Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene. Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.


The role of calcium in the inhibition of cAMP accumulation in epiphyseal cartilage cells exposed to physiological pressure. A hydrostatic pressure of 60g/cm sq (0.85 psi) inhibits the accumulation of cAMP in cells isolated from the proliferative zone of chick-tibia epiphyseal cartilage. The following findings indicate that this effect is mediated by a translocation of calcium: (i) the pressure enhances the cellular uptake of radiocalcium; (ii) the pressure effect on cAMP can be simulated by the calcium-ionophore A23187; (iii) the effects of pressure and A23187 are non-additive; (iv) the pressure effect is not produced in the presence of ethylenebis-(oxyethylenenitrilo)-pressure effect is not produced in the presence of ethylenebis-(oxyethylenenitrilo)-tetraacetic-acid (EGTA); (v) the particulate adenyl cyclase activity of the proliferative zone is susceptible to non-competitive calcium inhibition. Throughout this study cells from the hypertrophic zone of the same epiphyses were used as controls. In these cells the calcium uptake was enhanced by pressure, but the cAMP level was not affected by pressure, A23187 or EGTA. This change in responsiveness, which accompanies the maturation of the cartilage cells, was shown to be due to a decrease in the calcium-inhibition of adenylate cyclase.


The dynamics of antibody-induced redistribution of viral envelope antigens in the plasma membranes of avian tumour virus-infected chick embryo fibroblasts. Avian tumour virus-infected chick embryo fibroblasts express new antigens, identical with the viral envelope antigens, in their plasma membranes. Electron-microscopic examination of carbon-platinum replicas of cells labelled with haemocyanin-marked antibody has shown the distribution of these antigens to be diffuse over the cell surface with an increased concentration on peripheral cell processes. However, antigen-antibody complexes (AAC), resulting from reaction with specific antibody, may be redistributed into a variety of patterns. Observation of the time course of antibody-induced antigen mobility revealed a rapid and a delayed phase of redistribution. During the rapid phase (10 min or less) some of the antigen-bearing cells reorganized AAC into patches, while the remainder maintained a diffuse distribution. A fraction of the cells with either diffuse or patchy distribution also redistributed AAC into a pattern of 'marginal redistribution (MR)', consisting of linear aggreagation of AAC, at the cell edge. During the 'late' phase of redistribution (after about 20 min), AAC began to condense into one or more foci of coalescence (FC) on each cell. As the number of cells with FC increased with time, the fraction of cells which were labelled decreased. Electron-microscopic observation of thin sections of ferritin-labelled specimens indicated that AAC were lost by endocytosis and that this process was probably related to FC formation. Inhibitors of oxidative phosphorylation, protein synthesis, RNA synthesis, or microtubule assembly had no significant effect on the patterns or the course of redistribution. Iodoacetic acid (IAA), which depletes cellular ATP, and cytochalasin B (CB), which is believed to depolymerize microfilaments, partially inhibited MR and completely prevented FC formation and endocytosis. Paradoxically, IAA or CB-treated cells lost AAC very rapidly by some alternate mechanism not involving FC formation but which may entail a centrifugal migration of complexes to the cell extremities during the process of AAC disposal.


The morphology of green hydra endosymbionts as influenced by host strain and host environment. The ultrastructure of Chlorella-like algal endosymbionts from the Florida and English strains of green hydra was compared under different host feeding and photoperiodic regimes. Under standard conditions (host fed daily, 12-h photoperiod) the algae from the 2 strains exhibited considerable differences. The English symbionts had a pyrenoid, compact chloroplast membranes and vesiculated polyphosphate bodies. By comparison, Florida symbionts lacked a pyrenoid, had chloroplasts with less compact membranes and exhibited spherical polyphosphate bodies. When maintained in the dark, algae from English hydra lost their pyrenoids, showed great compaction of the chloroplast and developed large, shield-shaped, electron-dense bodies. In contrast, algae from Florida hosts did not exhibit gross ultrastructural modification. Reciprocal cross-transfers of symbionts were made by placing Florida algae in English aposymbiotic (algal-free) hosts and vice versa. After residence in Florida hosts, English symbionts appeared to undergo ultrastructural modifications resulting in a morphology indistinguishable from the native Florida symbionts. Florida algae showed no modifications resulting from residence in English hosts. It thus appears that the English symbiont has great morphological plasticity, as its structure is greatly modified depending upon the host in which it resides and the conditions under which the host is maintained. The results of these studies are discussed and compared with published accounts of free-living Chlorella and with reports dealing with other Chlorella symbionts.


Purification and characterization of commercial NADH and accompanying dehydrogenase inhibitors. The anion-exchange chromatography of commercial NADH using a potassium bicarbonate solution as eluent yields highly pure NADH with good stability. Twelve compounds are also separated which act as dehydrogenase inhibitors. The main impurities are further characterized. The compound mainly responsible for residual optical density in commercial NADH preparations is probably a stereoisomer of NADH which is in reversible equilibrium with NADH at pH values in the range 5-7. A method of thin-layer chromatography, to check commercial NADH preparations for impurities, is described.


Distribution of plasma glucagon immunoreactivity in a patient with suspected glucagonoma. Gel filtration of plasma from a patient with a clinical syndrome of glucagonoma and  a total plasma glucagon level of 2600 pg/ml, revealed the four glucagon immunoreactive fractions found in normal subjects. The total hyperglucagonemia observed was due to high levels of true glucagon and proglucagon moieties. The so-called "big plasma glucagon" (BPG) measured 190 pg/ml (normal average 113 +/- 79 pg/ml, Mean +/- SD, N = 10); the large glucagon immunoreactivity, LGI (9000 mol wt), measured 625 pg/ml (normal average 11 +/- 16 pg/ml); the true glucagon accounted for 1435 pg/ml (normal average 31 +/- 29 pg/ml); and the small glucagon immunoreactive fraction (approximately 2000 mol wt) measured 35 pg/ml (normal average 26 +/- 18 pg/ml). The high levels of LGI, considered a candidate for proglucagon, may reflect the increased secretory activity of the tumor.


The role of growth hormone and cortisone on glucose and gluconeogenic substrate regulation in fasted hypopituitary children. Panhypopituitarism may be associated with spontaneous hypoglycemia and marked insulin sensitivity. Five children with both growth hormone (GH) and adrenocorticotrophin (ACTH) insufficiency were studied in three periods: a) on no therapy; b) during cortisone acetate; and c) during GH and cortisone acetate replacement. With total caloric restriction prior to therapy, all patients became hypoglycemic (109 +/- 18 leads to 37 +/- 3.5 mg/dl, mean +/- SEM) and ketonemic (beta-hydroxybutyrate 0.10 +/- 0.02 leads to 3.04 +/- 0.63 mM and acetoacetate 0.05 +/- .01 leads to 0.80 +/- 0.15 mM) within 30 hours. Glutamine and alanine concentrations fell with fasting (511 +/- 13 leads to 293 +/- 26 muM and 394 +/- 58 leads to 137 +/- 12 muM, respectively) but to levels lower than in normal children. However, only alanine was significantly lower (P less than 0.05). With cortisone plus GH therapy, fasting glycemia was improved (73 +/- 6 mg/dl) at 30 hours fasting and was associated with increased alanine and glutamine concentrations (206 +/- 28 muM and 448 +/- 40 muM, respectively) and less ketonemia (beta-hydroxybutyrate 1.13 +/- 0.39 mM). Cortisone therapy alone resulted in intermediate improvement of these values. Only combined therapy resulted in increased lactate and pyruvate concentrations, which fell to normal with fasting. Fasting urinary ammonia excretion was unchanged whereas urea nitrogen excretion decreased significantly with therapy. The responses to alanine infusions following each study period in one patient were normal. The glycemic response to iv glucose was similar during each study period; however, post-prandial and glucose-stimulated insulin responses were increased with cortisone and cortisone plus GH therapy. We suggest that the hypoglycemia observed in hypopituitary patients is a substrate-mediated phenomenon, and that cortisone and growth hormone replacement therapy improve fasting glucose homeostasis, increase circulating alanine and glutamine concentrations, and decrease hepatic gluconeogenesis. These effects may be mediated through an increase in fat catabolism.


The effect of medroxyprogesterone acetate on the pituitary-adrenal axis. Twelve cancer patients and one patient with diabetes mellitus were treated with medroxyprogesterone acetate (MPA) by intramuscular injection in a total weekly dose of 400, 700, or 1200 mg. The treatment reduced the plasma cortisol concentration by 76% in the AM hours (21 leads to 5.0 mug/dl) and by 75% in the PM hours (12.8 leads to 3.2 mug/dl). Cortisol production rate decreased by 67% (19 leads to 6.2 mg/24 hrs). The 24 hour profile of plasma cortisol concentration measured in 3 patients showed zero secretion over this period. Low plasma ACTH values prevailed during treatment, and a blunted response to maximal ACTH stimulation was found. No evidence of adrenal insufficiency was observed in any patient, even though in some patients the plasma cortisol concentration remained at zero for many weeks. MPA has cortisol-like effects and the suppression of adrenal function is probably mediated by a negative feedback action on the hypothalamus or pituitary.


Pregnenolone, 17-OH-pregnenolone, and testosterone in plasma of patients with congenital adrenal hyperplasia. Both pregnenolone and 17-OH-pregnenolone were found to be higher in the plasma of patients with poorly controlled congential adrenal hyperplasia than in normal subjects. The plasma levels of these precursor steroids were significantly correlated with urinary 17-ketosteroid and pregnanetriol excretion and with plasma testosterone. The mechanism where by plasma pregnenolone and 17-OH-pregnenolone levels are elevated in patients with 21-hydroxylase deficiency is unknown, but the phenomenon of product inhibition is suggested as a possible explanation. As 17-OH-pregnenolone in plasma is almost entirely of adrenal origin, its measurement promises to be useful in the management of patients with congenital adrenal hyperplasia. Acute stimulation with ACTH caused negligible changes in the plasma levels of pregnenolone and 17-OH-pregnenolone and failed to distinguish between overly, appropriately, and under-treated patients. However, following repeated stimulation with repository ACTH, the steroid levels rose. These findings indicate limited adrenal responsiveness to ACTH following chronic glucocorticoid treatment of congenital adrenal hyperplasia, even in under-treated patients, and suggest that normal precursor  steroid levels in plasma and normal 17-ketosteroid and pregnanetriol excretion can only be achieved by the suppression of total steroidogenesis to less than that occurring in normal subjects.


