Mouse hepatitis virus (MHV-2). Plaque assay and propagation in mouse cell line DBT cells. Various factor sinfluencing the plaque formation of mouse hepatitis virus (MHV-2) in DBT cell monolayers were studied and a practical method for plaque assay was developed. Infected DBT cells yielded high-titered virus and were a satisfactory source of complement-fixing viral antigen. The predominant cytopathic effect of MHV-2 in DBT cells was cell rounding and detachment, but no syncytial formation was observed. Fluorescent antibody staining revealed specific fluorescence only in the cytoplasm of infected DBT cells. In one-step growth experiment, newly formed virus was first recognized within 4-hr postinfection and showed subsequently a rapid exponential increase. Release of newly formed virus from the cell was rapid, and a continuous release lasted for a certain period of time. The average per-cell yield of active virus was estimated to be about 6-7 X 10(2) plaque-forming units.


Preferential phosphorylation of NP-protein of influenza A2 virus by virion-associated protein kinase. Influenza A2 virions were found to contain protein kinase activity which was stimulated, like in other virion-associated kinases, with Mg++ and Nonidet-P 40 but not with cyclic AMP. The kinase phosphorylated only the NP-protein fraction of the influenza virions in the in vitro reaction. In contrast, none of the influenza virion proteins were phosphorylated significantly during the process of virus production in infected chorioallantoic membranes. The in vitro and in vivo phosphorylations of influenza viral proteins were compared with those of Sendai virus (HVJ).


[Disorder of the regulation of metabolic processes in coronary arteriosclerosis]. The activity of adenylcyclase was determined in the thrombocytes of 54 male patients with Stage III coronary atherosclerosis. In 34 patients without signs of ischaemic heart disease the activity of adenylcyclase and phosphodiestherase and the amount of cortisol were determined in blood plasma. In 40 rabbits the same enzymes activity was determined in the thrombocytes and lipid tissue, as well as the amount of lipoproteins of low and very low density was determined in blood plasma prior to and following cortisol administration. The results of clinical and experimental studies have demonstrated that cortisol affects the activity of the adenylcyclase system enzymes in the thrombocytes and lipid tissue, as well as the transformation of lipoproteins in blood plasma.


[Lipid composition of lipoproteins and excretion of 17-ketosteroids in coronary arteriosclerosis]. Blood plasma cholesterol and triglycides concentration lipids composition of lipoproteins as contrasted to the amount of 17-KS and their hormonally active fractions excreted were investigated in 87 patients with myocardial infarction in their history and in 49 practically healthy individuals with no clinical manifestations of ischemic heart disease. In patients with coronary atherosclerosis exhibiting normal blood plasma lipids level, the excretion of 17-KS and of their fractions did not differ from that in healthy individuals. Patients with hyperlipoproteinemia of the IIa, IIb and IV types demonstrated a significantly reduced excretion of 17-KS, etiocholanolone, androsterone and dehydroepiandrosterone. No differences in the excretion of androgens depending upon the type of hyperlipoproteinemia were recorded. A significant negative correlation between the blood plasma cholesterol concentration and the amount of ethocholanolone excreted, the level of hypertriglyceridemin and the passage of dehydroepiandrosterone with urine was noted. In patients with hyperlipoproteinemia and a reduced androgens excretion a deranged lipids composition of lipoproteins was disclosed. A decrease in the amount of androgens leads to disruption of the synthesis and metabolism of lipoproteins and exerts a marked influence on the emergence and further development of hyperlipoproteinemia.


[Adenyl cyclase activity in the tissues and excretion of cyclic adenosine monophosphate in experimental arteriosclerosis]. The activity of adenylcyclase in the tissues and the elimination of 3',5'-AMP with urine were studied in rabbits and rats with experimentally induced atherosclerosis. The relationship between the concentration of steroid hormones and the activity of adenylcyclase in the adrenals was studied after varying periods of atherogenic diets. The activity of adenylcyclase was shown to decrease in the liver and the adrenal glands, as well as the concentration of corticosterone in the adrenals and the urine excretion of 3',5'-AMP, when the animals were kept on the atherogenic diet for long periods of time. No changes in the activity of adenylcyclase were noted in the lipid tissues of the animals with atherosclerosis. The concentration of the steroid hormones in the animals with experimental atherosclerosis was directly dependent on the activity of adenylcyclase in the tissues and the urine excretion of 3',5'-AMP. The administration of Cortisole resulted in a sharp increase of 3',5'-AMP excretion and an activation of adenylcyclase in the liver and lipid tissues of both normal animals, and those kept on an atherogenic diet.


[Hormonal regulation of lactation (author's transl)]. Lactation is controlled by hormones from several endocrine glands. An undisturbed function of the anterior pituitary, of the adrenals, and of the ovaries is a prerequisite for a normal morphogenesis of the mammary gland. The epithelial ducts proliferative under the combined influence of estrogens, glucocorticoids and growth hormone, whereas the lobuloalveolar development depends on progesterone and prolactin in addition to the fore-mentioned hormones. During pregnancy pituitary prolactin may be substituted by placental lactogen. Milk synthesis begins in the second half of pregnancy. It is supported by prolactin and cortisol, which directly act on enzyme activities and processes of differentiation of the alveolar cells. The sudden surge in the secretion of milk after parturition is most likely due to the rapid decline of the serum levels of progesterone. The ejection of milk from the lactating mammary gland is controlled by a neuroendocrine reflex mechanism. Suckling is the appropriate stimulus for the release of oxytocin from the posterior pituitary. Oxytocin increases intramammary pressure by inducing contraction of the myoepithelial cells and thus aids in expelling the milk from the mammary glands. Maintenance of normal postpartum lactation depends on frequent and intensive suckling. Suckling does not only stimulate the release of oxytocin, but also provokes secretion of prolactin and ACTH. This increase in prolactin caused by suckling guarantees galactopoesis. Influencing secretion of prolactin has been proven to be a useful tool for regulating lactation. The experimental ergot derivative 2-Brom-alpha-ergocryptine is a potent suppressor of prolactin secretion from the anterior pituitary. In contrast to estrogens, alone or in combination with progestagens or androgens, this drug is not only effective in suppressing the onset of lactation, but also in inhibiting lactation once milk secretion had started. As to stimulating lactation in the human there is no effective drug available up to now.


[Cerebellar vincristine toxicity (author's transl)]. The diagnosis of Wilms tumor stage IV was established in a girl of 5 1/2 years of age in July 1972. Nephrektomy was followed by radiotherapy of the tumorbed and the lung metastasis. Thereafter the child was treated with Vincristine (VCR) and Actinomycine D (AMD) for two years. The child has been off treatment for the last 14 months and is free of disease. An intention tremor of the right hand was noted 4 weeks after the 32nd application of VCR and 2 weeks after a viral infection of unknown etiology. The writing proved to be hypermetric. There was no loss of proprioception and thus the symptoms were localized into the right cerebellum. Having excluded other causes (hemorrhage, metastasis) as far as possible and considering unilateral cerebellar encephalitis as very unlikely the cerebellar symptoms were thought to be VCR toxicity.


[Adriamycin cardiomyopathy in a child with Wilms tumor and liver metastases (author's transl)]. Nephrectomy with irradiation was performed in a 3;2 year old boy with Wilms tumor stage IV (livermetastases (liver metastases). Six months later a hemihepatectomy was performed on account of the liver metatases which persisted under a combined Actinomycin Vincristin therapy. A monotherapy with Adriamycin was instituted. The patient developed acute congestive heart failure. The patient developed acute congestive heart failure and showed typical EKG changes after a total dose of 700 mg/M2 of Adriamycin. In comparison to adults children reportedly tolerate somewhat higher total doses of Adriamycin. Therefore it is discussed, if in this case a limitation of liver function by hemihepatectomy and by irradiation has favored the appearance of the Adriamycin-induced cardiomyopathy.


[Combination of the syndrome of Sturge-Weber and the syndrome of Klippel-Trénaunay (author's transl)]. Up until now 39 cases of combined Klippel-Trénaunay syndrome and Sturge-Weber syndrome have been described. Here follows the report of a girl, now 4 years of age, displaying a full combination of these syndromes. Only a small part of the body surface is not covered with naevi teleangiectatici laterales. The patient has clear hypertrophy of the left cheek and of the left lower extremity, less noticeable on the left upper extremity. For therapeutic reasons the left side of the head and the left lower extremity were thoroughly angiographically examined--this revealed typical abnormalities. The vessel-alteration of the lower extremity are not extremely far developed and arteriovenous fistulas on a large scale are also absent. This allows us to dismiss the F.P. Weber syndrome on the one hand, while it explains the absence of complications of the Klippel-Trénaunay syndrome, as described in literature, on the other. The significance of the alterations of lymph nodes in this disease, which we are the first to describe, is at present not fully clear. The cerebral attacks have until now showed only a temporary response to medication.


An NZB virus or NZB mice with viral infections? Evidence for and against the possibility that the autoimmune disease of NZB mice has a viral aetiology is presented. It is considered that the case for a viral aetiology is unproven, although the possibility exists that virus may be present in incomplete form. Widely varying experimental results can be expected in experiments involving NZB mice until acceptable standards of mouse strain definition are laid down. Thus the comparison of results obtained from studies of NZB mice of diverse origin may invite misleading conclusions.


Black alcoholism. A comment on NIAAA's plan to combat the problem. The National Institute on Alcohol Abuse and Alcoholism's Interim Guidelines for the Establishment of Black Alcoholism Projects are criticized for their possible latent function of redirecting treatment energies away from the primary problem of alcoholism.


[Enzyme deficiencies in glycolysis and nucleotide metabolism of red blood cells in nonspherocytic hemolytic anemia (author's transl)]. The detection of enzyme deficiencies in glycolytic and nucleotide metabolism of human red blood cells has enriched the pathophysiological knowledge on the origin of nonspherocytic hemolytic anemias (NSHA). So far for 11 of 13 glycolytic enzymes deficiencies have been described which are connected with alterations of biochemical enzymatic properties. The most frequent enzyme deficiencies are those of GPI and PK. By performance of special electrophoretic techniques genetic studies allow the demonstration of homozygote and double heterozygote defect carriers. Up to now only adenylate kinase and pyrimidine 5' nucleotidase deficiencies have been detected as genetically determined in altered nucleotide metabolism. The metabolic alterations of several enzymopathies have been characterized so well, that the pathophysiological relations between enzyme deficiency and NSHA probably have been found to be a sufficient explanation.


