Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.


Studies on the chlorinating activity of myeloperoxidase.

Two methods were utilized to demonstrate the peroxidation of chloride ion to a free species (HOCl or Cl2) by myeloperoxidase. The peroxidase caused the volatilization of radioactivity from soultions containing hydrogen peroxide and [36Cl]NaCl, and catalyzed the formation of HOCl when solutions contianing these components were passed through a Millipore filter to which the peroxidase was adsorbed. In this flow system, 90 mug of canine myeloperoxidase generated 80 muM HOCl in the presence of 200 muM H2O2 at a rate corresponding to a turnover of 100 min-1. Under these conditions, o-tolidine, whose oxidation can be coupled to Cl- peroxidation in free solution, did not accelerate turnover. In contrast to chloroperoxidase and horseradish peroxidase, myeloperoxidase does not utilize chlorite for chlorination reactions. This oxidant inactivates the enzyme. At low pH, chloride ion suppresses the oxidation of myeloperoxidase (to the stable compound II) by both hydrogen peroxide and hypochlorite. Acceptor chlorination is therefore not a rate-controlling reaction in the myeloperoxidase mechanism, and the potential of the functional peroxidase couple is higher than the HOCl/Cl- couple under chlorinating conditions. The product-forming step may be a reverse of compound I formation at the expense of HOCl, rather than the chlorination of Cl- by a chloroperoxidase-like chlorinating intermediate.


Role of cortisol on the glycogenolytic effect of glucagon and on the glycogenic response to insulin in fetal hepatocyte culture.

The effects of insulin and glucagon on glycogen metabolism were studied in cultured fetal hepatocytes transplanted from 15-day-old fetuses. The effects of these hormones were examined just after transplantation, when the cells contained only minute amounts of glycogen, and during the 3 to 4 day culture period, when the hepatocytes were exposed to 10 muM cortisol and actively accumulated glycogen. At all stages of the culture, glucagon addition (10 nM) was followed by a rapid depletion of labeled glycogen, previously synthesized during a pulse labeling with [14C]glucose: this effect was mimicked by N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) (0.3 to 1 nM). Such a glycogenolytic effect of glucagon was observed even 6 hours after transplantation, i.e. at a time when cortisol was not present. In addition, glucagon clearly induced cyclic adenosine 3':5'-monosphosphate (cyclic AMP) accumulation in cells grown for 18 hours in the absence of cortisol. With cells grown for 3 days in the presence of cortisol, glucagon-dependent glycogenolysis was also obtained when cortisol was removed from the medium 20 hours before hormone addition. Thus the presence of cortisol is not necessary either to maintain a response to glucagon or for the onset of the glycogenolytic effect of glucagon. Insulin addition (10 nM) stimulated [14C]glucose incorporation into glycogen at all stages of the culture when grown in the presence of cortisol; no glycogenic response to insulin was observed 6 hours after transplantation where cortisol was not previously introduced. In addition, if the hepatocytes were grown in the presence of insulin alone (i.e. in the absence of cortisol) no significant storage of glycogen occurred. Maximal storage (or labeling) of glycogen was observed when hepatocytes were grown in the presence of both cortisol and insulin. The presence of cortisol was therefore necessary for the expression of the glycogenic effect of insulin. These data show that marked difference exist between the onset of developmental responses towards glucagon and insulin. The glucagon-dependent regulatory pathway should be present very early in fetal development and should not depend on cortisol. On the contrary, the onset of the insulin-dependent regulatory pathway seems to be induced during culture, and it is likely that this is caused by cortisol.


Tissue-specific chromosomal non-histone protein interactions with DNA.

A group of chromosomal non-histone proteins with affinity for DNA (NP) was isolated from rat liver and Novikoff hepatoma. This fraction, which represents less than 5% of the total chromatin protein content, binds preferentially to unique, double-stranded sequences of fractionated homologous DNA. The interactions are strong at low ionic strength (Km = 6.7 X 10(-9) M) and decrease with rising salt concentration. Complexes of the NP protein fraction with homologous DNA are immunologically tissue-specific. As determined by microcomplement fixation, the NP proteins in Novikoff hepatoma are associated with the transcriptionally active, diffuse fraction of chromatin.


Comparison of mode of activation of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase (protein kinase G) partially purified from silkworm pupae was selectively activated by cyclic GMP at lower concentrations. Nevertheless, the enzyme seemed to differ from adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) with respect to the mode of response to cyclic nucleotides. The catalytic activity and cyclic GMP-binding activity were not dissociated by cyclic GMP in a manner similar to that described for protein kinase A. The enzyme was not inhibited by regulatory subunit of protein kinase A nor by protein inhibitor. A sulfhydryl compound such as 2-mercaptoethanol or glutathione was essential for the activation by cyclic GMP, and an extraordinary high concentration of either Mg2+ (100 mM) or Mn2+ (25 mM) was needed for maximal stimulation by cyclic GMP. A polyamine such as spermine, spermidine, or putrescine could substitute partly for the cation. Kinetic analysis indicated that Km for ATP was decreased whereas Ka for cyclic GMP was increased significantly at high concentrations of the cation. The effect of the cation to decrease Km for ATP was not evident in the absence of a sulfhydryl compound. These characteristics of protein kinase G described above were not observed for protein kinase A which was obtained from the same organism.


Influence of glucagon and cyclic adenosine 3':5'-monophosphate on the phosphorylation of rat liver ribosomal protein S6.

The administration of glucagon, adenosine 3':5'-monophosphate, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate caused an increase in the phosphorylation of rat liver ribosomes. The increase (approximately 3-fold) was in the protein of the small ribosomal subunit. The proteins were separated by two-dimensional polyacrylamide gel electrophoresis and radioautographs were made of the gels. The effect of the hormone and of the nucleotides was entirely due to an increase in the phosphorylation of the 40 S ribosomal subunit protein S6.


Effects of epinephrine and insulin on phosphopeptide metabolism in adipocytes.

Isolated adipocytes, incubated in the presence of extracellular 32Pi to steady state 32P incorporation into cellular phosphopeptides, were exposed to hormones for 5 min. Epinephrine (10(-6) M) stimulated 32P incorporation into at least 12 major phosphopeptides, distributed in the cytoplasm, endoplasmic reticulum, and plasma membrane. Quantitatively pre-eminent among these were peptides of molecular weight 123,000 and 69,000, each located both in the cytoplasm and endoplasmic reticulum. The effect of epinephrine (10(-7) M) on 32P incorporation into these two peptides was augmented by theophylline (10(-3) M) in a synergistic fashion. Norepinephrine, dibutyryl N6,O2'-dibutyryl adenosine 3':5'-monophosphate, adrenocorticotropic hormone (ACTH) (synthetic 1 to 24 fragment), and glucagon mimicked the effect of epinephrine. Insulin modified adipocyte peptide phosphorylation in two ways. When present as the sole hormone, insulin (100 microunits/ml) consistently and selectively stimulated the 32P incorporation into a peptide of molecular weight 123,000 (endoplasmic reticulum, cytoplasm) without significant alteration in the 32P content of any other major peptide. A second effect of insulin was evident when epinephrine (10(-6) M) was present simultaneously. Insulin significantly inhibited the epinephrine-stimulated phosphorylation of the molecular weight 69,000 (endoplasmic reticulum, cytoplasm) and 26,000 (plasma membrane) peptides. Nevertheless, persistence of insulin-stimulated phosphorylation of the 123,000 peptide in the presence of epinephrine was shown by a 32P content of this peptide that was greater in the presence of both hormones than with either individually. These findings indicate that in intact adipocytes: (a) epinephrine acutely alters the phosphorylation of a large number of adipocyte peptides, partly at least, via activation of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase; (b) insulin opposes several epinephrine-stimulated phosphorylations in a manner consitent with its ability to lower epinephrine-stimulated intracellular cyclic AMP accumulation in adipocytes; and (c) insulin, in addition, exerts a unique stimulatory effect on adipocyte peptide phosphorylation that is independent of its effects on cyclic AMP metabolism and may be medicated by the generation of an as yet undefined intracellular "messenger" unique to insulin.


Biochemical properties of rat liver mitochondrial aldehyde dehydrogenase with respect to oxidation of formaldehyde.

The oxidation of formaldehyde by rat liver mitochondria in the presence of 50 mM phosphate was enhanced 2-fold by exogenous NAD+. Absolute requirement of NAD+ for formaldehyde oxidation was demonstrated by depleting the mitochondria of their NAD+ content (4.6 nmol/mg of protein), followed by reincorporation of the NAD+ into the depleted mitochondria. Aldehyde (formaldehyde) dehydrogenase activity was completely abolished in the depleted mitochondria, but the enzyme activity was restored to control levels following reincorporation of the pyridine nucleotide. Phosphate stimulation of formaldehyde oxidation could not be explained fully by the phosphate-induced swelling which enhances membrane permeability to NAD+, since stimulation of the enzyme activity by increased phosphate concentrations was still observed in the absence of exogenous NAD+. The Km for formaldehyde oxidation by the mitochondria was found to be 0.38 nM, a value similar to that obtained with varying concentrations of NAD+; both Vmax values were very similar, giving a value of 70 to 80 nmol/min/mg of protein. The pH optimum for the mitochondrial enzyme was 8.0. Inhibition of the enzyme activity by anaerobiosis was apparently due to the inability of the respiratory chain to oxidize the generated NADH. The inhibition of mitochondrial formaldehyde oxidation by succinate was found to be due to a lowering of the NAD+ level in the mitochondria. Succinate also inhibited acetaldehyde oxidation by the mitochondria. Malonate, a competitive inhibitor of succinic dehydrogenase, blocked the inhibitory effect of succinate. The respiratory chain inhibitors, rotenone, and antimycin A plus succinate, strongly inhibited formaldehyde oxidation by apparently the same mechanism, although the crude enzyme preparation (freed from the membrane) was slightly sensitive to rotenone. The mitochondria were subfractionated, and 85% of the enzyme activity was found in the inner membrane fraction (mitoplast). Furthermore, separation into inner membrane and matrix components indicated a distribution of aldehyde dehydrogenase activity similar to malic dehydrogenase.


