Effects of anaerobiosis and inhibitors on O2-production by human granulocytes. A study was carried out on the effect of a number of inhibitors as well as of anaerobiosis on the production of superoxide (O2 minus) by human granulocytes. Several metabolic inhibitors, including bazide, cyanide, antimycin A, and 2,4-dinitrophenol had no effect on O2 minus production. Methimazole, which inhibits myeloperoxidase, was also without effect. The sulfhydryl reagents N-ethylmalemide and iodoacetamide greatly inhibited O2 minus production. O2 minus production did not take place under N2. Agents that affect cell motility were also examined. Colchicine and vinblastine, alkaloids which affect microtubule function, had little effect on granulycote O2 minus production. On the other hand, cytochalasin B, a fungal antimetabolite thought to affect microfilaments, inhibited O2 minus production by resting granulocytes and stimulated its production by cells incubated with bacteria. The mechanism and significance of this effect remain to be elucidated.


The estimation of 3,4-dihydroxyphenylacetic acid, homovanillic acid and homo-isovanillic acid in nervous tissue by gas-liquid chromatography and electron capture detection. 1 A gas chromatographic method using electron capture detection is described for the estimation of three acidic metabolites of dopamine, 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-hydroxy-4-methoxyphenylacetic acid (homo-isovanillic acid, iso-HVA). The method is based on the formation of the trifluoroacetyl-hexafluoroisopropyl derivatives of the three acids. 2 The method has been applied to the estimation of DOPAC, HVA and iso-HVA in tissues from the central and peripheral nervous systems.


Possible role of a beta-adrenoceptor in the regulation of noradrenaline release by nerve stimulation through a positive feed-back mechanism. 1 The effects of isoprenaline, propranolol and phentolamine, were studied on tritiated noradrenaline overflow elicited by postganglionic nerve stimulation in guinea-pig isolated atria. 2 Isoprenaline (1.2 times 10-minus 8M) increased while propranolol (1.0 times 10-minus 7M) reduced the overflow of tritiated noradrenaline evoked by nerve stimulation. These effects were less than those of phentolamine (3.1 times 10-minus 6M), which increased by approximately three-fold the overflow of [3H]-noradrenaline elicited by nerve stimulation. 3 Neuronal accumulation of tritiated noradrenaline in guinea-pig atria was not affected by isoprenaline, propranolol or phentolamine at the concentration employed in this study. 4 Isoprenaline (1.2 times 10-minus 8M) induced a positive chronotropic effect of about 80 percent of the maximum. On the other hand, propranolol produced a shift to the right in the frequency-response curve to nerve stimulation and in the concentration-response curve to exogenous noradrenaline in guinea-pig atria. 5 In the isolated nictitating membrane of the cat, the frequency-response curve to nerve stimulation was not modified by propranolol, while in the presence of 3.9 times 10-minus 6M of N,-2-(2,6-dimethylphenoxy)propyl-N,N,N-trimethylammonium (beta-methyl-TM 10) there was a shift to the right and a depression of slope. Neither propranolol nor beta-methyl-TM 10 affected responses to exogenous noradrenaline. 6. The effects of isoprenaline and of propranolol on transmitter release are compatible with the view that in addition to the presynaptic negative feed-back mechanism for noradrenaline release by nerve stimulation mediated via alpha-adrenoceptors a positive feed-back mechanism exists in adrenergic nerve endings which is triggered through the activation of presynaptic beta-adrenoceptors.


Two types of insomnia: too much waking or not enough sleep. The stability of sleep was examined in two kinds of induced insomnia, namely after caffeine administration and after hypnotic drug withdrawal. The duration of each episode of any one sleep stage or any episode of intervening wakefulness plus drowsiness was determined. After caffeine there was an increase in longer episodes of intervening wakefulness plus drowsiness, but no significant change in the episode duration of any of the sleep stages. In the case of drug withdrawal there was no change in the episode duration of intervening wakefulness plus drowsiness, but there was a significant shortening of episode duration in sleep stages 2 and 3+4, with a similar trend for REM sleep episodes. Caffeine 'insomnia' thus seems characterized by increased stability of wakefulness, and hypnotic withdrawal 'insomnia' by decreased stability fo sleep. The type of analysis undertaken in this study could increase understanding of other types of insomnia.