[Malignant case of angiofibroma (author's transl)]. Various opinions about the etiopathogenesis of angiofibroma are mentioned. The authors, however, have not succeeded in confirming the frequently mentioned theory of hormonal formation of these tumours. It appears that surgical therapy is the only efficient way today of providing a clear view and a rapid radical removal of tumours, thus reducing the usually profuse haemorrhage during surgical treatment. Pre-operative ligation of the external carotid artery has no noticable effect on the reduction of haemorrhage during the operation. X-rays have no effect on the growth of the tumours. There is no great difference between the histological picture of a primary tumour and that of its recurrences. The malignant course of an angiofibroma in an adult is described which recurred several times and led to the involvement of the endocranium with a letal ending. The histological picture remained unchanged all the time. A spreading of the tumour was noticed in areas distant from the primary localisation of the tumour which we consider to be due to implantation and which indicates great potential growth of tumour cells of this angiofibroma.


[Lymphographic investigation of the face-neck-region (author's transl)]. Indirect lymphography is shown to be a technically uncomplicated and easy to use method of displaying the lymph nodes and -vessels of the face and neck regions. "Lipiodol-Ultra-Fluid" an oily contrast medium which has used in experiments with minipigs can be radiologically and histologically demonstrated in nodes for months. The histological findings in the course of a year are, at the beginning a phase of non-specific reaction followed by a phase of granulomatous reaction between two weeks and six weeks and finally a third phase where in the medium is stored virtually without histological reaction and is only reduced very slowly. Irreversible destruction of lymph-node structure as not infrequently follows the direct method - due to high-pressure application of the fluid was not evident. It may be assumed that disturbance of lymph node function or changes in lymph flow do not follow the application of the indirect method of lymphography.


[Simultaneous estimations of flowrate, total protein,- lysozyme- and immunglobulin-A-secretion in parotid glands with mixed tumours (author's transl)]. In comparison to normal gland secretions 28 patients with mixed tumors of the parotid glands are examined. Thereby fractionated secretions under rest and stimulation are collected and estimations of flowrate, protein-, lysozyme- and immunglobulin A-secretion are done. The following results are remarkable: 1. After pilocarpinstimulation response of the glands 20 to 30 min later than in normal glands. 2. Protein-secretion is diminished in rest and under stimulation. 3. Immunglobulin A and lysozyme secretion show a greater range than normal glands under rest and stimulation.


[The epidemiology of gastric cancer (author's transl)]. Gastric cancer is a highly fatal, common form of cancer in many countries and its medical, surgical, radiologic or combined treatment is very problematic. Thus, a search for its cause and prevention may be more promising than a search for its cure. Epidemiologic data from international studies suggest that environmental factors are more influential than genetic ones in the pathogenesis of gastric cancer, although its causes have not yet been identified. It is likely that the disease has a multi-factorial pathogenesis and that an ingested carcinogen may exert its influence at a time of enhanced susceptibility (damaged mucosa, older age, etc.), particulary in individuals with inherent, elevated risk (males, selected hereditary background etc.). In general, dietary studies have been less rewarding than occupational and geographic-pathologic analyses; such investigations may eventually identify the crude sources of carcinogens which then can be isolated by chemical means.


A study of the heparin-manganese chloride method for determination of plasma alpha-lipoprotein cholesterol concentration. To assess the limits of the heparin MnCl2 precipitation method for quantitation of alpha-lipoprotein cholesterol (C-HDL), effects of varying final McCl2 and heparin concentrations were studied, and the precipitation method was compared to preparative ultracentrifugation. In 65 parallel plasma aliquots, C-HDL (X+/-SE) determined by ultracentrifugation (54.3+/-1.8 mg/dl) correlated significantly (r=0.98, P less than 0.001) with the precipitation method (56.0+/-1.9 mg/dl). C-HDL by ultracentrifugal and precipitation methods were also similar in 16 subjects with triglycerides ranging from 150 to 312 mg/dl (41.4+/-2.6, 43.4+/-2.8, r=0.97, P less than .001). A constant amount of cholesterol in the supernatant was measured over a final McCl2 range of 0.046-0.23 M, and cholesterol values in the supernatant at final McCl2 concentrations of 0.046, 0.05, and 0.055 M did not differ from each other, P greater than 0.1. However, cholesterol levels in the supernatant at final MnCl2 concentration of 0.042 M differed from those at concentrations of 0.046, 0.05, and 0.055 M, P less than 0.05 and the amount of supernatant cholesterol increased as the final McCl2 concentration was reduced from 0.042 to 0.02 M. A constant amount of cholesterol in the supernatant was measured over a heparin concentration range of 92-734 USP units/ml. The final MnCl2 and heparin concentrations of 0.046 M and 184 USP units/ml, which are incorporated in widely used procedures, gave C-HDL values for the precipitation method which were in close agreement with the ultracentrifugal method. There is no evidence for a heparin-Mn++ precipitation of HDL and systematic underestimation of HDL by the precipitation method. However, the final MnCl2 concentration is very near the minimum required for accurate measurement of C-HDL. To preclude incomplete precipitation of low and very low density lipoproteins by insufficient manganese concentration, an increase of the manganese concentration should be considered.


Effect of chronic administration of propranolol on lipoprotein composition. Chronic effects of propranolol on plasma lipids and lipoprotein composition were examined in ten patients who had previous strokes and normal plasma lipids. Although plasma triglyceride and total cholesterol were not affected by propranolol, a slight decrease of free cholesterol and phospholipids and a significant increase of free fatty acids were observed in the eighth week of propranolol treatment. Reciprocal changes were observed in lipoprotein composition; these were an increase in lipids of very low-density lipoprotein and a decrease in lipids of both low-density and high-density lipoproteins. Postheparin lipolytic activity was significantly suppressed by the administration of propranolol. Inhibition of lipoprotein lipase by propranolol was considered to have played a role in the reciprocal changes of lipoprotein composition.


Renal cyclic adenosine monophosphate: an accurate index of parathyroid function. Measurement of total urine cyclic 3':5'-adenosine monophosphate (cyclic AMP) only incompletely discriminates between normal, hyperparathyroid, and nonparathyroid hypercalcemic patients. Only a fraction of total urine cyclic AMP is contributed by parathyroid hormone (PTH) action on the proximal nephron (renal cyclic AMP); the remainder is derived from plasma by glomerular filtration. We dtermined total urine and plasma cyclic AMP and PTH (by carboxy-terminal specific radioimmunoassay) in control, hyperparathyroid, nonparathyroid hypercalcemic, and surgically hypoparathyroid patients. Renal cyclic AMP was calculated as total urine cyclic AMP minus the filtered component. Of these determinations, only renal cyclic AMP segregated normal from hyperparathyroid, and hyperparathyroid from nonparathyroid hypercalcemic patients with complete accuracy. These data suggest that measurement of renal cyclic AMP provides an accurate index of parathyroid activity and allows clinical discrimination and appropriate treatment of the sub-groups of patients with malignancy and nonparathyroid hypercalcemia from those with hyperparathyroidism.


Calcium-dependent regulation of guanosine 3',5'-monophosphate in renal cortex: effects of ionophore A23187 and tetracaine and evidence for independent control of adenosine 3',5'-monophosphate. The effects of carbamylcholine (Cch), the divalent cation ionophore A23187 and Ca2+ on the cyclic 3',5'-guanosine monophosphate (cGMP) and cyclic 3',5'-adenosine monophosphate (cAMP) content of rat renal cortical slices were examined. In both the presence and absence of 10 mM theophylline, Cch detectably increased cGMP within 15 sec, with peak responses noted by 2 min. The maximal cGMP response to Cch alone (0.05 mM) was an increase of two- to three-fold over control. Theophylline, which was routinely present in the incubations and which alone increased cGMP of the slices two-fold over basal during 20 min incubations, potentiated the response to Cch (maximal increase, five- to sixfold over theophylline alone). The action of Cch to increase renal cortical cGMP was blocked by prior addition of atropine and was dependent upon the presence of Ca2+ in the incubation media. Exclusion of Ca2+ lowered basal cGMP and abolished increases mediated by Cch, while exclusion of Mg2+ was without detectable effect on cGMP. In slices incubated initially without Ca2+, reexposure to Ca2+ for 1min partially restored the cGMP response to Cch, and reexposure for 3 min completely restored this response. Since prior incubation of tissue in Ca2+-free buffer for only 2 min was sufficient to block the cGMP responses to Cch, depletion of tissue Ca2+ did not appear to be involved. A23187 also increased renal cortical cGMP fivefold in the presence of Ca2+. Its effects were not additive with those of Cch and were not additive with those of Cch and were not expressed by Mg2+ in Ca2+-free media. By contrast, tetracaine, which blocks Ca2+ transport across or binding to biologic membranes, reduced basal cGMP and inhibited the actions of Cch and A23187 to increase cGMP in cortical slices incubated with Ca2+. The action of 1 mM tetracaine to block Cch-mediated increases in cGMP was partially reversed by increasing media Ca2+ from 1.5 to 5 mM, but not by increasing media Mg2+ to 5 mM. In contrast to their effects on cGMP, Cch, A23187, Ca2+ exclusion, and tetracaine did not detectably alter basal renal cortical cAMP or cAMP responses to parathyroid hormone (PTH). Conversely, concentrations of PTH, glucagon, and isoproterenol which maximally increased renal cortical cAMP did not alter cGMP. Furthermore, prior incubation of slices with Cch did not alter their subsequent cAMP response to PTH at a time when cGMP levels were still elevated, while prior incubation with PTH did not affect the subsequent cGMP response to Cch at a time when cAMP was increased. These studies demonstrate modulation of renal cortical cGMP by cholinergic stimuli and Ca2+. They also indicate that cGMP and cAMP in renal cortex can be regulated independently.


Mechanism of inhibition of organic acid transport in rabbit renal cortex by cyclic AMP. The mechanism by which isoproterenol and cyclic AMP inhibit organic acid transport was examined. Accumulation of 131I-Hippuran (S/M) was used as an index of organic acid transport. Propranolol, an antagonist of isoproterenol's beta-adrenergic response, not only inhibited accumulation of Hippuran but, when combined with isoproterenol, caused further inhibition. PTH elicited larger increases than isoproterenol in cortical-slice cyclic AMP content but did not inhibit accumulation of Hippuran. This lack of correlation between a positive effect on the adenylate-cyclase-cyclic AMP system and inhibition of transport was also seen when other agents were tested. Cyclic GMP, which has been postulated to work in opposition to cyclic AMP, was as potent as cyclic AMP in decreasing S/M. The decreasing of S/M by adenine and uric acid could not be related to any known effect of these agents on tissue cyclic AMP content. Similarly, although 0.1 mM theophylline significantly decreased S/M, cortical cyclic-AMP content was not increased until a 100-fold greater concentration of theophylline was employed. The data suggest that the inhibitory effect of these agents was attributable to their molecular configurations, which interact directly with the organic acid transport system rather than indirectly via the adenylate-cyclase-cyclic AMP system.


