decreased, whereas activity (ammonia dependent) characteristic of the free large (alpha) subunit increased. Extracts of mutant cells contain free small (beta) subunits, as demonstrated directly by in vitro complementation using purified alpha subunits from wild type. The mutant enzyme from cultures grown in the presence of arginine had a markedly decreased affinity for adenosine 5'-triphosphate. Mutations in argR that cause depressed synthesis of OTCase suppressed the phenotype, and a certain mutation in argI, the gene encoding OTCase, enhanced it. In vitro experiments using purified enzyme confirm the stimulatory effect of OTCase on the activity of mutant CPSase.


Iron-chelating compound from Mycobacterium avium. A iron-chelating monohydroxamate was isolated from cultures of Mycobacterium avium grown on an iron-limiting medium. The hydroxyamate metabolite was characterized by chemical degradation and spectral measurements as L-alpha-asparaginyl-L-alpha-(N-hydroxy)-asparagine.


Stimulatory effect of lithium ion on proline transport by whole cells of Escherichia coli. The effect of monovalent cations on proline transport in whole cells of Escherichia coli K-12 has been examined. Lithium ion added to the uptake medium stimulated proline transport severalfold and K+ and Na+ were slightly effective, whereas Rb+, Cs+, and NH4+ were completely without effect. The stimulatory effect of Li+ on proline transport was not due to an increase in osmolarity of the uptake medium, and d 5 mM p-chloromercuribenzene sulfonic acid completely blocked this effect of Li+ without having any effect on the basal rate of proline transport. The Arrhenius plots for Li+-stimulated transport showed a clear transition point at 35 degrees C in addition to 20 degrees C which was also detectable in the basal transport. Lithium ion stimulated proline transport synergistically in the presence of glucose and succinate as a carbon source. The addition of 2.5 mM KCN or 0.5 mM arsenate did not inhibit this synergistic effect, although the presence of these inhibitors inhibited completely the stimulation of proline transport induced by the addition of carbon source. Carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol blocked both the basal and Li+-stimulated proline transport. When membrane potential of E. coli cells was measured by the dibenzyldimethylammonium uptake method, the incubation of Li+ with the cells did not affect the preexisting membrane potential. These results suggest that Li+ stimulates proline transport by intact cells of E. coli in a manner somewhat affecting membrane component(s) different from the transport carrier of proline. It is uncertain whether the effect of Li+ is directly involved in the mechanisms of energy coupling of proline transport.


Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation. Competent Haemophilus influenzae Rd recipients, either as phage HP1 restricting (r+) or nonrestricting (r-) nonlysogens or defective lysogens, were exposed to deoxyribonucleic acids from various wild-type phage HP1 lysogenic H. influenzae serotype strains (non-encapsulated derivatives of serotypes a,b, c, d, and e), to DNA from lysogenic Haemophilus parahaemolyticus, and to DNA from modified and nonmodified phage HP1. Transformation of antibiotic resistance markers and of prophage markers in homospecific crosses was observed to be unaffected by the recipient restriction phenotype, whereas the transfection response was much reduced in r+ recipients. Heterospecific transformation of prophage markers was reduced by only 80 to 90%, whereas antibiotic resistance marker transformation was 1,000 to 10,000 times lower. Heterspecific transfection was at least 100 times lower than homospecific transfection in both r+ and r- recipients. The general conclusion is that neither class I nor class II restriction enzymes affect significantly the transformation efficiency in homospecific and heterospecific crosses. The efficiency of heterospecific transformation may depend mainly on the deoxyribonucleic acid homology in the genetic marker region.


Isolation and partial characterization of an argR mutant of Salmonella typhimurium. An arginine regulatory mutant (i.e., mutated in the argR gene) has been isolated from a strain of Salmonella typhimurium LT2. The argR mutant was found to excrete arginine into the growth medium with glycerol but not glucose as carbon source. Constitutive synthesis of arginine biosynthetic enzymes was observed. Whereas previous results (A. T. Abd-E1-A1 and J. L. Ingraham, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, K169, p. 175) have shown constitutive synthesis of carbamyl phosphate synthetase in the argR mutant, the regulation of the synthesis of the last five enzymes of the pyrimidine pathway was unaffected. However, in pyrH mutants, known to exhibit derepressed synthesis of the pyrimidine enzymes, a 10-fold derepression of ornithine transcarbamylase was observed.