The acute thyroidal response to iodixed oil in severe endemic goiter. The acute changes in serum thyrotropin (TSH) triiodothyronine (T3) and thyroxine (T4), were measured in 14 subjects, 4 to 10 (mean = 6) days following intramuscular iodized oil injection. In 8 subjects with small or absent goiter there was a significant fall in T3 and a rise in TSH concentrations, suggesting an acute inhibitory effect of the iodine. In 6 subjects with large multinodular goiters there was a marked rise in both T3 and T4, and reduction in the elevated basal TSH. Biochemical hyperthyroidism occurred in 3 of these subjects. The acute thyroid hormone response to iodized oil, whether inhibitory or stimulatory in a particular subject, depends both on goiter size and on the serum concentration of TSH.


Acute and chronic estrogen effects upon serum somatomedin activity, growth hormone, and prolactin in man. Estrogen (E) reduces bioassayable GH-dependent serum somatomedin (SM) activity in acromegalics without affecting plasma growth hormone (GH) levels and inhibits the rise of SM activity normally produced by GH administration in GH-deficient subjects. We have now investigated the effect of E administration on serum SM activity and on plasma GH and prolactin (PRL) in 6 adult male subjects without pituitary pathology. Chronic E administration (ethinyl estradiol 0.5 mg/day for 7 to 70 days) reduced serum SM activity by 40 to 62% in each of 4 subjects (P less than 0.02 to less than 0.001). In 3 of the subjects, basal GH levels increased by 75 to 300% (P less than 0.05 to less than 0.001) and basal PRL levels increased by 90 to 200% (P less than 0.01 to less than 0.001). While iv administration of normal saline did not significantly affect either SM or GH, iv administration of E (bolus injection of 25 mg conjugated estrogens, USP) to 5 subjects resulted in: a) a 46 to 80% decrease in serum SM activity in all subjects, proceeding with an apparent half-life of 2 hours, becoming significant (P less than 0.05) at 2 hours (1 subject) to 3 hours (4 subjects), maximal at 6 hours, and persisting for 12 to 24 hours; b) GH elevation to 3 to 16 times baseline level (P less than 0.01) at 2 to 3 hours in 4 subjects; and c) no significant change of PRL levels in any subject. The mean GH response to iv E was maximal at a time (2 hours) when the mean SM activity had decreased only 20% and subsided well before the nadir of SM activity. The one patient without GH response to chronic or acute E administration may have been affected by absorption of triamcinolone being applied topically during the study. These results demonstrate that in males with normal pituitary function, E reduces serum SM activity, enhances basal GH and PRL secretion, and, upon iv injection, stimulates acute GH release. Although opposite chronic E effects upon GH and SM activity support a putative negative SM-GH feed-back mechanism, iv E administration apparently provokes acute GH release by a different mechanism. The half-life of serum SM activity in the human is probably much shorter than previously estimated.


Use of biological characteristics to type Herpesvirus hominis types 1 and 2 in diagnostic laboratories. Fifty clinical isolates of Herpesvirus hominis were typed by pock size on chicken embryo chorioallantoic membranes, sensitivity to heparin, and plaque formation on chicken embryo cell cultures. Of 19 isolates that were typed serologically, there was 100% concordance with respect to plaque formation. Pock size was incorrect in one instance and indeterminate in another. Heparin sensitivity was incorrect for one. Of 31 strains that were only biologically characterized, there were four for which heparin sensitivity did not agree with the other two characteristics. Of the three biological tests, the ability to form plaques in chick cell monolayer was the most reliable indicator type.


Diagnosis of herpes simplex virus infection by immunofluorescence. The utility of the indirect immunofluorescent antibody (IFA) technique for diagnosis of herpes simplex virus (HSV) infection was examined by testing specimens for this agent from 31 patients with encephalitis or meningitis, 17 with conjunctivitis, 19 with genital disease, and 1 with genital disease and meningitis. Brain biopsy tissue from four patients with encephalitis was positive by IFA and virus culture for HSV. Leukocytes in cerebrospinal fluid from these four patients and one with HSV meningitis were also positive by IFA, but virus isolation attempts on the fluid were all negative. Conjunctival scrapings from two patients with conjunctivitis were positive for HSV by both IFA and virus culture. Eleven of 12 culture-positive lesions of herpes progenitalis were positive by IFA, and 1 dark field-positive syphilitic chancre was also positive for HSV by both IFA and culture. Evidence for specificity of the results was provided by internal controls in each test and negative results from patients with other diagnoses. Thus, the IFA technique constituted a rapid, sensitive, and specific diagnostic method for the diagnosis of HSV infections.


Immunoperoxidase technique for detection of antibodies to human cytomegalovirus. The indirect immunoperoxidase antibody technique (IPA) has been applied to determine immunoglobulin (Ig)G to humans cytomegalovirus (CMV) antibodies in 114 blood donor sera, four cases of congenital cytomegalic inclusion disease, and four cases of acquired CMV infection. The results have been compared with those obtained with the CMV complement fixation (CF) test and indirect fluorescent antibody technique (IFA) for broad spectrum CMV antibody (sigmaAb) detection. IgG antibody has been detected by both CF and IPA. In healthy adult people IPA titers are usually higher than CF titers. In addition, IFA sigmaAb titers are generally higher than CF titers. Some sera negative by CF and IPA are positive at low dilutions by IFA sigmaAb antibody determination, due to the detection of small amounts of IgA or noncomplement-fixing IgG. Nonspecific results seem unlikely, since only nuclear inclusion fluorescence was interpreted as specific, as demonstrated by blocking tests. In acute CMV infection, the IFA sigmaAb and IPA IgG titers are essentially the same, except during the first weeks of infection, when IFA titers are higher and IgM is detectable. No cross-reactivity with other herpes group viruses, herpes simplex and varicella-zoster, was observed. Although some problems of nonspecific staining of cytoplasmic inclusions are shared by both methods, the IPA technique seems to possess the same degree of sensitivity and specificity as the IFA technique, but interpretation is easier and various procedural steps can be delayed without the technical problems associated with fluorescence microscopy.


Detection of neonatal calf diarrhea virus, infant reovirus-like diarrhea virus, and a coronavirus using the fluorescent virus precipitin test. Thirty-four calf and five infant fecal specimens were tested for the neonatal calf diarrhea virus (NCDV) and for the reovirus-like infantile diarrhea agent; respectively. The procedures used were the fluorescent virus precipitin test and immune electron microscopy. Fourteen of the calf stools contained detectable NCDV, and four of the five infant stools contained the reovirus-like human agent. Infectious NCDV was detected in four of the 34 calf fecal specimens when Madin-Darby bovine kidney cell cultures that had been inoculated with supernatant fluids from stool suspensions were stained with fluorescent antibody. The 20 calf stools that did not have detectable virus were examined for the bovine corona diarrhea virus. Coronavirus was found in two of these specimens.


Control of cell division: a unifying hypothesis. A constant feature of the initiation of cell division in a number of different cells is a rise in the intracellular level of calcium. The importance of cyclic nucleotides may depend on the way they interact with calcium. Cyclic AMP is apparently not an essential regulator of cell division but through its ability to modulate the intracellular level of calcium this cyclic nucleotide can exert profound effects on cell growth. In some systems (liver and salivary glands) cyclic AMP seems to augment the calcium signal whereas in others (lymphocytes and fibroblasts) it opposes calcium and can thus inhibit cell division. A rise in the level of calcium may be responsible for the parallel increase in cyclic GMP level which is usually associated with the stimulus to divide. An appealing feature of this calcium hypothesis is that it can account for the growth characteristics revealed by fibroblasts in tissue culture or embryonic cells during development. In both cases there is an initial phase of exponential growth during which I have proposed that the high level of calcium at mitosis persists into early G1 to provide the signal for the next division. In order to account for the sudden cessation of cell division at confluency, or at a specific stage during development, it is necessary to postulate that there is something different about the final mitosis which sets it apart from earlier mitoses. It is proposed that as the cells leave the last mitosis the level of calcium falls much more rapidly than it did during preceeding mitoses perhaps as a result of a more rapid rise in the level of cyclic AMP. This rapid rise in cyclic AMP level may have a dual function. Not only will it lower the level of calcium thus preventing further division, but it may also stimulate differentiation. Many of the embryonic cells which differentiate into specialized cells (lymphocytes, liver, salivary gland) retain the ability to divide if provided with appropriate stimuli. Although the nature of these stimuli vary considerably, they all seem to act by elevating the intracellular level of calcium.


Phosphoprotein phosphatase-catalyzed dephosphorylation of the 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum. The present study demonstrated the presence within the myocardium of phosphoprotein phosphatase activity which can account for dephosphorylation of a 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum that has been associated with the stimulatory effects of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase on calcium transport (Tada, M., Kirchberger, M. A., and Katz, A. M. (1975) J. Biol. Chem. 250:2640-2647). Dog cardiac microsomes, consisting mainly of fragmented sarcomplasmic reticulum, were phosphorylated by incubation with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, and subsequently washed with trichloroacetic acid or buffered KCl. Phosphorylated microsomes contained approximately 1 nmole of 32P bound per mg of microsomal protein, 32P labeling occurring almost exclusively at the 22,000 dalton component. Soluble phosphoprotein phosphatases, isolated from the cytosol, catalyzed dephosphorylation of 32P-labeled microsomes. The existence of a phosphoprotein phosphatase that is associated with the microsomes was demonstrated by the ability of the microsomes to dephosphorylate 32P-histone. This membrane-associated phosphatase activity can also account for a rapid decrease in the amount of 32P-labeling of the 22,000 dalton protein. The dephosphorylation of the phosphorylated 22,000 dalton protein by phosphoprotein phosphatase satisfies an important requirement for the phosphorylation of the 22,000 dalton protein to serve a physiological role, namely, its reversibility.