High density lipoproteinuria in nephrotic syndrome. Intravenous administration of the aminonucleoside of puromycin produces the nephrotic syndrome (proteinuria, hypercholesterolemia, hypoproteinemia and edema) in rats. This model is very similar to human nephrotic syndrome caused by various disease states. The current study was designed to assess the nature of urinary lipoproteins in the urine of nephrotic rats, including studies related to the urinary loss of the "activator" apolipoproteins for the lipoprotein lipase-triglyceride interaction. Sprague-Dawley rats were given a single intravenous injection (10 mg/100 g) of puromycin aminonucleoside. Plasma and urine were collected before and 7, 18, 29, 36, and 53 days after injection of puromycin. Urine was fractionated in the preparative ultracentrifuge into density (d) fractions less than 1.006 (very low-density lipoproteins), d = 1.006-1.063 (low-density lipoproteins), and d = 1.063-1.210 (high-density lipoproteins--HDL). The cholesterol, triglyceride, phospholipid, and protein content of these fractions was analyzed. Lipoprotein electrophoresis was performed in agarose agar. Urine from normal and nephrotic rats was added to an in vitro system containing lipoprotein lipase and triglyceride. The free fatty acids (FFA) liberated were then measured as an index of urinary activator property on this system. Measurable urinary lipoproteins were present only on days 7 and 18 after induction of the nephrotic syndrome. Coelectrophoresis of these urinary lipoproteins with rat plasma revealed a single band having alpha- (HDL) electrophoretic mobility. The total mean protein content of day-7 urinary lipoproteins (64.3%) was greater than the content of plasma HDL (52.9%). The protein content of urinary lipoproteins also increased with time. When day-7 and day-18 postinjection urine at nephrotic rats was added to the lipoprotein lipase system, the hydrolysis of triglyceride yielded a mean of 0.320 and 0.235 muEq FFA/ml/20 min, respectively. Control rat urine yielded 0.030 muEq FFA/ml/20 min and 0.000 muEq FFA/ml/20 min 7 and 18 days after injection of normal saline, respectively. It is inferred that in this experimental model (1) high-density lipoproteins are probably excreted in the glomerular filtrate, (2) alterations in the composition of the excreted lipoproteins may occur during their passage through the nephron. The possibility that only a selective portion of the HDL spectrum is excreted into the glomerular filtrate cannot be excluded. It is suggested that the urinary or renal loss of this functionally important lipoprotein may contribute to the pathophysiology of hyperlipoproteinemia in the nephrotic syndrome.


Diagnostic value of serum lipoprotein X in jaundice. The presence of lipoprotein X (LPX) was assessed in the serum of 61 jaundiced patients and of 41 patients without liver disease. The results are compatible with the reports of other authors that the presence of LPX is a more sensitive indicator of cholestasis than the serum alkaline phosphatase (SAP) level. However, the latter was more useful in excluding the presence of extrahepatic obstruction.


[Experiences with drug therapy of advanced metastatic carcinoma of the breast (author's transl)]. A group of patients with metastatic breast cancer treated by a distinct drug regimen is analyzed with special respect to clinical and psychological problems. The range of remissions in this study was similar to literature dates. General limitations of indication for cytostatic drugs and of long-time-therapy are discussed. The possible relations of histologic type of primary carcinoma to the efficiency of chemotherapy is mentioned.


Lipoproteins versus structural glycoproteins: atherosclerosis as an analogue and competitor of normal connective tissue interactions. There is a striking resemblance, in amino-acid composition and properties, between one class of structural glycoproteins from connective tissue and the apo low density lipoproteins (LDL) of blood plasma. It is suggested that there is a structurally specific analogy between the early atherogenic accumulation of LDL (and more particularly Lp(a), in arterial connective tissue and the normal interactions of structural glycoproteins with other connective tissue components, particularly proteoglycans and collagen.


Methionine-repressible homoserine dehydrogenase of Serratia marcescens: purification and properties. Serratia marcescens Sa-3 possesses two homoserine dehydrogenases and neither has any aspartokinase activity unlike the case of Escherichia coli enzymes. The two enzymes have been separated. One of them is active with either NAD+ or NADP+ and has been purified about 180-fold to homogeneity. This enzyme is completely repressed by the presence of 1 mM methionine or homoserine in the growth medium, but its activity is unaffected by any amino acid of the aspartate family either singly or together. In many of its properties (such as pH optimum, Km for substrate and cofactors), it resembles its counterpart in E. coli K12. Potassium ions stabilize the enzyme but are not essential for activity. Its molecular weight is around 155,000 as determined by gel filtration and approximately 76,000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme has two subunits (polypeptide chains) in the molecule: 8 M urea has no effect on enzyme activity. This enzyme represents approximately 30% of the total homoserine dehydrogenase activity of S. marcescens unlike in Salmonella typhimurium and E. coli K12 where it is a minor or a negligible component.


The effect of p-hydroxymercuribenzoate and congeners on microsomal glucose-6-phosphatase. Iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate (p-MB) and HgCl2 were tested as inhibitors of microsomal glucose-6-phosphatase. Iodoacetamide had no effect at 2 mM. N-ethylmaleimide inhibited only crude, but not purified microsomal preparations (M2) or crude microsomes exposed to deoxycholate. 14C-labelled N-ethylmaleimide was not bound by the M2 protein fraction. p-MB inhibited all types of preparations and the inhibition was not counteracted by detergent. A more detailed study was carried out with the purified M2 fraction (specific activity: 2-4 mumoles Pi/min/mg protein). Glucose-6-phosphate hydrolysis was inhibited 50% by 5 X 10(-5) M p-MB. The inhibition was completely reversible by dithiothreitol except when the enzyme was pre-incubated with p-MB in the absence of substrate. Then p-MB accelerated the temperature-dependent inactivation of glucose-6-phosphatase. Binding studies showed that around 3 mumoles 14C-p-MB were incorporated into 100 mg M2 protein regardless of the concentration of mercurial in the incubation mixture. That is, over a 25 fold range of p-MB concentration, causing up to 80% inhibition of enzyme activity, no difference was seen in the amount of labelled p-MB which was irreversibly bound to M2 protein. Kinetically p-MB behaved like a reversible inhibitor and this was confirmed by dilution experiments. Several compounds, including some amino acids, antagonized the inhibition by p-MB. The order of effectiveness was EDTA greater than barbital greater than tryptophan greater than histidine greater than lysine greater than other amino acids. Glycine, Tris and urea were ineffective competitors of p-MB inhibition. Double reciprocal plots showed that the Km for glucose-6-phosphate was increased and the Vmax reduced in the presence of p-MB. HgCl2 was a more effective inhibitor than p-MB with a Ki of 6 X 10(-6) M. We conclude that a reaction of p-MB with M2 sulfhydryls does not play a part in the inhibition of enzyme activity. It is suggested that p-MB may interact with one or more amino acid side chains in such a way that enzyme conformation is altered.


The action of insulin and dibutyryl cyclic AMP on the biosynthesis of polyunsaturated acids of alpha-linolenic acid family in HTC cells. Incubation of HTC cells (7288 C) with 114C-alpha-linolenic acid in Swim's 77 medium during 24 hours converted the fatty acid to octadeca-6,9,12,15-tetraenoic acid, eicosa-11,14,17-trienoic acid, eicosa-8,11,14,17-tetraenoic acid, eicosa-5,11,14,17-tetraenoic acid, eicosa-5,8,11,14,17-pentaenoic acid and unsaturated acids of 22 carbons. The existence of two pathways was recognized: one initiated by a delta6-desaturation and the other by an elongation of alpha-linolenic acid. Incubation of the cells with insulin and dibutyryl cyclic AMP modified both pathways in different ways. HTC cells were sensitive to insulin which enhanced the desaturating route increasing eicosapentaenoic acid synthesis and depressed the elongating route decreasing eicosatrienoic acid. In an opposite way, dibutyryl cyclic AMP decreased eicosapentaenoic acid synthesis and increased eicosatrienoic acid.


[Treatment of primary hyperlipoproteinemias. Comparison of N 041 with clofibrate]. The effect of essential phospholipids in combination with Clofibrate (N 041) was compared with that of Clofibrate alone in a double blind study in 96 patients with hypertriglyceridemia and/or hypercholesterolemia. The serum triglycerides fell more markedly with N 041 than with Clofibrate therapy alone. The greater cholesterol-lowering effect of N 041 was significant after treatment lasting 3 weeks and just failed to reach the 5% significance level after 6 weeks. Both preparations were well tolerated.


Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers. The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.


Studies on chemically induced dominant lethality. II. Cytogenetic studies of MMS-induced dominant lethality in maturing dictyate mouse oocytes. Young adult female mice were injected intravenously with either 50- or 100- mg/kg doses of methyl methanesulfonate. The females were superovulated and mated to untreated males at intervals ranging from 0.5 to 14.5 days after treatment. The fertilized ova were collected and cultured to the first cleavage mitosis, at which time the female chromosome complement was analyzed for structural chromosomal damage. Chromatid-type aberrations were observed, but at a much lower frequency than previously reported for treatment of post-meiotic male germ cells. The time after treatment at which chromosomal damage was observed and the frequency of affected cells agree, qualitatively, with existing dominant-lethal data derived from treatment of maturing oocytes. Parallel experiments in which metaphase I oocytes were analyzed indicate a lack of MMS-induced chromosomal damage in the meiotic stages. This observation is consistent with the suggestion that an intervening round of DNA synthesis is necessary for MMS-induced lesions to be translated into chromosomal damage. The low yield of chromosomal damage is consistent with the idea that maturing oocytes, unlike later spermatids and spermatozoa, are capable of performing macromolecular repair of premutational lesions.


Concentration of 3', 5' cyclic adenosine monophosphate in ventricular cerebrospinal fluid of patients with prolonged coma after head trauma or intracranial hemorrhage. A previous study showed that cerebrospinal fluid from the lateral ventricle of patients without disturbance of sensorium or intracranial pressure contains 15 to 30 nm 3', 5' cyclic adenosine monophosphate. We measured the concentration of this cyclic nucleotide by radioimmunoassay in cerebrospinal fluid from the lateral ventricle of six patients with prolonged coma (20 days or longer) after head trauma (four), or spontaneous intracranial hemorrhage (two). Coma was graded IV to I in order of decreasing severity. Fluid was removed at intervals of six to 72 hours from a Rickham reservoir placed in the lateral ventricle. Concentration of the cyclic nucleotide (mean +/- S.E.M.) in coma of Grades IV, III, II and I was 2.1 +/- 0.3, 4.6 +/- 0.5, 6.3 +/- 1.4 and 12.5 +/- 2.4 nM respectively. After sensorium became normal, cAMP was 21.0 +/- 1.4 nM. Correlation between grade of coma and concentration was -0.89 (P less than 0.01). Thus, prolonged coma appears to be associated with a disturbance of cyclic AMP metabolism within the central nervous system.