Kinetics of light-dark CO2 fixation and glucose assimilation by Aphanocapsa 6714. Cells of Aphanocapsa 6714 were subjected to alternating ligh-dark periods (flashing-light experiments). The corresponding activation (in the light) and inactivation (in the dark) of the reductive pentose cycle was measured, in vivo, from initial rates of 14CO2 incorporation and also by changes in the total concentration of 14C and 32P in soluble metabolites. Two principle sites of metabolic regulation were detected: (i) CO2 fixation was inactivated 15 to 20 s after removal of the light source, but reactivated rapidly on reentering the light; (ii) hydrolysis of fructose-1,6-diphosphate (FDP) and sedoheptulose-1,7-diphosphate (SDP) by their respective phosphatase(s) (FDP + SDPase) was rapidly inhibited in the dark but only slowly reactivated in the light. The time required for reactivation of FDP + SDPase, in the light, was on the order of 20 to 30 s. As a consequence of the timing of these inactivation-reactivation reactions, newly fixed CO2 accumulated in the FDP and SDP pools during the flashing-light experiments. Changes in the concentrations of the adenylate pools (mainly in the levels of adenosine 5'-triphosphate and adenosine diphosphate) were fast in comparison to the inactivation-reactivation reactions in the reductive pentose cycle. Thus, these regulatory effects may not be under the control of the adenylates in this organism. The activation of CO2 fixation in the light is at least in part due to activation of phosphoribulokinase, which is required for formation of ribulose-1,5-diphoshate, the carboxylation substrate. Phosphoribulokinase activity in crude extracts was found to be dependent on the presence of strong reducing agents such as dithiothreitol, but not significantly dependent on adenylate levels, although adenosine 5'-triphosphate is a substrate.


Effect of polymyxin on the outer membrane of Salmonella typhimurium: freeze-fracture studies. Polymyxin-caused projections on the cell surface of Salmonella typhimurium were seen as depressions in the outer concave fracture face and as protrusions in the outer convex fracture face, indicating participation of both leaflets of the outer membrane in these projections.


Aminoacyl transfer RNA formation. VII. Lack of correlation between aminoacylation and PPi-ATP exchange catalyzed by isoleucyl-tRNA synthetase of Escherichia coli in the presence of various divalent cations. Isoleucyl-tRNA formation and isoleucine-dependent PPi-ATP exchange catalyzed by purified isoleucyl-tRNA synthetase [EC 6.1.1.5] of Escherichia coli were studied in the presence of various amounts of either Mg2+, Ca2+, Fe2+, Ni2+, or Cu2+. In the presence of Mg2+, isoleucine-dependent PPi-ATP exchange was observed in parallel with isoleucyl-tRNA formation, while in the presence of Ca2+, isoleucyl-tRNA formation was observed without isoleucine-dependent PPi-ATP exchange. Moreover, isoleucine-dependent PPi-ATP exchange was much more in the presence of Fe2+ than in the presence of Mg2+, while little isoleucyl-tRNA was formed in the presence of Fe2+. In the presence of Ni2+ or Cu2+, neither reaction was observed. These data, indicating that formation of an isoleucyl-AMP-enzyme complex is not a necessary step in isoleucyl-tRNA formation, support the existence of a concerted mechanism of isoleucyl-tRNA formation in E. coli.


Induction of ornithine decarboxylase in cultured mouse L cells. I. Effects of cellular growth, hormones, and actinomycin D. The activity of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells in the confluent state was induced within 4 hr by cyclic AMP (cAMP) or by insulin. During growth of L cells the concentration of cAMP increased first, then induction of ODC occurred and finally the cell number increased: the levels of cAMP and ODC increased only transitorily and returned to the basal levels when the cells become confluent. In growing cultures, however, the presence of cAMP reduced induction of ODC and cell growth. These results suggest that cAMP is involved in induction of ODC and that its concentration may be important for enzyme induction as well as for cell growth. Actinomycin D with or without these inducers stimulated induction of ODC in L cells, whereas cycloheximide inhibited it, suggesting that these hormones affect the translational level of ODC synthesis. The effect of actinomycin D on induction of ODC was much greater in non-growing cells than in growing cells. It was also found that the half life of ODC was 81 min in non-growing cells and 112 min in growing cells. This suggests that turnover of the enzyme is more rapid in the non-growing than in the growing state and that there may be an RNA fraction which controls its turnover and which also has a very short half life.


3-Dehydroquinate synthase in germinating Phaseolus mungo seedlings. Dehydroquinate synthase, an enzyme catalyzing the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to 3-dehydroquinate, was detected in cell-free extracts of etiolated Phaseolus mungo seedlings. The reaction product, 3-dehydroquinate, formed from [1-14C]DAHP was identified by paper-radiochromatography. The enzyme required NAD+ and Co2+ for activity.