Binding of nitric oxide to reduced L-tryptophan-2,3-dioxygenase as studied by electron paramagnetic resonance.

Ferrous L-tryptophan-2,3-dioxygenase reacts with nitric oxide both in the presence and in the absence of L-tryptophan. Electron paramagnetic resonance studies suggest that the proximal ligand of the heme is a nitrogen atom, probably from an histidyl residue. The interaction of the protein with substrate changes both the symmetry of the paramagnetic center and the mode of interaction of the iron atom with its two axial ligands, NO and the proximal nitrogen atom. Optical absorption and EPR spectra suggest that the affinity of NO for tryptophan dioxygenase increases in the order: tryptophan dioxygenase, tryptophan dioxygenase + alpha-methyltryptophan, tryptophan diogenase " 5-hydroxytryptophan, tryptophan dioxygenase + L-tryptophan. A possible correlation between the number of superhyperfine lines in the EPR spectrum and the affinity of the enzyme for NO is discussed.


Apparent dispensability of the carbohydrate moiety of human interferon for antiviral activity.

Human leukocyte and tritium-labeled fibroblast interferons, prepared by induction with Sendai virus and with double-stranded polyinosinic acid.polycytidylic acid respectively, have been studied in relation to the carbohydrate moieties attached to them. These interferons were partially purified by immunoabsorbance and by gel filtration. On treatment with glycosidases, about 80% of the 3H-labeled sugar moieties in this glycoprotein-containing fraction was removed without detectable alteration of the antiviral activity or antibody-binding properties characteristic of interferon. The molecular weight of leukocyte interferon was reduced by about 4000. As others have reported, the heterogeneous character of interferon revealed by isoelectric focusing was greatly reduced by the enzyme treatment.


Studies on regulatory factors of ornithine decarboxylase activity during development of mouse mammary epithelium in vitro.

The activity of ornithine decarboxylase increases markedly in a biphasic manner during the hormone-dependent development of mouse mammary epithelium in vitro. The first peak of activity occurring at 3 to 4 hours of culture was elicited by incubating mammary explants in a culture medium without any added hormones, although addition of insulin or prolactin, or both, caused a greater increase. The emergence of the second peak of activity at about 12 hours depended on the actions of both insulin and prolactin. A second increase in activity could also be effected postmitotically by the delayed addition of prolactin. Studies with actinomycin D and cycloheximide suggest that the first increase in enzyme activity may be effected at a post-transcriptional level, whereas a second increase may be at both transcriptional and translational levels. During the first 3 hours of incubation, there was a rapid, transient increase in cyclic AMP concentration in mammary epithelium. The presence of insulin or prolactin in culture did not affect the change in epithelial cyclic AMP concentration. Addition of several derivatives of cyclic AMP, 0.1 to 0.5 mM, as well as prostaglandin E1, a stimulator of adenylate cyclase, resulted in enhancement of the first increase in enzyme activity. The effect of cyclic nucleotide was additive to that of insulin and prolactin and appears to be mediated at a post-transcriptional level. The stimulatory effect of a lower concentration of both the cyclic nucleotide and prostaglandin E1 was augmented by theophylline, an inhibitor of phosphodiesterase. These results may suggest possible involvement of cyclic AMP in the first increase in enzyme activity that occurs in the absence of any added hormones.


Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in hepatoma tissue culture cells by pure cholesterol and several cholesterol derivatives. Evidence supporting two distinct mechanisms.20l.

Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.


Purification and general properties of guanosine 3':5'-monophosphate-dependent protein kinase from guinea pig fetal lung.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was purified from the guinea pig fetal lung, a tissue shown to be the richest in this enzyme in all mammalian sources examined, and its general properties studied. The enzyme was purified 150-fold from crude extract by steps of pH 5.4 isoelectric precipitation, Sephadex G-200 filtration, hydroxylapatite treatment and DEAE-cellulose chromatography. The purified enzyme, free from contamination with adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase, had a specific activity at least equivalent to 600-fold purification of the enzyme from the adult lung. The pulmonary enzyme exhibited an absolute requirement of protein kinase modulator (prepared from various mammalian tissues with an exception of skeletal muscle) for its activity. Inhibitor protein of cyclic AMP-dependent protein kinase purified from rabbit skeletal muscle could not stimulate nor inhibit the cyclic GMP target enzyme, indicating the factors from mammalian sources regulating the two classes of protein kinases may not be the same. The enzyme had Ka values of 1.3 times 10(-8) and 3.3 times 10(-8) M for 8-bromo cyclic GMP and cyclic GMP, respectively, compared to 3.0 times 10(-6) M for cyclic AMP. Cyclic GMP lowered the Km of the enzyme for ATP from 6.3 times 10(-5) M in its absence to 2.1 times 10(-5) M in its presence, accompanied by an approximate doubling of the Vmax. The molecular weight of the enzyme (assayed by its catalytic and cyclic GMP-binding abilities) was estimated to be 123,000, corresponding to a sedimendation coefficient of 7.06 S, by means of sucrose density gradient ultracentrifugation. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity with optimal concentrations of about 30 and 0.7 mM, respectively. The maximal activity seen in the presence of Mg2+, however, was nearly twice as high as that seen in the presence of Co2+. Histones were generally effective substrates for the enzyme, whereas protamine, casein, phosvitin, phosphorylase kinase, and activator protein of phosphodiesterase were not. The cyclic GMP-dependent enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent enzyme in the presence of Mg2+.


Essential arginyl residues in mitochondrial adenosine triphosphatase.

Treatment of either beef heart or rat liver mitochondrial ATPase with the arginine reagent, 2,3-butanedione, resulted in enzyme inactivation. The reaction followed pseudo-first order kinetics until 90 to 95% of the enzyme had been inactivated, and prolonged incubation with butanedione resulted in complete inactivation. When the modification reaction was performed in the presence of ATP, the rate of inactivation was significantly decreased. The kinetics of inactivation indicates that the reaction of 1 molecule of reagent per active site of beef heart mitochondrial ATPase is necessary for inactivation. The loss of ATPase activity was also observed when submitochondrial particles were treated with butanedione. Studies with beef heart mitochondrial ATPase indicated that the inactivation was not due to enzyme dissociation into subunits. Kinetic studies with partially inactivated enzyme demonstrated that the Km values of ITP and of ATP in the presence of HCO3-were similar to the same constants for the control enzyme. When ATP was used as the substrate in the absence of anion activator, the partially inactivated enzyme still exhibited negative cooperativity. Inactivation was also observed when beef heart mitochondrial ATPase was treated with another arginine reagent, phenylglyoxal. The loss of ATPase activity was analyzed in terms of [14C]phenylglyoxal incorporation. From the present studies it is concluded that arginyl residues play an essential role in mitochondrial ATPase, probably at the hydrolytic site.


Basement membrane procollagen is not converted to collagen in organ cultures of parietal yolk sac endoderm.

Basement membrane procollagen biosynthesis was studied in organ cultures of embryonic rat parietal yolk sac endoderm by following [14C]proline incorporation into nondialyzable proteins. After reduction with 2-mercaptoethanol the 14C-proteins synthesized were characterized by agarose gel filtration and disc electrophoresis in the presence of sodium dodecyl sulfate. The labeled procollagen was identified by its content of hydroxy[14C]proline, its sensitivity to digestion with bacterial collagenase, and its resistance to digestion with pepsin. In cultures which were continuously labeled for periods from 6 hours to 4 days, the pro-alpha chains consistently eluted as a single peak with an apparent molecular weight of 160,000. After pepsin digestion the resultant alpha chains had an apparent molecular weight between 125,000 and 140,000. This suggests that basement membrane procollagen either contains non-triple helical pepsin-resistant regions or a triple helical region which is larger than the corresponding region of interstitial procollagen. Two experiments were performed to determine whether the chains of newly synthesized basement membrane procollagen were cleaved to a smaller molecular species. In the first, the hydroxylation and secretion of procollagen were blocked with alpha, alpha'-dipyridyl, and the resulting intracellular chains of basement membrane protocollagen were found to co-elute with fully hydroxylated and secreted pro-alpha chains. In the second, cultures were labeled for 1 day and chased for 3 days with unlabeled medium. Autoradiography had shown that most of the label was chased into new basement membrane. Agarose chromotography showed that after 3-day chase the pro-alpha chains still eluted with an apparent molecular weight of 160,000. Thus, the data indicated that basement membrane procollagen was deposited in new basement membrane without undergoing a time-dependent extracellular conversion.


Complications of arterial puncture in anticoagulated patients.

Thirteen patients who were receiving heparin in therapeutic doses had complications after femoral or brachial arterial puncture as follows: hematomas resulting in skin slough or infection in two, neuropathies of the median nerve in two and of the femoral nerve in two, and ischemia of the forearm muscles in seven. Since arterial puncture to determine blood gases in patients anticoagulated for pulmonary emboli can be dangerous, alternate means to establish the diagnosis are recommended. Early recognition and treatment of complications are essential.


[Success or failure factors in the treatment of insulin-secreting nesidioblastomas].

The authors report 5 cases of insulin- secreting nesidioblastoma which is not an exceptional tumour but may be suspected clinically before being confirmed in the laboratory. Fasting hypoglycemia or hyper-insulinism may be present, leading to arteriography and surgical exploration. The only valid forms of treatment are enucleation of the tumour or pancreatectomy, depending on whether it is situated on the right or on the left. The authors consider removal must be complete, if not, medical treatment should be preferred.


[Tumors of the carotid body. Diagnostic and therapeutic considerations. Apropos of a case].

