Preparation of polypeptide subunits of cytochrome oxidase from Neurospora crassa. Cytochrome oxidase was purified from Neurospora crassa by ammonium sulfate fractionation in the presence of bile salts. The enzyme preparations contained 10-13 nmol of heme a per mg of protein; no other hemoproteins could be detected. Dodecylsulfate gel electrophoresis resolved the enzyme complex into seven major bands, representing seven polypeptide subunits. A procedure is described that allows the isolation of these enzyme subunits on a large scale starting from a single batch of oxidase preparation. It involves dissociation of the enzyme complex by dodecylsulfate and subsequent separation of the obtained polypeptides by chromatography in the presence of various dodecylsulfate concentrations. Purification of subunits 3, 4, 5, 6 and 7 was achieved by column chromatography using molecular sieves (Sephadex G-100, Bio Gel P-60) and hydroxylapatite. For the purification of subunits 1 and 2 an electrophoretic separation on a preparative polyacrylamide gel was required. The advantages and disadvantages of the separation procedure of the enzyme polypeptides are discussed. As a special point of interest, the conservation of antigenic determinants of the polypeptide chains during the dodecylsulfate treatment is considered.


Protein components of very low density lipoproteins from hen's egg yolk. Egg yolk lipoproteins of very low density were found to contain proteins with cofactor activity for lipoprotein lipase. When delipidated very low density lipoproteins were dissolved in 10 mM HCl and fractionated by gel filtration about two thirds of the protein were in several components with estimated molecular weights of 60000 to more than 170000. The major low-molecular-weight proteins were the dimeric and monomeric forms of a previously characterized 9000-dalton peptide. The cofactor activity was not associated with any of these major proteins. A large-scale fractionation method was developed by which two proteins fractions with cofactor activity for lipoprotein lipase were purified more than thousand-fold. One fraction had a molecular size of about 9000 daltons and the other had a size of about 5000 daltons. Both these fractions could be further separated on the basis of charge into several fractions with cofactor activity. The cofactor proteins were relatively soluble both at high and at low pH. The retained their cofactor activity after denaturation in guanidinium hydrochloride and after reduction. During the initial steps in the purification of the cofactor proteins another low-molecular-weight protein followed the cofactors. It had a single 17500-dalton peptide chain and was present in four variants, three of which contained carbohydrate.


DNA unwinding enzyme II of Escherichia coli. 1. Purification and characterization of the ATPase activity. A DNA-stimulated ATP-gamma-phosphohydrolase of molecular weight 75000 was purified from Escherichia coli cells. The ATPase, a globular molecule (identical probably with an ATPase described previously by Richet and Kohiyama in 1976) shows specificity for adenine nucleotides, it prefers single-stranded DNA as the cofactor, it exhibits a complicated mode of response to variations of the cofacter concentration and it is devoid of nuclease activity. Preparations derived from rep3 mutant cells yield widely varying amounts of an apparently normal ATPase.


DNA unwinding enzyme II of Escherichia coli. 2. Characterization of the DNA unwinding activity. The DNA-stimulated 75000-Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180000-Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme - DNA complex. The enzyme binds specifically to single-stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single-stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells.


Detection of hepatoma in liver cirrhosis. The analogic liver scintigram using 99mTc sulfur colloid in cirrhotic patients does not permit determination of the nature of the areas of decreased uptake. Scintigrams with 67Ga citrate generally show increased activity in cases of hepatoma. In some cases, however, 67Ga citrate is less concentrated in neoplasic tissue, and it is not possible to detect a tumoral lesion in a cirrhotic liver. This is why we used double isotope scintigraphies with 67Ga citrate and 99mTc sulfur colloid, with digital subtraction, after simultaneous recording of 99mTc and 67Ga data on magnetic tape by means of an interface. In our series of 22 patients, the comparison of the results obtained by this double isotope technique with histology showed no false positive in substraction scintigrams. There was one false negative because of the lack of significance in the subtracted image for one of the six patients with cancer of the liver. For three of the six patients with hepatoma, the gallium scintigram showed an increased uptake in the tumor area. For the three other cases, the gallium uptake was equilibrated throughout the liver scintigraphy. It was therefore in cases where the gallium scintigram showed no increased activity that the subtraction technique was of greatest value, for it permitted the diagnosis of hepatoma in two cases.


Apallic syndrome in chronic mercury poisoning. This report includes five cases afflicted by chronic mercury poisoning which was observed in Iraq in 1972. All five cases showed the symptomatology of a severe cerebral damage combined with peripheral nerve lesion. The clinical picture reveals an apallic syndrome or a prestage ensuring in the full-blown picture. The combination of CNS lesions with polyneuropathy is typical of mercury poisoning with failure of all brain functions and the appearance of brain stem automatism, combined with severe muscular atrophy. When such conditions are established the remission seems to be impossible. The historical as well as the clinical and morphological facts of the Minamata disease is reviewed. The different stages of chronic mercury poisoning in Iraq are described.


Ascending vestibular projections: further results at cortical and thalamic levels in the cat. The purpose of this work was to precisely delineate the vestibular projection area in the cat's cortex and to trace electrophysiologically its afferent pathways at thalamic and mesencephalic levels. On anaesthetized animals, macropotentials evoked by electrical stimulation of the ampullar nerve from the horizontal semi-circular canal were recorded at three levels: on the cortex, the vestibular zone is restricted to the lower lip of the suprasylvian gyrus; in the thalamus, our results demonstrate the vestibular character of the magnocellular part of the medial geniculate body; at the mesencephalic level, our recordings indicate the existence of an ascending vestibular pathway situated laterally and ventrally with respect to the medial longitudinal bundle. The discussion focuses on two main points: 1. the precise cortical projection area of the horizontal ampullar nerve in relation to cytoarchitectonic areas and its possible functional role in motor programming. 2. the thalamic relays of the vestibulo-cortical pathway with regard to the hypothesis of two parallel input channels.


Changes of interhemispheric hippocampal responses during conditioning in the awake rat. The responses of the hippocampus of the awake rat to stimulation of one of its afferent pathways were measured during classical conditioning. It was found that when the contralateral hippocampus is stimulated concurrently with the presentation of a conditioned stimulus preceding either food or an aversive shock, a late (30-40 msec) negative component in the averaged evoked response can be recorded. This late component was absent when the interhemispheric stimulation was applied prior to presentation of a conditioned stimulus or when the rat was satiated or pretreated with drugs which interfere with noradrenergic or serotonergic neurotransmission. It is suggested that classical conditioning changes neurotransmission in certain  pathways in the brain and that the monoamines are involved in mediation of this change.


The action of a single toxic dose of 2-acetylaminofluorene or allylic alcohol on the adrenals. II. Effect on adrenal cyclic 3',5'-adenosine monophosphate concentration during the 1st and 2nd day. In the adrenals of male Sprague-Dawley rats the concentration of cyclic 3',5'-adenosine monophosphate (cAMP) was studied 5, 10, 14, 24, 30 and 39 hours after single intoxication by 2-acetylaminofluorene (AAF) or allylic alcohol (ALL). Beside diurnal changes a transient elevation of adrenal cAMP concentration occurred when compared with the controls 10 hours after intoxication. Thereafter, up to the 30th hour of investigation the values of the experimental groups as well as those of the control groups were within the same range. In the final stage of the experiment (30-39 hours after application) ALL intoxication was associated with a significant increase in adrenal cAMP concentration whereas it was reduced to subnormal level following AAF intoxication. In an earlier experimental study (Danz et al. 1976) we found inverse behaviour of the proliferative activity in the adrenal cortex: cell division was intensified by AAF whereas ALL was ineffective. These results indicate that division growth and functional activities are alternating conditions also in the adrenals. The possible causes of the different effects of intoxication by carcinogens or by noncarcinogenic substances are discussed.


Adrenoceptor sensitivity in disease as assessed through response to temperature alteration. Drug receptors are usually defined in terms of specific agonists and blocking agents. The adrenoreceptor occurs in two forms, alpha and beta. From a pharmacologic point of view, this receptor concept has proved useful by allowing the development of new, selective agonists and blocking agents. From a physiologic viewpoint, there is no need for two receptors. Clinically, it is important to find out whether the pharmacologic adrenoceptors are immutable. Evidence is accumulating, both pro and con, regarding this point. Some of the effects of temperature on the response of the isolated dilator muscle of the rabbit iris to catecholamines are described. The following questions are asked: Is it not possible that there is only one adrenoceptor whose specific agonists are epinephrine or norepinephrine? Do the alpha and beta configuration depend only on the local environment? While no answers are given, it is concluded that these are clinically relavant questions.


Cardiac adrenoceptors at low temperature: what is the experimental evidence for the adrenoceptor interconversion hypothesis? Isolated heart preparations of frog and rat were used to test the validity of the adrenoceptor interconversion hypothesis. This hypothesis claims that low temperature converts the inotropic beta-adrenoceptors in isolated frog and rat heart to alpha-adrenoceptors. The present results do not support the adrenoceptor interconversion hypothesis. In the isolated frog ventricle, lowering the temperature from 24 C to 14 C did not significantly alter the inotropic potency of the sympathomimetic drugs isoprenaline, epinephrine, and phenylephrine and did not reduce the potency of the beta-adrenoceptor blocking drug propranolol as an epinephrine antagonist. In the isolated rat left atrium, lowering the temperature from 31 C to 17-19 C did not significantly change the inotropic potency of isoprenaline, norepinephrine and phenylephrine, did not diminish the potency of propranolol, and did not increase the potency of the alpha-adrenoceptor blocking drug phentolamine.--Benfey, B. G. Cardiac adrenoceptors at low temperature; what is the experimental evidence for the adrenoceptor interconversion hypothesis?


[Character of metabolism and regulating role of cholinoreactive tissue systems during hypercapnia, hypoxia and cooling]. The content of the free fatty acids, ketone bodies, total glycogen, glucose, adrenaline and noradrenaline and morpho-histochemical picture of the tissues of neuro-endocrinal system (hypophysis and adrenal) in the brain, heart, liver, skeletal muscles and blood of the white non-linear rats, were studied 2-3 min adaptation to complex atmosphere changes: gradual increase of the CO2, decrease of the O2, and cooling (in the condition of deep hypothermia the rectal temperature was--RT--19.1 +/- 0.1 degrees C). The same parameters were studied in 48 hrs after the same training (at normothermia) and in 2-3 min. after the same repeated training in 48 hrs after the first one, at RT--20.2 +/- 0.1 degrees C. The fluctuating character of the metabolism and of the regulating systems was shown.