Oppositional effects of acetylcholine and isoproterenol on isometric tension and cyclic nucleotide concentrations in rabbit atria. The effects of acetylcholine chloride and isoproterenol on myocardiial cyclic GMP, cyclic AMP and on isometric tension were studied in isolated electrically driven rabbit atria. Acetylcholine (0.5 muM) produced a significant decrease in isometric force that was associated with a significant elevation in atrial cyclic GMP. Cyclic AMP was significantly lowered at 15 seconds after the addition of acetylcholine, but was only slightly decreased at earlier time periods. Both the negative inotropic action and increase in cyclic GMP after addition of acetylcholine were blocked by atropine. Isoproterenol (0.1 muM) produced a significant increase in isometric tension that was associated with a significant elevation in atrial cyclic AMP levels, whereas cyclic GMP levels were not changed. These effects were blocked by practolol. The increases in atrial cyclic GMP and cyclic AMP following addition of acetylcholine and isoproterenol, respectively, preceded the changes in isometric tension in response to these agents. These data support the hypothesis that changes in intracellular levels of cyclic AMP and cyclic GMP may mediate the positive and negative inotropic effects of adrenergic and cholinergic agents.


Influence on adipocyte plasma membrane bound protein kinase by feedback regulator. Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.


Insulin as an activator of cyclic AMP accumulation in rat fat cells. In rat fat cells incubated with lipolytic agents and insulin for 30 or 60 minutes the increase in cyclic AMP accumulation due to norepinephrine and theophylline or adenosine deaminase added during the last 2-5 minutes of the incubation period was much greater as compared to cells incubated in the absence of insulin. Protaglandin E1 or nicotinic acid were just as anti-lipolytic as insulin but prior incubation with these agents markedly decreased the subsequent rise in cyclic AMP accumulation due to late catecholamine addition. The ability of insulin to increase cyclic AMP accumulation appeared to be secondary to inhibition of lipolysis. These results indicate that prostaglandin E1 and nicotinic acid are inhibitors of cyclic AMP accumulation while insulin acts by another mechanism to reduce lipolysis.


Parellel inhibition of LH-RH-induced cyclic AMP accumulation and LH and FSH release by LH-RH antagonists in vitro. The relative potencies of seven antagonists of LH-RH to inhibit LH-RH-induced cyclic AMP accumulation and LH and FSH release were measured using rat hemipituitaries in vitro. At appropriate concentrations, [Des-His2, D-Ala6] LH-RH, [Des-His2, D-Ala6, des-Gly-NH210] LH-RH ethylamide, [Des-His2, D-Leu6] LH-RH, [D-Phe2] LH-RH, [Des-His2, Des-Gly-NH210] LH-RH propylamide, [D-Phe2, D-Leu6] LH-RH and [D-Phe2, D-Phe6] LH-RH led to parallel inhibition of cyclic AMP accumulation and LH and FSH release. [D-Phe2, D-Leu6] LH-RH and [D-Phe2, D-Phe6] LH-RH can inhibit 50% of LH-RH action at molar ratios of 100 and 30, respectively. These findings of parallel changes of cyclic AMP levels and LH and FSH release add strong support to the already obtained evidence for a mediator role of the adenylate cyclase system in the action of LH-RH in the anterior pituitary gland.


Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts. Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.


Cyclic AMP in human blood platelets: relation to platelet prostaglandin synthesis induced by centrifugation or surface contact. Exposure of blood platelets to physical stimuli such as centrifugation, filtration through Sepharose gel, or stirring with particulate material leads to production of prostaglandins E2 and F2alpha and to a reduction in cAMP. These effects resemble those produced by ADP, epinephrine, collagen, and thrombin in association with the platelet release reaction but are less  effectively inhibited by aspirin.


Multiple forms of glycogen synthase kinase: isolation of forms which are independent of cyclic AMP. Rabbit renal cortex was found to contain three types of glycogen synthase kinase (GSK). Cylic AMP-dependent protein kinase (GSK-C) accounted for only a small fraction of the total GSK activity. The predominant type of GSK (GSK-P) could be adsorbed to phosphocellulose, but not to DEAE cellulose. The other major type (GSK-D) could be adsorbed to DEAE cellulose and exhibited several peaks when eluted with a linear NaC1 gradient. GSK-P and GSK-D were not affected by cyclic AMP or by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. This suggests that cyclic AMP-independent mechanisms may play a major role in regulation of GSK. Neither GSK-P nor GSK-D were associated with the major peak of histone, kinase, casein kinase, protamine kinase or phosvitin kinase. Therefore it cannot be assumed that these protein kinase activities can be used to monitor GSK activity.


Effect of pregnancy and lactation on triglycerides of very-low-density lipoproteins of rat plasma. Triglyceride in the blood of rats increased during pregnancy, decreased to control during lactation, and increased again on weaning. The triglyceride content of the very-low-density lipoproteins (d less than 1.006 g/ml) changed in parallel with that of the plasma, and its magnitude indicated that it was chiefly responsible for the transport of triglycerides in the blood. These changes were accompanied by changes in the electrophoretic pattern of the lipoproteins of rat plasma, but no such changes were observed in lipoproteins of ovine and caprine serum.


Effect of stress on blood leucocyte and milk somatic cell counts in dairy cows. Blood and milk samples from Holstein cows were examined for total blood leucocyte count, differential blood leucocyte count, milk quality test, and somatic cell count in milk while the cows were stressed by corticotropin injection, confinement in a heat-humidity chamber, or environmental-heat stress by exposure during the hot summer months of June through November in southern Arizona. All three stressing conditions resulted in a moderate blood leucocytosis. Modest increases in somatic cell counts of milk were associated with corticotropin injection and environmental-heat stress. Positive correlations were recorded between blood leucocytes and somatic cell counts of milk in mastitis-free cows injected with corticotropin and between percent blood neutrophils and somatic cell counts of milk in environmental-heat stressed cows with no evidence of current mastitis.


Age-related changes in the flight muscle mitochondria from the blowfly Sarcophaga bullata. Flight muscle mitochondria have been isolated from female blowflies (Sarcophaga bullata) of different ages, alpha-Glycerophosphate and pyruvate-proline respiration rates increase during development. Only pyruvate-proline respiration declines toward senescence (30%). This decline can be overcome by ATP-NaHCO3. Cytochrome concentrations and hydrogen peroxide generation rates per protein increase during development but remain constant thereafter. Total NAD+ of metabolically completely oxidized mitochondria decreases during development; a small decline occurs between mature and senescent mitochondria. Respiring young mitochondria do not swell in potassium isethionate, very little in potassium chloride, and relatively slowly in potassium acetate. Mature and senescent mitochondria do swell in these three salts but cannot be differentiated from each other on this basis. None of the preparations swells in sodium chloride, sodium- or potassium Mops. While many differences exist between young and mature mitochondria, only the decline in pyruvate-proline respiration distinguishes mature from senescent mitochondria.


Nuclear inclusions: a study of aging in Drosophila. Increased numbers and distribution of nuclear inclusions were correlated with aging in Drosophila imagoes. The various types of nuclear inclusions observed in the present study and those reported in the literature were categorized to their morphologies. It was noted that they may be located (a) in the nucleus proper, such as parallel fibrillar bundles, virus-like particles, tubulo-membraneous systems and amorphous fibrillo-granular deposits, (b) between the inner and outer nuclear membranes, such as cytoplasm-like intrusions, multilocular bodies and electron opaque vacuoles, and (c) outside the nucleus proper, such as those cytoplasmic invagination that interdigitate with infoldings of the nuclear membrane. A possible explanation of the relationship of these nuclear inclusions with aging and death is proposed.


Fructose-1,6-bisphosphatase from young and adult rats. Fructose-1,6-bisphosphatase (E.C. 3.1.3.11) was purified from the livers of young (69-86 days) and adult (370-386 days) Fisher rats. The enzyme preparations were examined for increasing amounts of missynthesized proteins by means of heat-inactivation as well as for differences in regulatory properties. No significant difference with respect to the fraction of rapidly heat-inactivated enzyme or Km- and Ki-values was found. These results do not support the hypothesis that error accumulation resulting in an error catastrophe is a general phenomenon underlying senescence and death.


Endodermally-derived and neural crest-derived differentiation antigens expressed by a human lung tumor. The plasma membrane antigens of an undifferentiated small cell (oat cell) carcinoma of the lung were studied by the indirect immunofluorescence method on frozen section substrates with a rabbit antiserum prepared to the tumor plasma membrane fraction. After appropriate absorption of the antiserum, at least two differentiation antigens present on the tumor cells but undetectable on normal lung surface or glandular epithelial cells were identified. One antigen(s) was characteristic of certain normal, endodermally-derived epithelial cells of the digestive system, including those of colonic mucosa, hepatic ducts, pancreatic ducts and acini, and islets of Langerhans. The other antigen(s) was characteristic of certain normal, neural crest-derived cells in the peripheral nervous system, including cells in peripheral nerve, dorsal root ganglion, and anterior roots of the spinal cord; parasympathetic ganglion cells in the colon; and small nerves and nerve processes in the lung, colon, and skin. It was concluded that the presence of these differentiation antigens on the tumor cells resulted from the expression of gene products repressed in the normal cell of origin of the tumor.


Studies on the terminal stages of antibody-complement-mediated killing of a tumor cell. I. Evidence for the existence of an intermediate, T. The mechanism of the terminal steps in the lysis of antibody-sensitized tumor cells by complement (TAC) was studied. It was shown that once complement has reacted, lysis proceeded even in the absence of fluid-phase complement. Transformation of TAC to dead cells was found to be at least a two-step process: one of the steps was temperature dependent whereas the other was reversibly inhibited by EDTA. In analogy to the hemolytic system, TAC has been designated T.