Plasma immunoreactive melanotrophic hormones in patients on maintenance haemodialysis. Circulating levels of melanotrophic horomones and ACTH were determined  in patients treated by maintenance dialysis for chronic renal failure. Plasma melanotrophic hormone levels were greatly increased in all patients studied (125-1100 ng/1 as compared with 12-36 ng/1 in normal adults) and were correlated with the duration of treatment. Skin pigmentation, especially in exposed areas, was notably increased, particularly in those patients with the highest plasma melanotrophic concentrations. Plasma ACTH levels were normal or only slightly raised and circulating corticosteroid concentrations, as determined by a fluorimetric method, all lay within the physiological range. The dissociation between ACTH and melanotrophic hormone levels in these patients may have been the result of a slower metabolic clearance of the latter.


A comparison of the peptide composition of human serum low and very low density lipoprotein. We have examined the apoprotein structure of human very low density lipoprotein (VLDL) and two subfractions of human low density lipoproteins, LDL-2 (d 1


Mammalian fatty acid synthetase. II. Modification of purified human liver complex activity. Highly purified human-liver fatty acid synthetase complex was used to study the effect of several potential modifiers. Adenosine 3',5'-phosphate did not alter the activity of either purified synthetase or of multienzyme present in 700 times g supernates. Its dibutyryl derivative was also ineffective when incubated with liver slices. Fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6-phosphate stimulated significantly the activity of the purified enzyme. Fructose 1,6-diphosphate, which was most effective, decreased the Km of the synthetase for NADPH. Phosphoenolpyruvate, rac-glycero-3-phosphate and potassium phosphate were ineffective; All longg-chain fatty acyl-CoA thioesters tested were inhibitory, but this effect was not observed until the regions of their critical micellar concentrations were reached. Free myristate, palmitate, and stearate did not inhibit synthetase activity up to the highest concentration tested (1 mM)qn enzyme preparation derived from livers of fasted rats inactivated purified rat-liver 4'-phospho[14-C]pantetheine-fatty acid synthetase by releasing its prosthetic group. It also decreased the activity of the purified human-liver complex.


Lysolecithin-cholesterol interaction. A spin-resonance and electron-micrographic study. Another publication (rand, R. P., Pangborn, W., Purdon, A. D., and Tinker, D. O.(1975) Can. j. Biochem. 53, 189-195) has established that lysolecithin and cholesterol interact to form an equimolar complex. We have investigated this complex using the techniques of electron spin resonance (e.s.r) and electronmicroscopy. By varying the cholesterol concentration with lysolecithin in both thin films and dispersions studied by these techniques, we have observed the interaction between lysolecithin and equimolar complex below 50 mol % cholesterol, and between crystalline cholesterol and equimolar complex above 50 mol % cholesterol. We have observed an interesting alteration in morphology by electron microscopy, and an isotropic to anisotropic spectral change using 3-dosylcholestane and 12-doxylstearic acid spin-labelled probes when the cholesterol concentration is increased from 20 to 33 mol %. The equimolar complex is stable in the presence of crystalline cholesterol, and exhibits no phase changes in the physiological temperature range. Implications for membrane structure are discussed.