Alterations in enzyme and cytochrome profiles of Rana catesbeiana liver organelles during thyroxine-induced metamorphosis. Changes in membrane-localized phosphohydrolases, oxidoreductases, and cytochrome levels in response to in vivo thyroxine administration. A primary objective of the present study has been to determine the changes which occur in Rana catesbeiana liver organelle membranes during thyroxine-induced metamorphosis. To this end, enzyme and cytochrome profiles were determined for mitochondria, microsomes, and nuclear membrane fractions isolated from livers of R. catesbeiana tadpoles which had been fasted for 6 days at 15 +/- 0.5 degrees and then immersed in thyroxine, 2.6 X 10(-8) M, for periods of up to 12 days at 23.5 +/- 0.4 degrees. The ratio of total succinate-cytochrome c reductase activity in the initial homogenate fraction to the total activity of this mitochondrial "marker" enzyme recovered in the final mitochondrial fraction remained constant, approximately 0.5, throughout the course of thyroxine treatment; however, after a 3- to 4-day latency the mitochondrial protein mass recovered per unit mass of initial homogenate protein was found to increase significantly (approximately 2-fold by Day 10 of thyroxine treatment). A similar increase was also observed in the yield of microsomal, but not nuclear membrane, protein mass as a function of thyroxine treatment. Prolonged thyroxine treatment (12 days) resulted in approximately 50% decreases in tadpole liver homogenate and microsomal NADH-cytochrome c reductase specific activities; in contrast, mitochondrial and nuclear membrane NADH-cytochrome c reductase specific activities were not altered under the same conditions. In addition, homogenate and microsomal NADPH-cytochrome c reductase specific activities were found to have increased significantly after 12 days of thyroxine treatment; however, the specific activity of NADPH-cytochrome c reductase in the mitochondrial fraction was unchanged. It was also observed that thyroxine treatment resulted in increases in homogenate and microsomal glucose-6-phosphatase specific activities, whereas the mitochondrial as well as nuclear membrane glucose-6-phosphatase specific activities remained unchanged. Furthermore, in contrast to homogenate and mitochondrial monoamine oxidase specific activities, which decreased 30 and 40%, respectively, as a consequence of thyroxine treatment (12 days), the succinate-cytochrome c reductase and oligomycin-sensitive Mg2+ ATPase specific activities determined for these fractions increased significantly. In all instances, changes as a result of thyroxine treatment in membrane-localized homogenate or organelle enzyme specific activities were apparent only after a 3- to 4-day initial latent period. The in vitro effects of thyroxine (10(-10) - 10(-5) M) on the membrane-localized enzyme activities examined in this study were either negligible or, as in the case of mitochondrial succinate-cytochrome c reductase and microsomal NADH-cytochrome c reductase, opposite to the changes observed in response to in vivo thyroxine treatment, with the exception of microsomal NADPH-cytochrome c reductase activity which was enhanced approximately 2-fold by 10(-5) M thyroxine...


Interactions of the actin and nucleotide binding sites on myosin subfragment 1. The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-1) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCl, pH 7.0 at 4 degrees. The association constants K'A, KN, and K'N in the scheme (see article), were determined for the magnesium salts of ADP, adenyl-5'-yl imidodiphosphate AMP-P(NH)P, and PPi. The nucleotide binding site on S-1 was "mapped" with respect to its interaction on the actin binding site. The subsites were the beta- and gamma-phosphoryl groups of ATP bind had the largest effects. A quantitative measure of the interaction, the interaction free energy, was defined as -RT ln (KA/K'A). For ADP, K'A was 2.7 X 10(5) M-1 and the interaction free energy was -4.67 kJ M-1. For AMP-P(NH)P and PPi it was much larger. A ternary complex was shown to exist for ADP, S-1, and actin in the presence of Mg2+ and evidence from AMP-P(NH)P and PPi measurements indicated that ATP also likely forms a ternary complex. The mechanism of (S-1)-actin dissociation is discussed in light of these results.


Inhibition of gluconeogenesis and lactate formation from pyruvate by N6, O2'-dibutyryl adenosine 3':5'-monophosphate. N6,O2-Dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) inhibits gluconeogenesis and lactate formation but increases ketogenesis by isolated liver cells incubated with high concentrations of pyruvate. The inhibitory effects can not be explained on the basis of an inhibition of the pyruvate dehydrogenase complex nor by a change in the NAD+ oxidation-reduction potential of the mitochondrial compartment. Both oleate and 3-hydroxybutyrate substantially increase the rates of gluconeogenesis and lactate formation from pyruvate but do not overcome the inhibition caused by Bt2cAMP. A decreased effectiveness of pyruvate kinase is proposed to account for the inhibition of both gluconeogenesis and lactate formation by Bt2cAMP. This enzyme catalyzes a step required in the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasm and participates in the formation of glucose and lactate from pyruvate by the overall reaction: 2 pyruvate- + 2 NADHmito + 4 ATP4- + 4 H2O leads to 1/2 glucose + lactate- + 2 NAD+ mito + 4 ADP3- + 4 HPO4(2)- + H+. Inhibition of pyruvate kinase promotes gluconeogenesis with most substrates but inhibits gluconeogenesis from pyruvate for want of cytoplasmic reducing equivalents.