[Acceleration of the initial stages of thrombin clearance in the bloodstream of rats during stress]. In a stress due to 30-min immobilization, the initial stages of the thrombin clearance in the blood flow of rats are considerably accelerated. Thus, in 5 min after i.v. administration of thrombin I131, The blood relative radioactivity in experimental rats decreases by about 40% while in the control animals--by 20%. The same stress against the background of exogenous ACTH leads to a still greater acceleration of the thrombin clearance and the blood relative radioactivity within the 5 min decreases by 55%. The accelerated clearance of the thrombin I131 from the blood during immobilization and combination of the latter with ACTH administration is followed by its accumulation in the liver and its reduction in the lungs. The activating effect of ACTH on the thrombin I131 clearance is not, apparently, mediated through activation of the adrenal glucocorticoid function as the clearance initial stages are not accelerated after hydrocortisone administration. The clearance depends on the heparin contents in the blood: binding of endogenous heparin with protamine--sulphate leads to abolishing of the stress activating effect, and the speed of clearance in immobilized animals decreases to basal level of control animals.


[Effect of ACTH on plasma renin activity, aldosterone level and deoxycorticosterone level in humans (author's transl)]. Plasma aldosterone level (PAL) and deoxycorticosterone level (PDL) were determined in 5 subjects of normal sodium intake and 5 subjects of low sodium intake after the intramuscular administrations of ACTH (Upjohn, Cortrosyn) 40 IU, using radioligand methods. PAL and PDL reached their peaks at one hour after the administrations of ACTH, and then decreased in their normal ranges. Pretreated with the oral administrations of Dexamethasone 2 mg/day for 2 days, these subjects had, when ACTH were injected, such a similiar pattern in PAL and PDL as the peak of one hour and the consecutive falling without the pretreating of Dexamethasone. PAL and PDL of these subjects were not different between normal sodium intake and low sodium intake during the course of the experiment.


[Effect of biogenic amines on rat anterior pituitary cyclic AMP]. The effects of biogenic amines on the formation of adenosine-3',5' monophosphate (cAMP) in the anterior pituitary of rats were investigated. Pituitary halves were preincubated in a Krebs-Ringer bicarbonate buffer (pH 7.4) containing theophylline (10(-2)M) at 37 degrees C for 30 minutes and incubated in a fresh buffer for 15 minutes after the addition of test substances. Noradrenaline at 4 X 10(-4)M and serotonin at 3.3 X 10(-4)M were effective in elevating cAMP, but dopamine at 2 X 20(-4)M was ineffective. The beta adrenergic blocking agent, propranolol, effectively antagonized the cAMP response to noradrenaline, but the alpha adrenergic blocking agent, phentolamine, did not. The serotonin antagonist, methysergide, remarkably antagonized the cAMP response to serotonin, but the alpha and beta adrenergic agents did not.


[Lipid composition of circulating lipoproteins in various types of hyperlipoproteinemia (author's transl)]. By means of simple and selective precipitation methods with polyanions, three lipoprotein fractions rich in HDL (F1), LDL (F2) and VLDL (F3) were isolated from serum of normal subjects, of normolipemic patients with atherosclerotic disease and of patients with type IIA, IIB or IV hyperlipoproteinemia. Cholesterol content was used to quantify lipoprotein fractions and to measure relative triglycerides abundance (cholesterol/triglyceride ratio and regression coefficients). Findings in the atheroscerotic patients without hyperlipoproteinemia resembled those of the controls. No correlation between cholesterol and triglycerides was obtained in F1 of the various groups. However, a significant relation was established in F2 and the relative content of triglycerides was increased progressively from type IIA, through type IIB to type IV. In all types of hyperlipoproteinemia comparable relationships were found in F3 mainly containing VLDL, the precursor of LDL. In fractions F2 and F3, types IIB and IV were clearly distinguishable from type IIA.


Polygraphic studies of kitten development: sleep state patterns. The sequence of developmental changes in sleep pattern organization was derived from 12-hr recordings in 10-, 20 and 40-day-old kittens. Each characteristic of sleep patterning exhibited a distinct maturational sequence. Earlier changes (10-20 days) included quiet sleep (QS) increments and somatic activity decrements; later changes (20-40 days) included emergence of EEG slow waves and spindles, decrements in active sleep (AS), and diminution of QS respiratory variability. Changes in the lengths of state epochs and the types of state transitions spanned the period studied. We suggest that the differential development of the characteristics of sleep patterns reflects differential maturation of distinct nervous system elements.


Serum high density lipoprotein in diabetic patients. The purpose of the present investigation was the study of HDL lipoprotein changes in patients with diabetes mellitus. The comparison was made between 40 normal and 109 diabetic subjects and the following data was obtained: relative HDL concentration (polyacrylamide gel electrophoresis), HDL-cholesterol and apolipoprotein A concentrations. We found significant decreases in HDL (18-28%) and HDL-cholesterol (31-40 mg/100 ml) in most diabetics except in those with normalized serum levels of glucose and lipids (34% and 50 mg/100 ml respectively). There was a statistically significant difference in HDL and HDL-cholesterol concentrations between patients in the latter group and other diabetic patients. There was a negative correlation between HDL and HDL-cholesterol and serum glucose levels. No statistically significant difference was found when apolipoprotein A was compared in normal and diabetic subjects. Our results suggest that a deficient binding of cholesterol to apoprotein A might be present in diabetes.


Incorporation characteristics of uracil, uridine, and orotic acid into ribonucleic acid of neoplastic cells. Incorporation of uracil and uridine into ribonucleic acid (RNA) was compared among the ascitic and solid forms of Ehrlich mouse tumor, Morris hepatoma, Rhodamine sarcoma, gastric cancer and ulcer from human patients, and several normal rat tissues. Of these cells tested, the cells of Ehrlich ascites and solid tumors, human gastric cancer and ulcer, and certain tissues of a normal rat showed a considerably high activity. Furthermore, Ehrlich ascites tumor cells indicating a high incorporation activity was also high in activities of both phosphorylase and kinase for uridine, while Rhodamine sarcoma as a representative having a low incorporation activity was considerably low in these two enzymic activities. RNA synthesis from uridine phosphates by Rhodamine sarcoma was maintained to a fairly high extent contrary to its low activities of the phosphorylase and the kinase. Consequently, the low utilization of uracil and uridine by certain tumors was suggested to be due to the extremely low activities of both enzymes.


Detection of the estrogen receptor and response to endocrine therapy in male breast cancer patients. The estrogen receptor was measured in breast cancer of two male patients. Both patients had a positive receptor content in their tumors. The dissociation constant and number of binding sites were both very similar to those in the female counterpart. In one of the patients, the number of binding sites in the metastatic lesions of the liver was similar to that in the primary site. The patient responded well to the endocrine ablation therapy with a remission period of 8 months. The estrogen receptor was found to be positive in 12 out of 14 cases of male breast cancer reported previously including the present 2 patients. Three out of 4 cases with positive receptor contnet, including the present case, responded to orchiectomy.


Stimulation of a reconstituted, microsomal NADH oxidase system by carboquone, a quinoid anticancer chemical. Carboquone was found to greatly stimulate the aerobic NADH oxidation in the presence of both the partially purified NADH-cytochrome b5 reductase and cytochrome b5 prepared from hepatic microsomes by acting as an electron carrier from cytochrome b5 to molecular oxygen. Other quinoid anticancer chemicals, mitomycin-C, adriamycin, and daunomycin practically failed to do so.


Cytomegalovirus infection of the gastrointestinal tract in a patient with late onset immunodeficiency syndrome. An adult with the late onset immunodeficiency syndrome developed intractable diarrhea. Widespread cytomegalovirus (CMV) infection of the gastrointestinal tract was detected antemortem with detailed morphological studies and viral culture. The CMV-type cells were especially numerous in his severely ulcerated colon. Electron microscopy of infected cells in rectal biopsy material revealed the characteristic features of CMV infection. It is likely that the CMV infection contributed to the symptom complex and the mucosal injury. Unusual opportunistic infections as a cause of diarrhea should be considered in patients with late onset immunodefociency, especially if Giardiasis is ruled out.


Clinical recovery owing to target parietal cell failure in a patient with Zollinger-Ellison syndrome. A patient is presented with Zollinger-Ellison syndrome, in whom spontaneous disappearance of gastric hypersecretion and peptic ulcer disease occurred subsequent to an intercurrent illness causing acute nonspecific inflammation of the gastric mucosal lining. The dramatic clinical improvement after subsiding of the intercurrent illness was obviously linked to pronounced failure of the parietal cell mass for acid secretion and not to infarction of the gastrinoma because gastrin secretion by the tumor was unchanged.


Pancreatic acinar cell metabolism and function. This review outlines progress made during the past 11 years in research related to pancreatic acinar cell metabolism and function. We have reviewed information gained at the cellular level concerning structural and functional relationships, and effects of fasting and feeding, as well as the action of gastrointestinal hormones and cholinergic agonists on acinar cells. In toto, this information outlines a significant role for gastrointestinal hormones as mediators of secretion, synthesis, and control of trophism. This information provides a basis for more sophisticated inquiries as to the mechanisms of injury of alcohol and drugs. The information may prove helpful in developing diagnostic modalities for pancreatic disease, as well as understanding the processes involved in neoplastic transformation.


Studies of human malignant lymphomas after treatment with endoxan. I. Cytochemical studies in the respiratory enzymes succinic dehydrogenase and cytochrome oxidase. The respiratory enzymes succinic dehydrogenase and cytochrome oxidase were studied in normal lymph node and malignant lymphoma cells under the effect of endoxan treatment. It is concluded that the decrease in the respiratory enzymes in lymphoma cells may be taken as one of the outstanding differences between the neoplastic and normal tissues.