The authors report a new case of carotid body tumour operated on, in a 26 year old woman. Recalling the main clinical and histological characteristics of these tumours, they emphasize the importance of pre-operative assessment and the decisive value of arteriography. Modern techniques of surgery of the carotid bodies permit one to face the risk of damage during removal. They re-emphasize the necessity of surgical treatment able to ensure a cure. Finally, emphasizeing the special gravity of advanced tumours (group III), they condemn a conservative attitude and recommended operation as early as possible.


Rapid and sensitive liquid chromatographic determination of carbamazepine suitable for use in monitoring multiple-drug anticonvulsant therapy.

A rapid, sensitive and selective method for the determination of carbamazepine and its major metabolite in plasma has been developed. Other commonly used anticonvulsants can be determined in the same procedure without interference. After extraction with dichloromethane, the components are separated by high-pressure liquid chromatography without further clean-up or concentration on a column packed with small-particle silica gel. The mean recovery from plasma is 98.6% with a relative standard deviation of 1.6%. The detection limit for carbamazepine is approximately 2 ng/ml, requiring 1 ml of plasma.


Metabolic effects of parathyroidectomy in asymptomatic primary hyperparathyroidism.

Effects of parathyroidectomy on parathyroid function and calcium (Ca) metabolism were carefully evaluated in 6 patients with primary hyperparathyroidism without symptoms normally attributed to the disease and in 7 with bone disease or nephrolithiasis. Before parathyroidectomy, both groups of patients demonstrated evidence of the sequelae of parathyroid hormone (PTH) excess, since they presented one or more of the following features: low bone density by 125I-photon absorption, hypercalciuria (urinary Ca greater than 200 mg/day on an intake of 400 mg/day), negative Ca balance (absorbed Ca less than urinary Ca), elevated fasting urinary Ca greater than 0.2 mg/mg creatinine for a night-time sample after a 6-hour fast), and decreased renal function (creatinine clearance of less than 65 ml/min). Following parathyroidectomy, most of these deleterious effects were reversed commensurate with the return of immunoreactive serum PTH, serum Ca, and urinary cyclic AMP toward normal. These quantitative non-invasive techniques may be useful for the initial evaluation and follow-up of patients with asymptomatic primary hyperparathyroidism.


Effect of angiotensin II on cyclic guanosine monophosphate and cyclic adenosine monophosphate in human plasma.

Infusion of alpha-adrenergic catecholamines increases plasma cyclic guanosine monophosphate (pcGMP), raising the possibility that the pressor effect of these agents may be mediated by cyclic GMP. We infused pressor doses of angiotensin II in 10 studies in 8 normal subject and measured pcGMP and plasma cyclic adenosine monophosphate (pcAMP) by radioimmunoassay. After 120 minutes of infusion, mean pcGMP was 128 +/- 31% (SE) higher than baseline values (P less than 0.01) while pcAMP was increased 30 +/- 10% (P less than 0.05).


The urinary excretion of glucosiduronates of cortisol and cortisone.

Cortisol and cortisone glucosiduronic acids were synthesised in a 14C-labelled from and utilised in a double-isotope derivative procedure for the analysis of cortisol glucosiduronate (FG) and cortisone glucosiduronate (EG) in human urine. Normal adults were found to excrete between 16 and 100 mug/24 h of FG (n = 14) and between 55 and 120 mug/24 h of EG (n = 15). Elevated values were observed in subjects with Cushing's syndrome and following ACTH stimulation. Abnormal excretion was noted in one patient with hepatic cirrhosis and in one case of cholestatic jaundice. The ratio FG/EG was markedly increased after ACTH stimulation and, in the normal group, was positively correlated to a highly significant degree (P less than 0.001) with FG excretion. These two observations suggest that EG excretion is less sensitive than FG excretion to variations in cortisol production.


Normal gestation and diminished androgen responsiveness in an untreated patient with 21-hydroxylase deficiency.

A 22-year-old woman with 21-hydroxylase deficiency but minimal clinical evidence of androgenization was studied. She had conceived twice and had borne a normal male infant without therapy of any kind. The diagnosis of 21-hydroxylase deficiency was substantiated by the findings of 17-ketosteroid and pregnanetriol excretions of 18.1 and 8.1 mg/24 hours, respectively. Adequate basal compensation was indicated by a fasting plasma cortisol of 17.5 mug/dl. Plasma ACTH (207 pg/ml), testosterone (216 ng/dl) delta4-androstenedione (649 mg/dl), progesterone (249 ng/dl) and 17alpha-hydroxyprogesterone (4820 ng/dl) were all significantly elevated.


Angiotensin I-converting enzyme and angiotensin II levels in women receiving an oral contraceptive.

In 11 healthy, normotensive young women taking contraceptive medication (Enovid) for at least one year, plasma levels of angiotensin II were significantly higher than in healthy male and female controls. No significant difference was seen in the serum activity of angiotensin I-converting enzyme measured in vitro. Although serum angiotensin I converting enzyme activity is stimulated in several conditions in which other components of renin-angiotensin-aldosterone system are increased, this is not the case during administration of estrogens.


Comparison of a microneutralization test in cell culture and virus neutralization test in embryonated eggs for determining infectious bronchitis virus antibodies.

A microneutralization test (MNT) system utilizing cytopathic effect end points was effective in determing neutralization indexes for infectious bronchitis virus antibodies. The system is reproducible within 1 index unit at the 95% level of probability. Comparison of the MNT to tests in eggs resulted in a positive correlation (B =0.81), which was significant (P greater than 0.01). The quantitative dose-response relationship of the MNT is linear (P greater than 0.005), with the 95% prediction limits fitting between one 10-fold dilution.


Plaque assay of Sendai virus in monolayers of a clonal line of porcine kidney cells.

The MN strain of Sendai virus formed distinct plaques in monolayers of PS-Y15 cells, an established porcine kidney cell line. The plaque-forming ability was neutralized by specific antibody to the virus. A linear relationship was found between the concentration of virus and the number of plaques. The sensitivity of this assay was about equal to that of the in ovo titration. When applied to the serum neutralization test, the end points obtained were comparable to those of the hemagglutination-inhibition and complement-fixation tests.


Evidence for a parathyroid hormone-independent calcium modulation of phosphate transport along the nephron.

To disclose a parathyroid-independent calcium modulation of phosphate transport along the nephron, the effect of increasing plasma calcium concentration to subnormal levels in rats 6 days after parathyroidectomy (chronic PTX) was studied. Fractional phosphate reabsorption was significantly increased. The whole kidney response to calcium infusion was similar whether or not the thyroid gland was removed, which suggests that calcitonin is not involved. The micropuncture study indicated an increase in the reabsorptive capacity for phosphate (absolute reabsorption/absolute delivered phosphate per nephron segment) in the proximal tubule, the loop, and the terminal nephron when calcium was infused. Thus, the level of plasma calcium or some related factor affects the phosphate transport by the tubule independently of parathyroid hormone. With calcium infusion, the profile of phosphate reabsorption along the nephron became close to that of acutely parathyroidectomized rats, but with persisting differences. The level of plasma calcium concentration may partly account for the differences between the acute and the chronic steps of parathyroidectomy. The role of possible interferences between alterations of extracellular calcium concentration or some related factor and the adenylate cyclase-cyclic AMP system in such an action of calcium was evaluated. Cyclic AMP was infused so as to achieve a 10(-6) M plasma concentration. Combined infusions of calcium and cyclic AMP were also performed. The results are compatible with calcium inhibition of adenylate cyclase, although they do not rule out a direct action of calcium.


Effect of adenosine deaminase inhibition upon human lymphocyte blastogenesis.

The biochemical mechanisms by which a genetically determined deficiency of adenosine deaminase leads to immunodeficiency are still poorly understood and prompted this study. We have examined the effects of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) upon the response of human peripheral blood mononuclear cells to the mitogen concanavalin A (Con A). Cells isolated from normal volunteers were incubated in microtiter plates in the presence of various inhibitors, and the incorporation of tritrated thymidine or leucine into macromolecular material was measured after 64 h. EHNA at a concentration of 0.3 muM, which inhibited 90% of the adenosine deaminase (ADA) activity in a mononuclear preparation, impaired the incorporation of tritrated leucine into protein; 100 muM EHNA was the minimal concentration that inhibited thymidine uptake. The addition of 15 muM adenosine or 10 muM cyclic AMP to Con A-stimulated lymphocytes inhibited leucine uptake, while millimolar concentrations were required to inhibit thymidine uptake. Lower doses of adenosine and cyclic AMP stimulated thymidine incorporation. The inhibition of thymidine uptake observed with millimolar concentrations of adenosine was independent of the type of mitogen (pokeweed or Con A), the concentration of mitogen, or the medium used, but could be increased if the cells were cultured in a serum with reduced levels of adenosine deaminase. Washout experiments failed to demonstrate a critical period early in immune induction during which adenosine exerted its inhibitory effects. Noninhibitory doses of EHNA potentiated the effects of adenosine and cyclic AMP on leucine and thymidine uptake. EHNA at a concentration of 50 muM also potentiated the inhibitory effects on thymidine uptake of dibutyryl cyclic AMP, butyric acid, norepinephrine, and isoproterenol, but not theophylline. When mitogenesis was assayed by leucine incorporations, no synergy between EHNA and these compounds was apparent. Uridine relieved to some extent the inhibition of blastogenesis produced by adenosine and cyclic AMP, but not by dibutyryl cyclic AMP, norepinephreine, isoproterenol, or theophylline. Neither uridine alone nor uridine plus adenosine protected lymphocytes from the inhibitory effects of EHNA.


Patterns of phosphoribosylpyrophosphate and ribose-5-phosphate concentration and generation in fibroblasts from patients with gout and purine overproduction.