Studies of the regulation of purine nucleotide catabolism. Ehrlich ascites tumor cells containing radioactive ATP were incubated in vitro with a range of concentrations of 2-deoxyglucose in order to produce different rates of ATP catabolism. Concentrations of all radioactive products of ATP catabolism were measured, and apparent rates of adenylate deaminase and inosinate dehydrogenase and of adenylate and inosinate dephosphorylation were calculated. It was concluded that these processes were reggulated primarily by the rate of formation of substrate, and to a lesser extent in some cases, by substrate concentration. No evidence was obtained for regulation of these processes by the concentration of ATP. The deoxyglucose-induced catabolism of radioactive GTP was also studied. When ATP catabolism was induced by incubation with 2,4-dinitrophenol, time courses of accumulation of purine nucleoside monophosphates and rates of alternative pathways of their metabolism were quite different than when deoxyglucose was used.


Rat liver nuclerar protein kinases.

Rat liver nuclerar protein kinases. We have shown that nuclei isolated by two methods contain grossly different amounts of cyclic AMP-dependent histone kinase activity. Repeated washing of the isolated nuclei with a low ionic strength buffer removed the majority of the cyclic AMP-dependent histone kinase and cyclic AMP binding activity. Nuclear cyclic AMP-dependent histone kinase activity accounted for only 0.42% of the total cytoplasmic enzyme activity. Similarly, the lactate dehydrogenase activity associated with liver nuclei represented only 0.07% of the total cytoplasmic activity. The isolated liver nuclei contained only 0.27% of the total homogenate glutamate dehydrogenase activity and 1.7%of the total homogenate  glucose-6-phosphatase activity. The cyclic AMP-dependent histone kinase behaves as a cytoplasmic rather than a nuclear enzyme. We have also shown that using crude extracts, one can achieve separation of the two nuclear casein kinases, NI and NII, on sucrose density gradients in the presence of 0.5M NaCl. Nuclear casein kinases NI and NII had sedimentation coefficients of 3.0 and 593 S, respectively, in the presence of 0.5 M NaCl. Under conditions of low ionic strength, all of the casein kinase activity in the crude nuclear extract sedimented as one peak with a seminentation coefficient of 7.3 S. The aggregation-disaggregation which occurred in the crude extract was reversible and was mainly due to the aggregative and disaggregative properties of casein kinase NII. The two nuclear casein kinases have different affinities for chromatin. When nuclei were disrupted in a hypotonic solution and extracted with a buffercontaining 0.14 M NaCl, casein kinase NII could be completely extracted from the viscous nuclear material. Although a significant amount of casein kinase NI was extracted by the buffer containing 0.14 M NaCl, re-extraction of the nuclear material with a buffer containing 0.5 M NaCl yielded substantial amounts of casein kinase NI, and a final extraction with a buffer containing 1.0 M NaCl yielded measurable amounts of casein kinase NI. No casein kinase NII activity could be detected in the 0.5 M and 1.0M NaCl extracts.


The orientation of cytochrome c oxidase in coupled phospholipid membrane vesicles--a spin-label study. Cytochrome oxidase, an enzyme containing six different subunits, has been shown to span the inner mitochrondrial membrane. The arrangement of the subunits within the membrane is unknown. Wh have specifically labeled the 25 000 molecular weight subunit with a spin-label derivative of N-ethylmaleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (NEM-SL(5)). NEM-SL(5)-lebeled cytochrome oxidase can be incorporated into phospholipid membranes to form coupled vesicles of the Hinkle, Kim & Racker ((1972) Jriol. Chem; 247, 1338-1399) type. The resonance spectrum of NEM-SL(5) is similar in both soluble and vesicular cytochrome oxidase. Since ascorbate has been shown to reduce only spin label that is exposed to the exterior surface of a closed vesicle, we have used ascorbate to determine the NEM-SL(5)-binding site in the coupled vesicles; NEM-SL(5)-labeled cytochrome oxidase vesicles are reduced by 10 mM ascorbate with tau 1/2 of 1 min at 22 degrees C; The rate of reduction is relatively independent of temperature. We conclude that (1) cytochrome oxidase is unidirectionally or preferentially oriented in the vesicle membrane, and (2) the NEM-SL(5)-binding site on the 25 000 molecular weight subunit is exposed to the external aqueous medium.