Enzyme histochemistry of cultured ovarian cells. III. Histochemical characteristics of bovine granulosa and theca interna cells grown separately in cell culture. Granulosa and theca interna cells were isolated from bovine preovulatory ovarian follicles. They were cultured separately but in the same conditions of cell culture. Both cell types, grown as monolayers, were investigated histochemically with special regard to the activity of several hydroxysteroid dehydrogenases: delta53betaOH-SDH, 17betaOH-SDH, 20alphaOH-SDH and G6P-DH. Bovine granulosa and theca interna cells during in vitro culture showed high activity of delta53betaOH-SDH and G6P-DH, the enzymes essential to progesterone biosynthesis. Enzyme pattern of cultured cells indicated continuation in vitro of luteinization, which in the normal preovulatory follicle of the bovine ovary begins prior to ovulation. There was investigated as well the influence of single doses of gonadotrophic hormones and estradiol on growth, lipid contents and enzymic activity of cultured in vitro bovine granulosa and theca interna cells.


[Modification of chromosome aberrations caused by kilham virus in rat embryo cells]. Mutagenic effect of Kilham virus on the frequency of chromosome aberrations in the cells of primary and continous rat embryo cultures and the modification effect of cadmium salt on the mutagenic potential of this virus was studied. The frequency of chromosome aberrations increased in the primary rat embryo culture after Kilham virus enfection. Rat embryo culture chronically infected with Kilham virus did not differ from control continuous cells in the frequency level of chromosome aberrations. Isertion of cadmium in the process of cultivation increased the mutagenic effect of kilham virus in the primary rat embryo culture.


Anterior interosseous nerve paralysis: report of 13 cases and review of Japanese literatures. Fifty-six cases of anterior interosseous nerve paralysis including our thirteen cases were collected by reviewing the Japanese literature. The causes of the paralysis were similar to those in the cases reported in other countries. Paralysis is often only partial, and this is of importance in the differential diagnosis.


[Double controlled regulation of renin release by the sympathetic nervous system. Stimulation via beta receptors and inhibition via alpha receptors]. The beta-sympathomimetic amine isoprenaline increases the plasma renin concentration by a stimulation of beta-receptors which control renin release. Isoprenaline also lowers systemic blood pressure and causes a reflex-mediated activation of the sympathetic nervous system. In these investigations it has been tested to see whether the catecholamines released by this activation modulate renin release by stimulation of certain alpha-receptors. Pretreatment of rats with reserpine or with the ganglionic blocking agent Trimethidinium enhanced the increase in plasma renin concentration induced by isoprenaline. So did pretreatment with the irreversible alpha-receptor antagonist phenoxybenzamine. Renal denervation also increased the effect of isoprenaline on plasma renin concentration. It is concluded that catecholamines released from the sympathetic nervous system can decrease renin secretion by an activation of certain alpha-receptors. The sympathetic nervous system may thus exert control of renin release by beta-receptors which stimulate and by alpha-receptors which diminish renin secretion.


[The mechanism of action of histamine on the myocardium and the smooth vascular muscles]. Histamine-H1-receptors are only present in the left atrium of guinea-pig hearts, whereas the ventricular myocardium contains histamine-H2-receptors exclusively. Although the stimulation of both receptor types causes positive intropic effects, only the H2-receptor response is closely associated with increases of cyclic AMP. H1- as well as H2-receptors are present in the rabbit mesenteric artery. Cyclic AMP serves as a messenger for H2-receptor mediated relaxation only and not for H1-receptor mediated contraction. Thus, the different effects on the cyclic AMP-system must also be considered in H1- and H2-receptor differentiation.


[Measurement of liver circulation by means of an ultrasonic Doppler flow measuring device]. The ultrasonic-Doppler technique is a very simple method for flow measurement. With this method the generally accepted mechanism of hepatic arterial flow increase after reduction or interruption of the portal vein flow could be confirmed. The increase was not related to cardiac output. Previous doubts about the portal vein flow behaviour after suppression of hepatic artery flow were decided, joining the little group of authors, who did not observe a variation of the portal vein flow in this condition. The concept of the autoregulation of the liver blood supply seems to be connected to the hepatic arterial system only. Although an increase of the hepatic artery flow after infusion of isoproterenol supports the existance of beta-receptors in the hepatic arterial system, it could be proved, that there was no reduction after propranolol infusion. This suggests, that autoregulation of the liver blood supply is not connected to the beta-receptors only.


Delivering Mental Health Services to an ambulatory, low-income population. Lincoln Community Health Center, Durham, North Carolina. The social work and mental health unit of a comprehensive neighborhood health center delivers mental health services in coordination with general health services to ambulatory patients in a predominantly low-income black community. The unit works with other general health, mental health, and social service resources to provide a broad continuum of coordinated care. Services are provided to individuals, families, and groups, along with consultation, education, training, and referral. Before the unit was established in July 1971, the patients it serves were seen only for crisis intervention and in emergency rooms and did not have the benefit of a thorough evaluation or follow-up. Staff of the unit now identify potential medical and emotional problems before they reach the crisis stage, and they maintain contact with the patients to ensure that the prescribed regimen is carried out.


[Fundamentals of anti-NADase reaction in A-streptococcus infections and its determination in microtiter test (author's transl)]. A-streptococci of known serotypes were investigated for their ability to produce NADase. For this, a microtiter-dilution technique was employed. The results were compared with those of Lazarides and Bernheimer who had determined the NADase-activity in an other way. It seems, that there is no fixed relation of NADase-production and growth curve for all of the strains. At the moment, anti-NADase can be determined only with a macrotechnique, which is, with respect to costs, time work and others, difficult to handle. A microtiter-technique is described, which is adapted to the macrotechnique. With 426 young men in the age of 18-20 years titers were found between less than 50 and 800 with a 95% level between 200 and 300. The reproducibility of the micromethod is like that of the macromethod. It is of great importance to redetermine the titer in the upper limit of normal and those which are expected to rise. The possibility to seize also infections with G- and C-streptococci is mentioned.


[Adjuvant activity of gram-negative bacteria and their structural components (author's transl)]. Regarding the adjuvant activity of gram-negative bacteria we have to distinguish at least 4 different potencies, i.e., 1) increase in the production of circulating antibodies during the primary and secondary immune responses; 2) induction of susceptibility to systemic anaphylaxis; 3) prompt production of experimental "allergic" diseases, and 4) increase in resistance to infections. Although all gram-negative bacteria contain several structural components with adjuvant potencies, the immunopotentiating effectiveness of the corresponding whole bacteria becomes--with the exception of killed cells of Bordetella pertussis--only detectable to a weak degree.


Relationship between the Epstein-Barr virus and undifferentiated nasopharyngeal carcinoma: correlated nucleic acid hybridization and histopathological examination. In order to examine critically the closeness of association between Epstein-Barr virus (EBV) DNA and nasopharyngeal carcinoma (NPC) a correlated histopathological and nucleic acid hybridization study was performed on 51 undifferentiated NPC, 4 NPC with some signs of squamous differentiation, 7 nasophayngeal tumors of other histological types and 14 head and neck carcinomas located outside the nasopharynx. All 51 undifferentiated NPCs contained significant numbers of EBV-genome copies per cell. Two of the somewhat differentiated NPCs were also EBV-DNA-positive, whereas 2 were negative. Of the 7 other nasopharyngeal tumors, 1 was EBV-DNA-positive. Histological examination, however, showed that this was a typical Burkitt lymphoma. The other 6 tumors were all EBV-DNA-negative lymphoproliferative malignancies. All 14 had head and neck carcinomas located outside the nasopharynx were EBV-DNA-negative. The sera of undifferentiated NPC patients had elevated antibody titers against the EBV-determined antigens, the EA (D) componet in particular. These findings confirm that there is a regular association between EBV-DNA and undifferentiated NPC.


Cell mediated immunity during infectious mononucleosis to Epstein-Barr virus associated antigens. Twenty-three patients with recent infectious mononucleosis were studied for cell-mediated immunity to Epstein-Barr virus (EBV)-associated antigens. By means of a virion-containing antigen preparation, (P3J), leukocyte migration inhibition was demonstrated in 6 of 13 patients with acute infectious mononucleosis (IM) and in 6 of 10 patients studied during the convalescent period. Inhibition with a soluble antigen (S) preparation was seen in only a few patients at all stages of the illness. Lymphocyte blast transformation on exposure to P3J occurred in 4 of 9 patients with acute IM; no other study group reacted. Control donors lacking anti-EBV antibodies did not demonstrate migration inhibition or blast transformation with either P3J or S. Control donors with evidence of previous EBV infection did demonstrate migration inhibition with P3J (8 of 12) and S (3 of 10). These studies indicated that cell-mediate immunity to EBV-associated antigens could be detected by either leukocyte migration inhibition or lymphocyte blast transformation, but neither assay is diagnostic of infectious mononucleosis.


Production of hybrid cells by fusion of human malignant tumour cells and primary mouse embryo cells. Hybrid cells with a subtetraploid mouse chromosome complement were produced by fusion of three types of human tumour cells with primary mouse embryo cells. The most frequently present presumptive human chromosome was 21. Numerous chromosome rearrangements were present. Some hybrid cells produced regressing tumours in mice.


Bovine leukemia virus specific antibodies among French cattle. II. Radioimmunoassay with the major structural protein (BLV p24). A radioimmunoassay (RIA) for the major internal protein of the bovine leukemia virus (BLV p24) was established using anti-BLV p24 natural antibodies and purified 125I-labelled BLV p24. The final precipitation of the immune complexes was realized by a preparation of inactivated Staphylococcus aureau Cowan I. Sera from 363 cows belonging to (1) leukemic herds, (2) non-leukemic but BLV-exposed herds, and (3) apparently unexposed herds were studied comparatively in BLV p24 RIA, complement fixation and immunodiffusion. The BLV p24 RIA appeared much more senstive than the two other methods in the detection of positive sera. With this method 100% of the leukemic animals, excluding those with juvenile lymphosarcoma, presented very high antibody titers (greater than or equal to 10,000). Practically all cows with persistent lymphocytosis were also positive with slightly lower levels of antibodies, confirming the relationship between BLV infection and the persistent lymphycytosis. Moreover, about two-thirds of the hematologically suspect animals and one-third of the normal animals from BLV-exposed herds were found positive, whereas 100% of the sera from unexposed cows remained negative for anti-BLV p24 antibodies.