In the majority of patients with gout and excessive uric acid production, underlying enzyme abnormalities have not been identified. In the present study, measurement of both the rate of generation and concentration of phosphoribosylpyrophosphate (PP-ribose-P) and the concentration of ribose-5-phosphate in cultured cells were undertaken to establish a classification of purine overproducers to direct study of additional enzyme defects. Fibroblasts were cultured from 24 individuals assigned to 4 groups: group 1, 5 normal controls; group 2, 5 patients with gout and normal dialy urinary uric acid excretion (gouty controls); group 3, 7 patients with well-defined enzyme abnormalities and excessive urinary acid excretion (4 with hypoxanthine-guanine phosphoribosyltransferase deficiency and 3 with excessive PP-ribose-P synthetase activity); and group 4, 7 patients with gout and excessive uric acid excretion but without grossly abnormal activities of the above enzymes in erythrocyte lysates. In all 14 fibroblast strains from patients showing excessive production of uric acid (groups 3 and 4), rates of purine synthesis de novo and PP-ribose-P concentrations exceeded values for cells from control groups. Cells from group 3 patients with hypoxanthine-guanine phosphoribosyltransferase deficiency showed normal PP-ribose-P generation, while those with excessive PP-ribose-P synthetase activity demonstrated increased generation of this regulatory substrate. All strains from group 3 patients had normal ribose-5-phosphate concentrations. Five cell strains from group 4 patients showed one of the two patterns of abnormalities in these measurements seen in strains from group 3 patients: two resembled hypoxanthine-guanine phosphoribosyltransferase-deficient cells, and three resembled cells with excessive PP-ribose-P synthetase activity. Analyses of erythrocyte enzyme preparations from two of these patients in group 4 have led to identification of a kinetic variant of each enzyme as predicted from the foregoing patterns. Two additional group 4 cell lines that showed increased ribose-5-phosphate concentrations in addition to increased PP-ribose-P concentrations and generation were classified in a separate subgroup, since in the individuals excessive purine synthesis appeared to result from increases ribose-5-phosphate concentration, leading to increased availability of PP-ribose-P. No abnormality in either hypoxanthine-guanine phosphoribosyltransferase or PP-ribose-P synthetase has been found in erythrocyte preparations from one patient so classified.


Effects of a 3-day fast and of ethanol on splanchnic metabolism of FFA, amino acids, and carbohydrates in healthy young men.

Splanchnic metabolism was studied to quantify changes underlying the fatty liver, hyperlipemia, and hypoglycemia produced by ethanol. Four subjects fasted for 15 h were compared with five subjects fasted for 69 h under basal conditions and during continuous intravenous infusion of sufficient ethanol to give a concentration of 3-5 mM in arterial blood plasma. Splanchnic storage of fatty acids was estimated from the difference between uptake of FFA and secretion of derived products. Basal values for splanchnic uptake of FFA were twofold higher after the 69-h fast while splanchnic storage of fatty acids and production of ketone bodies increased threefold. Values for basal secreation into the blood of triglycerides derived from FFA were similar in the two groups. In both nutritional states, the fraction of FFA taken up in the splanchnic region oxidized to ketone bodies and to CO2 fell when ethanol was given because of preferential oxidation of ethanol to acetate, and the fraction esterified rose. However, systemic transport and splanchnic uptake of FFA fell with ethanol in subjects fasted 15 h, so that neither storage of triglycerides in splanchnic tissues nor secretion into the blood increased. In subjects fasted 69 h, ethanol increased transport of FFA and splanchnic storage of fat. In all but one subject it also increased secretion of triglycerides into the blood. The concentration of glucose in blood fell during ethanol infusion in all five subjects undergoing the 69-h fast. Mean splanchnic glucose production was maintained at about one-half of the pre-ethanol value, despite virtual cessation of splanchnic uptake of lactate and of those amino acids that are metabolized via malate. Quantitative estimates of extrasplanchnic metabolism suggest that enhanced formation of alpha-glycerophosphate from glucose, in addition to impaired hepatic gluconeogenesis, may contribute to ethanol-induced hypoglycemia in man.


Reduced sensitivity of the hepatic adenylate cyclase-cyclic AMP system to glucagon during sustained hormonal stimulation.

Hormone-induced desensitization of hormonal regulation of cyclic AMP (cAMP) content has been described in a number of tissues. In the present study, we examined responses of rat liver to glucagon after periods of sustained exposure to the hormone in vivo and in vitro. In intact anesthetized rats infused with glucagon (50 ng/min) for 1 h or more and in liver slices incubated with the hormone (10 muM) for this period, hepatic cAMP responsiveness to glucagon was significantly blunted compared with that of tissue exposed to the hormone for shorter periods. The reduction in hepatic cAMP responsiveness to glucagon appeared to be fully expressed by 2 h. With the doses of hormone employed, the sequential alterations in hepatic responsiveness seemed to be limited to the cAMP system, since other parameters of glucagon action did not wane with time. Diminished hepatic cAMP responsiveness during sustained hormonal exposure could not be attributed to decreased glucagon availability, accelerated extracellular release of cAMP, hepatic ATP depletion, or enhanced phosphodiesterase activity. Studies in vitro suggested that modulation of the cAMP response occurred at the level of adenylate cyclase (AC). During sustained exposure of hepatic slices to glucagon, reductions in glucagon-responsive AC correlated temporally with those in cAMP and both changes were reversible. Alterations in glucagon-responsive AC were demonstrated over a wide range of ATP (10 muM-0.1 mM) and glucagon (10 nM-5 MM) concentrations in the cyclase reaction mixture, and appeared to be a noncompetitive phenomenon relative to glucagon. Maximal NaF-responsive AC did not fall concomitantly with time. Thus, the reduction in glucagon-responsive AC was probably not related to a reduction in the catalytic unit of the enzyme, but could have been due to an alteration in glucagon binding to its receptor sites, or in the coupling mechanism involved in transmission of the hormonal signal to the catalytic unit.


Activation of adenylate cyclase by cholera toxin in rat liver homogenates.

The effect of cholera toxin on adenylate cyclase from rat liver has been studied in a broken cell preparation. The activation of the enzyme in this in vitro preparation requires the addition of nicotinamide adenine dinucleotide (NAD) to the incubation medium and the presence of cell components other than the membrane-bound adenylate cyclase. Once the activation of the cyclase is produced, the effect persists despite repeated washing or solubilization of the enzyme. The effect can be obtained with concentrations of cholera toxin as low as 0.4 nM after 15 min of incubation at 22 degrees C, and stimulation can be detected after only 5 min of incubation at 37 degrees C. The activation of the enzyme is still apparent after at least 2 h at 0 degrees C. Preincubation with choleragenoid in vitro does not interfere with this effect of the toxin. Animals pretreated by an intravenous injection of cholera toxin do not respond to the in vitro addition of cholera toxin and NAD to the same extent as untreated animals; i.e., the effects overlap to suggest that the in vitro effect is the same as that in vivo. Responses to isoproterenol, glucagon, and NaF were also similar in the in vitro broken cell-activated system, as previously reported for the enzyme activated in vivo.


Cell electrophoresis research directed toward clinical cytodiagnosis.

The application of cell electrophoresis to cytodiagnosis requires that a scientifically established basis exists for identifying abnormal cells electrophoretically, that research to detect such differences in the cytodiagnostic setting is possible and that a rapid and simple method of cell electrophoresis is adaptable to the clinical setting. Data are presented indicating modifications of electrophoretic mobility due to herpes simplex virus type 1 infection and Rous sarcoma virus transformation of culture cells. A simple apparatus for electrophoretically separating cells on a density gradient and collecting them for subsequent analysis is described, and results of experiments with this apparatus are consistent with those obtained by microscopic electrophoresis. Laser-doppler spectroscopic electrophoresis is suggested as a rapid method adaptable to clinical application.


Effects of neoplasms on inflammation: depression of macrophage accumulation after tumor implantation.

The local accumulation of macrophages at sites of neoplasms may be a critical event in immunologically mediated tumor killing. Individuals with neoplasms, however, have been noted to have depressed monocyte chemotactic responsiveness in vitro. To determine the effect of neoplasms on macrophage migration, mice were implanted subcutaneously with either sarcoma or hepatoma cells and their macrophage migratory function quantified in vivo and in vitro. The ability of tumor-bearing animals to mobilize macrophages to an inflammatory site in vivo was depressed by as much as 61% by 6 days after tumor implantation. The in vitro chemotactic responsiveness of macrophages recovered from the peritoneal cavities of tumor-bearing animals was also markedly depressed. Macrophage migration was not affected by implantation of normal syngeneic or allogeneic tissues. In addition, the accumulation of polymorphonuclear leukocytes in vivo was not depressed in tumor-bearing animals. These findings suggest that neoplasms themselves may depress the host's ability to localize macrophages at inflammatory sites in vivo and thereby hinder immunologically mediated tumor destruction.


Antibody in the sera of tumor-bearing mice that mediates spleen cell cytotoxicity toward the autologous tumor.

Pretreatment of MTV-induced BALB/cfC3H mammary tumor cells with autologous serum results in increased spleen cell cytotoxic activity and the recruitment of previously inactive spleen cells to cytotoxic activity against the target cells. These recruiting antibodies are tumor-specific for individual tumors; pretreatment with such serum of target cells of an MTV-induced mammary tumor obtained from a different BALB/cfC3H female results in blocking of spleen cell activity. The autologous recruiting factors are active at dilutions of 1000 or more of whole serum are found in the 19S fraction after gel filtration.


Complexity of factors in sera of different mice that affect MTV-induced mammary tumor cells.

Specific spleen cell activity in microcytotoxicity assay can be altered by pretreatment of target mammary tumor virus (MTV)-induced mammary tumor cells with serum. Serum from both BALB/cfC3H females neonatally infected with MTV and BALB/c females horizontally exposed to MTV antigens will block specific spleen cell activity against isologous mammary tumor cells. On fractionation of sera, blocking factors are localized in the 7s fraction. The 19s fraction contains recruiting factors that are not detectable in the unfractionated serum; these factors are active against isologous tumors and are thus distinct from the tumor-specific recruiting factors previously described in the sera of tumor-bearing females, which are active only against the autologous tumor. Antibodies mediating complement-dependent cell lysis are also detectable after serum fractionation.