Four variables affecting magnitude of dentrifice abrasiveness. A five-factor study was done on human dentin abrasion that involved two abrasives, either alone or in dentifrice form, two brush brands, two brush hardnesses, two concentrations, and two temperatures of testing. The hard brushes caused 3.6 times as much wear as the soft brushes, a greater influence than the abrasives compared. A brush X hardness interaction indicated that there is a wide variation among the stiffness gradings of toothbrushes. Tests run at 37 C were 28% less abrasive than those at room temperature, suggesting a softening of bristles because of the warmer temperature. A highly significant abrasive X concentration interaction showed that dentifrices became more than twice as abrasive on dilution form 100 to 50%, but that abrasives alone did not show such changes on dilution. The second study included two hardnesses, two abrasives, three diluents, four concentrations, and two replicates. An interaction was found between abrasive and diluent, showing that glycerine inhibited abrasion by 88% in comparison with saliva and CMC. A hardness X concentration interaction showed that wear varied widely with concentration for the hard brush but scarcely at all for the soft brush. These findings suggest that abrasiveness of dentifrices depends strongly on testing conditions and that no single set of conditions is suitable for evaluating dentifrice abrasiveness.


Studies on the terminal stages of antibody-complement-mediated killing of a tumor cell. II. Inhibition of transformation of T to dead cells by 3'5' cAMP. Transformation of T to dead cells was prevented by 3'5' cAMP. The effect of 3'5' cAMP was dose, time, and temperature dependent. T washed free of 3'5' cAMP after short-term incubation proceeded to die to the same extent as control cells. After 3 hr of incubation of T with 3'5' cAMP the level of killing was significantly reduced. The 3'5' cyclic nucleotides of uridine, guanine, cytosine, and thymidine and the 2'3' cyclic adenosine nucleotide were not effective. It was concluded that prolonged treatment of T with 3'5' cAMP either irreversibly blocked the damage-producing process or facilitated the reapir of damaged sites.


Naturally soluble tumor antigens from guinea pig hepatomas: isolation and partial characterization. Naturally soluble tumor antigens were detected in the ascites fluid of guinea pigs bearing an ascites tumor and from exhausted tissue culture media of cultured tumor cells. Two antigenically distinct cell lines of diethylnitrosamine-induced strain-2 guinea pig hepatomas (line-10 and line-1) served as the source of tumor antigens. Tumor antigen activity was detected by four different techniques: immunodiffusion, inhibition of complement-mediated cytotoxicity, inhibition of membrane immunofluorescence, and delayed cutaneous hypersensitivity. With syngeneic tumor-specific antiserum, line-10 guinea pig tumor antigens were detected by immunofluorescence in the concentrated ascites and tissue culture fluids. With a xenogenic antiserum, demonstrated to be tumor specific, line-10 tumor antigens were detected not only in the concentrated ascites and tissue culture fluids but also in two of the partially purified fractions of these fluids. When the line-10 concentrated ascites and its fraction I were subjected to ultracentrifugation at 300,000 x G for 1 hr, the antigen activity was retained in the supernatant and thus by this criterion the tumor antigens detected in these samples are soluble. Immunodiffusion data indicate that more than one antigen is present in the line-10 system since three lines of precipitation were detected when line-10 concentrated ascites was reacted with the line-10 tumor-specific antiserum. In contrast to this, the line-10-concentrated tissue culture fluid displayed only one line of precipitation. Although tumor antigens could not be demonstrated in the other antigenically distinct tumor cell line, line-1, by immunodiffusion or inhibition of membrane immunofluorescence, inhibition of complement-mediated cytotoxicity was able to detect tumor antigens in the line-1 concentrated ascites and tissue culture fluids.


Regulatory serum lipoproteins: regulation of lymphocyte stimulation by a species of low density lipoprotein. Low density lipoproteins (LDL) containing apolipoprotein B were separated from 15 fresh normal human serum pools by three independent isolation methods including sequential ultracentrifugal flotation, affinity chromatography, and polyanion precipitation. A discrete subpopulation of LDL (LDL-In) was isolated which possessed comparable inhibitory activity for PHA, PWM, and allogenic cell stimulated human lymphocytes in vitro at concentrations of 1 to 10 mug protein/1 x 10(5) lymphocytes/0.25 ml culture. LDL-In was characterized by a mean buoyant density of 1.055 g/ml in KBr, a m.w. of 2 to 3 x 10(6) daltons and a composition of 20 to 25% protein and 75 to 80% lipid with beta electrophoretic mobility. The biologic activity of LDL-In was non-cytotoxic, independent of mitogen concentration, and dependent upon the concentration of serum in the culture assay. The effect was temporally dependent requiring approximately 24 hr for induction of a stable suppressed state. Suppression was reversible with shorter periods of exposure to LDL-In. LDL-In did not inhibit lymphocytes at periods greater than 19 hr after stimulation, suggesting that LDL-In may influence metabolis events associated with the inductive phase of lymphocyte activation by lectins and allogeneic cells. LDL-In was clearly distinguishable from T lymphocyte E rosette inhibitory factor since it did not influence E rosette function of lymphocytes. The physicochemical and biologic properties of LDL-In clearly distinguish this reguloratory lipoprotein from previously described immunoregulatory factors.


Effect of murine cytomegalovirus on the in vitro responses of T and B cells to mitogens. Both in vitro and in vivo murine cytomegalovirus (MCMV) infection depressed the responses of lymphocytes to both B and T cell mitogens. The possibilities that macrophages or nonspecific T cell inhibition of B cells might account for the depressed responses were eliminated. In vitro data suggested that B cell responses are more susceptible to this depression than T cell responses. The possibility that the depression of T cell responses is not a direct effect of viral infection of lymphocytes is discussed. To investigate further the interaction between B and T lymphocytes and MCMV, mice with B and T cell deficiences were studied. A comparison of the susceptibility of athymic Nu/Nu mice and T cell competent Nu/+ littermates to MCMV showed that the LD50 for Nu/Nu mice is 10-fold lower than that for Nu/+ mice, but Nu/+ mice given an LD50 of virus died much sooner after infection than Nu/Nu mice given an LD50. Pathogenic mechanisms responsible for death may be different in these two groups of mice. Similarly the MCMV LD50 for B cell-deficient mice (treated with goat anti-mouse IgM serum) was 10-fold lower than the LD50 for mice treated with normal goat serum, but given an LD50 of virus, the latter died sooner after infection than the former. In contrast, there was little difference between the LD50 or time of death after MCMV infection of CBA x DBA F1 male mice (which are deficient in their response to thymic independent antigens) and their normal littermates, the CBA x DBA F1 female mice.


Enhancement of DNA synthesis and cAMP content of mouse thymocytes by mediator(s) derived from adherent cells. Supernatants of adherent mouse peritoneal exudate cells or human mononuclear cells were used as the source of lymphocyte activation factor (LAF). LAF was found to potentiate the effect of mitogens such as PHA and Con A on DNA synthesis by mouse thymocytes. However, LAF also was capable of reducing vigorous thymosyte reactions to Con A. Thus, LAF usually enhanced the effect of PHA on DNA synthesis by BALB/c thymocytes to a relatively greater degree than that of Con A. This change in the ratio of Con A to PHA response of thymocytes suggests that LAF can serve as a regulator of thymocyte DNA synthesis. Moreover, in the presence of LAF, allogeneic thymocytes developed the ability to have bidirectional mixed thymocyte reactions. Exposure to LAF not only improved the ability of parental thymocytes to act as responder cells, but, in addition, led to increased stimulatory activity of F1 thymocytes, presumably by promoting the differentiation of stimulator cells. These indications that LAF affected differentiation were investigated further by studying its effect on the cAMP content of thymocytes. LAF stimulated significant immediate but transient elevations of intracellular cAMP and adenylate cyclase activity in thymocyte membranes. In contrast, the mitogens themselves failed to elevate or to influence the effect of LAF on the content of intracellular cAMP of thymocytes. Furthermore, the potentiating effect of LAF on mitogen-induced thymocyte DNA synthesis at times was enhanced by exogenous cGMP, carbachol, or imidazole. These findings suggest that LAF, through its stimulation of cAMP levels in thymocytes may in turn promote thymocytes to differentiate sufficiently to become competent to proliferative in response to mitogens.


Studies on T cell clonal expansion. II. The in vitro differentiation of pre-killer and memory T cells. Spleen cells from C57BL/6 mice immunized with a DBA/2 mastocytoma (P815) were harvested at various stages of the immune response and cultured in vitro in the presence and absence of antigen. Killer T cell activity in immune spleens could not be demonstrated until 6 or 7 days after antigen, but spleen cells harvested as early as 3 or 4 days and cultured for 24 hr at 37 degrees C showed significant cytotoxicity. This increased activity was not augmented further by culturing with antigen. "Memory" T cells, whose in vitro differentiation into killer cells required the presence of antigen, could not be demonstrated until 9 or 10 days after alloantigenic stimulation. Once produced, however, these cells persisted for at least 6 months. Memory cells, like killer T cells bound avidly to homologous allogeneic monolayers. There were indications that the memory T cell pool was heterogeneous. On one hand, when cells harvested 10 days after stimulation were exposed to antigen in vitro, their lytic activity increased within 24 hr but showed no further increases when the culture period was extended. In contrast, 45-day-old immune cells showed increasing lytic activity throughout a 4-day exposure to antigen. Augmentation of lytic activity in both cell populations was independent of DNA synthesis through the first 24 hr of culture. Subsequent increases in the activity of 45-day cells was dependent upon cell proliferation. Both the antigen-independent augmentation of lytic activity which followed culturing of immune cells, and the antigen-induced differentiation of memory cells were reversibly inhibited by a series of drugs which raised lymphocyte cAMP levels.


Heterologous sera: a target for in vitro cell-mediated cytotoxicity. During the course of studies in inbred Strain 2 Guinea pigs on in vitro cell-mediated cytotoxicity to viral induced fibrosarcomas, it was observed that fetal calf serum in the medium in which the target cells were grown was responsible in some instances for much of the cytotoxicity observed. This fetal calf serum-specific cytotoxicity seemed mainly mediated by T cells and appeared to be independent on histocompatibility between target and killer cells.


The development by cytomegalovirus-infected cells of binding affinity for normal human immunoglobulin. After infection with human cytomegalovirus (CMV), cells develop an affinity for normal human immunoglobulin G (IgG). This was demonstrated using 125iodine-labeled purified IgG. It was further demonstrated that the immunoglobulin molecule binds to CMV-infected cells via its Fc portion, and competition for binding to infected cells occurred between purified preparations of human IgG and the Fc fragment of human IgG. Whole sera from individuals with or without a high titer of anti-CMV antibody were labeled with 125iodine and it was demonstrated that serum from individuals with no anti-CMV antibody had an affinity for CMV-infected cells which probably reflected binding of IgG via its Fc fragment. The possible significance of these results in immunologic studies of human CMV is considered.