Adenylate cyclase activity and the cAMP level are not directly correlated with transformation by avian sarcoma viruses. The adenylate cyclase activity was measured in chick embryo fibroblasts (CEF) infected with temperature-sensitive mutants (ts) of avian sarcoma virus (ASV). When CEF transformed with a (ts) mutant at 36 degrees C were incubated at the non-permissive temperature (41 degrees C), recovery from the low adenylate cyclase activity detectable in the transformed state was slower than the disappearance of signs of morphological transformation. After a downward shift of the temperature the activity decreased and this change was also slower than the alteration of cell morphology. The affinity of the enzyme system for ATP also changed after, and not during, morphological alteration. No significant difference was observed between the cAMP levels in ASV-transformed and non-infected CEF. These findings are consistent with the idea that adenylate cyclase is not involved in cell transformation and that the change in its activity is secondary to cell transformation.


Development of a congeneic line of the GR mouse strain without early mammary tumours. Based on the previous observation that Mtv-2 controls the appearance of pregnancy-dependent mammary tumours and the early expression of mammary tumour virus (MTV)-antigens in the milk, an attempt was made to develop a congeneic GR line, without this locus, by introducing genetic material of the C57BL/10 strain. The cross-intercross system was used for this purpose. The mice of the even-numbered generations were selected for the absence of the Mtv-2 locus with the early maammary tumour (EMT) test and during the later cycles with the immunodiffusion (ID) test for MTV-antigens in the milk. After six cycles of cross-intercross, brother-sister mating was started with mice selected for the absence of Mtv-2. After two brother-sister matings the milk-transmitted MTV was eliminated by foster-nursing. Of the foster-nursed subline 233 mice (16 of the first, 118 of the second and 99 of the third generation) were tested for the presence of Mtv-2 with one or two of the following three methods: (1) ID-test, (2) EMT-test and (3) by examination of the development of spontaneous mammary tumours in breeding females. The tests were negative in 226 mice. This indicates that Mtv-2 was absent in nearly all mice of the congeneic subline. The positive reactions in the seven other tested mice were weaker than that observed in the GR strain and in GR mice foster-nursed by BALB/c. The presence of the Mtv-2 gene in the seven positive mice of the congeneic line is doubtful. With the ID test, viral antigens were detectable in 50% of the milk samples from the first lactation period of mice of the segregating backcross I population [GR congeneic X (GR congeneic X GR)], indicating a one-gene difference between the congeneic GR line and the progenitor GR strain.


Resistance of guinea-pig hepatoma cells to complement-mediated lysis induced by ascites fluid or serum from tumor-bearing animals. The mechanisms of tumor cell susceptibility and resistance to cytotoxic antibodies were investigated in the guinea-pig ascites hepatoma (line-1 and line-10) system. Treatment of line-specific rabbit antibody-coated tumor cells by ascitic fluid, by serum of tumor bearers or by tumor extract inhibited subsequent complement-mediated lysis. Inhibition by ascitic fluid and serum was not line-specific, but inhibition by tumor extract was line-specific. Treatment of tumor cells with ascitic fluid or tumor extract prior to exposure to specific cytotoxic antibody and complement did not inhibit lysis. Incubation of cytotoxic rabbit antisera with ascitic fluid, tumor-bearer serum or tumor extract neutralized their complement-dependent cytotoxic activity on tumor cells. Tumor-immune guinea-pigs exhibited line-specific delayed cutaneous reactions after injection with ascitic fluid or tumor extract. Studies with indrect immunofluorescence revealed that exposure to ascites fluid or tumor extract caused a rapid shedding of rabbit antibodies from the tumor cell surface. Evidence is presented indicating that the active fraction of ascites fluid was associated with immune complexes consisting of IgG and tumor antigen in excess. The relevance of these findings to tumor escape from immune destruction in vivo is discussed.


In vivo and in vitro TSTA-inducing activity of temperature-sensitive (ts) mutants of SV40. In vivo TSTA induction in Syrian hamsters was studied with the use of SV40 ts mutants (A, B, C, BC and D). The ts A30, TS A239 and possibly also the ts BC210 mutants were defective in resistance-inducing activity in hamsters in contrast to wild type SV40 and other ts mutnats. At the permissive temperature ts A30 and ts A239 did not induce TSTA in hamster cells during abortive infection in vitro, while they did so in green monkey cells at both permissive and non-permissive temperatures. In hamster cells transformed by ts A30, ts A239 and ts A209 mutants none at all or very little TSTA was detected by in vivo transplantation immunological tests. Thus, expression of TSTA induced by these three SV40 ts A mutants was found to be dependent from the species of infected cells and was being a temperature independent viral function.


The electrophysiological effects of lithium in the rat. The effects of lithium on the tail nerve conduction velocity of the rat were studied. No effect of lithium on the conduction velocity was found, even at toxic serum lithium levels. No effect of lithium on repetitive stimulation of the distal forelimb flexor muscles was seen. The results suggest that electrophysiological measurements do not provide an effective means of titrating serum lithium levels in the rat. The results may have clinical signficance since they provide evidence against neuropathic or neuromuscular transmission defects as the cause of the transient weakness of some patients taking lithium.


Increased intraocular pressure following topical azide or nitroprusside. In rabbits the topical administration of sodium azide (NaNs) or sodium nitroprusside (SNP) increased intraocular pressure in a dose-response manner. These agents, which activate guanylate cyclase, elevated cyclic GMP in the aqueous humor. Systemic blood pressure and pulse were not altered. Tonographic outflow facility was unchanged, suggesting an increase in aqueous humor flow as the mechanism for the elevation of intraocular pressure. Posterior chamber aqueous humor ascorbate concentration was decreased in the eye receiving the NaN3 or SNP. Systemic pretreatment with phenoxybenzamine, an alpha-adrenergic blocking agent, prevented the elevation of intraocular pressure observed following NaN3 and SNP. Pretreatment with systemic indomethacin, propranolol, or acetazolamide or the topical application of atropine or epinephrine failed to alter the elevation of intraocular pressure by either NaN3 or SNP.


[Vesicular fluid of hereditary bullous dystrophic epidermolysis splits alpha 1-antitrypsin]. In epidermolysis hereditaria bullosa dystrophica increased collagenase activity can be detected and seems to be one of the pathogenetic mechanisms of this disease. Neither the origin nor the mechanism of increased collagenolysis is known. Whether the cause of the enzymatic imbalance is the increased collagenase production or decreased collagenase-inhibitor activity cannot be decided. Factors of decreased protease inhibitor activity could be the quantitative or qualitative defect or the inactivation of the inhibitor. Clear, sterile vesicular fluid was incubated with alpha-1-antitrypsin, which is known to inhibit collagenase. By means of an immunoelectrophoretic method the cleaving of the inhibitor into two antigenic split products was found. We suggest that this might be responsible for the increased collagenolysis in this form of epidermolysis.


A study of Cytomegalovirus, Epstein-Barr virus and Herpesvirus hominis (types 1 and 2) antibody in institutionalized and non-institutionalized children. In an attempt to demonstrate differences in antibody prevalence between free-living and institutionalized children, antibodies to Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Herpesvirus hominis (HVH), types 1 and 2, were assayed in 123 children. The children comprised three groups consisting of 41 institutionalized patients with Down's syndrome (all non-disjunctive trisomy-G karyotype and equal numbers of age-, sex-, and race-matched non-mongoloid institutionalized subjects and non-institutionalized normal controls. CMV antibody titer values were statistically similar in the three groups. However, fewer mongoloids (21.9%) were seropositive than other institutionalized retardates (39.0%) and normal control subjects (43.9%). Antibody titer values to EBV were also similar; however, in comparison to the other groups, significantly more mongoloids were seropositive at younger ages. More mongoloids were seropositive to HVH-1 and had higher antibody titers than the other two groups. Antibody to HVH-2 was more prevalent in institutionalized subjects, 85.4% in mongoloids and 65.8% in other institutionalized retardates, than in normal non-institutionalized children (26.8%).


Biochemical analysis of genetic recombination in eukaryotes. Recent studies concerning molecular mechanisms of genetic recombination in eukaryotes are reviewed. Since many of these studies have focused on the testable predictions arising from the hybrid DNA theory of genetic recombination, this theory is summarised. Experiments to determine the time of meiotic crossing-over and the structure of the synaptonemal complex which facilitates meiotic crossing-over are described. Investigations of DNA nicking and repair events implicated in recombination are discussed. Properties of proteins which may facilitate hybrid DNA formation, and biochemical evidence for hybrid DNA formation are presented. Finally, a nuclease which has been implicated in gene conversion is described.


A simple technique for supporting single cells in electron microscopic immunocytochemistry and embedding. Cell suspensions of rat anterior pituitaries were filtered with a polycarbonate filter (pore size 3 micron) and fixed on the filter. After fixation the cells were adherent to the filter and immunocytochemical staining could be accomplished by simply dipping the filter into the different incubation media. The cells could be dehydrated and embedded in Epon 812 on the filter. After polymerization the embedded filter was sawn into small blocks and the cell layer was sectioned tangentially on an ultramicrotome. This method also seems to be applicable to other histochemical studies on single cells.


Role of conducting airways in partial separation of inhaled gas mixtures. A positive (hollow) cast of the bronchial tree was made from a pig's lung. Gas mixtures containing sulfur hexafluoride (SF6) and helium (He), and SF6 and argon (Ar), were blown down the cast at two different flows, the cast having first been filled with air. Gas was sampled by a mass spectrometer probe from 1-mm-diam branches situated on short, medium, and long pathways. The front of the SF6 appeared in advance of the fronts of the He and the Ar. This relative advancement was greater a) with the SF6/He mixture than with the SF6/Ar mixture; b) at slower flows; and c) on longer pathways. With reverse flow up the cast using SF6/He there was little differences between the arrival times of the two gas fronts at either flow. These results could be explained by the effects of Taylor dispersion on gases having different diffusion coefficients.