Inhibition of mitogen-induced lymphocyte transformation by local anesthetics.

Chlorpromazine (CPZ) and lidocaine were added to cultures of mouse spleen cells stimulated by concanvalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). Concentrations of CPZ greater than 5 x 10(-6)M and concentrations of lidocaine greater than 2 x 10(-3)M totally inhibited the mitogenic responses to all four mitogens. Minimal inhibitory concentrations of neither drug interferred with cell viability as determined by trypan blue uptake or 51Cr release. The effects were totally reversed by the removal of the drugs from the culture. Addition of the drug at intervals after mitogen exposure demonstrated that the inhibited event occurred relatively soon after exposure to the mitogen. For example, the addition of lidocaine or CPZ more than 24 hr after Con A stimulation had no effect on tritiated thymidine incorporation. Elevated concentrations of cyclic AMP, cyclic GMP (or their derivatives) or calciunown membrane active actions of these drugs and the rapid reversibility of the effect strongly support the idea that the local anesthetics act on the surface membrane of lymphocytes. Binding of radiolabeled Con A or LPS to lymphocyte membranes in the presence of lidocaine or CPZ was not inhibited. The possibility exists that CPZ and lidocaine disorganized cell membranes so as to interfere with the surface membrane elaboration or action of a second messenger, or interfere with cell-cell interactions.


Immunity to C-type RNA viruses: antibody formation after natural or deliberate infection.

A combined virologic and immunologic study was done in mice that were either naturally or deliberately infected with C-type RNA viruses. In about 60% of B10. A mice, natural infection was accompanied by detectable anti-VEA antibodies in the serum. The absence of such antibodies was a reliable indicator of the absence of virus in B10 mice. Deliberately infected young adult B10 mice failed to eliminate the virus completely, despite high titers of anti-VEA antibodies. B10 mice exposed to B tropic viruses at birth made anti-VEA antibodies but failed to eliminate the virus. By contrast, some NIH Swiss mice exposed to N-tropic virus at birth made anti-VEA antibodies and eliminated the virus.


Lysis of tumor cells by antibody and complement. VI. Enhanced killing of enzyme-pretreated tumor cells.

The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase.


Effect of lectins on the levels of cAMP and cGMP in guinea pig lymphocytes: early responses of lymph node cells to mitogenic and non-mitogenic lectins.

Guinea pig lymph node lymphocytes were reacted with two mitogenic lectins (LcL-A or PHA) and two nonmitogenic lectins (AbL or WGA)m and the levels of cAMP and cGMP were determined during 30 min of culture. Various concentrations of LcL-A and PHA caused modest (1.5 to 2-fold) rises in cAMP levels. However, the rise in cAMP did not correlate with the degree of mitogenicity. AbL caused a greater rise in cAMP than the mitogenic lectins whereas WGA had no effect on cAMP levels. None of the four lectins causes an appreciable (i.e., greater than 1.5-fold) change in cGMP levels. There does not seem to be any positive correlation between early changes of cAMP and/or cGMP and mitogenesis.


An IgG-Fc receptor induced in cytomegalovirus-infected human fibroblasts.

Cytomegalovirus- (CMV) infected cultured human fibroblasts incubated with normal human serum were shown to react with IgG by direct and indirect fluorescent antibody tests. The binding of IgG to infected cells was unrelated to the presence of anti-CMV complement fixing (CF) antibodies and was not observed with uninfected cells. The reaction was first observed 36 hours after infection as diffuse cytoplasmic staining which by 72 hr became localized into a dense perinuclear structure. The cytoplasmic fluorescence was not detected with anti-IgA or anti-IgM fluorescent conjugates. The reaction was seen with purified IgG and Fc fragments but was only minimally detectable with Fab fragments. It occurred in fibroblasts infected with three standard laboratory strains of CMV and seven recent CMV isolates from patients and was observed in six separate lots of human foreskin fibroblast cultures as well as in WI-38 cells. We conclude that CMV infection induces the formation of a IgG receptor in human fibroblasts.


Release of xenotropic type C RNA virus in response to lipopolysaccharide: acitivity of lipid-A portion upon B lymphocytes.

The present studies demonstrate that lipopolysaccharide (LPS) causes the release of endogenous xenotropic type-C RNA virus from BALB/c spleen cells. The evidence suggests that virus release is stimulated by the lipid-A portion of LPS and primarily involves an action of LPS on B lymphocytes. LPS had little or no effect on virus release by T lymphocytes, macrophages, or fibroblasts. These results indicate that the differentiated state of the cell plays an important role in the regulation of endogenous virus release.


Mitogen induction of murine C-type viruses. I. Analysis of lymphoid cell subpopulations.

We reported previously in vitro induction of endogenous C-type viruses from normal mouse spleen cells by lipopolysaccharide (LPS) as well as by combination treatment with concanabalin A and 5-bromo-2'-deoxyuridine (Con A/BrdU). To identify the cell types sensitive to virus induction and to study the relationship of mitogenicity to virus induction we have compared T cell populations (BALB/c thymus cells and cortisone-resistant thymus cells), B cell populations (nu/nu spleen cells and lymph node cells), adherent BALB/c peritoneal cells and mixed populations (BALB/c spleen cells, macrophage-depleted BALB/c spleen cells, and lymph node cells). LPS-induction occurred only in B cell-containing populations. In contrast, induction by Con A/BrdU depended on the presence of both T and B cells. In both instances, neither macrophages nor hemopoietic cells appeared to be a major source of virus. Treatment with anti-Ig serum and complement reduced virus induction by LPS/BrdU but not by Con A/BrdU suggesting that different cell populations produce virus after stimulation with these two different mitogens.


Effect of cross-immunization on monotypic antibody responses to herpes simplex virus types 1 and 2.

New Zealand White rabbits were immunized subcutaneously with partially purified UV-inactivated preparations of herpes simplex virus (HSV-1 or HSV-2) in complete Freund's adjuvant. After the initial immunizations, designated groups of animals received additional amounts of either HSV-1 or HSV-2 at 35-day intervals. Sera were absorbed with lysates of cells infected with heterotypic virus and the residual monotypic antibodies were detected by 51Cr-release assay using HSV-infected target cells. A positive correlation was found between the ratio of neutralizing antibodies to HSV-1 and HSV-2 (II/I index) and the content of monotypic antibodies. Results showed that production of monotypic antibodies to HSV-1 And HSV-2, under the conditions employed, was independent of previous exposure to HSV.


The seroepidemiology of reovirus, coxsackievirus infections among Panamanian Indian children.

Rates of hepatitis B virus infection varied significantly among Panamanian Indian tribes. Chocó and Mainland Cuna Indians had a greater prevalence of antibody to hepatitis B surface antigen than Guaymi and Island Cuna Indians. The village water supply appeared to be the major environmental difference that distinguished the tribes from one another. Since contaminated water plays a role in the transmission of enterovirus, an attempt was made to assess relative exposure to contaminated water by determination of the prevalence of antibodies to reovirus and coxsackievirus B in children 10 years old or younger. Infection with coxsackievirus B occurred more frequently in Chocó and Mainland Cuna Indians than in Guaymi or Island Cuna Indians. Although essentially all children tested were exposed to reoviruses, Mainland Cuna Indians had the highest geometric mean titers of antibody, followed by Chocó, Guaymi, and Island Cuna Indians in descending order. This parallel in rates of hepatitis B and enterovirus infection supports the hypothesis that fecal-oral transmission of hepatitis B virus is important in certain populations.


Mechanism of activation adenylate cyclase in vitro by polymyxin-released, heat-labile enterotoxin of Escherichia coli.

Heat-labile enterotoxic material released from Escherichia coli by polymyxin B activates the adenylate cyclase of pigeon erythrocyte ghosts in a time- and concentration-dependent manner. The activation requires nicotinamide adenine dinucleotide, adenosine triphosphate, and another component of the erythrocyte supernatant. The active species has a molecular weight of about 23,000-24,000 daltons, is inhibited by antibodies to the toxin of Vibrio cholerae, and is not irreversibly denatured by sodium dodecyl sulfate. Thus in many respects the active species from E. coli behaves the same as peptide A1 of cholera toxin.


Mode of action of Vibrio cholerae enterotoxin in cultured adrenal tumor cells.

The effects of Vibrio cholerae enterotoxin on steroidogenesis and on formation of adenosine 3':5'-cyclic phosphate (cyclic AMP) in two adrenal tumor cell lines were compared. Steroidogenesis was half-maximal at concentrations of 1 ng of cholera toxin/ml in the mutant OS-3 cells and 3 ng of cholera toxin/ml in the parent Y-1 cells. At the end of an 8-hr incubation, toxin-induced formation of cyclic AMP in the mutant cell line was reduced by 90%. A molar ratio of GM1 ganglioside (galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide; GGnSLC) to cholera toxin of 3:1 caused half-maximal inhibition of steroidogenesis in both cell lines. When equine antiserum to choleragenoid was added to adrenal cells 15 min after cholera toxin, there was marked inhibition of cyclic AMP formation and of steroidogenesis. Pretreatment of Y-1 cells with adrenocorticotropin rendered them unresponsive to hormonal induction of cyclic AMP formation, but these cells had an unimpaired response to cholera toxin. These studies, utilizing two adrenal cell lines, suggest important differences between the mode of action of cholera toxin and that of adrenocorticotropin in cultured adrenal tumor cells.


Multiple roles of erythrocyte supernatant in the activation of adenylate cyclase by Vibrio cholerae toxin in vitro.

Peptide A1 of Vibrio cholerae toxin, nicotinamide adenine dinucleotide, adenosine triphosphate, and a soluble protein present in erythrocyte supernatant are required for the activation of pigeon erythrocyte ghost adenylate cyclase but are not required to maintain the activated state. The compounds are all required simultaneously, and when all are added to ghosts, adenylate cyclase activity increases at a linear rate without delay. Under optimal conditions significant activation of cyclase is given by less than one molecule of toxin per ghost. Intact cholera toxin may be inactive in vitro. There is a delay of about 1 min between the addition of intact toxin and the attainment of the final rate of increase of adenylate cyclase activity. During this period, glutathione reduces the disulfide bond between peptides A1 and A2. The delay is eliminated if the toxin is reduced before addition. More A1 is liberated if the toxin is also denatured with sodium dodecyl sulfate.