Characteristics of action of prostaglandins on cyclic AMP accumulation in rat anterior pituitary gland. Prostaglandins (PGs) were found to lead to a marked stimulation of cyclic AMP accumulation in rat anterior pituitary gland in vitro in the following decreasing order of potency: PG E-1 E-2 GREATER THAN A-1 A-I GREATER THAN F-1ALPHA F-2ALPHA. The effect of PGs is potentiated by theophylline. The stimulatory effect of PGs on cyclic AMP accumulation is already detected 2min after the addition of 1-x 10-7 to 1-x 10-6 M PG E-2 and its maximal effect is reached after approximated 30 min of incubation, with a progressive decrease toward basal cyclic AMP levels at later time intervals. Increased intracellular cyclic AMP concentrations are accompanied by an increased release of the nucleotide into incubation medium. Complete removal of Ca-e+ from the incubation medium by addition of EGTA was found to increase the stimulatory effect of PG E-2 ON CYCLIC AMP accumulation. The action of PGs on hormonal release and cyclic AMP accumulation support the hypothesis of a role of PGs in the mechanism of anterior pituitary hormone (particularly growth hormone) release.


The conformational states of oxidized and oxygenated beef-heart cytochrome c oxidase. 1. The "oxygenated" form of cytochrome has been generated by treatment of the enzyme with ascorbic acid. 2. "Oxygenated oxidase" so generated is stable over long periods (24 h). 3. Sedimentation velocity experiments have shown the "oxygenated" oxidase to be a less compact molecule than the oxidized.


The conformational states of cytochrome oxidase in the mitochondrion. THE Soret spectrum of "resting" cytochrome oxidase in cytochrome-c depleted mitochondria has been determined. The spectrum obtained is dependent on the rate at which the oxidase is turning over. In the least active preparations, the spectrum is almost pure "oxidized"  oxidase. With increasing activity the spectrum is converted to a mixture of "oxidized" and "oxygenated" oxidases. It is concluded that the same conformational differences between the two non-reduced forms that are found in the purified enzyme also occur in these cytochrome-c depleted mitochondria.


Effects of Lubrol detergents on adenylate cyclases. The nonionic detergent Lubrol WX showed diverse, concentration-dependent effects onbasal and stimulated adenylate cyclases. Above concentrations of 0.001-0.01% Lubrol WX, the basal activity of cyclase from Ehrlich ascites cells was inhibed about 50%, and that from rat fat cells was doubled. In both cases, hormonal sensitivity was lost at 0.01%. These effects were reversed upon dilution of the detergent. It is suggested that solubilization of adenylate cyclases at such low concentrations of Lubrol should be attempted since it is conceivable that loss of hormone sensitivity may then be reversible. Different Lubrol-type detergents may also offer centain advantages, since Lubrol PX effects were not identical with those of Lubrol WX.


Effect of Eimeria acervulina infection on nutrient retention with special reference to fat malabsorption in chickens. Nutritional balance studies were conducted to determine the effect of Eimeria acervulina infection on the retention of nutrients and the efficiency of utilization of protein and energy in the diet during the course of intestinal coccidiosis in chickens. Both light and electronmicroscopic studies were conducted to elucidate the pathogenesis of fat malabsorption in the infected chickens. The infection with E. acervulina during the acute phase of the disease (day 4 to day 8 postinfection) caused a reduction (P smaller than 0.01) in the retention of percent protein, percent ether extract and percent gross energy of the diet with a concomitant increase (P smaller than 0.01) in the concentration of ether extract and gross energy of the excreta. The metabolizable energy value of the diet was also depressed during this phase. During the recovery phase of the disease (day 9 to day 13 postinfection) infected chickens voided excreta containing a slightly greater concentration of ether extract when compared to the controls but differences in the retention of percent protein and percent gross energy were no longer observed. An increased retention of percent phosphorus also occured during the recovery phase. Studies involving the carcass composition revealed that the efficiency of utilization of protein and metabolizable energy of the diet for tissue deposition was reduced (P smaller than 0.05) during day 0 to day 8 postinfection but only the efficiency of metabolizable energy utilization was found to be depressed (P smaller than 0.05) when data for day 0 to day 14 postinfection were analyzed. Both light and electronmicroscopic studies of the duodenal villi of infected chickens during day 5 to day 6 postinfection revealed accumulation of large globules of fat in the villus epithelial cells parasitized by the gammonts of E. acervulina indicating a possible blackade of "fat exit" from these cells.