Penetration of inhaled He and SF6 into alveolar space at low tidal volumes. To study mixing of inspired gas with lung gas, penetration of simultaneously inspired helium (He) and sulfur hexafluoride (SF6) into alveolar space was determined in normal subjects at low tidal volumes (from 50 to 500 ml) and at varied lung volumes and speeds of inspiration/expiration. The volume of inspired gas reaching the alveolar space, termed alveolar-tidal volume, VTA, was calculated from preinspiratory lung volume, inspired volume, and inspired and expired alveolar test gas concentrations. The difference between the VTA values calculated for He and SF6, VTA(He) - VTA(SF6), was influenced by tidal volume, lung volume, and the speed of inspiration/expiration, but it was always positive. The results are qualitatively explainable on the basis of easier diffusive mixing of He in lung airways compared with SF6. Since Taylor dispersion would produce deeper penetration, and therefore higher VTA, for a less diffusible gas the results provide no evidence for its implication in pulmonary gas exchange.


Control of liver size in heat-acclimated hamsters. In the hamster, heat acclimation reduces liver weight more than it does body weight. Therefore, liver weight constitutes a lower percentage of body weight during exposure to high ambient temperature. This change is not a result of dehydration since water content of the whole body and of the liver is not altered during heat acclimation. However, changes in lactic dehydrogenase isozyme proportions indicate a higher rate of liver degradation during the first 2 wk of heat exposure. These changes are accompanied by enhancement of DNA synthesis which is found to be elevated during the early period of heat exposure and later to fall to the control levels. The enhanced DNA synthesis might be a result of a high rate of tissue regeneration which probably takes place in the organ following the commencement of the degradative processes as was suggested in partial hepatectomy. Since the activity of DNA synthesis is negatively correlated with cyclic AMP levels, it is suggested that cyclic AMP plays some role in controlling hepatic DNA synthesis during heat acclimation.


Isolated, ventilated, perfused newborn rabbit lung preparation and its assessment. To achieve an isolated, ventilated, perfused newborn rabbit lung preparation, newborn rabbits are anesthetized and ventilated. The pulmonary vasculature is perfused with a Krebs-Ringer bicarbonate solution containing glucose and plasma expanders, and the lungs are isolated in a 37 degrees C chamber at 100% humidity. The success of a perfusion is evaluated on three levels. 1) Visual. 2)mechanical: a, drop in flow of greater than 20% indicates failure; b, an increase of more than 25% of the initial lung weight indicates fluid accumulation and failure. 3) Biochemical: a, the endogenous ATP concentration after perfusion; b, the level of the enzyme choline phosphotransferase (CPT) after perfusion. Isolated, perfused, ventilated newborn rabbit lungs maintained for 4 h with no changes in the monitored physical and mechanical parameters have an endogenous ATP level of 1.06 +/- 0.06 (2 SD) mumol ATP/mg wet weight and a CPT level of 1.34 +/-0.15 (2 SD nmol [14C]CDP-choline per milligram protein in 30 min. These values are stable at a level lower than observed in intact, hypoxic, newborn rabbit's lungs. In constrast, if perfusion is maintained after any of the monitored criteria indicate failure, the endogenous ATP concentration and CPT activity are significantly depressed.


Respiratory competence of Dictyostelium discoideum spores. Analysis of the respiratory chain of spores of Dictyostelium discoideum, which lack a cyanide-sensitive respiration, indicated that cytochromes a-a3, b, and c-c1 are present at levels identical to those found in the vegetative amoebae. The specific activities of enzymes of both the respiratory chain and the citric acid cycle in the 600 x g supernatant fraction of sonically treated spores were at least as high as in similar preparations of amoebae. The activities of glutamic dehydrogenase and oligomycin-sensitive adenosine triphosphatase were reduced in the spores 30 and 56%, respectively. Intact spores appeared to lack a cyanide-sensitive respiration as a result of inadequate quantities of respiratory substrate and, more importantly, as a result of a lack of the cofactor nicotinamide adenine dinucleotide. The emergence phase of spore germination was sensitive to the antibiotic chloramphenicol, which is a specific inhibitor of mitochondrial protein synthesis. It is concluded that germination requires the early synthesis of oxidized nicotinamide adenine dinucleotide and generation of respiratory substrates and one or more mitochondrially synthesized proteins.


Characterization of a novel thiosulfate-forming enzyme isolated from Desulfovibrio vulgaris. An enzyme that formed thiosulfate from bisulfite and trithionate was purified from extracts of Desulfovibrio vulgaris. This enzyme, designated as "thiosulfate-forming" enzyme, required the presence of both bisulfite and trithionate. Various 35S-labeling studies showed that thiosulfate was formed from bisulfite and the inner sulfur atom of trithionate. This involved a nucleophilic attack by the bisulfite ion, resulting in the displacement of the two outer sulfonate groups of trithionate that recycled to participate as free bisulfite in subsequent reactions. This reaction required a reduction, presumably by a concerted mechanism with thiosulfate formation. The natural electron carrier cytochrome c3 participated in this reductive formation of thiosulfate. This reaction was coupled to the bisulfite reductase-catalyzed reaction, which resulted in the reconstruction of a thiosulfate-forming pathway from bisulfite.


Intracellular distribution of enzymes of phospholipid metabolism in several gram-negative bacteria. Cell-free extracts of Salmonella typhimurium, Serratia marcescens, Enterobacter aerogenes, and Micrococcus cerificans contained the following enzymatic activities related to phospholipid metabolism: cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):l-serine O-phosphatidyltransferase (phosphatidylserine synthase), phosphatidylserine decarboxylase, CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase), phosphatidylglycerophosphate phosphatase, and CDP-diglyceride hydrolase. The intracellular distribution of these enzymatic activities as determined by sucrose density gradient centrifugation of cell-free extracts was shown to be similar in each species investigated. The phosphatidylserine decarboxylase, phosphatidylglycerophosphate synthase, and CDP-diglyceride hydrolase activities were all associated with the cell envelope fraction, whereas the phosphatidylserine synthase activity was associated mainly with the ribosomal fraction. These enzymatic activities are comparable and have an intracellular distribution similar to those found in Escherichia coli cell-free extracts. Therefore, the pathways established for phospholipid biosynthesis in E. coli can also account for the synthesis of the major phospholipids (phosphatidylethanolamine and phosphatidylglycerol) in several other gram-negative organisms. In addition, the unusual ribosomal association of the phosphatidylserine synthase from E. coli (Raetz and Kennedy, J. Biol. Chem. 247:2008-2014, 1972) appears to be a general property for this activity in several other bacterial species.


Sulfate uptake in Saccharomyces cerevisiae: biochemical and genetic study. Sulfate uptake is the first step of the sulfate assimilation pathway, which has been shown in our laboratory to be part of the methionine biosynthetic pathway. Kinetic study of sulfate uptake has shown a biphasic curve in a Lineweaver-Burk plot. The analysis of this plot indicates that two enzymes participate in sulfate uptake. One (permease I) has a high affinity for the substrate (K(m) = 0.005 mM); the other (permease II) shows a much lower affinity for sulfate (K(m) = 0.35 mM). Regulation of the synthesis of both permeases is under the control of exogenous methionine or S-adenosylmethionine. It was shown, moreover, that synthesis of sulfate permeases is coordinated with the synthesis of the other methionine biosynthetic enzymes thus far studied in our laboratory. An additional specific regulation of sulfate permeases by inhibition of their activity by endogenous sulfate and adenosyl phosphosulfate (an intermediate metabolite in sulfate assimilation) has been shown. A mutant unable to concentrate sulfate has been selected. This strain carried mutations in two independent genes. These two mutations, separated in two different strains, lead to modified kinetics of sulfate uptake. The study of these strains leads us to postulate that there is an interaction in situ between the products of these two genes.


Ribonucleic acid polymerase mutant of Escherichia coli defective in flagella formation. Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.


2-Hydroxypyridine metabolism and pigment formation in three Arthrobacter species. Three species of the genus Arthrobacter, A. crystallopoietes, A. pyridinolis, and A. viridescens, have the capabilities to utilize 2-hydroxypyridine (2-HP) as the sole source of carbon and nitrogen for growth and to produce an extracellular crystalline pigment from this substrate. Degradation of 2-HP by cell-free extracts requires the presence of both reduced nicotinamide adenine dinucleotide and molecular oxygen and is stimulated by flavin mononucleotide, suggesting the presence of a monooxygenase activity in the extract. Loss of the ability to produce pigment at a high spontaneous frequency, 0.26% loss per generation, is observed only with A. crystallopoietes and can be visualized by the presence of sectored and fully nonpigmented colonies on solid media containing 2-HP. Concomitant with the loss of pigment-producing character are both loss of ability to utilize 2-HP for growth and oxidation of 2-HP by cell-free extracts. These three 2-HP-associated characteristics also are lost simultaneously by treating cultures of A. crystallopoietes with curing agents, such as acridine orange and mitomycin C, but are not curable in A. pyridinolis or A. viridescens. All nonpigmented strains of A. crystallopoietes are nonrevertible for these properties. These data suggest that 2-HP-related characteristics are plasmid determined in A. crystallopoietes but not in A. pyridinolis and A. viridescens. A survey for the presence of plasmids in these three species and two physiologically unrelated species, A. globiformis and A. atrocyaneus, revealed plasmid material only in A. globiformis and A. crystallopoietes.


Requirement of cyclic adenosine 3',5'-monophosphate for the thermosensitive effects of Rts1 in a cyclic adenosine 3',5'-monophosphate-less mutant of Escherichia coli. Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.


Membrane phospholipid synthesis and phenotypic correlation of an Escherichia coli pss mutant. A pair of putatively isogenic pss(Ts) and pss+ (phosphatidylserine synthetase structural gene) strains was constructed and analyzed, together with the revertants, for the physiological consequences of cessation of the optimal synthesis of phosphatidylethanolamine (PE). Their in vivo and in vitro abilities to synthetize PE and the growth rates at different temperatures were determined. The rate of PE synthesis by OS2101 pss(Ts) was inversely related to the culture temperature. OS2101 in a low-salt broth medium stopped division and formed filamentous cells with declining viability upon the elevation of culture temperature from 27 to 42 or 44 degrees C, whereas the syntheses of deoxyribonucleic acid, ribonucleic acid, and protein were not affected. Proper concentrations of cations such as Na+, K+, NH4+, and Mg2+ or of sucrose could remedy the division and growth of OS2101 at the restrictive temperature without restoring normal PE synthesis. A remedial effect other than osmotic protection of these effectors and an adaptive regulatory mechanism for PE formation are suggested.