Cholera toxin and the adenylate cyclase-activating signal.

Studies with chemically modified cholera toxin derivatives showed that all treatments that decreased the ability of toxin to bind to mouse thymus cells or to polystyrene-coupled GM1 ganglioside caused a concomitant reduction in the toxin's ability to increase adenosine 3':5'-cyclic phosphate (cyclic AMP) in thymus cells and skin vascular permeability in rabbits. Dissociation of the H (heavy) and L (light) subunits abolished the biologic activity without inhibiting receptor binding, as did treatment with arginyl-specific reagents (which did not change the aggregation state of the toxin). When thymus cells were incubated with 125I-labelled toxin at 37C,only about 1% of the total cell-bound radioactivity was recovered in the cytosol supernate. Similar values were found for cells incubated with toxin at 0C, and with 125I-labelled choleragenoid at 37C or 0C. Thymus cells rapidly bound less than or equal to equal to 5 X 10(4) cholera toxin molecules per cell at both 0C and 37C. Much less, however, of the radioactive toxin bound at 37C than of that bound at 0C was displaced by addition of unlabelled toxin or choleragenoid. Similar temperature-related irreversible binding was noted with 125I-labelled choleragenoid. The relative amounts of H and L subunits in the irreversibly cell-bound and in the displaced 125I-labelled toxin were indistinguishable. Treatment of thymus cells at 37C, but not at 0C, with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide caused a 10-fold reduction of adenylate cyclase stimulation by cholera toxin without inhibiting activation by epinephrine or prostaglandin E1, or appreciably altering the basal, unstimulated enzyme activity. The carbodiimide inhibited the cyclic AMP response to cholera toxin when added shortly after the toxin had bound to the cells (early in the lag phase).


Interaction of Vibrio cholerae toxin with sarcoma 180 cell membranes.

Three discrete phases are discernible in the activation, by Vibrio cholerae toxin, of adenylate cyclase in fragments of sarcoma 180 cell membranes. In the first, or preparatory, phase the toxin must be exposed to dithiothreitol or nicotinamide adenine dinucleotide (NAD) in the absence of the membranes. In the second phase, the prepared toxin is dissociated to yield a macromolecular cyclase-activating factor (MCAF) in the presence of the membranes. In the third phase, membrane basal adenylate cyclase is activated by MCAF in the presence of NAD. The integrity of the catecholamine or beta-receptor associated with sarcoma adenylate cyclase is irrelevant in the activation of cyclase by MCAF. This activation proceeds undiminished even if the beta-receptor is desensitized or blocked by propranolol.


Purification of the polymyxin-released, heat-labile enterotoxin of Escherichia coli.

The heat-labile enterotoxin of Escherichia coli strain H-10407 has been purified by use of a commercially available affinity gel (Affi-Gel 202). This gel possesses a strong and highly specific affinity for the enterotoxin released from intact E. coli cells by polymyxin B. The polymyxin-release technique could be used with fermenter-size batches of E. coli cells grown in a casamino acids-yeast extract medium. With a simple (NH4)2SO4 back-extraction step prior to affinity chromatography, large batches of E. coli enterotoxin could be processed rapidly. Affi-Gel 202-purified E. coli enterotoxin produced a single precipitin band in the presence of several different antisera against crude preparations of the toxin. The same antigen produced a precipitin band in the presence of both cholera antitoxin and antiserum to choleragenoid.


Malignant oncocytic tumour of the parotid salivary gland.

A 49-year-old man developed a tumour mass in his right parotid salivary gland nine years after a histologically proven benign mixed tumour of the same salivary gland had been surgically removed. Radical resection of the right parotid salivary gland and associated lymph nodes and soft tissues of the neck was performed. The parotid tumour was composed of oncocytic cells which infiltrated the surviving salivary gland tissue. Most of the excised lymph nodes contained metastatic deposits of oncocytic cells identical to the tumour seen in the parotid. There are no previous reports of the occurrence of both pleomorphic adenoma and malignant oncocytoma in the same salivary gland.


Unilateral multiple tumours of the parotid gland.

The occurrence of two morphologically distinct tumors in a single parotid gland is rare. Pathologic examination of a unilateral parotid mass removed from a 63-year-old male revealed the presence of a benign mixed tumor and an adenolymphoma. No other examples of this relationship were encountered in the literature.


Measurement of apolipoprotein A-I in rat high density lipoprotein and in rat plasma by radioimmunoassay.

A double antibody radioimmunoassay (RIA) for rat apolipoprotein A-I is reported. The ApoA-I isolated from delipidated HDL by gel filtration yielded a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), and its amino acid composition resembled that reported by others. ApoA-I was iodinated by lactoperoxidase and the resulting 125I-apoA-I was purified by gel filtration. Up to 93% of 125I-apoA-I was precipitable by antibody and greater than 99% of bound 125I-apoA-I was displaced by "cold" apoA-I. Other rat lopoproteins and apolipoproteins did not react in this system. Human plasma were also not reactive, nor were dog, goat, and sheep plasmas.


Apoproteins of the lipoproteins in a nonrecirculating perfusate of rat liver.

The apoproteins of serum lipoproteins and of lipoproteins present in a nonrecirculating perfusate of rat liver were compared by immunochemical, gel electrophoretic, and solubility techniques. Serum and perfusate very low density lipoprotein apoprotein composition were not different. No evidence for the presence of a lipoprotein resembling serum low density lipoprotein was obtained. However, the apoprotein composition of circulatory high density lipoprotein was quantitatively different from the secretory product in the density 1.06-1.21 range. As measured by stained sodium dodecyl sulfate gel electrophoretic patterns, the arginine-rich protein was the major secretory apoprotein while the A-I protein was the major apoprotein in circulating high density lipoprotein. A very similar pattern was seen in perfusates of orotic acid-fatty livers. It was concluded that although the liver secrets lipoproteins in the high density class, circulatory high density lipoprotein is largely a product of catabolic processes.


Competitive protein binding radioassay of plasma and urinary unconjugated cortisol.

In order to establish a more reliable profile of the function of the adrenal cortex, a study has been made of this simultaneous estimation of plasma and urinary unconjugated cortisol by competitive protein binding (CPB) radioassay. Both plasma and urinary cortisol were measured by CPB radioassay after solvent extraction. Normal ranges were found for plasma and urinary cortisol. The samples obtained in the morning (8:00 am) established a normal plasma cortisol range of 9.0-21.6 mug% with a mean of 15.3. range for urinary unconjugated cortisol was 12.0-41.5 mug/day (mean 26.9). Dexamethasone suppression and adrenocorticotrophic hormone stimulation of plasma and urinary cortisol were tested with individuals having normal and abnormal adrenal cortical function. RESULTS INDICATED A GOOD CLINICOPATHOLOGICAL CORRELATION. It was concluded that plasma and urinary unconjugated cortisol are reliable laboratory tests for adrenocortical function and would be the methods of choice rather than urinary 17-ketogenic steroids.


The effects of chemotherapy on the Wistar-Furth Wilms' Tumor.

An evaluation of single versus multiple therapy utilizing actinomycin D and vincristine to establish optimum therapeutic value on Wilms' tumor was completed in the present study. A Wistar/Furth Wilms' tumor model was treated with four dosage levels of actinomycin D and vincristine in single and multiple courses of treatment immediately after tumor injection. The results observed and analyzed indicated an optimum dose level for each drug and its most effective therapy course. Actinomycin D (at 300 mg/kg) in single or multiple dose was most effective, to an equal degree, in prolonging survival, decreasing the number of metastases, and the weight of the renal primary as well. Vincristine chemotherapy on a low-dose basis was associated chiefly with increased survival only.


Application of an in-vitro perifusion technique to studies of luteinizing hormone release by rat anterior hemi-pituitaries: Self-potentiation by luteinizing hormone releasing hormone.

A technique is described for the continuous perifusion of rat adenophypophyses. Exposure of the perifused glands to repeated equal 5 min stimuli with hypothalamic extract resulted in a series of equal peaks of corticotrophin secretion, the response was proportional to log dose over the range 0 - 25-2 - 0 rat hypothalamic equivalents/ml. Repeated equal stimuli with hypothalamic extract, or with luteinizing hormone releasing hormone (LH-RH) at concentrations of 2 or 10 ng/ml, resulted in a progressively increasing series of peaks of LH secretion, i.e. a self-potentiating or priming effect. The effect took between 30 min and 1 h to develop. A delayed increase in the responsiveness of the glands was also seen with continuous incubation of anterior pituitaries with LH-RH. The relevance of these observations to the physiological control of LH secretion is discussed.


Ability of various factors to oppose the stimulatory effect of dibutyrylcyclic AMP on the release of melanocyte-stimulating hormone by the rat pituitary in vitro.

Various agents were tested for their ability to oppose the stimulatory effect of dibutyryl cyclic AMP on the release of the melanocyte-stimulating hormone from the rat neuro intermediate lobe in vitro. Only dopamine exhibited an inhibitory effect; serotonin, gamma-amino-butyric acid, tocinoic acid, tocinamide, the tripeptide Pro-Leu-Gly-NH2 and dibutyryl cyclic GMP were all ineffective.


Tamoxifen as an anti-tumour agent: effect on oestrogen binding.

Tamoxifen (ICI 46,474) has been shown to possess anti-tumour properties in the dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model. During tamoxifen therapy the binding of [3H]oestradiol in vivo to uterine (P less than 0-001), vaginal (P less than 0 X 01) and tumour (P less than 0-001) tissues was significantly reduced. Tamoxifen therapy was without effect on the binding of [3H]oestradiol in heart tissue. The determination of specific oestrogen-binding components in vitro was significantly reduced (P less than 0-01) in tumours from tamoxifen-treated rats and tamoxifen inhibited the binding of [3H]oestradiol to 8S oestrogen-binding components, derived from rat uteri and DMBA-induced tumours, in vitro.