Activation and inhibition of fat cell adenylate cyclase by fluoride. Incubation (30 degrees) of fat cell particulate fractions with fluoride before assay in the effective absence of fluoride results in activation of adenylate cyclase. Whereas the effect of fluoride (1.3 to 7 mM) when added to the assay was maximal in less than 2 min, 10 or 15 min of incubation before assay was usually required to produce maximal activation with any given concentration of fluoride. Under both conditions 3 to 5 mM fluoride produced maximal activation. After incubation with fluoride for 5 to 20 min cyclase activity was constant for at least 15 min of assay without fluoride; maximal activity was greater than that produced by fluoride added to the assay system and the concentration required to produce significant activation was lower. Fluoride activation in the assay or during prior incubation could be prevented by pyrophosphate. When added during the early minutes of assays with fluoride, 1.5 mM pyrophosphate, which had little effect on the activity of enzyme previously incubated with fluoride, rapidly reduced activity to essentially basal levels; when added after 10 min its effect was equally rapid but much smaller. It appears that activation is initially reversible by removal of fluoride as it is by addition of pyrophosphate, but becomes largely irreversible by these means with continued exposure to fluoride. Fluoride in the assay system inhibited cyclase activated by isoproterenol or choleragen or by incubation with fluoride prior to assay; inhibition, dependent on fluoride concentration, was maximal at 5.3 mM. Since maximal activity produced by incubation with fluoride before assay exceeded that of nonincubated preparations assayed with fluoride, and was reduced to the latter level when assayed in the presence of fluoride, we infer that inhibition is reversible at a time when fluoride activation is relatively irreversible. Pyrophosphate (1.5 mM), which prevented fluoride activation, did not reduce fluoride inhibition of isoproterenol-, fluoride-, or choleragen-activated cyclase. When 3 mM MnCl2 was present in the assay, inhibition by fluoride was not observed. In descriptive terms, MnCl2 appeared to cause rapid reversal of fluoride inhibition. Thus, fluoride inhibits, in an apparently similar manner, fat cell adenylate cyclase whether it is activated by isoproterenol, fluoride, or choleragen. Although fluoride activation and inhibition can apparently be dissociated or modified differentially, until the mechanism(s) of action of fluoride is elucidated it cannot be concluded that these are totally independent processes.


Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases. Adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) purified from silkworm pupae phosphorylated five major fractions of calf thymus histone, whereas guanosine 3':5'-monophosphate-dependent protein kinase (protein kinase G) purified from the same organism reacted preferentially with H1, H2A, and H2B histones. Amino acid analysis of the phosphopeptides which were obtained by proteolytic digestion revealed that both protein kinases A and G showed the abilities to phosphorylate the same serine hydroxyl groups in H1 and H2B histones. Both protein kinases reacted with Ser-38 in H1 histone. With H2B histone as substrate protein kinase A phosphorylated Ser-32 as well as Ser-36, whereas protein kinase G reacted preferentially with Ser-32 and the reaction with Ser-36 was very slow. H3 and H4 histones were practically inactive substrates for protein kinase G. Although H2A histone has not been analyzed, the evidence has raised a possibility that protein kinase G utilizes a portion of the substrate proteins for protein kinase A.


Inactivation of porcine heart cytoplasmic malate dehydrogenase by pyridoxal 5'-phosphate. Pyridoxal 5'-phosphate (pyridoxal-5'-P) has been found to act as a bifunctional reagent during the inactivation of porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37). The biphasic kinetics and X-azolidine-like structure formed were similar to those observed for mitochondrial malate dehydrogenase (Wimmer, M.J., Mo, T., Sawyers, D.L., and Harrison, J.H. (1975) J. Biol. Chem. 250, 710-715). In the cytoplasmic enzyme, however, irreversible inactivation representing X-azolidine formation was found to be the dominant characteristic of the interaction with pyridoxal-5'-P. Spectral evidence indicated that at total inactivation 2 mol of pyridoxal-5'-P were incorporated per mol of enzyme or one pyridoxal-5'-P per enzymatic active site. The presence of NADH protected the enzyme from inactivation suggesting interaction of pyridoxal-5'-P at or near the enzymatic active centers of this enzyme. Fluorometric titrations indicated that pyridoxal-5'-P-inactivated enzyme failed to bind NADH or at least failed to bind NADH in the same fashion as native enzyme.


Quantitative aspects of the conversion of 5 beta-cholestane intermediates to bile acids in man. The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.