Icosahedral inclusions (carboxysomes) of Nitrobacter agilis. The icosahedral bodies of Nitrobacter agilis are about 120 nm in diameter and, as viewed by electron microscopy, consist of an outer shell enclosing 10-nm particles. The inner 10-nm particle is the enzyme D-ribulose 1,5-bisphosphate carboxylase. The bodies isolated from cells incubated 1 month without nitrite had a specific activity for the enzyme of 0.54 mu mol of CO2 fixed per min per mg of protein.


Production of large amounts of acetate during germination of Bacillus megaterium spores in the absence of exogenous carbon sources. When Bacillus megaterium spores germinate in the absence of an exogenous carbon source, the first minutes of germination are accompanied by production of large amounts (approximately 70 nmol/mg of dry spores) of acetate and much smaller amounts of pyruvate and lactate. The majority of these compounds are excreted into the medium. Exogenous pyruvate and alanine are also converted to CO2 and acetate by germinating spores, presumably by using the pyruvate dehydrogenase that is present in dormant spores. These data suggest that the 3-phosphoglyceric acid stores in the dormant spore and alanine generated by proteolysis early in germination can be catabolized to acetate during germination with production of large amounts of reduced nicotinamide adenine dinucleotide, acetyl coenzyme A, and adenosine 5'-triphosphate.


A new method for measurement of cyclic AMP phosphodiesterase activity. The extreme sensitivity of chicken muscle fructose 1,6-bisphosphatase to inhibition by 5'-AMP has been utilized to develop a new method for the assay of cAMP phosphodiesterase activity. In this method, the substrate (cAMP) is first incubated with phosphodiesterase and the amount of 5'-AMP formed is then determined by measuring the degree of inhibition of fructose 1'6-bisphosphatase activity. The present method conveniently employs the spectrophotometric technique and is sensitive enough to detect the conversion of 50 pmol of cAMP to 5'-AMP in 1 ml of reaction mixture. This method is considered particularly valuable for those laboratories that are not equipped with facilities for measuring radioactivity.


Phosphatidylcholine substrate specificity of lecithin: cholestrol acyltransferase-in high density lipoproteins and in lipids dispersions. Lecithin: cholesterol acyltransferase (LCAT) was more highly activated by apolipoprotein A-I (apoA-I) with dimyristoyl phosphatidylcholine (DMPC) than with dilinoleoyl phosphatidylcholine (DLPC) when lipid dispersion of cholesterol and each phosphatidylcholine was used as a substrate. When the enzyme reactions were activated by whole apolipoproteins of high density lipoproteins (HDL), DLPC was more available to the LCAT reaction than DMPC with high concentrations of apoHDL in an incubation mixture. However,  no detectable enzyme reaction was observed with dipalmitoyl phosphatidylcholine (DPPC) under both conditions. On the other hand, all of these phosphatidylcholines acted as substrates of LCAT when they were incorporated into HDL coupled to Sepharose. The order of their relative reactivities to cholesterol was DMPC, DPPC, AND DLPC under the conditions used.


Purification and some properties of cytochrome c-552 from an extreme thermophile, Thermus thermophilus HB8. A c-type cytochrome, cytochrome c-552, from a soluble fraction of an extreme thermophile, Thermus thermophilus HB8, was highly purified and its properties investigated. The absorption peaks were at 552, 522, and 417 nm in the reduced form, and at 408 nm in the oxidized form. The isoelectric point was at PH 10.8, the midpoint redox potential was about +0.23 V, and the molecular weight was about 15,000. The cytochrome c-552 was highly thermoresistant. The cytochrome reacted rapidly with pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2], but slowly with bovine cytochrome oxidase [EC 1.9.3.1], yeast cytochrome c peroxidase [EC 1.11.1.5], or Nitrosomonas europaea hydroxylamine-cytochrome c reductase [EC 1.7.3.4].


Inhibition by Mg2+ of the interaction of Ca2+ with spin-labeled g2 bound to myosin. According to the measurement of ESR spectrum, Ca2+ induced conformational change of spin-labeled g2 bound to myosin in the presence of 1 mM Mg2+. The half-maximal changes were observed at pCa 6.8 and at pCa 3.7. Spin-labeled phosphorylated g2 bound to myosin showed one transition at pCa 4.5, which shifted to pCa 6.5 after the dephosphorylation  with E. coli alkaliphosphatase.


Studies on the role of adenosine 3':5'-monophosphate in the activation of liver phosphorylase. Crude extracts of rabbit liver, preincubated to promote the dephosphorylation of enzymes or other regulatory proteins, were used to study the role of cyclic AMP in the activation of glycogen phosphorylase. Inasmuch as endogenous liver phosphorylase was irreversibly altered by the preincubation procedure, crystalline skeletal muscle phosphorylase was used as the substrate in these studies. In the presence of magnesium ions and ATP, phosphorylase b was converted to phosphorylase a, and in an apparent biphasic process the phosphorylase a formed was subsequently converted to phosphorylase b. In the presence of adenosine 3':5'-monophosphate the rate of phosphorylase a formation and the maximal amount of phosphorylase a formed were increased. The cyclic AMP effect was enhanced by glucose-6-P and required the presence of glycogen. The catalytic subunit of cyclic AMP-dependent protein kinase could replace cyclic AMP in the stimulation of phosphorylase a formation. The effects of cyclic AMP or the catalytic subunit were shown to be due to stimulation of phosphorylase kinase rather than to inhibition of phosphorylase phosphatase. Preliminary fractionation experiments showed that it is possible to separate phosphorylase kinase catalytic activity from a factor or factors required for stimulation of its activation by the catalytic subunit.


Synthesis and release of low density lipoproteins by the isolated perfused pig liver. In previous studies, we have shown that a relatively large amount of low density lipoproteins is released into the perfusate during isolated pig liver perfusion. The present studies were done to determine the source of these lipoproteins. Breakdown of the very low density lipoproteins to low density lipoproteins by the perfusion apparatus or by hepatic catabolism was excluded by adding 125I very low density lipoproteins to the perfusate in the presence and absence of a liver and then measuring the radioactivity in the low density lipoprotein fraction after rate-zonal ultracentrifugation. Release of preformed low density, lipoproteins from the liver was investigated by injecting iodine-labeled low density lipoproteins in vivo several hours prior to perfusion of the liver and then measuring the release of labeled low density lipoproteins into the perfusate. It was shown that intact labeled low density lipoproteins were released by the perfused liver. De novo synthesis of the low density lipoproteins was established by measuring the incorporation of [1-14C]leucine into this lipoprotein fraction. The radioactivity in the low density lipoprotein fraction increased with time and accounted for 20 to 25% of the total radioactivity incorporated into all the lipoprotein fractions. The incorporation of [1-14C]leucine into the low density lipoproteins was confirmed by rate-zonal analysis. We conclude that the low density lipoproteins in the perfusate from pig liver perfusions were derived mainly from a preformed liver pool, but also partly from de novo synthesis by the liver.


Prolyl hydroxylase of earthworms. Substrate specificity of an enzyme from the subcuticular epithelium. A relatively crude enzyme preparation derived from the subcuticular epithelium of earthworms catalyzed the formation of 4-hydroxyproline from prolyl residues in unhydroxylated natural collagens and in several synthetic collagen-like polypeptides. The specificity of hydroxylation differed from that of all vertebrate polyl hydroxylases in that (Gly-Pro-Ala)n was a much better substrate than (Gly-Ala-Pro)n. In contrast, however, only the so-called Y position proline (Gly-X-Y) was hydroxylated in Gly-Pro-Pro sequences derived either from natural collagen or from synthetic polypeptides; specificity of hydroxylation for the latter sequence is identical with that of the vertebrate enzymes. Little or no formation of 3-hydroxyproline could be demonstrated in preparations of the enzyme active as a 4-hydroxylase. In contrast with an earlier report from another laboratory, using a crude extract of earthworm body wall, we were unable to demonstrate either significant 3-hydroxyproline formation or efficient 4-hydroxylation of X position prolyl residues in synthetic polypeptides with the internal sequence Gly-Pro-Pro.


Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. A protein kinase which phosphorylated histone and protamine was partially purified from bovine cerebellum. Casein and phosvitin were inert as substrates. The enzyme did not require any cyclic nucleotide. A sulfhydryl compound such as 2-mercaptoethanol, glutathione, or cysteine was necessary for the reaction. The optimum pH was 8.5 to 9.0 Km values for ATP and whole histone were 3.3 X 10(-6) M and 150 microgram/ml, respectively. The optimum concentration of Mg2+ varied with histone fractions employed; with H2B histone as substrate the enzyme was most active at 50 to 100 nM Mg2", whereas with H1 and H2A histones the maximum activity was observed at 5 to 10 mM Mg2+ and with H3 and H4 histones the enzyme was active over a range of 5 to 75 mM Mg2+. The enzyme phosphorylated Ser-32 and Ser-36 in H2B histone and Ser-38 in H1 histone, although the reaction with Ser-36 in H2B histone was very slow. The molecular weight was 6.4 X 10(4). The sedimentation coefficient and Stokes radium were about 4.5 and 29 A, respectively. The enzyme showed heterogeneity upon isoelectrofocusing electrophoresis with isoelectric points of 5.6, 6.0, and 6.6. The enzyme was not inhibited by protein inhibitor nor by the regulatory subunit of cyclic AMP-dependent protein kinase. Preliminary analysis suggested that the enzyme was produced from its precursor protein by a limited proteolytic reaction.


Protein synthesis in 3T3 and SV40-transformed 3T3 cells. Activity of ribosomes and cytosol proteins in cell-free protein synthesis. The activity of specific components involved in protein synthesis in 3T3 cells and its SV40-transformed derivative, SV3T3, were examined in a cell-free protein synthetic system, and the results correlated with previous studies, indicating that a decreasing rate of protein synthesis does not accompany the stationary phase of growth. We found that 3T3 and SV3T3 polysome preparations containing endogenous mRNA were equally efficient in supporting cell-free protein synthesis in this system. Further, the net protein synthesis observed was not altered by an increase in the population density of the cellular polysome source. The activity of the aminoacyl-tRNA synthetase enzymes from 3T3 and SV3T3 cells was examined in vitro after isolation by pH 5 precipitation and by ammonium sulfate fractionation. The activity of these preparations from stationary phase 3T3 and nonexponential phase SV3T3 cells was found to be approximately 3 times higher than the activity of fractions from the homologous exponential phase cell. However, at both growth stages, the SV3T3 preparations were 30 to 40 times more active than the 3T3 preparations. These findings may have implications for the different growth properties observed in the two cell types.