Control of oestrogen production in human pregnancy: effect of trophic hormones on steroid biosynthesis by the foetal adrenal gland in vitro.

[14C]Acetate was incorporated into dehydroepiandrosterone, pregnenolone and their sulphates and into cholesterol during incubations of adrenal tissue from three human foetuses of 10-18 weeks of gestation. No incorporation of [14C]acetate into cortisol or other 4-en-3-oxo steroids could be demonstrated. Porcine ACTH stimulated the incorporation of [14C]acetate into both dehydroepiandrosterone sulphate and cholesterol when added to incubations of adrenal tissue from foetuses of 18 weeks gestation. Such an effect was not observed with tissue from a 10-week-old foetus. Stimulation of steroid biosynthesis from [14C]acetate was also achieved by addition of human growth hormone chorionic somato-mammotrophin. These trophic hormones may therefore play some part in regulating the provision of precursors for oestrogen biosynthesis in pregnancy.


Alpha-adrenergic receptor activity, cyclic AMP and lipolysis in adipose tissue of hypothyroid man and rat.

Lipolysis and intracellular levels of cyclic AMP of adipose tissue from man and rat in both hypothyroid and euthyroid states were studied in response to stimulation by catecholamines in vitro. Hypothyroid patients were studied before and after treatment, and were also compared with euthyroid obese controls. The experimental group of rats was rendered hypothyroid by the addition of 2.9 mM-propylthiouracil to their drinking water, and their status confirmed by plasma thyroid function tests. Evidence for alpha-adrenergic receptor activity was found in rat adipose tissue, but was less marked than the pronounced alpha-adrenergic activity in human adipose tissue. Glycerol release from adipose tissue in response to noradrenaline stimulation was less marked in hypothyroidism in both species, and was related to an increased alpha-adrenergic activity. No evidence was found for increased alpha-adrenergic effects on cyclic AMP level in hypothyroid subjects, and little evidence was found in adipose tissue from hypothyroid rats. This discrepancy may be due to the presence of the phosphodiesterase inhibitor, theophylline, in the incubation system. The possible modulatory role of thyroid hormones on receptor and phosphodiesterase activity, and on lipolysis, is discussed.


Mechanisms of genetic resistance to Friend virus leukemia in mice. II. Resistance of mitogen-responsive lymphocytes mediated by marrow-dependent cells.

Friend leukemia virus suppresses the proliferative responses of normal thymus-dependent (T) and bursa equivalent-dependent (B) lymphocytes from spleen, thymus, lymph node, and bone marrow to mitogens. The suppressive effect of Friend virus complex (FV) requires fully infectious virions. Friend erythroleukemic cells, washed to removed extracellular virus, fail to suppress concanavalin A (Con-A)-induced mitogenesis of normal spleen cells. This indicates that FV does not mediate its immunosuppressive effect via transformed erythropoietic cells. The in vitro suppressive effect of FV on lymphocyte mitogenesis is under host genetic control. Spleen, bone marrow, and thymus cells from strains of mice susceptible to FV-induced leukemogenesis in vivo were quite susceptible to the suppressive effects of FV in vitro. On the other hand, similar cells from strains of mice such as C57BL/6 resistant to Friend erythroleukemia, were quite resistant to in virto immunosuppression by FV. Mitogenesis of splenic T cells from resistant B6 mice, previously treated with 89Sr, became susceptible to suppression by FV. This indicated that the in vitro resistance of lymphocytes to FV-induced suppression is not an intrinsic property of T cells, but is controlled by marrow-dependent (M) cells which are selectively eliminated by treatment with 89Sr. M-cell function does not develop in mice less than 3-wk old. The Con A response by thymus cells from 2-wk-old B6 mice was susceptible to suppression by FV, further supporting the concept that M cells may regulate the genetic resistance to FV.


The role of glucose limitation in the regulation of the transport of glucose, gluconate and 2-oxogluconate, and of glucose metabolism in Pseudomonas aeruginosa.

The pathway of glucose metabolism in Pseudomonas aeruginosa was regulated by the availability of glucose and related compounds. On changing from an ammonium limitation to a glucose limitation, the organism responded by adjusting its metabolism substantially from the extracellular direct oxidative pathway to the intracellular phosphorylative route. This change was achieved by repression of the transport systems for gluconate and 2-oxogluconate and of the associated enzymes for 2-oxogluconate metabolism and gluconate kinase, while increasing the levels of glucose transport, hexokinase and glucose 6-phosphate dehydrogenase. The role of gluconate, produced by the action of glucose dehydrogenase, as a major inhibitory factor for glucose transport, and the possible significance of these regulatory mechanisms to the organism in its natural environment, are discussed.


Extracellular cyclic AMP-phosphodiesterase accelerates differentiation in Dictyostelium discoideum.

Extracellular cyclic AMP-phosphodiesterase accelerates the development of aggregation competence in Dictyostelium discoideum when present during the preaggregation stage. The effect on development appears to depend only on hydrolysis of extracellular cyclic AMP and not on other properties of the phosphodiesterase molecule. Extracellular cyclic AMP-phosphodiesterase, as a promoter of differentiation, acts mainly throughout the first half of interphase. Our evidence supports the proposal that cyclic AMP oscillations control the rate and possibly the initiation of development. Since extracellular cyclic AMP-phosphodiesterase acts from the beginning of interphase cyclic AMP oscillations may also occur from early interphase, at least in the presence of this enzyme. This would imply that the cyclic AMP oscillator is a determinant, but not a product, of the developmental programme.


Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida.

Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment. The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-alpha-glutamokinase/N-acetylglutamate-gamma-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-delta-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for ornithine carbamoyltransferase). No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P. putida A90, but a 1-5- to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P. putida A90. Aspartate carbamoyltransferase in crude extracts of P. putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate.


Morphogenesis of picornaviruses: characterization and assembly of bovine enterovirus subviral particles.

Bovine enterovirus-I (BEV-I) infection results in the production of a low amount of infective virus. A large number of non-infectious virus particles can be detected in BEV-I lysates by haemagglutination. Attempts to isolate DI particles that might be responsible for this effect failed. However, infected cells were shown to contain large amounts of 80S particles as well as lesser amounts of 160S, 130S, 45S, 14S and 5S particles. The proportion of these subviral particles detectable by density gradient sedimentation depended on the ionic strength of the gradient buffer. At high ionic strength 130S particles were transformed into 160S particles, and 45S into 80S particles. The polypeptide composition of each virus particle was examined. Pulse-chase experiments confirmed that 80S particles were the predominant virus particles accumulating. No precursor-product relationship could be established for the 80S particle, although 5S and 14S particles were shown to be precursors of mature virus particles.


Characterization of an enterovirus associated with acute infectious lymphocytosis.

An enterovirus (EVU-16) previously isolated from children with acute infectious lymphocytosis has been further characterized. The EVU-16 virus sediments as a 135S particle in sucrose gradients, has a density of 1-335 g/ml in CsCl, contains 4 polypeptides and has a single stranded RNA genome sedimenting at 35S. These structural features as well as the presence of a virus-related particle, the procapsid, are similar to those of other enteroviruses. However, the largest polypeptide of EVU-16 is 49000 daltons, which is considerably larger than the corresponding polypeptide from poliovirus; the sizes of the other three viral polypeptides were similar in both viruses. Attempts to induce lymphocytosis by the inoculation of EVU-16 into various animals, including immunologically aberrant 'nude' mice, were unsuccessful.


Infectivity, oncogenicity and transforming ability of BK virus and BK virus DNA.

The human papovavirus, BK, appeared weakly oncogenic in newborn hamsters and was able to induce in vitro transformation of rat kidney cells. The infectivity of BK virus DNA was determined by employing the DEAE-dextran method. In human embryonic cells the infectivity was approx. 10(5) p.f.u./mug of DNA. The transforming ability of BK virus in primary rat kidney cells was measured by employing the calcium method and appeared to vary from 1 to 10 foci per mug of DNA.


Electrophysiological changes during episodes of the Kleine-Levin syndrome.

Diurnal EEGs of a 17 year old male with the Kleine-Levin syndrome revealed moderate diffuse abnormalities and stage REM at sleep onset during attacks. Overnight, stages 3, 4, and REM of sleep were decreased, but sleep onset REM stage was not seen. These records returned to normal between attacks.


Motor nerve conduction study in patients on diphenylhydantoin therapy.

Mean motor conduction velocity of posterior tibial nerves was significantly reduced in epileptic patients treated with diphenylhydantoin formore than 10 years or in patients with serum diphenylhydantoin level above 20 mug/ml. Subnormal serum folate was not responsible for this reduction and clinical peripheral neuropathy was infrequent.


Pure motor hemiplegia secondary to brain-stem tumour.

'Pure motor hemiplegia' is a common stroke  syndrome defined by Fisher as paralysis of face, arm, and leg on one side, unaccompanied by sensory signs, visual field defect, aphasia, or apractognosia. It occurs almost exclusively in hypertensive patients and carried a good prognosis. We report a case of a normotensive patient in whom pure motor hemiplegia was the presenting feature, not of a cerebrovascular syndrome, but of a pontine glioblastoma. We note that brain-stem tumours may masquerade as brain-stem strokes.


Adenyl cyclase system and cerebral energy state.