Yeast cytochrome c messenger RNA. In vitro translation and specific immunoprecipitation of the CYC1 gene product. An assay based upon indirect immunoprecipitation has been developed for yeast cytochrome c and apocytochrome c. The specificity of this assay was demonstrated by its ability to selectively precipitate cytochrome c from an autolysate of yeast cell proteins. Translation of the polypeptide chain of cytochrome c in a wheat germ extract programmed with yeast poly(A) RNA was demonstrated using this immunoprecipitation assay. Translation of poly(A) RNA from yeast strains carrying nonsense mutations in the cyc1 gene yielded in vitro cytochrome c polypeptides which were shorter than the wild type protein by the amount expected for polypeptide chains which had terminated at the nonsense codon. The in vivo rate of cytochrome c synthesis was shown to be 6-fold greater in derepressed cells than in glucose-repressed cells. The 6-fold difference is sufficient to account for the 6-fold higher level of cytochrome c in derepressed than in repressed cells. The level of translatable cytochrome c mRNA is at least 4 times as high in derepressed as in glucose-repressed cells, suggesting that regulation occurs at some step in the synthesis of this messenger.


Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles. Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.


In vitro partial relipidation of apolipoproteins in plasma. In vitro recombination of lipids with apolipoproteins is achieved when a concentrated solution of plasma lipids in petroleum ether is mixed with delipidated plasma. Combination of phospholipids and unesterified fatty acids are observed in amounts comparable with those originally present in the native unextracted plasma; triglycerides combine partially and cholesterol only slightly. On agarose gel immunoelectrophoresis, a component in the delipidated plasma which is reactive with high density lipoprotein antibodies migrates more slowly than high density lipoprotein in the undelipidated control plasma. However, the component reacting with low density lipoprotein antibodies in the delipidated plasma moves more rapidly than low density lipoprotein in the native plasma. These changes are reversed by recombination of lipid with delipidated plasma. All lipids present in the plasma phase after relipidation travel with the lipoproteins during zonal electrophoresis. The apparent concentrations of proteins reacting with high density and low density lipoprotein antibodies decrease when no lipid is present in plasma on assay by single radial immunodiffusion and immunoelectrophoresis, using commercially available lipoprotein antibodies. On relipidation, full immunochemical properties of high density lipoprotein are restored, but relipidated low density lipoprotein exhibits only partial immunochemical restoration.


Superoxide anion as a mediator of drug-induced oxidative hemolysis. The superoxide dismutase inhibitor diethyldithiocarbamate (DDC) was utilized to study the toxic effect of 1,4-naphthoquinone 2-sulfonate (NQ), a structural analog of the hemolytic drug, menadione, on red cells. NQ was shown to react with hemoglobin and result in the generation of superoxide anion (O2-). Red cells treated with NQ were found to undergo a gradual disappearance of their oxyhemoglobin and also hemolyze. Red cells pretreated with DDC to inhibit cellular superoxide dismutase were found to be markedly sensitive to oxyhemoglobin destruction and hemolysis in the presence of NQ. Superoxide dismutase-inhibited red cells were also found to undergo a slow autohemolysis in the absence of NQ. No evidence for lipid peroxidation was obtained for red cells treated with NQ either in the presence or the absence of DDC. Ghosts prepared from superoxide dismutase-inhibited red cells exposed to NQ were found to retain a green hemoglobin-derived pigment.


Phosphorylation in vitro of eukaryotic initiation factors IF-E2 and IF-E3 by protein kinases. Purified protein synthesis initiation factors IF-E2 and IF-E3 from rabbit reticulocytes were phosphorylated in vitro with protein kinases isolated from the same source. The highest levels of phosphorylation resulted from incubation of the factors with a cyclic nucleotide-independent protein kinase previously shown to have specificity for acidic proteins. The extent of phosphorylation of initiation factor IF-E2 was between 0.3 and 0.4 mol of phosphate per mol of factor complex, with either ATP or GTP as phosphoryl donor. Initiation factor IF-E2 is composed of three nonidentical polypeptides; only the polypeptide with a molecular weight of 52,000 was phosphorylated. The extent of phosphorylation of initiation factor IF-E3 was between 0.7 and 1.0 mol of phosphate per mol of factor complex with GTP as phosphoryl donor; with ATP, less phosphorylation of the factor was obtained. Initiation factor IF-E3 is composed of 9 to 11 nonidentical polypeptides; only 2 of these, with molecular weights of 120,000 and 70,000, were phosphorylated. A lower level of phosphorylation of initiation factor IF-E3 was found with the cyclic AMP-dependent protein kinase; the polypeptide of molecular weight 140,000 was the major site of phosphorylation.


Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis. Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.