Partial purification and characterization of rat liver phenylalanine hydroxylase phosphatase. A phosphatase which catalyzes the release of 32Pi from 32P-labeled phenylalanine hydroxylase has been purified about 50-fold from rat liver extracts. The phosphatase is able to catalyze the removal of only 70 to 80% of the 32Pi leaving about the same amount of Pi still bound to the hydroxylase as was originally present in the native enzyme. Dephosphorylation is accompanied by a corresponding loss in phenylalanine hydroxylase activity when the activity is measured with the natural cofactor, tetrahydrobiopterin, but not when measured with the synthetic cofactor, 6-methyltetrahydropterin. The partially purified phosphatase has very low activity toward p-nitrophenylphosphate, histone phosphate, and phosphorylase a. The activity toward these substrates has not been purified to the same extent as the phenylalanine hydroxylase phosphatase activity.


Lactoperoxidase-catalyzed iodination of horse cytochrome c:monoiodotyrosyl 74 cytochrome c. Iodination of horse cytochrome c with the lactoperoxidase-hydrogen peroxide-iodide system results initially in the formation of the monoiodotyrosyl 74 derivative. This singly modified protein was obtained in pure form by ion exchange chromatography and preparative column electrophoresis. It shows an intact 695 nm absorption band, the midpoint potential of the native protein, a nuclear magnetic resonance spectrum which indicates an undisturbed heme crevice structure, a normal reaction with antibodies directed against native horse cytochrome c, and circular dichroic spectra in which the only changes from those of the native protein can be ascribed to the spectral properties of iodotyrosine itself. This conformationally intact derivative reacts with the succinate-cytochrome c reductase and the cytochrome c oxidase systems of beef mitochondrial particle preparations indistinguishably from the unmodified protein, showing that the region including tyrosine 74 is not involved in these enzymic electron transfer functions of the protein. The circular dichroic spectra of this derivative indicate that the minima observed at 288 and 282 nm in the spectrum of native ferricytochrome c originate from tyrosyl residue 74.


Nereis cuticle collagen. Isolation and properties of a large fragment resistant to proteolysis by bacterial collagenase. Native cuticle collagen, obtained from Nereis virens, was incubated with purified bacterial collagenase (EC 3.4.4.19). The kinetics of proteolysis were monitored by viscometry, in parallel with similar digestions of calf skin collagen. Comparison of the kinetics of digestion of the two collagens, at similar enzyme to substrate ratios (w/w), showed that the native cuticle collagen was relatively refractory to digestion by bacterial collagenase. Characterization of the cuticle collagen digest by sodium dodecyl sulfate-polyacrylamide electrophoresis and agarose gel filtration in CaCl2 showed a large polypeptide, of about 300,000 daltons, to be a major product. The native form of this product, a unique fragment, was isolated from the digest by ethanol precipitation. It was found to have an intrinsic viscosity of 120 dl/g, to have an optical rotary dispersion curve characteristic of collagen, to undergo a typical collagenous thermal transition with a Tm of 23.2 degrees, and to have a calculated molar mass of 900,000 g with molecular dimensions of 9,000 X 13 A. It had an amino acid composition which was similar, but not identical with the native cuticle collagen. Although the original substrate contained two dissimilar chains, A and B, in a molar ratio of 1:2, the collagenase-resistant product appeared to be composed of only one type of polypeptide fragment. Possibly, the original subunits contain similar, if not identical collagenase-resistant regions.


Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. VI. Isolation and sequences of eighteen fragments from the cyanogen bromide digest. The 1030-residue polypeptide chain of the NAD-specific glutamate dehydrogenase of Neurospora crassa was fragmented by treatment with cyanogen bromide. The isolation and sequences of 18 fragments ranging in size from 4 to 51 residues are described. Some of these peptides proved to be cleavage products resulting from hydrolysis at acid-sensitive aspartyl-prolyl bonds. Some overlaps could be deduced on the basis of known sequences of peptides obtained by tryptic hydrolysis.


Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. VII. Isolation and sequences of three large cyanogen bromide peptides. The isolation and sequences of three peptides of large size from a cyanogen bromide digest of the NAD-specific glutamate dehydrogenase of Neurospora crassa are reported. These three peptides comprise 86, 117, and 134 residues, respectively, and represent approximately 30% of the estimated 1030 residues in the peptide chain.


Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. VIII. Isolation and sequences of peptides from a tryptic hydrolysate of the maleylated protein. The NAD-specific glutamate dehydrogenase of Neurospora crassa was S-carboxymethylated with [14C]iodoacetate, maleylated, and hydrolyzed with trypsin. The isolation and sequences of the resulting peptides are described. These peptides gave information on the structure of the protein that was previously unknown and gave many overlaps of previously isolated segments of the protein.


Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. IX. Isolation and sequences of several large cyanogen bromide peptides. The isolation and sequences of several large peptides from cyanogen bromide cleavage of the 1030-residue polypeptide chain of the NAD-specific glutamate dehydrogenase of Neurospora crassa are described. One of these is in the 669-residue sequence of the COOH-terminal end of the protein. The remaining peptides have been aligned in two partial sequences in the NH2-terminal portion of the polypeptide chain.


Subcellular fractionation and morphology of calf aortic smooth muscle cells. Studies on whole aorta, aortic explants, and subcultures grown under different conditions. A comparative biochemical and morphological study was made of calf aortic smooth muscle cells found in situ and grown in vitro under various conditions. Striking alterations in enzyme contents, physical properties, and morphological appearances of lysosomes, endoplasmic reticulum, plasma membranes and, to a lesser extent, mitochondria were observed upon culturing of calf aortic smooth muscle cells. These changes first appeared in cells growing out of tissue explants. They developed further upon subculturing of the cells and depended greatly on the culture conditions used. The alterations included increases in specific activities of some 5- to 25-fold of four acid hydrolases, an average ninefold increase in 5' -nucleotidase, sevenfold increase in cytochrome oxidase, and fourfold increase in neutral alpha-glucosidase in subcultured smooth muscle cells compared to aortic cells in situ. Cell fractionation studies showed significant shifts in the equilibrium densities of plasma membranes, microsomes, and lysosomes, but not of mitochondria, in smooth muscle cells growing out from explants and in subcultured cells, compared to cells isolated from intact aortas. Although the cells grown in vitro exhibited typical phenotypic features of smooth muscle cells such as abundant myofilaments and surface vesicles, alterations in the morphological appearance of the endoplasmic reticulum, Golgi apparatus, and, especially, lysosomes were observed. These results demonstrate significant differences in specific cellular characteristics and functions of aortic smooth muscle cells grown in vitro compared to aortic cells in situ.


Macrophage deformability and phagocytosis. The influence of several metabolic inhibitors and pharmacologic agents on macrophage deformation (induced by fluid shear stress) was examined in relationship to changes in ATP content and phagocytosis of latex beads. Two relatively specific inhibitors of glycolysis (iodoacetate [IA], and sodium fluoride [NaF]) and a sulfhydryl-binding agent (N-ethylmaleimide [NEM] markedly inhibited phagocytosis and reduced cell deformability. A microtubule-disrupting agent (vinblastine) and a highly specific inhibitor of glycolysis (2-deoxyglucose) markedly inhibited phagocytosis without influencing cell deformability. An organomercurial sulfhydryl binding agent p-chloromercuribenzene (PCMBS) and a microfilament-disrupting agent (cytochalasin B) inhibited phagocytosis and increased cell deformability. The effects of these agents on phagocytosis and cell deformability bore no consistent relationship to alterations in cellular content of ATP. The observation that 2-deoxyglucose, the most specific inhibitor of glycolysis examined, reduced ATP content to levels far lower (15 percent of control values) than those achieved by any other agent examined and inhibited phagocytosis without altering cell deformability, suggests that alterations in cell deformability induced by NaF, IA, NEM, PCMBS, and cytochalasin B are not due to inhibition of glycolysis per se, but instead result from direct or indirect effects of these agents on cell constituents, possibly contractile proteins, which are determinants of cell deformability. The finding that cytochalasin B, NEM, PCMBS, and IA interfere with phagocytosis and alter cell deformability, together with evidence that these agents interact with isolated actin and myosin, suggests that contractile proteins are important both in phagocytosis and as determinants of cell deformability. The observation that vinblastine, colchicines, and heavy water (D(2)O) did not alter cell deformability, even though vinblastine caused formation of intracellular crystals of microtubular protein, indicates that microtubules are not major determinants of cell deformability. The observations that beads adhered normally to surfaces of cytochalasin B- and of PCMBS-treated cells and that shear-stress induced deformation was increased whereas phagocytosis was markedly inhibited, suggest that deformation of cells around beads associated with ingestion depends on some form of cellular (contractile?) activity, whereas deformation of cells by fluid shear stress is a passive phenomenon.


High-pressure liquid chromatography of amino acids and dipeptides on a tripeptide bonded stationary phase. A bonded tripeptide (L-valyl-L-phenylalanyl-L-valine) phase has been used as a stationary phase in liquid chromatography. Unique retention orders and retention variations are shown for phenylthiohydantoin (PTH) derivatives of amino acids and isomeric dipeptides using acidic and basic mobile phases. It is shown that the 25 PTH-amino acids studied here have different capacity ratios when 1% citric acid in water (ca. pH 2.5) is used as the mobile phase. The analysis time for these amino acid derivatives could be shortened without a loss in the resolution by adding 5% methanol to the above mobile phase. Of particular importance is the fact that the basic amino acids are eluted first. Isomeric dipeptides have been resolved by using deionized water (pH 5.5), and 1% sodium citrate in water (pH 7.9), as mobile phases. The data show that the nature of the mobile phases, the pH, and the ionic strength are the important factors affecting the selectivity and efficiency of the separations of amino acids and dipeptides.


Analysis of polybrominated biphenyls.