Big renin and biosynthetic defect of aldosterone in diabetes mellitus. To determine the cause of selective aldosterone deficiency in two patients with diabetes mellitus, studies of renin and of aldosterone-precursor metabolites were performed under conditions of sodium depletion and ACTH stimulation. Plasma renin concentration was elevated in both patients, and stimulated plasma renin activity was low in one and normal in the other. Fractionation of plasma extracts demonstrated the presence of "big renin," a relatively inactive precursor of renin. Metabolites of aldosterone precursors were increased, suggesting deficient 18-hydroxylase in one patient and dehydrogenase in the other. The results suggest that hypoaldosteronism in diabetic patients may result from combined defects in both renin and aldosterone biosynthesis.


Leydig-cell agenesis: a cause of male pseudohermaphroditism. We studied a 35-year-old patient with female external genitalia, primary amenorrhea and XY karytotype. Plasma testosterone was 10 ng per deciliter, which did not change after administration of human chorionic gonadotropin, increased to 22 ng per deciliter after ACTH, and decreased to 0.9 ng per deciliter after dexamethasone. Plasma delta 4-androstenedione, dehydroepiandrosterone and 17-hydroxyprogesterone were in the normal range. Plasma luteinizing hormone was high, but follicle-stimulating hormone normal (7.5 mlU per milliliter). There were two testes with epididymis and vas deferens, but no Mullerian structures. Microscopical examination showed hyalinization of tubules, which were lined by normal Sertoli cells and occasional immature germ cells. No Leydig cells were seen. After castration follicle-stimulating hormone increased to 43 mlU per milliliter. We conclude that this case of male pseudohermaphroditism was probably due to a Leydig-cell agenesis, that the epididymis and vas deferens can be developed in such a condition and the follicle-stimulating hormone secretion is regulated, at least in part, by a non-androgen substance secreted by Sertoli cells.


Correction of leukocyte function in Chediak-Higashi syndrome by ascorbate. Because ascorbate potentiates chemotaxis of normal leukocytes, we examined the effect of ascorbate on polymorphonuclear leukocytes from a patient with the Chediak-Higashi syndrome. Chemotactic migration was 104+/-16 leukocytes per 10 fields (mean+/-S.D.) initially and 258+/-44 (P less than 0.001) after ascorbate, as compared to 182+/-10 in controls. There was no bactericidal activity by 40 minutes in the patient's untreated leukocytes. After ascorbate bactericidal activity of patient and untreated control cells was the same. The addition of ascorbate reduced cAMP levels in the patient's cells from a mean of 34.5 pmoles per 10(7) polymorphonuclear leukocytes to 5.9, as compared to a control value of 3.1+/-1.4. The association of elevated cAMP and impaired function in the polymorphonuclear leukocytes of patients with the Chediak-Higashi syndrome may be related to abnormal microtubular assembly.


Effects of norgestrel and metyrapone on pituitary-adrenal-ovarian function. To assess the effects of d-norgestrel on pituitary-adrenal-ovarian function, basal levels and responses to metyrapone of urinary 17-ketogenic steroids (17-KGS) and 17-ketosteroids (17-KS), plasma cortisol (F), plasma delta4-androstenedione (A), plasma testosterone (T), plasma estrone (E1) and estradiol (E2), plasma and urinary LH and FSH were determined in 10 normal women before and while taking d-norgestrel 1 mg/day. Cortisol secretion rate (CSR) and binding capacities of cortisol binding globulin (CBG) and testosterone-estradiol binding globulin (TeBG) were also measured. Norgestrel did not significantly alter 17-KGS, 17-KS, F, LH, FSH, CSR, or the 17-KGS and 17-KS responses to metyrapone. Norgestrel reduced TeBG binding capacity but not CBG binding capacity. Norgestrel competitively inhibited the binding of dihydrotestosterone to TeBG under in vitro conditions. Levels of T, E2, and E1 were reduced by norgestrel. All measured hormone levels except FSH were increased following metyrapone prior to norgestrel administration. Norgestrel completely blocked the metyrapone-induced increases in LH and E2 and markedly reduced the E1 increase. Metyrapone reduced E2 during norgestrel treatment.


Myoepithelioma. Review of the literature and report of a case with ultrastructural confirmation. The myoepithelioma is a rare salivary gland tumor composed nearly exclusively of myoepithelial cells. A case occurring on the palate of a 22-year-old man is reported. Electron microscopic findings of occasional desmosomes, a basal lamina associated with the plasma memb;rane, and perinuclear cytoplasmic filaments confirmed the diagnosis. The myoepithelioma appears to resemble the pleomorphic adenoma clinically and may be a development variant of the pleomorphic adenoma.


The implantation of natural tooth form bioglasses in baboons. A preliminary report. This report describes a 6-month screening study comparing the tissue responses of several bioglass natural tooth implants in adult female baboons. The dental implants were designed to elicit osteogenesis at the interface of the implant and the oral tissues. This is in contrast to those types of implant materials that are merely biologically acceptable. Incisor teeth were extracted, dupplicated in bioglass, returned to the natural sockets, and splinted to the adjacent natural tooth, where they remained splinted for 3 months and then free standing for an additional 3 months. Microscopic evaluation revealed distinct histopathologic characteristics with certain  bioglass formulas, such as ankylosis, a pseudofibrous capsule with fibers running parallel to the root surface, or an apparently true periodontal membrane with properly oriented Sharpey's fibers. True cementogenesis was not found.


Abnormal tooth tissue in human odontodysplasia. The affected teeth in this case of odontodysplasia exhibited abnormal hypoplastic enamel, abnormal dentin containing extensive interglobular regions and completely lacking a peritubular matrix, and an abnormal irregular tissue consisting of highly calcified and partially fused granules located within the dentin of the tooth tips. Electron micrographs of the irregular tissue showed that the granules consisted of crystallites densely and radically packed in a noncollagenous amorphous matrix. Granules were surrounded by a slightly calcified, irregularly arranged, collagenous matrix. The irregular tissue formed in the pulp of the tooth tip befofe and independently of the dentin. It was at least partially formed by pulpal calcification. Abnormal dentin matrix was formed by odontoblasts which were less differentiated than normal. Odontodysplastic teeth showed abnormal differentiation of odontoblasts and ameloblasts resulting in defective enamel and dentin and, in extreme cases, in extensive pulpal calcification.


[Pathogenetic investigations on a case of mauriac syndrome (author's transl)]. The results of clinical and biochemical investigations on a girl with all obligatory signs of Mauriac syndrome already in infancy were compared with the different hypotheses suggested in order to explain the pathogenesis of this disease. One possible explanation for the origin of MS might be a decreased sensitivity of adenylate-cyclase to glucagon or adrenalin. Hypersensitivity to insulin, resulting in a decreased production of cyclic AMP and activation of glycogen synthetase could be excluded by measuring the urine excretion of cAMP with and without insulin. Furthermore no signs of dyspituarism were detectable on our case and the hypothesis of MS being a combination of primary glycogenosis and diabetes mellitus could also be refuted. Liver enzyme activities were normal.


Neoplasia in the rat induced by N-hydroxyphenacetin, a metabolite of phenacetin. N-hydroxyphenacetin, a phenacetin metabolite, was fed to rats as a 0.05-0.5% dietary supplement. After 9 months, tumours of the liver were found in 36 of 64 animals. One animal also developed a renal tumour. No tumours were found in control animals. The findings implicate phenacetin as a carcinogen and suggest that N-hydroxyphenacetin may be the metabolite responsible.


Unusual roentgenological findings in cytomegalic inclusion body disease: large and circumscribed calcareous deposits of the basal ganglia and scattered calcifications of the parieto-occipital cortex. The roentgenological and clinical findings of a five year old girl suffering from cytomegalic inclusion body disease are reported. In this disease unusual circumscript and large calcareous deposits of the basal ganglia and scattered gyriform calcifications of the occipito-parietal cortex were shown on plain skull x-rays and their paraventricular localization demonstrated on pneumoencephalotomogram.


The incipient Wilms' tumor.

The incipient Wilms' tumor. The diagnosis of Wilms' tumor is usually made by excretory urography after the patient presents with an abdominal mass. A diagnosis prior to the appearance of an obvious mass might result in an improved cure rate and less morbid therapy. The three cases described illustrate the difficulty in making an earlier diagnosis since pyelograms were "normal" prior to detection of the abdominal mass.


Bilateral nephroblastomatosis.

Bilateral nephroblastomatosis. Bilateral nephromegaly with a histological picture of diffuse Wilms' tumor or so-called bilateral nephroblastomatosis is a rare condition. We present an additional case documented by urography. To our knowledge, this is the first report of diffuse nephroblastomatosis recognized at 2 hours of age. A few cases are reported but urographic documentation is available in only 4 of those and in our own. The review of the urographic findings reveals features which seem seem to be nearly pathognomonic of this condition. Urographic appearance similar to that of adult type polycystic disease without the typical radiolucent cysts, should raise the suspicion of a diffuse bilateral process in the kidneys. Exclusion of other infiltrative diseases, like leukemia and glycogen storage disease, should lead to radiologic diagnosis of nephroblastomatosis. Renal biopsy is then indicated.


Segmental embryonic peripheral neuropathy. Based on a new interpretation of the radiology, the author has proposed the hypothesis that reduction deformities of the limbs are of sensory neuropathic origin. This hypothesis is examined in terms of the segmental sensory innervation of the skeleton. The predicted effect of sclerotome subtraction is compared with the radiological malformations, and is found to support the hypothesis.


Metastatic neonatal Wilms' tumor: a case report with review of the literature. An unusual case of congenital Wilms' tumor is discussed. Although the very existence of neonatal Wilms' has been challenged, this case represents a well documented example, rendered all the more unusual by concurrent metastatic disease and a second primary malignancy in the posterior fossa.


The effect of premature delivery on the development of gluconeogenic enzymes in the guinea pig. The activities of key gluconeogenic enzymes in the livers of newborn guinea pigs were monitored as a function of time following birth either vaginally at term or prematurely by cesarian section at 62 days of gestation. The activity of hepatic glucose-6-phosphatase rose dramatically from 1.40 +/- 0.26 mumol/min/g at birth to a maximum of 6.8 +/- 0.9 mumol/min/g at 24 hr in prematurely delivered animals although there was little significant change in activity in full term animals. The activity of hepatic fructose-1,6-diphosphatase and mitochondrial phosphoenolpyruvate carboxykinase changed little over the first 3 days of life in either full term or premature animals. Cytosolic phosphoenolpyruvate carboxykinase, on the other hand, had low activity at birth being 0.11 +/- 0.03 mumol/min/g in full term and 0.06 +/- 0.04 mumol in premature animals rising to values of 0.71 +/- 0.06 and 1.12 +/- 0.12 mumol/min/g, respectively, at 24 hr of life. Pyruvate carboxylase activities in the premature animals remained significantly lower than those in full term animals in the first 72 hr of life. Transient hypoglycemia was evident in the prematurely delivered animals, but not in the full term animals, the blood glucose values being 82 +/- 7 mg/100 ml for the full term animals and 20 +/- 8 mg/100 ml for the premature infants at 2 hr of life.