Human lymphocyte subpopulations: giant SRBC rosettes. Human thymus-derived lymphocytes have the ability to form rosettes with sheep red blood cells (SRBC) in vitro. In the investigation of rosettes of peripheral blood lymphocytes of 10 normal subjects, the number of SRBC adhering to the lymphocyte in each of 100 rosettes was assessed. The percentage of rosettes with SRBC greater than or equal to 36 per rosette was only 1.2 +/- 0.5. These were defined as giant SRBC rosettes. Peripheral blood lymphocytes were stimulated in vitro by four mitogens: sodium periodate, neuraminidase plus galactose oxidase, pokeweed mitogen, and concanavalin A. The lymphocytes were then cultured at 37 degrees C. The giant rosette-forming lymphocytes became significantly increased 4 to 24 hr after stimulation, prior to the appearance of lymphoblasts or increased incorporation of tritiated thymidine. The giant rosettes were not caused by the hemagglutinating properties of pokeweed mitogen and concanavalin A that were adsorbed on the lymphocyte surfaces. This was shown by the fact that, on removal of the receptors by trypsinization, they were regenerated on culture in vitro in the absence of the mitogens. It was concluded that giant SRBC rosettes constituted a marker for some of the activated lymphocytes. Their appearance was independent of the increase in size of the cells or of DNA synthesis. These receptors were intrinsic to lymphocytes and not caused by mitogens adsorbed on their surfaces.


Factors regulating growth and pigmentation of melanoma cells. Growth and melanization are intimately related in melanoma cells. MSH, by promoting elevated cyclic AMP levels, causes increases in melanization, cessation of growth, and gross morphologic changes in Cloudman S-91 melanoma cells. Growth inhibition results from high levels of cyclic AMP while growth stimulation occurs with lower levels. During melanization, oxidation products of tyrosine are generated which are toxic to the cells. Genetic studies have revealed that some of these processes are related through common biochemical pathways. This article reviews work of recent years on such regulatory mechanisms in melanoma.


Localization of adenosine o',5'-monophosphate in mouse epidermis by immunofluorescence. Adenosine 3',5'-monophosphate (cyclic AMP) has been localized in mouse epidermal cells using an immunofluorescent technique. Within 10 min following the intraperitoneal injection of isoproterenol or 30 hr following the topical application of croton oil in acetone, staining was clearly visible in the cytoplasm of the basal cells.


Effects of reserpine, epidermal growth factor, and cyclic nucleotide modulators on epidermal mitosis. The mouse ear G2 mitosis assay was modified for the screening of potential antimitotic agents. An inhibitory adrenergic influence, which maintains mitotic rate at a normally low level, was removed by pretreatment of mice with reserpine. This depletes endogenous catecholamines, produces a state of enhanced mitotic activity, and makes the epidermal cells particularly sensitive to mitotic inhibition by agents which elevate the levels of cyclic AMP. Isoproterenol [IC 50 approximately 1 X 10(-9) M], prostaglandins, dibutyryl cyclic AMP [IC 50 approximately 2 X 10(-5) M], papaverine, theophylline and 5' AMP were inhibitory in the assay, whereas dibutyryl cyclic GMP and the cholinergic stimulator carbamylcholine either stimulated or had no effect on mitosis. Epidermal growth factor was employed as an alternate means of stimulating cell division. Skin fron newborn mice or rats pretreated with this substance had increased epidermal mitotic activity which was inhibited cyclic AMP elevators.


Adrenocortical hydroxylase deficiencies in acne vulgaris. To investigate the possible occurrence of partial 11- or 21-hydroxylase deficiences in acne, an androgen-dependent disorder, 11 women with chronic nodulocystic acne were subjected to a 24-hr infusion of ACTH and their urine analyzed for tetrahydro S and pregnanetrio. The results obtained were compared to those found in 8 control women. Seven of the patients exhibited elevated excretion of either tetrahydro S or pregnanetriol, probably indicative of partial 11- or 21-hydroxylase deficiencies, respectively.


Urinary excretion of carnitine in man.

Urinary excretion of carnitine in man. A convenient assay procedure for urinary carnitine is described. Urinary excretion of carnitine is determined in human subjects in various experimental states. Average excretions by this procedure were 59.3+/-3.3 mg. per day in men and 44.1+/-2.9 mg. per day in women. Carnitine excretion had a wide variation in women during the menstrual cycle and reached a maximum at the time of ovulation, but the excretion was relatively constant in men. Marked increases in carnitine excretion were observed in the states with increased lipolysis induced by fasting or ACTH injection. This finding suggests that lipid mobilization might be reflected in the excretion of carnitine in urine.


Varicella-zoster virus and facial palsy. Two cases of Ramsey-Hunt Syndrome are reported, one of which presented features of the Guillain-Barré Syndrome. It is suggested that the varicella-zoster virus is an aetiological factor in the Guillain-Barré Syndrome.


Peripheral facial palsy and herpes zoster infection. A clinical and virological study of 133 consecutive cases of peripheral facial palsy has provided evidence for simultaneous infection with the varicella-zoster virus in 9 patients (6-8 per cent). Seven of these presented differing but typical manifestations of the Ramsay Hunt syndrome, and the diagnosis was therefore strongly suspected on the basis of the clinical findings alone. The remaining two patients had neither herpetiform rash nor involvement of the eighth cranial nerve, and had been diagnosed in the clinic as typical cases of Bell's palsy. In this series the incidence of zoster infection in apparent idiopathic facial palsy was 1-8 per cent.


Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias. Five glycosphingolipids (GSL), glucosylceramide, lactosylceramide, trihexosylceramide, globoside, and hematoside (GM3) were studied in serum from normal human subjects and patients with dyslipoproteinemia and found to be exclusively associated with the various classes of serum lipoproteins. Based on a unit weight of lipoprotein protein, the total amount of GSL in serum  normal subjects was twice as high in very low density lipoprotein (VLDL) (d less than 1.006 g/ml) and low density lipoprotein (LDL) (d 1.019-1.063 g/ml) as in high density lipoproteins HDL2 (d 1.063-1.125 g/ml) or HDL3 (d 1.125-1.21 g/ml). In abetalipoproteinemia the levels of serum GSL were slightly reduced when compared to normal serum and were all found in the only existing lipoprotein, HDL; this contained 2-3 moles of GSL/ mole of lipoprotein as compared to 0.5 GSL/mole in normal HDL. In hypobetalipoproteinemia and Tangier disease, the serum glycosphingolipids were 10 to 30% reduced in concentration compared to the 75% reduction in other lipids, and were again found to be associated only with the serum lipoproteins. The relative proportions of GSL did not vary substantially in the normo- and hypolipidemic subjects studied. Only in patients with homozygous familial hypercholesterolemia was there a significant (3-4-fold) elevation of all of the five GSL species and this elevation of all of the five GSL species and this elevation correlated well with that of the circulating cholesterol and LDL. On a molar basis the LDL of these patients contained the same amount of GSL as normal subjects (5 moles GSL/mole protein). It is concluded that: (1) glycosphingolipids are associated only with the major lipoprotein classes in both normal and dyslipoproteinemic serum; (2) the relative proportions of the five glycosphingolipids are not significantly affected by dyslipoproteinemia; (3) only in severe hypolipoproteinemia do the remaining serum lipoproteins carry a complement of glycosphingolipids greater than normal. Although our results establish that glycosphingolipids are intimately associated with serum lipoproteins, the mode of association or the structural and functional significance of such an association remains undetermined.


The effects of N-hexyl-O-glucosyl sphingosine on normal cultured human fibroblasts: a chemical model for Gaucher's disease. Normal human skin fibroblasts were grown in the presence of N-hexyl-O-glucosyl sphingosine (HGS), an inhibitor of aryl glucosidase and glucocerebrosidase. Tests of the cells with aryl glycosides showed that beta-glucosidase activity in the cells was drastically reduced while other enzyme activities (alpha-glucosidase, beta-galactosidase, and N-acetyl-beta-hexosaminidase) were normal or elevated. Exposure of cells to HGS for 28 days resulted in increased values for cell weight per plate, glucocerebroside concentration, and galactosyl-galactosylglucosyl ceramide concentration. The concentrations of total lipid, cholesterol, and protein were unchanged, as was the fatty acid distribution within the glycolipids. Chemically, the inhibitor-treated cells exhibited a model form of Gaucher's disease. Although many membranous cytoplasmic inclusions were induced by HGS, they were unlike the characteristic inclusions seen in individuals with the genetic disorder. Skin fibroblasts from a Gaucher patient showed no abnormalities in composition or appearance.


The effect of sex on the quantity and properties of the very low density lipoprotein secreted by the liver in vitro. Livers from normally fed male and female rats were perfused in vitro with different amounts of oleate, and the production and properties of the very low density lipoprotein (VLDL) were studied. The mobility of the VLDL in the zonal ultracentrifuge was dependent on the uptake of free fatty acid and on the sex of the animal from which the liver was obtained. A higher proportion of the VLDL secreted by livers from females displayed a more rapid mobility in the zonal ultracentrifuge and, in addition, contained less phospholipid and cholesterol per mole triglyceride than the VLDL from the male, suggestive of larger size of the VLDL secreted by livers from the female rats. Such differences were diminished when the VLDL was compared at equal output of triglyceride but unequal uptake of free fatty acid. These data suggest that the properties of the VLDL are only secondarily modulated by sex, and primarily result from differences in the capacities of livers from either male or female rats to synthesize triglyceride for transport as VLDL. The quantity of triglyceride secreted, regardless of sex, may be an important determinant of both size and number of the VLDL particles. The incorporation of endogenous hepatic fatty acid into VLDL triglyceride was diminished in livers from both sexes by increased uptake of oleate. The greater output of VLDL triglyceride by livers from female animals was dependent on both exogenous and endogenous fatty acids when relatively small quantities of exogenous oleate were available for uptake by the liver. The proportion of palmitate and oleate in the phospholipid of the VLDL secreted by livers from male rats decreased and the content of arachidonate increased with increasing uptake of oleate; no differences were observed in the composition of the phospholipid fatty acids among the various experimental female groups, although these contained more stearate and less oleate and linoleate compared to the male groups. The change of fatty acid composition of the VLDL phospholipid may reflect inclusion of specific types of phospholipid in the VLDL structure for transport of triglyceride from the liver under particular conditions.