Analysis of polybrominated biphenyls. High-performance liquid chromatography, gas chromatography-mass spectrometry, ultraviolet spectrometry and nuclear magnetic resonance spectrometry have been used to characterize the behaviour of a number of individual brominated biphenyls and to assess the composition of several mixtures of these compounds. The dependence of retention and spectral characteristics on the bromine content of the substituted biphenyls is discussed and the behaviour of polybrominated biphenyls is compared with that of polychlorinated biphenyls. The applicability of high-performance liquid chromatography to photodegradation studies is demonstrated.


Peptide separations using fluorescence detection. The formation of fluorophores by the action of o-phthalaldehyde with amino acids and peptides has provided a highly sensitive assay for these compounds. A relatively simple system for the analysis and separation of peptides, in the range 5 nmole to 10 micromole, normally derived from enzymic digestion of proteins, is described. The system comprises a gradient-generating device feeding volatile pyridine buffers via a pump to a column of cation-exchange resin. Eluate from the column is fed through a proportioning pump to a fluorocolorimeter, output from which is displayed on a recorder. For analytical runs the eluate is mixed with o-phthalaldehyde in borate buffer containing Brij 35 and 2-mercaptoethanol prior to its passage into the detector. For preparative work the eluate stream is split, one reacting with 0-phthalaldehyde, the other for collection. Results on the analysis and preparation of tryptic peptides derived from cytochrome c and Salmonella histidinol dehydrogenase are discussed.


Filter paper solid-phase radioimmunoassay for human rotavirus surface immunoglobulins. A filter paper solid-phase radioimmunoassay has been developed. Filter paper disks adsorbed a large amount of rotavirus and serum globulin and gave small mean variation of coating and low background binding. The rotavirus isolated from stools from infants with acute enteritis 1, 3, and 4 days after onset of symptoms was shown to be already covered with immunoglobulin G (IgG), IgA, and IgM antibodies by this radioimmunoassay, by immunoelectrophoresis, and by immune electron microscopy. The immunoglobulins covering the virus particle were partially separated during 125I labeling and eluted at the position expected for IgG during Sephadex G-200 gel filtration. Rabbit antiserum prepared against purified fecal rotavirus contained not only rotavirus antibodies but also a fairly large amount of immunoglobulin antibody, reflecting the antibodies on the rotavirus particle surface.


Use of sodium polyanetholesulfonate-CaCl2 for removal of serum nonspecific inhibitors of rubella hemagglutination: comparison with other polyanion-divalent cation combinations. By using trypsin-treated human type O cells as indicators, we compared the abilities of four polyanion-divalent cation combinations (heparin-MnCl(2); high-and low-molecular-weight dextran sulfate-CaCl(2); and sodium polyanetholesulfonate [SPS]-CaCl(2)) for removal of serum non-immunoglobulin (lipoprotein) inhibitors of rubella hemagglutination. The combination of SPS-CaCl(2) was found to be the most effective, precipitating completely the pre-beta and beta-lipoproteins and reducing the alpha-lipoprotein levels by more than 50%. Hemagglutination patterns after this treatment were clear and stable, and, when normal sera were tested, hemagglutination-inhibition (HI) titers were comparable to those obtained after standard heparin-MnCl(2) treatment. High-molecular-weight dextran sulfate-CaCl(2) removed serum lipoproteins almost as effectively as SPS-CaCl(2). However, problems of nonspecific agglutination and the heavy hemagglutination patterns resulting made this combination unacceptable for routine purposes. Neither low-molecular-weight dextran sulfate-CaCl(2) nor heparin-MnCl(2) removed the pre-beta lipoproteins completely, and occasionally traces of beta-lipoprotein also remained after treatment. The presence of pre-beta lipoproteins in normal sera after treatment may be of no consequence in the HI test since we have found that the very-low-density lipoprotein fractions obtained by ultracentrifugal methods from normal sera (those corresponding to the pre-beta fractions obtained by electrophoresis) had no HI activity. However, very-low-density lipoprotein fractions from all hyperlipemic sera tested had HI activity (titers ranging from 1:16 to 1:1,024) which, in the majority of cases, was not eliminated after heparin-MnCl(2) treatment. In every case, treatment with SPS-CaCl(2) removed this nonspecific activity completely. Since hyperlipemic sera may occasionally be encountered in routine rubella HI antibody testing, we recommend the use of SPS-CaCl(2) rather than heparin-MnCl(2) for pretreatment of sera.


Vesicular stomatitis virus causes abortion and neonatal death in ferrets. Vesicular stomatitis virus caused transplacental infection in ferrets, resulting in infant death. No clinically significant illness was observed in the adult, despite histological changes in the uterus, liver, and spleen.


Multiple symmetric lipomatosis: a defect in adrenergic-stimulated lipolysis. The cellularity of normal and lipomatous adipose tissue and its response to different lipolytic agents have been studied in a group of 10 patients with multiple symmetric lipomatosis (MSL). In MSL patients, fat cells from lipomatous tissue are smaller than normal, uninvolved adipocytes. Fat cells from lipomata show minimal variations in size following conspicuous increase of lipomatous masses. These findings suggest that the growth of lipomata can be attributed to the neoformation of adipocytes rather than to an enlargement in the single fat cells. The incidence of reduced glucose tolerance and of hyperlipoproteinemia is similar in MSL patients and in controls. A significant reduction in plasma free fatty acids was observed in MSL patients after a 24-h fast as well as after noradrenaline infusion. A specific insensitivity of lipomatous tissue to the lipolytic effect of noradrenaline and isoprenaline was observed in vitro, as indicated by glycerol release in the medium, whereas response to theophylline and to dibutyryl cyclic AMP was retained. The lipolytic response to catecholamines was retained. The lipolytic response to catecholamines was normal in the nonlipomatous adipose tissue of MSL patients. In basal conditions ATP concentrations were similar in normal and in lipomatous adipose tissue. However, incubation with noradrenaline induced a significant fall in intracellular ATP levels in normal tissue, whereas no variations were observed in lipomatous tissue. Theophylline, instead, induced a prompt and significant decrease in intracellular ATP levels in lipomatous tissue. These observations indicate that the block in catecholamine-stimulated lipolysis in lipomatous tissue of MSL patients can be localized at a level preceding the formation of cyclic AMP.


Thyroid hormonelike actions of 3,3',5'-L-triiodothyronine nad 3,3'-diiodothyronine. l-Thyroxine is converted to 3,5,3'-l-triiodothyronine (T(3)) as well as to 3,3',5'-l-triiodothyronine (reverse T(3)). One product of further deiodination is 3,3'-diiodothyronine (3,3'T(2)). The serum levels of reverse T(3) and 3,3'T(2) change considerably in various physiological and disease states. We previously found that reverse T(3) and 3,3'T(2) bind to the solubilized hepatic nuclear "receptors" for thyroid hormones. This led us to study binding and actions of these metabolites in cultured rat pituitary cells in which glucose consumption and growth hormone production are regulated by T(3) and l-thyroxine. Reverse T(3) and 3,3'T(2) stimulated growth hormone production and glucose consumption and inhibited nuclear binding of radioactive T(3). Either metabolite produced maximal effects that equaled those of T(3), and neither inhibited the T(3) response. Further, additive effects were observed when reverse T(3) was combined with submaximal concentrations of T(3). In serum-free and serum-containing media, concentrations of 3,3'T(2) 50- to 70- and 10- to 100-fold greater, respectively, than those of T(3) were required for equivalent stimulations and for inhibition of nuclear binding by T(3). The relative activity differences under the two conditions can be attributed to weaker serum protein binding of 3,3'T(2) than T(3). With cells in serum-free media, reverse T(3) was a less avid competitor than 3,3'T(2) for T(3) binding by the nuclear receptors, and was less potent than 3,3'T(2) (0.001 the potency of T(3)) in inducing growth hormone production or glucose oxidation. In incubations with serum-containing media, reverse T(3) was an ineffective competitor for T(3) binding, and had only 0.1 the inducing potency of 3,3'T(2) (0.001 the potency of T(3)). The weaker activity of reverse T(3) relative to 3,3'T(2) in serum-containing media could be explained by stronger serum binding of reverse T(3) than 3,3'T(2). In addition, after long-term incubation of cells with radioactive reverse T(3), much of the cell-associated radioactivity was recovered as 3,3'T(2). These studies suggest that reverse T(3) and 3,3'T(2) can stimulate thyroid hormone-regulated functions as weak agonists by acting via the same receptors that mediate T(3) actions. Moreover, some of the effects of reverse T(3) may be due to 3,3'T(2) produced by deiodination of reverse T(3).


Neurogenic coronary vasoconstrictor effects of digitalis during acute global ischemia in dogs. The rapid i.v. administration of digitalis has recently been shown to cause a substantial increase in coronary vascular resistance in the normal heart. This neurogenically mediated decrease in coronary blood flow would be potentially detrimental if it occurred during ischemia. The present study evaluates the effects of i.v. acetylstrophanthidin and digoxin on coronary vascular resistance during acute global ischemia in 29 dogs anesthetized with chloralose and urethane. Under these conditions, 0.5 mg of i.v. acetylstrophanthidin in 15 dogs resulted in erratic increases in coronary vascular resistance. The peak rise was 12+/-5% above control (P less than 0.01). In 7 of the 15 dogs, the initial erratic rise in coronary vascular resistance culminated in a steep rise associated with acute elevation in left ventricular end-diastolic pressure, which in four dogs terminated in ventricular fibrillation. During the nonischemic control periods, the peak rise in coronary vascular resistance with acetylstrophanthidin was 16+/-1% above control (P less than 0.01). In five dogs, prior alpha adrenergic receptor blockade with phenoxybenzamine prevented the rise in coronary vascular resistance with acetylstrophanthidin during ischemia. Similar erratic increases in coronary vascular resistance were observed with i.v. digoxin (1 mg) during ischemia in three dogs. In two of these dogs, there was a progressive rise in coronary vascular resistance associated with elevation of left ventricular end-diastolic pressure and ventricular fibrillation. The increase in coronary vascular resistance with digoxin during ischemia was abolished with phenoxybenzamine in two additional dogs. Thus, i.v. digitalis in the ischemic heart results in potentially detrimental increases in coronary vascular resistance mediated through alpha adrenergic receptor stimulation.