Insulin stimulation of heart glycogen synthase D phosphatase (protein phosphatase). Insulin rapidly produced an increase in per cent of total heart glycogen synthase in the I form in fed rats. In fasted rats the response was diminished and delayed. In diabetic animals there was no response over the 15-min time period studied. Since synthase phosphatase activity is necessary for synthase D to I conversion, the phosphatase activity was determined in extracts from these groups of animals. In the fasted and diabetic rats phosphatase activity was less than one-half of that in fed animals. Administration of insulin to fasting animals increased synthase phosphatase activity to a level approaching that of fed animals by 15 min. In diabetic animals insulin also stimulated an increase in synthase phosphatase activity but 30 min were required for full activation. Insulin had no effect in normal fed animals. Insulin activation of synthase phosphatase activity in heart extracts from fasted animals was still present after Sephadex G-25 chromatography and ammonium sulfate precipitation. Thus insulin had induced a stable modification of the phosphatase itself or of its substrate synthase D rendering the latter a more favorable substrate for the reaction. A difference in sensitivity of the reaction to glycogen inhibition was present between fed and fasted animals. Increasing concentrations of glycogen had only a slight inhibitory effect in extracts from fed animals but considerably reduced activity in extracts from fasted animals. Insulin administration reduced the sensitivity of the phosphatase reaction to glycogen inhibition. This could explain, at least in part, the increased phosphatase activity noted in the insulin-treated, fasted rats since glycogen was routinely added to the homogenizing buffer.


2-Fluoroadenosine 3':5'-monophosphate. A metabolite of 2-fluoroadenosine in mouse cytotoxic lymphocytes. 2-Fluoroadenosine (F-Ado) is a potent inhibitor of lymphocyte-mediated cytolysis studied in vitro. The inhibition of cytolysis by F-Ado was potentiated markedly by an inhibiotr (Ro 20-1724) of adenosine 3':5'-monophosphate (cAMP) phosphodiesterase and, unlike the inhibition caused by adenosine, was irreversible when the cytotoxic lymphocytes were incubated with F-Ado and were then washed free of exogenous nucleoside. Incubation of cytotoxic lymphocytes with F-Ado resulted in the rapid, dose-dependent formation of 2-fluoroadenosine 5'-triphosphate (F-ATP); the build-up of F-ATP within these cells was accompanied by a reciprocal depletion of ATP. Once formed intracellularly, the F-ATP was not diminished during a subsequent 30-min incubation of the cells in F-Ado-free medium. 2-Fluoroadenosine 3':5'-monophosphate (F-cAMP), a novel compound, was synthesized chemically. This cAMP analogue was found to be highly cross-reactive in a radioimmunoassay specific for cAMP and to be equipotent to cAMP in its ability to activate a crude preparation of protein kinase derived from rat brain. A column chromatographic procedure was devised whereby F-cAMP and cAMP could be purified simultaneously from tissue extracts. Treatment of cytotoxic lymphocytes with F-Ado resulted in the formation of presumptive F-cAMP in amounts greater than that of cAMP, as determined by the concentration of F-Ado added to the medium and was not observed when the lymphocytes were incubated with either adenosine or 2-chloroadenosine, two agents which caused large increases in cAMP. The simultaneous presence of Ro 20-1724 enhances greatly the formation of F-cAMP from F-Ado without affecting the pool size of F-ATP. Removal of exogenous F-Ado from cells previously incubated with this drug and subsequent incubation of these cells in drug-free medium did not result in a substantial reduction in intracellular F-Ado (via prior incubation with F-Ado); 2'-deoxyadenosine was also effective in this capacity, while 9-beta-D-arabinofulanosyladenine was without effect. The level of cAMP was elevated transiently, in a dose-dependent manner, by F-Ado, and returned to control value after removal of exogenous F-Ado from the cells. Ro 20-1724 enhanced greatly this transient elevation of cAMP caused by F-Ado.