Lactic acidosis in three sibs due to defects in both pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. A Canadian Indian family is described in which three of the children were mentally retarded, and had seizures and other neurological abnormalities. They had chronic metabolic acidosis associated with elevated blood levels of lactate, pyruvate, and alanine. Two of the children excreted large amounts of pyruvic and alpha-ketoglutaric acids in the urine and had elevated plasma levels of glutamic acid and proline. Hypoglycemia occurred with fasting in two of the children. Treatment with pharmacological doses of thiamine, lipoic acid, biotin, riboflavin, and various dietary regimes was without effect. One child died at 3 1/2 months and another at 4 1/2 months; the third is still alive at 23 months of age. Enzyme assays revealed a low level of activity of both the pyruvate and alpha-ketoglutarate dehydrogenase complexes in cultured fibroblasts of one of the sibs. These patients appeared to have partial defects in the oxidation of pyruvate, as well as of alpha-ketoglutarate within the tricarboxylic acid cycle.


Neoplasia of early life and its relationships to teratogenesis. The biologic peculiarities of tumors of early life are elucidated. The oncogenic grace period is emphasized, wherein infantile tumors tend to behave in a relatively benign fashion up until 3-6 months of age. A review of the types of congenital malformations associated with the development of neoplasms is presented. These associations appear to be of fundamental importance in developmental pathobiology. They are illustrated by the tendency for neoplasms to develop in anomalous or dysplastic tissues, such as developmental vestiges, undescended testes, dysgenic gonads and certain hamartoses. There is an increased incidence of tumor occurrence in: (1) specific teratologic disorders: aniridia, hemihypertrophy, Beckwith's syndrome, basal cell nevus syndromes and others; (2) cytogenetic abnormalities: Down's syndrome, 13q- syndrome (D-deletion), trisomy 18; (3) chromosomal instability syndromes: Fanconi's anemia, ataxia-telangiectasia, Bloom's syndrome. Finally, many agents, known to be carcinogenic when administered postnatally to animals, are teratogenic in the fetus. A few agents--urethan, alkylnitrosoureas, estrogens--are both teratogenic and carcinogenic when administered to the fetus transplacentally. It is suggested that the timing of intrauterine insult is important in determining whether the effect on the offspring is teratogenic, oncogenic or both. Teratogenesis appears to be the more primitive response. Other theories explaining the concurrence of tumors and anomalies are offered.


The nephroblastomatosis complex and its relationship to Wilms' tumor: a clinicopathologic treatise. 1. At least one third of all children with Wilms' tumors have evidence of aberrant metanephric differentiation, hamartomas, adenomas and nodules of blastema or Wilms' tumorlets in the subcapsular renal cortex. Only the presence of nodular renal blastema was found to correlate with younger age, suggesting that the other tumorlike lesions may be derivatives. 2. In our experience, all patients with bilateral and sequential bilateral Wilms' tumors have exhibited these characteristics in the "uninvolved" renal tissue removed at nephrectomy. 3. Pancortical (infantile), diffuse superficial (late infantile) and multifocal (juvenile) variants of nephroblastomatosis are defined. In the latter 2 forms of nephroblastomatosis the prognosis after modern therapy is at least as good as it is in patients with unilateral, unicentric Wilms' tumor arising in a histologically normal kidney. 4. The morphologic evidence presented strongly indicates that Wilms' tumor in patients with nephroblastomatosis develops either from nodular blastema or metanephric hamartomas. It is hypothetically possible that all Wilms' tumors develop from these precursors. The latent period between cessation of nephrogenesis and clinical presentation of Wilms' tumor is interpreted as favoring the hypothesis that carcinogenesis per se is the result of a postnatal event but that the substrate for tumor development, aberrant metanephric proliferation, has its inception during gestation.


[Adult Niemann-Pick disease: a 26 years follow-up. Report of a case with isolated visceral involvement, excess of tissue sphingomyelin, and deficient sphingomyelinase activity (author's transl)]. The case report of Niemann-Pick disease, in a 26 years old woman whose first symptoms appeared when she was 17 months old, is described. The disease, involving considerable hepatosplenomegaly and pulmonary infiltration, was diagnosed by the presence of lipid laden macrophages (resembling foam cells, sea blue histiocytes and kidney intermediate forms) in the bone marrow, liver and kidney, and an excess of tissue sphingomyelin and cholesterol, and a decrease in sphingomyelinase in circulating leucocytes. The results of ultrastructural, histochemical and biochemical studies on hepatic and renal lipids are reported. The relationship of the case to the sea blue histiocyte syndrome is discussed.


"Fingerprinting" high molecular weight RNA by two-dimensional gel electrophoresis: application to poliovirus RNA. Conditions are described under which complete RNase T1 digests of high molecular weight RNA can be separated into numerous components by two-dimensional gel electrophoresis. Small and large oligonucleotides (n = 1 - 2c0) can be resolved without losses. The procedure yields fingerprints which are diagnostic for a particular species of RNA and an index of its purity as will be shown for the genomes of poliovirus type 1 and 2.


Reduced DNA repair in mouse satellite DNA after treatment with methylmethanesulfonate, and N-methyl-N-nitrosourea. We have measured DNA repair in mouse satellite and main band DNA as resolved by Ag+-Cs2SO4 centrifugation in response to treatment with the alkylating agents, methyl methanesulfonate, and N-methyl-N-nitrosourea. We find that there is a statistically significant lower incorporation of 3H-Tdr into the satellite DNA as compared to the main band at varying periods after treatment with the alkylating agents. This suggests a reduced repair activity in the satellite DNA. We have measured the extent of binding of 14C-methyl methanesulfonate to the satellite, and main band DNA, and no difference in binding was observed, indicating that the reduced repair activity of satellite DNA is not due to a difference in binding of alkylating agents. We believe that the reduced incorporation of 3H-Tdr into satellite DNA may be due to its location in the condensed chromatin fraction.


Conformational analysis of polynucleotides. I. The favorable left-handed helical model for the poly(8,2'-S-cycloadenylic acid) with high anti conformation. 1) Energy calculations have shown that poly (8,2'-S-cycloadenylic aicd) can form left-handed helices owing to the high anti conformation. 2) Two favorable left-handed helices are characterized by axial translation per residue (Z=4.3 and 3.6A) and by rotations per residue (theta= 40 degrees and -25 degrees). 3) The proposed helical models might be stable in aqueous solution and is well explicable of the optical property of this compound.


Minichromosome of simian virus 40: presence of histone HI. In contrast to conclusions of previous studies /I-3/ claiming the absence of histone HI from the SV40 and polyoma viral minichromosomes we have found that a preparation of purified SV40 minichromosomes does contain histone HI. The content of HI in relation to other four histones in the SV40 minichromosomes is close to that in the cellular chromatin. Histone HI in the isolated SV40 minichromosomes is bound apparently to internucleosomal DNA stretches as was shown already for HI in the cellular chromatin /4/. In addition it was found that more than 90% of the purified SV40 minichromosomes migrated as a single discrete deoxyribonucleoprotein band upon agarose gel electrophoresis.


Satellite DNA sequence content of polylysine-titratable and nuclease-resistant fractions of mouse liver hepatoma chromatin. Micrococcal nuclease digestion of mouse TLT liver hepatoma chromatin proceeds rapidly to the point where approximately 35% of the DNA is recoverable by centrifugation of the chromatin DNA through 3M CsCl. The satellite DNA sequence content of this recoverable DNA is the same as whole chromatin DNA (10%). The 11s (penultimate digestion product) monomer, as well as intermediate multiples and relatively undigested large chromatin segments are separable on steep hlycerol gradients. The DNA isolated from these fractions also contains the normal 10% satellite DNA content. Progressive polylysine titration of chromatin followed by nuclease digestion gives anomalous recoveries of DNA but, nonetheless, the satellite sequence content titration of chromatin, followed by pronase and then nuclease digestion, again gave recoverable DNA with a satellite sequence content of 10%. These results are discussed in terms of the conclusion that nucleosome (or upsilon-body) structures are distributed in a random fashion over the genome.


Preparation of subcellular fractions from rat liver: comparison of the Polytron with the Dounce homogenizer. The PolytronR and Dounce homogenizers have been evaluated for preparation of homogenates of rat liver prior to isolation of subcellular fractions by differential centrifugation. Marker enzymes used to evaluate the subcellular fractions included cytochrome oxidase, monoamine oxidase, D-amino acid oxidase, acid phosphatase, glucose-6-phosphatase, ethyl morphine demethylase, and lactate dehydrogenase. No significant difference in the distribution of enzymes (percent recovery or specific activity) was observed between the two methods of homogenization. In addition, there were no significant differences in the ultrastructural appearances and respiratory control ratios of the mitochondrial fractions prepared by the two methods of homogenization.


Metalloprotein electron transfer reactions: analysis of reactivity of horse heart cytochrome c with inorganic complexes. The reactions of horse heart cytochrome c with Fe(ethylenediaminetetraacetate)2-, Co(1,10-phenanthroline)3(3+), Ru(NH3)6(2+), and Fe(CN)6(3-) have been analyzed within the formalism of the Marcus theory of outer-sphere electron transfer, including compensation for electrostatic interactions. Calculated protein self-exchange rate constants based on crossreactions are found to vary over three orders of magnitude, decreasing according to Fe(CN)6(3-) greater than Ru(NH3)6(2+) greater than Fe(EDTA)2-. The reactivity order suggests that the mechanism of electron transfer involves attack by the small molecule reagents near the most nearly exposed region of the heme; this attack is affected by electrostatic interactions with the positively charged protein, by hydrophobic interactions that permit reagent penetration of the protein surface, and by the availability of pi symmetry ligand (or extended metal) orbitals that can overlap with the pi redox orbitals of the heme group.