Effects of sub-total gastro-intestinal pancreatectomy of the rat foetus. Sub-total pancreatectomy in utero was performed in 18-day-old rat foetuses. Pancreatectomized, sham-operated and control foetuses were collected 3 days later and body weight, glucose and insulin levels in blood, and glycogen content and glucose-6-phosphatase (G-6-Pase) activity of the liver were determined. Pancreatectomized foetuses showed only very small pancreatic remnants (less than or equal to 1 mg) and accordingly their insulin levels were much lower (four to five times) than those of sham-operated or control foetuses; their blood glucose levels were slightly increased and liver glycogen content and G-6-Pase activity were slightly reduced; their body weights were also reduced. These results are discussed in relation to other relevant data in the literature. They afford direct experimental evidence of the endogenous origin of insulin in the foetal blood. It is suggested that during the last days of intra-uterine life insulin merely completes the action of the glucocorticoids on glycogen storage in rat foetal liver and probably contributes to foetal body growth. Its relative ineffectiveness on the foetal blood glucose level is not explained. As pancreatectomized foetuses develop sub-normal liver G-6-Pase activity, glucagon is probably not responsible for the increase in this activity occurring during normal development before birth.


Ultrastructural observations of lethal yellow (Ay/Ay) mouse embryos. Ay/Ay embroys were identified by the presence of large exluded blastomeres (Pedersen, 1974) and examined cytologically and ultrastructurally. Cell organelles, inclusions and junctions in the excluded blastomeres were compared with those of non-excluded cells of Ay/Ay embryos and control embryos. Excluded blastomeres always had the fine structural characteristics of earlier developmental stages and may have arrested at the 4- to 8-cell stage of slightly later. Interior cells (inner cell mass) were observed in all mutant blastocysts. Non-excluded cells of Ay/Ay embryos were normal until degenerative changes appear in the late blastocyst stage. The mode of action of the +Ay gene was not determined, but evidence from this study and others indicate that the effects of +Ay gene action occur over a wide range of time in early cleavage and implantation.


The pharmacology of an insect ganglion: actions of carbamylcholine and acetylcholine. 1. Methods for presenting dose-response data for the ganglionic actions of cholinergic agonists (e.g. carbamylcholine) are compared, using the mannitol-gap technique for electrophysiological recording of synaptic events at the cercal nerve, giant fibre synapse of the sixth abdominal ganglion of the cockroach Periplaneta americana. 2. At concentrations around 10(-5)M, carbamylcholine has no effect on ganglionic polarization but potentiates the monosynaptic EPSP. At 10(-4)M and higher concentrations, ganglionic depolarization is accompanied by a reduction of EPSP. 3. Pretreatment with eserine (10(-6) M) considerably shifts the dose-response curve for acetylcholine so that synaptic transmission is consistently sensitive to 10(-6) M acetylcholine.


The requirement for membrane sialic acid in the stimulation of superoxide production during phagocytosis by human polymorphonuclear leukocytes. The effect of desialylation on phagocytosis of latex particles and oxidative metabolism of human polymorphonuclear leukocytes was studied. Removal of 20% total leukocyte sialic acid by bacterial neuraminidase had no effect on phagocytosis of latex particles and phagocytosis-associated activation of hexose monophosphate shunt in human polymorphonuclear leukocytes. In contrast, desialylation prevented the stimulation of superoxide production either by phagocytosis or by concanavalin A. It is concluded that membrane sialic acid is essential for the stimulation of superoxide production by human polymorphonuclear leukocytes.


A quantitative assay for transformation of bone marrow cells by Abelson murine leukemia virus. A quantitative Abelson murine leukemia virus (A-MuLV) lymphoid cell transformation assay has been developed using a semisolid agarose culture system. Under these conditions lymphoid cell transformation was shown to vary linearly with the dose of A-MuLV used. The susceptibility of bone marrow cells from different strains of mice to A-MuLV-induced transformation can be estimated using the agarose assay. Strains with bone marrow cells of high, medium, and low susceptibility to A-MuLV can be identified. The assay has been used to study the susceptibility of cells from lymphoid organs of fetal and adult mice to A-MuLV. Cell suspensions from fetal liver, adult bone marrow, and adult spleen are susceptible to A-MuLV, while thymocytes are resistant to A-MuLV-induced transformation. Bovine serum albumin gradient fractionation of bone marrow cells before infection with A-MuLV demonstrates that the majority of A-MuLV-sensitive cells are recovered in a broad band partially overlapping the majority of the nucleated cells. The agarose assay system allows study of A-MuLV-lymphoid cell interaction at the level of single cell-single virus particle interaction.


Association between mitogenicity and immunogenicity of 4-hydroxy-3,5-dinitrophenacetyl-lipopolysaccharide, a T-independent antigen. Polymyxin B, which is a basic polypeptide produced by various strains of Bacillus Polymyxa, has previously been shown to prevent the lethal effect of LPS and to neutralize the Schwartzmann reaction. In this study we have investigated the interactions between polymyxin B and lipopolysaccharide (LPS) and hapten LPS conjugates. Polymyxin B was found to suppress mitogenicity of LPS and also to inhibit immunogenicity of the hapten conjugate 4-hydroxy-3,5-dinitrophenacetyl (NNP)-LPS. Inhibition was not due to interference with the expression of NNP determinants nor to cross-reactivity between PB and the hapten. Since mitogenicity and immunogenicity decreased in parallel, we conclude that B-cell activation in specific thymus independent responses does not take place in the absence of a nonspecific (non-Ig-mediated) signal.


Generation of superoxide anion and chemiluminescence by human monocytes during phagocytosis and on contact with surface-bound immunoglobulin G. Extent of O-2-release and chemiluminescence, attributed to singlet oxygen, has been compared in human monocytes and neutrophils during phagocytosis, stimulation by the surface-active agent phorbol myristate acetate, or contact with aggregated IgG in a model of immune complex disease. Monocytes generated O-2-and chemiluminescence with each of the three stimuli, although values were significantly less than those of neutrophils from the same individuals. Lymphocytes had no significant activity in either assay with any stimulus. Oxygen metabolites released from mononuclear phagocytes are highly reactive and could play a part in both the beneficial and detrimental aspects of chronic inflammation.


Effect of dithiothreitol on meiotic maturation of mouse oocytes in vitro: dependence of the effect on N6,O2'-dibutyryl Adenosine 3',5'-cyclic monophosphate. The inhibitory effect of dithiothreitol on meiotic maturation of mouse oocytes in vitro is a function of the intracellular dibutyryl cyclic AMP concentration. Inhibition of nuclear (germinal vesicle) breakdown by dibutyryl cyclic AMP is reversed upon transfer of oocytes to plain culture medium, whereas, transfer to medium containing dithiothreitol results in continued inhibition. Dithiothreitol significantly enhances the effectiveness of dibutyryl cyclic AMP as an inhibitor of meiotic maturation. These results suggest that the reported effect of sulfhydryl reducing agents on membrane dissolution and reconstitution, in both meiotic and mitotic cells, may be attributed to their influence on intracelular levels of cyclic AMP.


Beating intraocular hearts: light-controlled rate by autonomic innervation from host iris. The sympathetic adrenergic ground plexus of the rat iris is able to innervate whole embryonic hearts transplanted to the anterior eye chamber. The transplants beat continuously from the time of transplantation and for at least five months, initially at 150-200 beats/min, and later approximately 250  beats/min. From seven days postoperatively onwards increased light to the eye produced deceleration while decreased light produced acceleration of the transplants. The rate changes coincided with changes in pupil diameter. Topical atropine reduced the light dependent rate variations of the transplants, especically the dramatic decrease in rate at white light. Waking stress caused a large rate increase. The "waking-effect" was strongly reduced by sympathetic decentralization and completely abolished by propranolol. The high rate seen in red light was decreased to the low rate of white light by decentralization. Isoprenaline restored red light rate levels in the decentralized transplant. The intraocular heart thus receives a dual functional autonomic input from the host iris and becomes a sensitive monitor of an autonomic nervous activity that can easily be varied at will in a physiological way by changing the light influx to the eye.


Biochemical studies of taste sensation. III. Preparation of a suspension of bovine taste bud cells and their labeling with a fluorescent probe. A method to prepare suspensions of taste bud cells is described. Bovine circumvallate papillae, which contain most of the taste buds in this animal, are incubated in collagenase-containing medium and the epidermal sidewall tissue is then dissected from the inner gelatinous dermis. The sidewall tissue, which contains the taste buds, is gently homogenized by manual operation of an all-glass homogenizer with a loose-fitting pestle. The suspended material is separated on a discontinous Ficoll gradient (2%, 8%, 10%, 12% w/w). The material banding at the 8-2% interface is greatly enriched in spindle-shaped cells that are morphologically similar to taste bud cells as they appear in situ. These cells are not seen when the procedure is done with tissues devoid of taste buds, namely the upper surface of the circumvallate papilla or epithelium from the intermolar eminence. Fluorescence analysis indicates that the hydrophobic probe, 8-anilino-1-naphthalenesulfonate (ANS), binds to relatively nonpolar sites in the suspension. It is postulated that the probe is adsorbing onto the surface membrane of the cell. These preparations may be useful in studying specificity and transduction in taste sensation.