Escherichia coli acetate kinase mechanism studied by net initial rate, equilibrium, and independent isotopic exchange kinetics. The equilibrium constant of the reaction catalyzed by acetate kinase (E.C. 2.7.2.1.) was determined: K = (MgADP) (acetylphosphate)/(MgATP)(acetate) = 6.7 +/- 1.3 X 10 (-4) (pH 7.4, 25 degrees). The respective free nucleotides uncomplexed to magnesium inhibit the net reaction in both directions: competitively with the respective magnesium nucleotide (MgATP or MgADP) and noncompetitively with the co-substrate (acetate or acetylphosphate). Excess free magnesium also inhibits the net reaction in both directions. The inhibition is not competitive with the phosphoryl donor (MgATP or acetylphosphate) but is competitive with respect to the phosphoryl acceptor (acetate or MgADP). A 50-fold increase in concentration of reactants at equilibrium in 0.1 M Tris/HCl, pH 7.4, at 25 degrees resulted in a rise to plateau levels of the acetate equilibrium acetylphosphate exchange rate (measured with [U-14C]acetate) and of the ATP equilibrium ADP exchange rate (measured with [U-14C]ADP and 10-fold higher than acetate equilibrium acetylphosphate), suggesting that there is no compulsory order of binding of magnesium nucleotide and co-substrate and that all chemical transformation steps cannot be much slower than the dissociation steps. The ATP equilibrium ADP exchange rate was independent of the presence or concentration of co-substrate, whereas the acetate equilibrium acetylphosphate exchange reaction occurred only in the presence of magnesium nucleotide, and the rate was directly related to the degree of saturation of enzyme with magnesium nucleotide. The independent ATP equilibrium ADP exchange, which presumably involves a phosphoenzyme intermediate, was progressively inhibited by increasingly elevated MgADP concentrations (when MgADP/MgATP greater than or equal 5), but increasing MgATP/MgADP was not inhibitory, suggesting that MgADP may bind to nonphosphorylated as well as phosphorylated enzyme, but that MgATP cannot bind to phosphorylated enzyme. While direct transfer of the phosphoryl group between nucleotide and co-substrate in a concerted mechanism has not been ruled out, an "activated ping pong" mechanism can also be proposed which is compatible with the isotopic exchange and initial net rate kinetic results. This mechanism includes a phosphoenzyme intermediate and requires enzyme-bount MgADP for phosphorylation of the enzyme by acetylphosphate.


8-Alkylaminoadenyl nucleotides as probes of dehydrogenase interactions with nucleotide analogs of different glycosyl conformation. The nucleotides 8-amino-, 8-methylamino-, and 8-dimethylaminoadenylic acid have been synthesized and their preferred conformations about the glycosyl bond in qaueous solution have been determined by 1H nuclear magnetic resonance spectroscopy. Paramagnetic relaxation studies, nuclear Overhauser enhancement measurements, chemical shifts, and coupling constant comparisons indicate that their is rotation about the glycosyl bond and that preference for either the anti or syn conformation depends on the extent of alkyl substitution on the 8-amino group. The primary and secondary amines 8-amino- and 8-methylaminoadenylic acid adopt a perferential anti conformation about the glycosyl bond, while the tertiary amine 8-dimethylaminoadenylic acid exists predominantly in the syn form. These three analogs provide a system to study interactions of a dehydrogenase with coenzyme inhibitors having different glycosyl conformer populations. All three analogs are competitive inhibitors of NADH in reaction with chicken muscle lactate dehydrogenase, and the Ki values show little dependence on the nature of the amino substitution. This demonstrates that the distribution of conformations about the nucleotide glycosyl bond does not effect the competition of the nucleotide for lactate dehydrogenase apoenzyme. Several models for enzyme-coenzyme binding are discussed. The available data cannot distinguish whether the enzyme binds nucleotide in both the anti and syn conformations or in purely the anti conformation. However, at some stage of the enzyme-coenzyme interaction, there appears to be a strong stabilization of the nucleotide in the anti conformation about the glycosyl bond.


Incorporation of mitochondrial membrane proteins into liposomes containing acidic phospholipids. Cytochrome oxidase, QH2-cytochrome c reductase, and the oligomycin-sensitive adenosine triphosphatase were incorporated into liposomes by a new procedure which yielded unidirectional orientation of the proteins. Cytochrome oxidase was reconstituted in the mitochrondrial orientation and the adenosine triphosphatase in the submitochondrial orientation. Reconstitutions were achieved by incubating the proteins at room temperature with liposomes which contained phosphatidylcholine, phosphatidylethanolamine, and an acidic phospholipid (cardiolipin, phosphatidylinositol, or phosphatidylserine). The incorporation occurred without added detergent or sonication. This incorporation procedure may serve as a model for the insertion of proteins in vivo.


Spin label studies on conformational changes of aphohemoglobin due to heme binding. Human apohemoglobin (globin) was spin-labeled at the beta-93 sulfhydryl groups with 2,2,5,5-tetramethyl-3-aminopyrrolidine-I-oxyl. Spin-labeled globin exhibited an EPR spectra that is less immobilized than that of spin-labeled hemoglobin, indicating the conformational difference in the vicinity of the label between hemoglobin and globin. Spectrophotometric titration of spin-labeled globin with protohemin showed that 1 mol of globin (on the tetramer basis) combines with 4 mol of hemin, producing a holomethemoglobin spectrophotometrically indistinguishable from native methemoglobin. The EPR spectrum was also changed strikingly upon the addition of protohemin. This change, however, was not proportional to the amount of hemin added, but marked changes occurred after 3 to 4 mol of hemin were mixed with 1 mol of spin-labeled globin. The EPR spectrum of spin-labeled hemoglobin thus prepared was identical with that prepared by direct spin labeling to methemoglobin. These results suggest the preferential binding of hemin to alpha-globin chains in the course of heme binding by globin. This assumption was further confirmed by preparing spin-labeled semihemoglobin in which only one kind of chain contained hemin (alpha h betaO SL and alpha O beta h SL). The EPR spectrum of the alpha h beta O SL molecule showed a slightly immobilized EPR spectrum, similar to that of spin-labeled globin mixed with 50% of the stoichiometric amount of hemin. On the other hand, the alpha O beta h SL molecule showed a distinctly different EPR signal from that of globin half-saturated with hemin, and showed an intermediate spectrum between those of beta h SL and alpha h beta h SL. These results indicate that heme binding to globin chains brings about a major conformational change in the protein moiety and that chain-chain association plays a secondary role. We conclude that hemin binds preferentially to alpha-globin chains and that the conformation of globin changes rapidly to that of methemoglobin after all four hemes are attached to globin heme pockets.