Adenosine 3':5'-cyclic monophosphate deficiency in Neurospora crassa. Depending on growth conditions, the adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels of the fr mutant, a morphologically aberrant strain of Neurospora crassa, are reduced 2- to 5-fold. By taking advantage of the differences in phenotype of fr in liquid and agar cultures and the positive response of fr grown on solid support to exogenous theophylline, a relationship between the degree of morphological abnormality and intracellular cyclic AMP levels of the mutant is observed. Progressive restoration of the fr phenotype toward a normal state is paralleled by increases in cyclic nucleotide content. Striking differences in the sedimentation and thermal characteristics of the fr and wild-type adenylate cyclases [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] are observed. Approximately 50% of the normal activity sediments at 105,000 X g compared to 5% of the mutant enzyme. In addition, the overall stability of the fr adenylate cyclase is significantly decreased and its rate of inactivation at 37 degrees in the absence of substrate is 10-fold greater than the wild-type adenylate cyclase. Arrhenius plots also indicated that the Q10 (increase in rate per 10 degrees temperature increase) and the temperature of maximal activity of the fr enzyme are reduced. Supplementation of fr agar cultures with linolenic acid results in an elevated cyclic AMP content and a wild-type-like morphology similar to that obtained with inhibitors of phosphodiesterase (3':5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). An increased thermostability of the fr adenylate cyclase occurs on linolenate enrichment of the mutant. It is concluded that the cyclic AMP deficiency is at least partially responsible for the fr phenotype and that this reduction results from a membrane defect that affects adenylate cyclase function.


Identification of enzyme-bound activated CO2 as carbonic-phosphoric anhydride: isolation of the corresponding trimethyl derivative from the active site of glutamine-dependent carbamyl phosphate synthetase. The activated CO2 intermediate formed in the reaction catalyzed by glutamine-dependent carbamyl phosphate synthetase was identified as carbonic-phosphoric anhydride through the use of two independent procedures. The carboxy phosphate intermediate was reduced to formate by treatment with potassium borohydride. Although both free CO2 and the enzyme-bound activated CO2 are reduced to formic acid by borohydride, it was possible to selectively introduce a 14C label into the enzyme-bound activated CO2 and thus into the formic acid derived from it. Such [14C]formate formation required the presence of ATP, KCl, and the enzyme, and evidence was obtained that the [14C]formate found is not derived from carbamyl phosphate or from bicarbonate bound nonspecifically to the enzyme. When the enzyme was treated with L-2-amino-4-oxo-5-chloropentanoate (or cyanate), the formation of [14C]formate was increased about 2-fold, a finding consistent with the previous observation that such treatment effects a similar increase in the bicarbonate-dependent cleavage of ATP catalyzed by the enzyme. When reaction mixtures containing the enzyme, [gamma-32P]ATP, and [14C]bicarbonate were methylated by treatment with diazomethane, a labeled compound was formed which cochromatographed with authentic trimethyl carboxy phosphate. Equimolar quantities of 14C and 32P wer incorporated into the intermediate, thus confirming its identification as carboxy phosphate. Nonenzymatic transphosphorylation from ATP to bicarbonate to form carboxy phosphate was also detected by diazomethane trapping.


Competition between cellular and viral mRNAs in vitro is regulated by a messenger discriminatory initiation factor. Encephalomyocarditis viral RNA has previously been shown to outcompete host cellular mRNA has for translation in vitro in crude and fractionated protein synthesizing systems. In the present communication it is shown that the competition is regulated by an initiation factor or complex of factors, and not the 40S initiation complex per se. The factor primarily involved is the murine equivalent of a component present in a partially purified preparation of rabbit initiation factor M3. Both the murine and rabbit factors are clearly messenger discriminatory.


Importance of the third amino acid residue of oxytocin for its action on isolated rat uterus: study of relationship between hormone conformation and activity. In a continued effort to relate the three-dimensional structure of a peptide hormone to its biological activity, the dose-response relationships of [3-phenylalanine] oxytocin (oxypressin), with an aromatic amino acid residue in position 3, and [3-beta-cyclohexylalanine]oxytocin, with an aliphatic amino acid residue to position 3, were determined in the rat uterine assay in vitro and compared to that of oxytocin. Oxypressin has not only a lower affinity for the smooth muscle receptor than the natural hormone, but also a decreased maximal response (efficacy). [3-beta-Cyclohexylalanine]oxytocin exhibits an even lower affinity than oxypressin, but retains the same maximal response as oxytocin. A reorientation of the tyrosine sidechain, caused by the presence of a neighboring aromatic sidechain in position 3, away from the surface of the 20-membered ring is though to remove the phenolic hydroxyl group from its optimal position in the "active center" of oxytocin and give rise to the reduced efficacy of oxypressin.


Fate of mismatched base-pair regions in polyoma heteroduplex DNA during infection of mouse cells. Heteroduplex DNA has been constructed from two variants of polyoma virus that differ genotypically at four distinct sites. The genotypes of the progeny virus derived from infections of mouse cells with single heteroduplexes have been analyzed to determine how the genotypic markers of the parental heteroduplex segregate. Markers that are separated by a length of DNA greater than 600 nucleotides segregate independently. Segregation was not detected between two markers separated by only approximately 90 nucleotides. We interpret these results on the basis of the correction of mismatched base-pair regions in the heteroduplex before the completion of DNA replication. We suggest that this technique provides valuable information concerning gene conversion in mamalian cells and permits the transfer of genotypic markers from one virus strain to another.


Specificity of the protein kinase activity associated with the hemin-controlled repressor of rabbit reticulocyte. Highly purified preparations of hemin-controlled repressor of rabbit reticulocyte contain a 3':5'-cyclic AMP-indenpendent protein kinase activity that phosphorylates the low-molecular-weight (about 38,000) polypeptide chain of the initiation factor that forms a ternary complex with GTP and Met-tRNAf. These preparations also phosphorylate several polypeptide components of reticulocyte 40S ribosomal subunits. However, no significant levels of phosphorylation are observed when casein, histones, Artemia salina 40S ribosomal subunits, or other initiation factor fractions are used as substrates although high levels of phosphorylation are obtained with cruder preparations of the repressor. An antibody to these highly purified preparations of repressor has been obtained from the serum of immunized goats. Preincubation with immune goat IgG results in the neutralization of the inhibitory activity of the repressor, while normal IgG has no effect. Preincubation with immune IgG also abolishes the protein kinase activity responsible for the phosphorylation of the initiation factor and reticulocyte 40S subunits. Histone phosphorylation by crude repressor preparations, on the other hand, is unaffected by preincubation with immune IgG.


Autoregulation of simian virus 40 gene A by T antigen. During lytic infection by simian virus 40, gene A is transcribed into early RNA, which is translated into A protein (T antigen). Both the rate of synthesis and the intracellular amount of early RNA are higher in cells infected by temperature-sensitive A (tsA) mutants than in cells infected by wild-type virus. These differences are observed at permissive temperature (32 degrees) and are amplified greatly after a shift to restrictive temperature (41 degrees). For example, at 32 degrees cells infected by tsA mutants synthesize early RNA approximately twice as fast as cells infected by wild-type virus. After the shift to 41 degrees, the rate of synthesis in the tsA infection increases to 15 times the rate in the wild-type infection. In contrast, cells infected by tsA mutants do not overproduce late RNA. We suggest that the A protein regulates its own synthesis by negative feedback control of gene A transcription.


Regulation of protein synthesis in reticulocyte lysates: phosphorylation of methionyl-tRNAf binding factor by protein kinase activity of translational inhibitor isolated from hemedeficient lysates. A previous study demonstrated that the translational inhibitor from lysates of heme-deficient rabbit reticulocytes is associated with a protein kinase activity. Chromatography of this inhibitor preparation on phosphocellulose yields two distinct protein kinase activities, PC1 and PC2. PC1, which consitutes about 90% of the activity in the unresolved preparation, does not inhibit protein synthesis in lysates, but actively phosporylates calf thymus histone II in a 3':5'-cyclic AMP-denpendent reaction. PC2 contains the translational inhibitor, phosphorylates histone poorly, and is not cyclic AMP-dependent. While [gamma-32P]ATP as the phosphate donor, the two kinase fractions were analyzed with the putative substrates, salt-washed 40S ribosomal subunits, and the initiation factor that mediates the binding of Met-tRNAf to the 40S subunit. PC1 is inactive with the initiation factor, but phosphorylates 40S subunits at a single major site that migrates as a 31,000-dalton band in sodium dodecyl sulfate-acrylamide gels; phosphorylation requires cyclic AMP. Similar phosphorylation of the reticulocyte 40S site (31,000 daltons) can be demonstrated with other cyclic AMP-dependent kinases from reticulocytes, rat liver, and bovine heart muscle. PC2 phosphorylates the small subunit (38,000 daltons) but not the large subunit(s) of the initiation factor; the reaction does not require cyclic AMP. PC2 does not phosphorylate 40S subunits. In the presence of 40S subunits, the initiation factor appears to be rapidly bound in a manner that effectively blocks phosphorylation of the initiation factor by PC2; under the same conditions phosphorylation of the 40S subunit by PC1 is not affected. The initiation factor has been shown to reverse the inhibitions of protein chain initiation induced in lysates by heme deficiency, double-stranded RNA, oxidized glutathione, or the purified translational inhibitor. The observation that the Met-tRNAf binding factor is phosphorylated by PC2 supports the hypothesis that this initiation factor is a target for the action of the translational inhibitor activated in heme deficiency.


Incorporation of adenosine into ATP: formation of compartmentalized ATP. The incorporation of [3H]adenosine, [3H]adenine, and [3H]hypoxanthine into adenine nucleotides of nude (athymic) mouse liver and human hepatoma grown subcutaneously in nude mice was studied. 3H and 32P radioactive labeling in vivo of acid-soluble nucleotides followed by chromatographic procedures indicated that, in contrast to [3H]adenine and [3H]hypoxanthine, [3H]adenosine is preferentially incorporated into ATP in comparison with its incorporation into AMP and ADP. This phenomenon, as well as complementing the recently reported 3-fold increase in total cellular ATP upon treatmen- with 0.5-1.0 mM concentrations of adenosine, indicates the formation from adenosine of compartmentalized ATP that is not produced from either adenine or hypoxanthine. The observed effect is of larger magnitude in the growth-arrested normal liver than in the actively growing tumor.


Covalent modification of proteins by metabolites of NAD+. Covalently bound adducts of ply(L-lysine), bovine serum albumin, lysine rich histone (f1) and deoxyribonucleotidase I (DNase, EC 3.1.4.5) with adenosine diphosphoribose and ribose-5-phosphate were prepared at pH 7.4 and 9.5. Macromolecular adducts of bovine serum albumin and histone (f1) were isolated by gel filtration and electrophoresis. Reduction of products by NaBH4 did not dissociate the ribose-5-phosphate moiety from macromolecules. Specific introduction of 3H into the adducts also indicated Schiff base formation. The reaction of ribose-5-phosphate with epsilon-amino groups of histone (f1) approached 70-90% saturation. Spermine and spermidine also react with adenosine diphosphoribose and ribose-5-phosphate to form 1:1 Schiff bases. It is proposed that high turnover of cellular NAD+ is the source of aldehydic metabolites which may regulate macromolecular metabolism by covalent modification of nuclear proteins, whereas polyamines serve as modulators of this control cycle.


Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA. The translation of encephalomyocarditis virion RNA in extracts from interferon-treated L-cells is inhibited by the addition of double-stranded RNA (dsRNA) at 400 ng/ml. A similar inhibition in response to dsRNA is seen in control cell extracts supplemented with small amounts of a postribosomal supernatant fraction from interferon-treated cells (interferon cell sap): Neither interferon cell sap nor dsRNA alone is inhibitory in control systems. The inhibition is much reduced if translation is carried out at low ATP concentrations.Conversely, the inhibitory capacity of the interferon cell sap is increased 100-fold if it is preincubated with dsRNA and ATP prior to its addition to the protein-synthesizing system. After this preincubation all detectable dsRNA can be removed without any diminution of the inhibitory activity of the cell sap. These results are compatible with a two-step model for the inhibition in which a pre-inhibitor is activated by dsRNA, the activated inhibitor then interacting with the protein synthesis system to inhibit translation.


Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Cultured fibroblasts derived from patients with homozygous familial hypercholesterolemia, which lack functional low density lipoprotein (LDL) receptors, fail to bind, take up, or degrade the lipoprotein with high affinity; therefore LDL-cholesterol is not made available for suppression of cholesterol synthesis or activation of cholesteryl ester formation. When LDL was given a positive charge by reaction with N,N-dimethyl-1,3-propanediamine (cationized LDL), the rate of degradation of the lipoprotein was increased by more than 100-fold in the homozygous familial hypercholesterolemia fibroblasts. Degradation of cationized LDL was inhibited by chloroquine, suggesting that it occurred in cellular lysosomes. Although the cationized LDL entered the cell through a mechanism independent of the LDL receptor, the cholesterol liberated from the degradation of the lipoprotein became available for suppression of cholesterol synthesis and stimulation of cholesteryl ester formation in the homozygous familial hypercholesterolemia fibroblasts. The rate of degradation of albumin by fibroblasts was also increased by more than 100-fold when this protein was coupled to N,N-dimethyl-1,3-propanediamine. The ability to deliver a protein to lysosomes by giving it a strong positive charge may have potential relevance not only to familial hypercholesterolemia, but also to inborn errors of metabolism that involve deficiencies in lysosomal enzymes.


Effect of sugars on early biochemical events in development of Dictyostelium discoideum. Metabolizable sugars blocked development of the slime mold Dictyostelium discoideum; the same sugars also inhibited the formation of contact sites "A", of membranal 3':5'-cyclic adenosine monophosphate (cAMP)-binding sites, and of total cAMP phosphodoesterase (3':5'-cyclic-nucleotide phosphodiesterase; EC 3.1.4.17; 3':5'-cyclic-nucleotide 5'-nucleotidohydrolase). These inhibitory effects of the sugars on the synthesis of cellular components, required for the aggregation of developing amebae, were paralleled by an inhibition of the accumulation of cAMP which normally accompanies development. The inhibition by sugars could be overcome partially by pulsing the amebae with nanomolar concentrations of cAMP only after the amebae had acquired cAMP-binding sites. The findings suggest that metabolizable sugars inhibit development by blocking the formation of cAMP and, conversely, that development in D. discoideum may be related to the energetic state of the cell.


Regulation of tubulin synthesis in islets of Langerhans. Tubulin represents a major protein in rat pancreatic islets, which averages 0.5% of the total protein content and 6% of the noninsulin protein synthesized under basal metabolic conditions. Glucose increases the synthesis of tubulin twofold to threefold. Tubulin synthesis is also stimulated by adenosine 3':5'-cyclic monophosphate in both the absence and presence of glucose; this effect of adenosine 3':5'-cyclic monophosphate occurs preferential to noninsulin protein synthesis at physiological glucose concentrations. Tubulin synthesis was decreased more than 75% by fasting, an effect prevented by maintaining animals on glucose exclusively. The fasting-induced reduction in tubulin synthesis is corrected in vitro by increasing adenosine 3':5'-cyclic monophosphate levels. These findings parallel changes previously reported in insulin release and suggest that physiological agents can exert their insulin secretory action through an effect upon the rate of tubulin synthesis.


Control of growth of benzo(a)pyrene-transformed 3T3 cells. The growth controls observed in benzo[a]pyrene-transformed 3T3 cells (BP3T3) are compared with those of virus-transformed and normal 3T3 cells. Superficially, the chemically transformed BP3T3 cells have the same behavior as virus-transformed SV3T3 cells. Both grow to high cell density in culture medium with 10% serum, both form colonies in Methocel, and both are tumorigenic. Closer examination, however, has disclosed that BP3T3 cells exhibit "normal" growth controls at low serum concentrations. In contrast to the behavior of SV3T3 cells, the initiation of DNA synthesis in BP3T3 cells is still dependent on a serum factor. If BP3T3 cells are grown in medium with 0.2% serum, the cells become quiescent, with growth arrested in the Gu or G0 phase of the cell cycle. The addition of serum or the fibroblast growth factor (FGF) to such quiescent cells leads to the initiation of DNA synthesis and the resumption of growth. As with normal 3T3 cells, if the growth rate of BP3T3 cells is limited by a suboptimal concentration of serum, the growth rate of the cells is increased by the addition of FGF. Also, BP3T3 cells show density-dependent regulation of growth, if the medium contains a low concentration of serum. BP3T3 cells, therefore, have the behavior of "transformed" cells when cultured in medium with 10% serum, but behave as "normal" cells in medium with low serum. In comparison with normal 3T3 cells, the difference in growth behavior of BP3T3 cells appears to be due to a substantial decrease in the cells' requirement for a serum growth factor of the FGF type. Exploration of possible causes of this substantial decrease indicates that the primary cause is a lower rate of depletion of the serum growth factor from the culture medium by BP3T3 cells. The decrease in rate of depletion is sufficient to account for the uncontrolled growth of BP3T3 cells in medium with 10% serum. It is suggested that a decreased rate of depletion of a growth factor may contribute to tumorigenicity of cells in vivo.


Loss of mouse chromosomes in somatic cell hybrids between HT-1080 human fibrosarcoma cells and mouse peritioneal macrophages. Somatic cell hybrids between mouse peritioneal macrophages and HT-1080 human fibrosarcoma cells lose mouse chromosomes and retain the entire complement of human chromosomes. In contrast, somatic cell hybrids between cells derived from two different mouse continuous cell lines and HT-1080 human cells were found to lose human chromosomes preferentially. Loss of mouse chromosomes is not a general property of hybrids between mouse macrophages and transformed human cells; the hybridization of mouse macrophages with cells derived from five different human fibroblast lines transformed by simian virus 40 resulted in the production of hybrid clones that preferentially lost human chromosomes.


Neurophysiological defects in temperature-sensitive paralytic mutants of Drosophila melanogaster. A new temperature-sensitive paralytic mutant of Drosophila, comatose, is compared behaviorally and physiologically with the previously known types, para and shi. All three have different properties with respect to kinetics of paralysis at high temperature and recovery from paralysis; com is hypersensitive to paralysis by cooling. Neurophysiological experimeents indicate different mechanisms for paralysis in each of the mutants.


A fetal protein in chromatin of Novikoff hepatoma and Walker 256 carcinosarcoma tumors that is absent from normal and regenerating rat liver. Antibodies to chromatin proteins of Novikoff hepatoma cells formed precipitin bands in the double-diffusion immunoprecipitation assay with chromatin proteins of Novikoff hepatoma, Walker 256 carcinosarcoma, and 18-day fetal rat liver. The antigen used for preparation of antiserum was the chromatin proteins initially extracted with 3 M NaCl-7 M urea and soluble after dialysis to 0.14 M NaCl-0.35 M urea. The chromatin proteins used for analytical studies were extracted with 0.6 M NaCl containing 0.01 M Tris-HCl (pH 8) and 100 muM phenylmethylsulfonyl fluoride. Corresponding chromatin proteins of normal and 18-hr regenerating rat liver, heart, and kidney did not form precipitin bands. The antigen was purified from the chrmatin of Novikoff hepatoma cells by exclusion chromatography on Sephadex G-150 and preparative nondenaturing polyacrylamide gel electrophoresis. Its migration on denaturing sodium dodecyl sulfate-polyacrylamide gels corresponded to a molecular weight of 26,000. Amino acid analysis showed that the ratio of acidic to basic amino acids was 1.4 to 1.0. Evidence for its homogeneity included its migration as a single protein spot on two-dimensional polyacrylamide gel electrophoresis and its single lysine amino-terminal amino acid. This protein is a glycoprotein, as shown by the presence of 15 moles of galactosamine per mole of antigen. These studies demonstrate the presence of a fetal glycoprotein in the chromatin of two tumors that may have an important role in determining their gene products.


Effect of 3':5'-cyclic GMP derivatives on the formation of Caulobacter surface structures. Exogenous derivatives of 3':5'-cyclic GMP, 8-bromo- and N2,O2'-dibutyryl cyclic GMP, coordinately repress surface structure differentiation in Caulobacter crescentus. Growth in the presence of cyclic GMP derivatives resulted in the loss of flagella and pili formation and concomitant resistance to both DNA phage phiCbK and RNA phage phiCb5 infection without affecting growth rate, stalk formation, and equatorial cell division. The effect of cyclic GMP derivatives was shown to be the repression of synthesis of specific structural proteins. This effect could be reversed by exogenous N6,O2'-dibutyryl 3':5'-cyclic AMP, and mutants resistant to repression by cyclic GMP derivatives exhibited a pleiotropic phenotype affecting a cyclic AMP-mediated event.


Effect of human nasal secretions on the antiviral activity of human fibroblast and leukocyte interferon. Many normal human nasal secretions contain an inhibitor of human fibroblast IF. This inhibitor had no effect on human leukocyte IF. The amount of inhibition of fibroblast IF increased with increasing quantities of nasal secretions. Also, the inhibition could be overcome with increasing concentrations of IF.


Lutropin receptors from male and female tissues: different responses to a lutropin receptor binding inhibitor. An inhibitor for lutropin receptor site binding (LH-RBI), which strongly inhibited the binding of 125I-labeled ovine lutropin ([125I]oLH) to ovarian LH receptors, did not inhibit the [125I]oLH binding to testicular LH receptors. Preincubation of the LH-RBI with [125I]oLH did not affect the binding of preincubated ]125I]oLH to ovarian LH receptors. No inhibition of [125I]oLH binding to testicular LH receptors was observed even uhen the concentration of LH-RBI was significantly increased or when the testicular LH receptors uere first incubated with LH-RBI prior to the addition of [125I]oLH and a second incubation. Scatchard analysis revealed that the dissociation constant of [125I]oLH binding was essentially the same in the presence or absence of LH-RBI. The results suggest that: (i) the lutropin receptor of ovaries, but not of testes, has a specific LH-RBI binding site in addition to the lutropin binding site, and (ii) the binding of the LH-RBI produces an "allosteric" type of inhibition to the binding of lutropin at the lutropin binding site.