A comparison of gel diffusion, fluorescent antibody and virus isolation methods in experimental and natural cases of infectious bursal disease. In studies with chicks inoculated with the Sk-1 strain of infectious bursal agent the bursa of Fabricius was found to be the tissue of choice for virus isolation as well as for use in the fluorescent antibody test and the agar gel diffusion test. In separate experiments positive results were obtained until postinoculation days 3 or 4 by the agar gel diffusion test, 5 or 6 by the fluorescent antibody test and 14 by the virus isolation method, respectively. Bursas from chickens involved in seven natural outbreaks of infectious bursal disease were then examined by these three methods. Virus was isolated from six outbreaks and infectious bursal agent antigen was demonstrated in three by the agar gel diffusion test method and seven (three by direct examination and four after one passage in chicks) by the fluorescent antibody test method. Passage in chicks was required when nonspecific fluorescence complicated the interpretation of fluorescent antibody test results.


Investigation of factors of probable significance in the pathogenesis of pneumonic pasteurellosis in cattle. Six groups of ten beef calves six to eight months of age were shipped from western Canada and observed untreated for one week after arrival. The following parameters were measured daily: body temperature, plasma fibrinogen, nasal bacterial mean colony counts of Pasteurella hemolytica and Pasteurella multocida, total and differential leukoyte counts, packed cell volumes and the following, twice during the week: serum and nasal antibody titres to P. hemolytica and parainfluenza-3 virus. The lungs from 44 of the calves were obtained at post mortem and given a numerical score based on the degree of pneumonia present. Animals were designated SICK and WELL according to body temperature and plasma fibrinogen. The SICK animals had higher nasal mean colony counts of P. hemolytica than the WELL animals. The SICK animals had lower levels of serum antibody to P. hemolytica than the WELL on day 1 but had a greater rise in titre over the week than did the WELL animals. Both groups were similar with regard to serum antibody to parainfluenza-3 virus and there was little change in these titres. The SICK animals had a much greater degree of pneumonia than the WELL. The values of some of the parameters were combined with the data of previously studied animals in order to provide a comparison of SICK and WELL with larger numbers of animals.


Correlation between the serum neutralization test and the indirect immunofluorescent test for the detection of specific antibodies to infectious bovine rhinotracheitis virus. Comparative titrations show that immunofluorescence on infected rabbit kidney cells is relatively as sensitive as the neutralization test for the detection of antibodies to the infectious bovine rhinotracheitis virus. When calf testis cells infected with this virus are used for the detection of antibodies by the indirect immunofluorescent test nonspecific staining is high. This nonspecific reaction is absent when rabbit kidney cells are used.


Improvements in the modified direct complement fixation test and its application in the detection of bluetongue antibodies in cattle and sheep sera. In this study, improvements were made in the technique and the preparation of the antigen. It was possible to perform three extractions and elutions resulting in a soluble reactive preparation from each batch of infected mouse brain. This led to an appreciable increase in the yield of highly reactive antigen. The presence of bluetongue antibodies was not detected in 13,210 sheep sera. Of the 13,486 bovine sera tested, only three questionable reactions were obtained. It was possible to determine that two of these animals were imported. Various isolation methods, including transmission trials to susceptible sheep followed by serological tests on the sheep sera, failed to confirm the infection in the three reactors.