Cell surface receptors for insulin and human growth hormone. Effect of microtubule and microfilament modifiers. The receptors for the polypeptide hormones, insulin and growth hormone, are located on the cell surface. Since the cytoplasmic microtubules and microfilaments are involved in the mobility and distribution of surface receptors for immunoglobulins and lectins, we investigated the role of these structures in the binding of insulin and human growth hormone to their receptors on cultured human lymphocytes (IM-9). Cells preincubated with microfilament modifiers, cytochalasin A, B, and D (10 mug/ml), had decreased binding of insulin (30%) and human growth hormone (60%) under steady state conditions, which was not reversed by removing the cytochalasins from the medium and was due entirely to a reduced number of receptor sites on the cell surfact. The lost receptors were not detected in the medium, suggesting a redistribution within the cell. The cytochalasins failed to alter the affinity of the hormones for their receptors or the negative cooperativity of the insulin receptor. The anti-microtubule agents (vincristine, vinblastine, colchicine) had no effect on the binding of insulin and growth hormone to their receptors. Deuterium oxide, a stabilizer of microtubules and other proteins, decreased the affinity (40%) of insulin for its receptors under steady state conditions and accelerated moderately the spontaneous dissociation of 125I-insulin from its receptors. Since cytochalasin decreases the number of available insulin and human growth hormone receptor sites, cytochalasin-sensitive microfilamentous structures appear to modulate the exposure of cell surface hormone receptors, while microtubules do not seem to be involved.


In vitro epiphyseal-plate growth in various constant electrical fields. An in vitro epiphyseal-plate model was subjected to various electrical fields. At a voltage gradient of 1500 volts per centimeter, a consistent, highly significant acceleration of growth of the epiphyseal plate occurred as measured from photomacrographs and as indicated by incorporation of 45Ca, 35S, and 3H-thymidine. The growth acceleration was due to voltage gradients and not to current flow. Although the mechanism of action of the electrical field is not known, it is obvious that the voltage gradient, either directly or indirectly, incites a physiological response of the growth-plate chondrocyte.


Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment. Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.


Heterogeneity of phospholipid synthesis in rat liver endoplasmic reticulum during proliferation of smooth membranes. During proliferation of smooth endoplasmic reticulum (SER) induced by phenobarbital the specific activity of acyltransferases of the smooth microsomes increases, there is a transient rise in the phospholipid/protein ratio of these membranes, and an increased incorporation of [14C]glycerol into smooth-membrane phospholipid. Microsomes separated into subfractions on 2 gradients exhibited a heterogeneous distribution of these characteristics, indicating a non-uniform distribution of the site of phospholipid synthesis in the ER under these conditions. Cytochemical localization of acyltransferases on whole liver and smooth and rough microsomes confirmed this heterogeneity, and indicated that the distribution of this activity was not restricted to any morphologically distinct site in the ER of the intact cell. After 4 days of phenobarbital treatment the increased membrane is restricted to lighter subfractions and is similar in distribution to that of increased acyltransferase activity. These results indicate that the synthesis of membrane phospholipid and the growth of the SER in response to phenobarbital is not uniform but occurs at randomly dispersed sites in the SER while proteins may be added preferentially at these sites resulting in a final uniform distribution.


A theoretical study of the effects of cyclic AMP phosphodiesterases during aggregation in Dictyostelium. During aggregation the larger Dictyostelium species use cAMP as a chemoattractant and possibly also as a transmitter. In passage from cell to cell, cAMP levels are modulated by diffusion and by enzyme hydrolysis. It appears that the important cAMP-hydrolysing enzyme is a phosphodiesterase bound to the cell membrane, the main roles of which are (1) very fast hydrolysis of cAMP and (2) steepening of spatial cAMP gradients. An extracellular phosphodiesterase has no function, so far as can be conjectured from present data.