Role of converting enzyme in the cardiovascular and adrenal cortical responses to (des-Asp1)-angiotensin I. (Des-Asp1)-angiotensin I, angiotensin II and III were evaluated for pressor activities in conscious nephrectomized rats and for steroidogenic actions in rat adrenal zona glomerulosa. The pressor effect of this angiotensin nonapeptide was similar to that found with mole-equivalent doses of angiotensin III (one-third as active as angiotensin II) and was significantly attenuated by pretreatment with the 0. jararaca nonapeptide converting enzyme inhibitor. Hence, (des-Asp1)-angiotensin I is a substrate for converting enzyme in vivo, and the rapid conversion indicates that an alternate pathway for the formation of angiotensin III could exist. (Des-Asp1)-angiotensin I possessed only 0.1% of the activity of angiotensin III as a steroidogenic agent in cell suspensions of rat adrenal zona glomerulosa. Angiotensin I was a weak steroidogenic agent in vitro (1%) and was not blocked by an inhibitor of converting enzyme. Adrenal cells dispersed from the outer zone of the cortex would appear to be devoid of significant converting enzyme activity.


Characterization of extracellular particles released from continuous cell cultures derived from human leukemia. Extracellular particles, with a density of 1.18-1.22 g/cm3 in sucrose, were detected in the culture medium of a continuous cell line (JIII) derived from a patient with monocytic leukemia. These particles contained RNA, DNA, and a DNA polymerase. They synthesized DNA with endogenous templates and primers and also used exogenous DNA but not poly(rC) oligo(dG) as a template. Pretreatment with Nonidet P-40 stimulated DNA polymerase activity while treatment with ribonuclease partially inhibited the enzyme activity. Fluorescent antibodies made to the particles stained both JIII and Z-597 cells derived from human leukemias but not other types of human or nonhuman cultured cells tested. The particles do not appear to be oncornaviruses but may be a particulate antigen associated with malignant cells of hemopoietic and lymphoid origin.


Occurrence in human bone marrows of an antigen released from continuous cell cultures derived from human leukemia. Fluorescent antibodies prepared against extracellular particles from a continuous culture of cells derived from a monocytic leukemia stained JIII cells but not cells infected with Rauscher leukemia virus or simian sarcoma virus. These antibodies reacted with 38% of bone marrow preparations from patients with lymphoma, 26% of preparations from patients with nonmalignant blood disorders and 6% of preparations from patients with leukemia. Bone marrow films from patients with lymphoma over the age of 50 stained less frequently than those from patients under 50. These particles released from JIII cells are not antigenically related to two of the commonly studied oncornaviruses, but may be indicative of the etiology or disease process of lymphoma in young patients.


Acyl group composition of membrane phospholipids in mammary tissues and carcinoma induced by dimethylbenz(a)anthracene. Phospholipids and their acyl group composition in mammary adenocarcinomas and mammary tissue of the same tumor-bearing animals were investigated. Breast adenocarcinoma induced by dimethybenz(a)anthracene exhibited a phospholipid pattern which was different from that of the mammary tissue. Tumor phospholipids had higher proportions of diacyl-GPI, diacyl-GPE, and alkenylacyl-GPE and a lower proportion of diacyl-GPC than the controls. The acyl groups of most phospholipids in tumors showed a marked increase in the proportion of 18:1 and a decrease in the proportion of 18:2. The fatty acid composition of plasmalogen and triglyceride, however, remained unchanged. In spite of the decrease in the proportion of 18:2, there was no apparent difference in the proportion of 20:4 in most of the phosphoglycerides; however, a significant decrease in this fatty acid was noted in diacyl-GPI. Results of this study demonstrated that the membrane phospholipids of mammary adenocarcinoma were altered in respect to acyl group composition. Changes in physical properties of the cell membrane, in turn, could lead to abnormal manifestation of membrane regulated events in tumor cells.


[The influence of sulpirid on sleep. Results of polygraphic night sleep recordings (author's transl)]. Short and long term effects of sulpirid on the sleep EEG in humans were investigated in 8 patients aged 20-68 years (average 40.4) suffering from autonomic instability. In each patient seven polygraphic night sleep recording were performed. After an adaption night and two nights without medication each subject got 200-400 mg sulpirid i.m. for two days and then 300-600 mg orally for a three-week period. In both groups the short-term application of sulpirid caused a reduction of waking periods and a slight decrease of stage 3, while stage 2 was increased. In psychotic patients the long-term orally administered substance was followed by an increase of deep synchronous sleep (stage 4). As this alteration of sleep couldn't be revealed in the control group, we consider this finding as a result of the improvement of psychosis. In the patient group the amount of REM sleep was augmented during the short-term application as well as during the long-term application, where, as in the control group the amount of REM sleep decreased when sulpirid was given orally for three weeks. This result is discussed in comparison with the properties of the tricyclic antidepressant drugs and monoamine oxidase inhibitors and the clinical effects of sulpiride.


Experimental considerations in biological ESR studies. I. Identity and origin of the 'tissue lipid signal': a copper-dithiocarbamate complex. The complex ESR signal previously observed in fatty tissues has been identified. It is a copper low-molecular-weight sulphur compound complex similar to copper-dithiocarbamate. The usual source of this signal is contact of copper-containing tissue with surgeon's gloves. This result emphasizes the care that must be taken in obtaining and interpreting ESR spectra of tissues. The formation of this complex by intentionally adding dithiocarbamate to tissue can be used as a very simple and sensitive analytical method for tissue copper. Copper levels of less than 0-1 ppm can be detected by this method. This is an order of magnitude more sensitive than determinations by atomic absorption.


The effect of diethyldithiocarbamate on passive avoidance learning by chicks (Gallus domesticus). Diethyldithiocarbamate (DDC), a competitive inhibitor of dopamine-B-hydroxylase, produced a dosage-related depletion of neural NE and impairment of passive avoidance learning in young chicks. Retention was not impaired, however, as shown by normal relearning of the task a day later, when the drug was no longer active. Perhaps, neural NE depletion impairs ability to inhibit responding and, thus, impairs passive or inhibitory avoidance learning indirectly. Alternatively, NE depletion may slow down learning by interfering with consolidation, but if the task is well learned, it is remembered.


[Mineral metabolism of the growing bone and its disturbances--methods of radiological examinations (author's transl)]. New possible interactions of vitamin D and its derivates are discussed with reference to metabolism of calcium and phosphorous and its effects on the skeletal system. The degree of osteoporosis and osteomalacia determined only by means of roentgenology depends on subjective factors. A survey is presented about objective determination which includes skeletal mineral content by radiology and nuclear medicine examination. Following a survey of our own investigative methods and results the follow-ups in various forms of vitamin D refractory rickets are reported.


[Methods of radiological bone age assessment (author's transl)]. An assessment of the bone age can be made in different manners. Numerical methods calculating the number of existing ossification centers are to inaccurate. The use of "age-of-appearance" tables gives a more accurate evaluation. In both methods, however, x-ray films of several body parts must be made. Therefore, they are complicated and lead to a higher patient radiation exposure. Methods using hand and wrist as a representative area of the whole skeleton are of greater value for routine bone-age assessments. There is a wide-spread use of the Greulich-Pyle atlas. The atlas-method is fully sufficient in the great majority of cases when certain rules are considered. A more detailed information can be achieved by using the so-called "bone-by-bone" evaluation. A score system was introduced by Tanner and Whitehouse which should be used to a greater extent than is done up to now. Metrical methods give no real information about the bone age but additional informations which can be helpful in following examinations with short intervals.


[Significance of bone age estimation to the clinic (author's transl)]. 1. Typical alterations of bone maturation may be observed in many growth disturbances, e.g. consitutional delay of growth and development, early normal puberty and precocious puberty. They may be helpful for diagnosis and differential diagnosis. 2. Within certain limits it is possible to predict the definite adult stature of a child from his measured height and bone age. The prognosis frequently results in therapeutical consequences. 3. In order to correctly perform and evaluate hormonal treatment in endocrinopathies, regular controls of bone age are of utmost importance during childhood.


Prostaglandins and inflammation: enhancement of monocyte chemotactic responsiveness by prostaglandin E2. The effects of prostaglandins on human monocyte chemotaxis were studied in vitro. None of the prostaglandins tested, including members of the A, B, E or F series, were chemotactic for monocytes. Prostaglandin E2 however, enhanced the chemotactic responsiveness of monocytes to complement - activated human serum by almost 200%. The enhancement of chemotaxis was not directly related to the ability of PGE2 to raise intracellular cyclic AMP levels. These studies support a role for prostaglandins as modulators of the inflammatory response.


Effects of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells. Prostaglandins of the E-series (PGE1 and PGE2) may be involved in disease-related, localized loss of bone. E-prostaglandins increase the cyclic AMP content of many cells; and, to determine if their effects on bone are mediated by cyclic AMP, we examined the effects of E-prostaglandins and of other agents on the cyclic AMP content of cultured bone cells. PGE2 produced a rapid, marked and dose-related increase in the cyclic AMP content of confluent monolayers of bone cells isolated from newborn rat calvaria. At 2.8 X 10(-6) M, PGE1 and PGE2 had approximately the same effect, while the effect of PGF2alpha was much less pronounced. In the presence of theophylline, PGE2 had a more marked effect than parathyroid hormone (PTH) and the combination of PGE2 and PTH had a synergistic effect. The divalent, cationic, ionophore, A23187, produced an increase in cellular cyclic AMP and had an additive effect in combination with PGE2. Synthetic salmon calcitonin (CT), which inhibits the bone resorptive effect of PGE2, increased cellular cyclic AMP and had an additive effect in combination with PGE2. A prostaglandin antagonist, SC-19220, partially inhibited the resorptive effect of PGE2 and reduced its effect on cellular cyclic AMP. The calcium antagonist, D600, inhibited the bone resorptive effects of PGE2 but had no effect on increased cellular cyclic AMP produced by PGE2. The marked effect of PGE2 on bone cell cyclic AMP suggests that this action is involved in the mechanism of PGE2-related bone loss. The fact that agents with different effects on PGE2-induced increases in cellular cyclic AMP can inhibit its resorptive actions, suggests that PGE2-induced changes in cyclic AMP may be related less to its resorptive actions than to its inhibitory effect on bone formation.