Type 1 and type 2 herpes simplex uvietis in immune rabbits: importance of pure virus preparation. Systemic immunization with type 1 herpes simplex virus (HSV) protected rabbits from developing uveitis when they were challenged intravitreally with either purified type 1 or purufied type 2 HSV. But when an HSV preparation contaminated with a nonviral foreign antigen (fetal calf serum) was used for both systemic immunization and challenge, the immunization against HSV failed to exert a protective effect because the fetal calf serum had produced a nonviral immunologic reaction in the uveal tract. This clearly indicates the importance of preparing virus properly for use in experiments of this nature.


Clostridium welchii corneal ulcer--a case report. A case of corneal ulcer in a 10-year-old Nigerian boy in which clostridium welchii and achromobacter were isolated is reported. The absence of trauma and rarity of only corneal involvement in a clostridial infection is emphasized. The bacteriology of the clostridial infection is emphasized. The bacteriology of the clostridial ocular infections is reviewed, the media necessary for isolation of the organism and the symbiotic relationship with other bacteria are discussed. The clinical course of the disease with uneventful recovery of the patient is emphasized.


Hydrogen cyanide, a secondary metabolite of Pseudomonas aeruginosa. Seventy-four of 110 strains of Pseudomonas aeruginosa tested produced detectable amounts of HCN from growth in 2% peptone or nutrient agar. Of the 25 species of12 bacterial and fungal genera tested, other than P. aeruginosa, only P. fluorescens and P. polycolor gave positive HCN tests. Cyanide is produced after cessation of active growth. Iron was stimulatory to cyanogenesis in concentration above 1 muM, while copper, zinc, cobalt, and manganese at concentrations of 20 muM had no effect. Cyanogenesis id dependent on the temperature of incubation within ranges which allow complete growth. Inorganic phosphate in concentrations between 90 and 300 mM allows growth but inhibits HCN production. Growth of cells anaerobically, using nitrate as the electron acceptor, results in low cyanide yields, which can be partially reversed by subsequent aerobic incubation. These results indicate that HCN is a secondary metabolite of P. aeruginosa.


Relationship between the DNA content and mesosome number in cells of Bacillus. In cells of Bacillus there is evidence that deoxyribonucleic acid forms an association with some membranous structure within the cell, possibly mesosomes. Cells of varieties of Bacillus cereus and Bacillus subtilis were examined to see if any quantitative relationship existed between the numbers of mesosomes and DNA content. No direct relationship could be domonstrated. However, cells of Bacillus cereus var. alesti A(-) maintained a characteristic and constant DNA content and number of mesosomes regardless of growth conditions. During sporulation, a variant of A(-), termed A(-)3, SEQUESTERS ITS DNA at both ends of the cell, leaving a small amount of DNA but no mesosomes in the center compartment. Since this center compartment is capableof growth and division upon replacement in fresh medium (rejuventation) it was examinedfor mesosome content as DNA synthesis and division were initiated. In most cells, acentral mesosome was formed at the site of cell septum formation; however, the presenceof a mesosome was not an absolute prerequisite for cell division. We propose that atthe onset of cell growth, mesosomes primarily function in the process of cell septum formation. As growth and division proceed, mesosomes are produced in characteristicnumbers and may act as the site of DNA synthesis and (or) segregation.


Host response to infection by Coxiella burneti. A host response to infection by Coxiella burneti was investigated. Infectedyolk sacs were harvested from embryonated eggs and assayed for glycolytic activity. Assays of glycolytic enzymes included glucose isomerase, aldolase, phosphofructokinase,fructose-1,6-diphoshatase, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase. No significant differences in enzymatic activity between normal and infected tissues through the 12th day was observed. From the 13th day through the 16thday, the glycolytic activity of normal tissues decreased. Glycolytic activity of infected tissues did not decrease, but showed a gradual increase during this same time period. Embryos from infected eggs demonstrated a gradual decrease in total weight fromthe 12th day until death occurred on the 16th day.