Antibodies to Epstein-Barr virus and cytomegalovirus in Guillain-Barre syndrome. Twenty-three patients with Guillain-Barré syndrome (GBS) were investigated for serum antibody titres against Epstein-Barr virus capsid antigen (EBV-VCA), cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV1). The geometric mean EBV-VCA titre was 45 in GBS, compared with 15-22 in healthy adults and 60 in sarcoidosis. Nine of the 15 GBS patients (60%) from whom multiple serum specimens were available, displayed a four-fold variation in antibody titre to EBV-VCA during the course of their disease. The geometric mean titres against CMV and HSV were also slightly higher in GBS compared with controls, but no titre variations were observed. Serial samples of both serum and cerebrospinal fluid (CSF) were available from 2 patients. Serum antibody titres against EBV were significantly higher in both during the initial phase of the disease. Serum antibody titres against CMV were also significantly elevated in 1 patient, but not until the second month after onset. The CSF antibody titres against EBV were higher in the beginning of the disease compared with the values during convalescence, but the titres observed were low. No measurable CSF antibody titres against CMV were found. The observation of varying serum antibody titres against EBV during the course of GBS can be interpreted as primary infections or as manifestations of reactivated latent virus infections. Further studies of the pathogenetic significance of this for the aetiology of the disease are warranted.


Herpes encephalitis. Rapid diagnosis and treatment with antiviral drugs. The use of specific IgM antibodies and direct electron-microscopic examination of brain biopsies or vesicle fluid was tested as means of raped diagnosis in 6 cases of herpes simplex encephalitis seen consecutively in Montreal. In 2 of 3 biopsies herpes viruses were seen by negative staining of a cell extract within 1 hr. In the negative case, the biopsy was done almost 1 month after onset. In 2 additional cases herpes virus particles were found directly in the fluid of isolated vesicles. In the last 2 cases, who survived, the diagnosis of herpes encephalitis rested upon the demonstration of a greater than 4-fold rise in complement fixing herpes simplex virus antibodies in convalescent sera and upon the appearance late in the course of the encephalitis of specific antibodies in the cerebrospinal fluid. The early appearance of specific IgM antibodies contributed to the diagnosis in 4 of the 6 cases. Antiviral therapy was attempted in alternate cases (3 cases) but was not successful. Brain biopsy is rarely performed for diagnostic purposes but when prompt antiviral therapy is contemplated, the examination of the biopsy material for herpes virus particles by electron microscopy in negative staining and thin sections can rapidly and reliably confirm the diagnosis.


A comparison of the fine structure of motor end-plates in Duchenne dystrophy and in human neurogenic diseases. With the aim of investigating the pathogenesis of Duchenne dystrophy, and in particular the role of denervation, the fine structural features of motor end-plates in the limb muscles of 13 patients with Duchenne dystrophy were compared with those in 26 controls and 50 patients suffering from known neurogenic diseases. The wide variation in control end-plates es emphasised. The sole plates in Duchenne dystrophy tended to retract from the end-plate, and shared only sprout formation, enclodure of terminals in Schwann cell cytoplasm and (in one instance) close packing of neurofilaments in the terminal with the changes in denervating end-plates. In these latter, withdrawal of terminals from the sole and various degenerative figures were the rule. It was concluded that there is no adequate morphological evidence in motor end-plates to support the concept of Duchenne dystrophy as a neurogenic disease.


Peripheral nerve changes induced by methyl n-butyl ketone and potentiation by methyl ethyl ketone. A study of the sequential morphological changes in the peripheral nerve induced by experimental inhalation exposure of methyl n-butyl ketone (MBK) revealed that the earliest change was an increase in the number of neurofilaments in the large myelinated nerve fibers. This change occurred prior to axonal swelling or myelin thinning. As the duration of exposure lengthened the number of neurofilaments gradually increased and ultimately produced axonal swelling with secondary thinning of the myelin sheath. This appears to be the pathogenesis of the "giant axonal" neuropathy. Another change observed early in this neuropathy was the presence of inpouchings of the myelin sheath, which also increased in number in parallel to the duration of exposure. A careful study of the sequential changes in the entire motor unit did not show a predilection for early morphological changes at the axon terminal. Abnormalities at the neuromuscular junction occurred only after a full spectrum of changes were seen in the main nerve trunk, nerve roots and intramuscular nerves. An important observation was the marked potentiation of peripheral neurotoxicity observed when animals were exposed to MBK in combination with methyl ethyl ketone (MEK) at a ratio of 1:5, MBK:MEK. The latter solvent showed no neurotoxic effect alone. This might help explain a recent outbreak of a polyneuropathy affecting many workers. One further observation was that the sural nerve of a patient with prolonged exposure to MBK showed changes similar to those induced experimentally.


Experimental panencephalitis induced in suckling mice by parainfluenza type 1 (6/94) virus. I. Clinical and pathological features. Intracerebral inoculation of two strains of suckling mice with 6/94 virus, a parainfluenza type 1 virus originally isolated from two patients with multiple sclerosis, produced clinical disease 1-2 weeks after inoculation. Of 528 animals inoculated, 33% died (26% of the ICR strain and 76% of the BALB/c strain) usually between two or three weeks after injection. Animals that recovered appeared to develop normally. Pathological changes were of two types. Initially, there was a necrotizing panencephalitis with virus-specific intracytoplasmic inclusions in choroid and ependymal epithelial cells and neurons. The second major lesion appeared about 6 weeks post inoculation and consisted of a noninflammatory spongiform degeneration of white matter that primarily involved the cerebral hemispheres; a diffuse vacuolar encephalapathy primarily affecting the brain stem; and a persistent minimal inflammation.


Experimental panencephalitis induced in suckling mice, by parainfluenza type 1 (6/94) virus, II. Virologic studies. 6/94 virus, a parainfluenza type 1 isolate from multiple sclerosis brain tissue, produced a chronic panencephalitis when inoculated intracerebrally into suckling ICR mice. Immunofluorescent staining revealed 6/94 viral antigen in ependyma, meninges, choroid plexus, and perivascular parenchymal sites from day 3 to 128 days after infection. Hemadsorption-neutralizing antibody was first detected between 20-25 days after infection and remained at high titers for 7 months. Using embryonated chicken eggs, virus was recovered from mouse brains for only 8 days, but could be recovered from brains grown in vitro as explants for 37 days after infection. In cell lines established from explanted brain tissue, immunofluorescence was the most sensitive indicator of virus presence, although infectious virus was not produced. Fusion of these mouse brain cells with human (W138) indicator cells was the most effective means of rescuing 6/94 virus.


Experimental panencephalitis induced in suckling mice by parainfluenza I (6/94) virus. III. Ultrastructural studies. Intracerebral inoculation of newborn mice with Parainfluenza I (6/94) virus produces a chronic panencephalitis. Electron microscopic studies were carried out over 125 days of the infection. Productive infection of choroidal and ependymal epithelial cells was seen from postinoculation days 2nd to the 8th. Fusion of adjacent choroid and ependymal cells resulted in giant cell formation. Completed virions were seen adsorbed to circulating macrophages and these cells replicated intracytoplasmic nucleocapsids. Neuronal infection was evident on the 3rd postinoculation day, was widespread by the 6th day postinoculation and persisted to the 35th day postinoculation. Nucleocapsid alignment and budding from neuronal plasma membranes was never seen. An initially intense mononuclear cell infiltrate subsided by the 35th day but residual inflammation persisted throughout the study. Late in the course of the infection, vacuolation of the neuropil and a periventricular and deep cerebral spongiform change was seen which could not be directly associated with local viral replication. These ultrastructural findings are correlated with prior light microscopic, virological and immunofluorescent studies of the infection and compared to other experimental models of myxovirus central nervous system infections.


"Paramyxovirus-like" intranuclear inclusions occurring in the nervous system in diverse unrelated conditions. Filamentous intranuclear inclusions similar to the "paramyxovirus-like" intranuclear inclusions described in active multiple sclerosis (MS) were observed incidentally in four patients who had diverse disorders such as rabies, mannosidosis, metachromatic leukodystrophy, cerebral aneurysm and ischemic infarcts. The observation is in support of the mounting evidence that the "virus-like" intranuclear inclusions are not specific for MS and may occur frequently in a variety of diverse conditions. Moreover, neither virological nor immunological evidence as to the viral nature of the intranuclear inclusions has been presented to date, and the intranuclear inclusions have been observed mostly in degenerating or autolyzed cells in biopsied or autopsied tissue samples. In view of all the circumstantial evidence, it is suggested that the intranuclear "paramyxovirus-like" inclusions may represent an alternation of nuclear chromatin common to antemortem degeneration and postmortem autolysis in a variety of cells, the nature of which remains to be elucidated.


Organization of crustacean neuropil. II. Distribution of synaptic contacts on identified motor neurons in lobster stomatogastric ganglion. Identified neurons in the stomatogastric ganglion of the lobster were examined and reconstructed by serial section electron microscopy. Each motor neuron consists of a soma, a primary process leading directly from the soma to the motor axon which leaves the ganglion, and a group of secondary processes which branch from the primary process and ramify within the neuropil. Synapses are found only on small processes in the synaptic neuropil, never on the primary processes or on larger secondary processes in the coarse neuropil. Nearly every secondary process of every neuron examined makes both pre- and postsynaptic contacts. Hence these neurons are not polarized into distinct pre- and postsynaptic regions but have both input and output distributed over each of the secondary processes in the neuropil. The conncetion between a specific pair of neurons is also distributed over several branches of both the pre- and the postsynaptic neurons. The restriction of synapses to the more distal portions of the secondary processes suggests that no single contact or localized group of contacts can exert an overrriding influence on the neuron by virtue of an especially advantageous position. The close proximity of input and output on most secondary processes suggests that synaptic input may be capable of directly influencing output without the intervention of action potentials. The distribution of specific synapses over several branches of both pre- and postsynaptic neurons suggests that each neuron functions as a whole without differentiation into specialized branches.


Evaluation of mithramycin in the treatment of anaplastic gliomas. A controlled, prospective, randomized study evaluated the use of mithramycin in the treatment of anaplastic glioma compared to a similar group of patients receiving best conventional care. From a total of 116 patients in the study, 96 were within the valid study group. All patients were operated on, had histological confirmation of anaplastic glioma, and received radiotherapy at the discretion of the principal investigator. Fifty-two patients received mithramycin at a dose of 25 mug/kg/day for 21 days, while 44 patients were in the control group. There was no significant difference in the median survival from time of randomization in those receiving mithramycin (21 weeks) as compared to those not receiving mithramycin (26 weeks). There was no significant difference between the two groups in relation to age distribution, sex, location, diagnosis, tumor characteristics, signs or symptoms, or radiotherapy received. Duration of symptoms correlates positively with survival and was also significantly longer in the control group than in the treated group. This, however, did not account for the failure of mithramycin to be found an effective agent. Although the study was not designed to evaluate the efficacy of radiotherapy, patients who were so treated had a significant improvement in survival. The toxic complications of mithramycin included gastrointestinal symptoms, dermatological involvement, anemia, and liver dysfunction, indicating the need for close supervision.