The energy charge potential of the cerebral adenylate system, the cerebral lactate: pyruvate system, and the cerebral glycogen level were used to characterize the cerebral energy state of the brain. Cerebral adenyl cyclase activity and cyclic AMP concentrations were investigated to evaluate their possible influence on the regulatory processes in brain metabolism. These biochemical parameters were evaluated on the cortical motor area of the brain of the beagle dog in hypovolaemic hypotension during acute hypoxia (obtained by altering the composition of the inhalation mixture) and during the post-hypoxic recovery (three minutes after the restoration of normal ventilation). This experimental model of acute hypoxia was also used for investigating the action of some substances acting at beta-adrenergic receptor level or at vascular level. The substances were perfused into the carotid artery at the rate of 0.5 ml/min for three to six minutes. During the first stage of hypoxia, the adenyl cyclase system is probably an important biochemical regulatory factor. This system becomes less important when a critical threshold is crossed (oxygen arterial partial pressure less than 25-20 mmHg) below which other factors become rate-limiting. A beta-receptor stimulating agent enhanced the mechanisms of physiological post-hypoxic recovery; inhibition of this action was obtained with a beta-receptor blocking agent. Under these experimental conditions, no correlation existed between the vascular effects of the agents and brain metabolism.


Morphology and enzyme histochemistry of dorsal muscles of the cat neck.

An examination has been made of the five dorsal muscles of the cat neck which insert into the lamboidal crest. The three larger muscles, splenius, biventer cervicis, and complexus, are characterized by the presence of tendinous inscriptions which serve as the insertion points of shorter muscle fibers which do not run the length of the muscle. Longer fibers are bound by the inscriptions and, thus can transmit tension developed by shorter fibers. These three muscles are multiply innervater by nerves emerging from upper cervical spinal roots. Multiple innervation is not associated with multiple end plates but with arrangement so that lower roots innervate a high proportion of short fibers and higher roots, a high proportion of long fibers. Occipitoscapularis and rectus capitis major are, by comparison, simple muscles, each with a single motor nerve. Of the muscles examined, occipitoscapularis alone does not have its origin on the vertebral column but on the scapula, and is unlikely to have any major function in head movement.


Morphological correlates of synaptic transmission in lamprey spinal cord.

The dye Procion brown was used to identify in the light and electron microscope, synaptic contacts made between monosynaptically coupled neurons in the lamprey spinal cord whose synaptic interaction had been recorded. Synaptic contacts were made on different dendrites of the postsynaptic cell at different distances from the soma. Some of the contacts were made on dentritic spines and some on the smooth shaft of the dentrites. Serial sections through synaptic contacts made on dendritic processess of the postsynaptic cells were used for three-dimensional reconstruction of the synapses using computer graphics techniques. The computer reconstructions and detailed examination of the serial EM micrographs revealed the large proliferation of membrane involved in making these en passant synapses as well as the morphological changes due to stimulation of the presynaptic axon. These changes include depletion of synaptic vesicles and formation of complex vesicles and synaptic cisternae. Besides chemical synaptic contacts, four electrotonic contacts were located, confirming the mixed electrochemical synaptic response recorded from the postsynaptic cell. The mean quantum content was estimated and compared with the estimate of the available transmitter pool, assuming the quantal release hypothesis applies at these synapses. The total transmitter pool was estimated by counting all synaptic vesicles in all synaptic contacts. It was estimated that about 6% of the total transmitter pool is available for release at these synapses. This compares with less than 1% at the neuromuscular junction and about 20% at sympathetic synapses. These results support the hypothesis that synaptic vesicles may be recycled as described by Heuser and Reese (22) at the neuromuscular junction. Ongoing studies are investigating the effect on a variety of synaptic junctions to stimulation for different periods of time of presynaptic axons. The methods described in this study can also be used to test the models of synaptic interaction on dendritic trees described by Rall (39) and Jack and Redman (24).


Glioblastoma multiforme in children.

Of 488 children with central nervous system neoplasms, 43 (8.8%) had glioblastomas, 22 of which were in the cerebral hemispheres, 16 in the brain stem, two in the cerebellum, and three in the spinal cord. The male to female ratio was 3:2. Glioblastoma multiforme of the cerebral hemispheres occurred at a mean age of 12.7 years, and the frontal lobe was the most commonly involved. Main presenting symptoms included headache (85%), nausea or vomiting (65%), and seizures (35%). Papilledema (45%) was the most common physical finding. The longest survivals were achieved by a combination of operation and radiation (22 months). Brain stem glioblastomas occurred at a mean age of 6.7 years, with the pons as the most frequent site. Nausea or vomiting (50%) and headache (36%) were the main presenting symptoms; the major physical findings were ataxia (43%), cranial nerve palsies (28%), and paresis (28%). The length of survival was greatest with radiation alone (10.5 months). The period of survival of children with glioblastoma multiforme was significantly increased with steroid therapy. Glioblastoma multiforme behaves similarly in children and adults. Intracranial glioblastomas have a more rapidly fatal course than that of other similarly situated gliomas in childhood.


Localization of myocardial disorders other than infarction with 99mTc-labeled phosphate agents.

Myocardial studies with 99mTc-labeled phosphate agents were obtained in 20 patients without demonstrable cardiovascular disease, 24 patients with unstable arteriosclerotic heart disease (ASHD) without acute infarction, and six patients with myocardiopathy. The patients without cardiovascular disease showed no localization or tracer; the patients with unstable ASHD and without acute infarction showed nonfocal ill-defined accumulation of tracer; and the patients with myocardiopathy showed diffuse accummulation of tracer throughout the confines of an enlarged cardiac outline. Careful evaluation of both the distribution and intensity of the activity, in conjunction with the clinical picture, allows differentiation among these disease processes. Since ischemic areas around infarcted tissue may show increased activity, the value of this technique for sizing acute myocardial infarction may be limited.


Bone scan patterns of patients with diffuse metastatic carcinoma of the axial skeleton.

Bone scan findings (using 99mTc-stannous pyrophosphate) in five patients with diffuse metastatic carcinoma of the axial skeleton are reviewed. Although there were few visually recognizable asymmetries of tracer localization, the diffuse involvement was diagnosed through abnormally elevated counting rates in the axial skeleton, decreased visualization of the kidneys, and faint or absent visualization of the appendicular skeleton.


Nature of the dietary carbohydrate and metabolism of glycosaminoglycans and glycoproteins in rats.

The polysaccharide from blackgram (Phaseolus mungo) has been previously reported to cause lower cholesterol, phospholipids and triglyceride levels in rats fed either low-or high-fat diets containing cholesterol. The effect of this polysaccharide fraction as compared to that of glucose and sucrose on the metabolism of glycosaminoglycans and glycoprotein has been studied. The pattern of change in the levels of different glycosaminoglycans varied in the different tissues. Sucrose fed animals gave lower levels of sulphated glycosaminoglycans in the aorta and liver. The polysaccharide and glucose fed animals gave comparable values in the aorta except in the case of chondroitin sulfate B which was higher and heparin lower in the polysaccharide group. L-glutamine:D-fructose-6-phosphate amino transferase and UDPG dehydrogenase were lowest in the sucrose fed animals and highest in the polysacchride group with the animals in the glucose group showing intermediate values, but UDPG pyrophosphorylase, while highest in the polysaccharide group, was similar in the glucose and sucrose groups. Some of the degrading enzymes studied-beta-glucuronidase, hyaluronidase and aryl sulphatase-were highest in the sucrose group and generally lowest in the polysaccharide group. Levels of 3'-phosphoadenosine-5'-phosphosulphate, the biological sulphating agent, the sulphate activating system which includes ATP sulphurylase and APS kinase and sulphotransferase activity were also lowest in the sucrose fed group and highest in the polysaccharide group. The glycoprotein concentration was highest in the liver and lowest in the kidney in the sucrose group.


Salivary gland tumors: review of 643 cases.

643 cases of salivary gland tumors constitute two series of histological sections that were studied from hospitals and dental schools in Southeast Scotland and Southern Ontario. The Scottish series represented epithelial tumors of the parotid and intra-oral salivary glands, but the Canadian series also included tumors of the submandibular and sublingual glands. Classification was based on that recommended by the World Health Organisation (Thackray 1972). While direct statiscal comparisons between the two series are not appropriate, the differences between them suggest that malignant tumors are more common in Canada. The Scottish series contains the largest proportion of benign salivary tumors so far reported. In the Scottish series, 88.7% of parotid tumors were benign compared with 51.9% of Canadian series. In the Canadian series from the submandibular glands, 21.2% only were benign. Of the intra-oral salivary tumors, 62.2% from the Scottish series were benign compared with only 34.7% from the Canadian series.


Histochemical characteristics of muco-substances in three soft-tissue tumours.

The mucosubstances present in myxoid liposarcomas, malignant fibrous histiocytomas and intramuscular myxomas were investigated. The results suggest that such investigations are unlikely to provide a useful diagnostic aid in these three lesions.


Retardation of dissolution and growth of calcium oxalate monohydrate.

The kinetics of dissolution and growth of calcium oxalate monohydrate were examined in the presence of small concentrations of pyrophosphate, chlorophyll, and other agents. Data presented show that the retardation in mass transport in both processes is controlled by the nature of the additive, its concentration, and the way the additives are combined in the dissolution medium. Dissolution was studied using a particle counter method, and growth was conducted in a gel system under the slow diffusion of the reacting ions. Results obtained show that chlorophyll is more active than other inhibitors studied and suggest a higher surface adsorption intensity on the primary sources of the crystal surface.


Antinociceptive effects of lanthanum and cerium in nontolerant and morphine tolerant-dependent animals.

Intracerebroventricularly (i.vt.) administered lanthanum chloride was found to produce antinociceptive effects as measured by the mouse tail-flick and hot plate tests and the rat tail-flick test. These antinociceptive effects were reduced by peripheral administration of the narcotic antagonist naloxone or by i.vt. administration of calcium chloride. In addition, animals made tolerant to morphine were also tolerant to the effects of La+++. In morphine-dependent mice, the incidence of both abrupt and naloxone-precipitated withdrawal jumping was reduced by La+++ administration. In view of the known inhibitory effects of La+++ on Ca++ binding and movement, these findings suggest that alterations in Ca++ localization may be involved in the analgetic action of narcotic drugs as well as the development of narcotic tolerance and dependence.