Unusual bone-scan findings in acute osteomyelitis: case report. In osteomyelitis, bone-scan findings precede the appearance of bone changes on radiographs. In cases where focal ischemia occurs, the earliest scan finding may be a "cold" area that later becomes "hot" as active periostitis develops.


Gallium-67  uptake by a malignant fibrous histiocytoma: case report. Avid 67Ga-citrate uptake was observed in a malignant fibrous histiocytoma of the mediastinum. The realtionships of the tumor to the heart and liver was shown by 99mTc-sulfur colloid liver-spleen scanning and 99mTc-pertechnetate angiography performed in conjunction with a 67Ga-citrate whole-body scan. This is the first report of 67Ga-citrate uptake by this unusual tumor.


Mechanism of localization of 99mTc-labeled pyrophosphate and tetracycline in infarcted myocardium. The gross and subcellular localizations of 99mTc-labeled pyrophosphate and tetracycline in myocardial infarcts were studied in a rabbit model. Experiments utilizing double-nuclide labeling were carried out using a useful mapping technique. Concentration of the various chelates decreases in an expected manner from the center of the infarcted area toward its periphery, but it is higher near the epicardial surface than toward the endocardium. Technetium-99m-pyrophosphate is concentrated in the same infarcted areas as 45Ca ion or 32P-pyrophosphate, but to a much greater degree. The uptake is dependent on both the degree of necrosis and residual blood flow. Gel filtration experiments with rabbit serum indicate that 99mTc-tagged pyrophosphate, tetracycline, and diphosphonate are mainly protein-bound, whereas 32P-pyrophosphate is not. Subcellular localization studies show that 99mTc-tetracycline and 99mTc-pyrophosphate are bound primarily to soluble protein, and only a small fraction is associated with nuclei, mitochondria, and microsomes. The uptake of technetium chelates in myocardial infarcts may be due to the formation of polynuclear complexes with denatured macromolecules rather than to the deposition of calcium in mitochrondria.


Biological activity of 24,25-dihydroxycholecalciferol in chicks and rats. The ability of 24R, 25- and 24S, 25-dihydroxycholecalciferol to stimulate intestinal calcium transport and bone calcium mobilization in chicks was measured. Enhancement of intestinal calcium transport by 325 or 130 nmoles of either compound was maximal by 24 hours. The effects of these compounds on bone calcium mobilization were also maximal by 24 to 36 hours. When lower doses were tested, 2 nmoles of the 24R, 25-dihydroxycholecalciferol significantly stimulated intestinal calcium transport, whereas 130 nmoles of the S isomer were required for a significant response. Neither steroid had a significant effect on bone calcium mobilization when doses of less than 130 nmoles were given. When chicks received orally 32.5, 325 or 1,625 pmoles of 24R, 25-dihydroxycholecalciferol daily from hatching for 4 weeks, several parameters showed a dose-related response. These included growth, serum calcium, bone ash, renal 25-hydroxycholecalciferol-1-hydroxylase and intestinal vitamin D-dependent calcium binding protein. Rats given 32.5 to 1,625 pmoles of 24R, 25-dihydroxycholecalciferol for 3 or 6 weeks were equivalent to vitamin D-treated controls in terms of growth and serum calcium levels. It is concluded that within the lower dose ranges (2 to 30 pmoles) the R isomer of 24,25-dihydroxycholecalciferol is more active in stimulating intestinal calcium transport than the S isomer but that neither compound increases bone calcium mobilization at these dose levels. Also, the rat is more responsive in terms of growth and serum calcium, to small dialy doses of 24R, 25-dihydroxycholecalciferol than is the chick.


Effects of fluoride and ethane-1-hydroxy-1,1-diphosphonate on bone metabolism in the growing chick. White Leghorn cockerols were fed a semipurified diet (containing fluoride at 0 to 800 ppm) from the day of hatching. The birds were also injected daily with solutions containing ethane-1-hydroxy-1,1-diphosphonate (EHDP) at concentrations ranging from 5 to 20 mg as phosphorus/kg of body weight or isotonic saline. Changes in bone mineral composition and plasma Ca, Mg and F were determined as well as alterations in bone pyrophosphatase and the amounts of bone cyclic-3',5'-adenosine monophosphate (cAMP). Since dietary fluoride in high amounts is known to stimulate new bone formation, the purpose of the study was to assess whether a potent inhibitor of mineralization, such as EHDP, might alter the response to high dietary fluoride. Although bone fluoride and magnesium were increased in relationship to dietary fluoride, the administration of EHDP had little effect on these changes at low to moderate levels of fluoride. The levels of soluble bone pyrophosphatase were not greatly influenced by changes in dietary fluoride. Administration of EHDP at 20 mg P/kg, however, typically decreased the levels of bone pyrophosphatase. Bone cAMP did not appear to be influenced by either dietary fluoride or EHDP. Using tissue culture techniques, the effects of fluoride on calcium uptake and release from embryonic chick bone were also studied. The presence of 1 mM fluoride in the medium appeared to stimulate calcium uptake and reduced calcium release from chick bone. In general, the results were in keeping with previous suggestions that the major effect of fluoride on bone is the formation of fluoroapatite and subsequent effects this may have on other metabolic alterations.


Binding of 7-dehydrocholesterol to sterol carrier protein and vitamin D3 effect. It was confirmed that delta 5,7-sterol delta 7-reductase activity was suppressed by cholecalciferol (vitamin D3) in the enzyme system consisted of microsomes and sterol carrier protein (SCP). The enzyme activity was significantly decreased in the combination with microsomes obtained from either vitamin D-deficient or vitamin D3-treated rat liver and with SCP obtained from vitamin D3-treated rat. It was also demonstrated by the binding assay of the dextran-charcoal technique that 7-dehydrocholesterol binding to SCP could be specifically displaced by vitamin D3. The inhibition of cholecalciferol on 7-dehydrocholesterol binding to liver SCP was confirmed to be non-competitive inhibition.


Carbon-13 nuclear magnetic resonance spectra of vitamins D and related compounds. 1. The natural abundance carbon-13 nmr of vitamins D (D2 and D3) and several isomers (5, 6-trans-vitamin D2, isotachysterol2 and isovitamin D2)  have been completely assigned by employing off-resonance noise-decoupling, acetylation shifts, and lanthanide-induced shifts experiments. The last two techniques were especially useful for the present study. 2. Carbon-13 nmr spectral characteristics of the three main conjugated triene moieties (SE-Z-SZ, SE-E-SZ, or SE-E-SE), involved in the molecules of vitamin D and its isomers, were revealed. Thus, the striking dependence of the shieldings on molecular geometries and high sensitivity of the resonances to the environments of conjugated systems were surveyed. 3. Conformational preferences in solutions of the hydroxyl groups in vitamins D2 and D3 as well as 5, 6-trans-vitamin D2 were conveniently determined.


Quantitative studies on cell-mediated immunity in the pleural cavity of guinea-pigs. Intrapleural injection of PPD into CFA-sensitised guinea-pigs produced a reaction having many of the characteristics of classical delayed hypersensitivity. Quantitative investigations of the time course of this reaction showed that it was typified by an initial delay in the onset of increased vascular permeability, which reached its peak between 12 and 18 hr. The cellular response in the pleural cavity followed a similar pattern, attaining maximum levels between 18 and 24 hr. Mononuclear cells dominated the reaction from 12 to 48 hr. The histological appearance of the lung tissue was also studied. Treatment with Cobra venom factor, a complement-depleting agent, failed to modify this reaction. Cyclic AMP levels were assayed for both the cellular and cell-free exudates.


25-Hydroxycholecalciferol inthe management of rickets associated with extrahepatic biliary atresia. In children with extrahepatic biliary atresia, impaired hydroxylation and defective intestinal absorption of cholecalciferol may lead to a deficiency of vitamin D and rickets. The data presented herein demonstrate that in such patients serum levels of vitamin D measured as 25-hydroxycalciferol are reduced. A moderate therapeutic oral dose of 25-hydroxycholecalciferol, by circumventing the hepatic conversion of cholecalciferol to 25-hydroxycholecalciferol, will replete vitamin D stores and maintain the serum concentration of 25-hydroxycalciferol required to prevent or heal rickets in these patients.


Evaluation of a live, attenuated respiratory syncytial virus vaccine in infants. Respiratory syncytial virus ts-1 is a live attenuated experimental vaccine which was administered intranasally to 25 infants 11 to 19 months of age. Clinical evaluation was carried out following a controlled, double-blind protocol which eliminated observer bias, assessed intercurrent illness, and was designed to detect virus transmission. At the low dose of virus of virus used (100 TCID50) 8 of the 25 recipients were successfully infected with RS virus ts-1 as determined by virus shedding or antibody response.


HCG + ACTH stimulation of in vitro dehydroepiandrosterone production in human fetal adrenals from precursor cholesterol and delta5-pregnenolone. Fetal adrenal glands were obtained from legal abortions in the 14--22 gestational weeks. The adrenal cortex was separated from the medulla using a micromanipulator. The cortex was homogenized in a glass-teflon homogenizer and the microsomal fraction was isolated by centrifugation. Tissue slices were also prepared by the method of Deutsch. The microsomal fraction or the slices were incubated with (4-14C) pregnenolone or (4-14C) cholesterol in the presence of an NADPH regenerating system and oxygen. ACTH or HCG was added. After extraction and paperchromatography radioactivity was determined in a scanner. The conversion of pregnenolone to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone was increased by ACTH. In the presence of HCG dehydroepiandrosterone production only was increased,--and this occurred in slices only. In the microsomal fraction HCG was without effect on steroid biogenesis from dehydroepiandrosterone.