[Rubella virus.  III. Purification of infected or non-infected Vero cell membranes]. Separation of Vero cell membrane in a discontinuous sucrose gradient reveals five fractions. After infection a sixth fraction appears. It contains virioins but mostly modified membranes with subunits 5-6 nm in diameter, probably the hemagglutinin. None of the enzymes used was associated with this fraction. No modification of the other fractions was observed after infection.


Solitary liver nodules.

Solitary liver nodules. There has been confusion in the literature over the nomenclature of solitary liver nodules. Several such lesions have recently been reported in patients taking oral contraceptives. Similarities exist between these cases that suggest they may be examples of focal nodular hyperplasia. Here three further cases are presented. The criteria for making the diagnosis and its importance are discussed.


Medical Research Council of Canada therapeutic trial of human growth hormone: first 5 years of therapy. The Medical Research Council of Canada has initiated human growth hormone (hGH) therapy in 151 patients with documented complete hGH deficiency that was idiopathic in 76% of cases, secondary to craniopharyngioma (organic) in 17% and of varied cause in 7%. Approximately 50% of the patients with idiopathic disease had isolated hGH deficiency; during therapy thyroid deficiency developed in five patients and cortisol deficiency in three. A similar increase in mean height velocity occurred in the first treatment phase for patients less than 12 years old (0.93 plus or minus 0.30 cm/mo) and those 12 years and older (0.86 plus or minus 0.29 cm/mo). Although subsequent courses of hGH therapy yielded significantly diminished response in both age groups, this diminution was not progressive: the height velocity of the younger patients returned to 0.82 plus or minus 0.26 cm/ml in the fifth therapy phase. The mean height velocity attained at the optimal dosage (0.20 to 0.29 units/kg three times per week) for each age group did not differ significantly. Despite therapy being carried out for only 6 months of the year, normal increment ratios for height age and bone age against chronologic age were observed in the patients with idiopathic disease. In only four patients did treatment failure occur, and three of these were more than 20 years old. The addition of fluoxymesterone (10 mg/d) to the hGH therapeutic regimen (15 units/wk), when diminished response to hGH alone became evident, promoted an enhanced growth response in 9 of 11 older patients. These data indicate that age of the patient and dosage of hGH, but not diagnostic category, were important influences on the response to therapy. Younger patients responded best and maintained a higher mean growth velocity than older patients during intermittent hGH therapy


The structure of a sulfated glycoprotein of chick allantoic fluid: methylation and periodate oxidation. The structure of an antigenic, sulfated glycoprotein from chick chorioallantoic fluid has been investigated by exogalactosidase digestion, methylation and mass spectral analyses, periodate oxidation, and Smith degradation. The main carbohydrate chains are composed of D-galactosyl residues linked at C-3 and 2-acetamido-2-deoxyglucose residues linked at C-4. Fucose and N-acetylneuraminic acid residues are nonreducing terminal groups, and the N-acetylneuraminic acid groups are linked to the D-galactose residues at C-3. Most of the sulfate groups (91% of the sulfate) are located on C-6 of the 2-acetamido-2-deoxyglucose residues, and the rest on C-6 of the D-galactose residues. A large number of the D-galactose residues (36.9% of the total) are present as nonreducing terminal groups and another 21.7% of the D-galactose residues are in penultimate position to the nonreducing terminal N-acetylneuraminic acid residues. Although mild periodate oxidation indicates the presence of D-galactose in furanoside form (5.5% of total D-galactose), no 5-O-methyl derivative of D-galactose was observed on methylation.


Methylation of rat liver mitochondrial deoxyribonucleic acid by chemical carcinogens and associated alterations in physical properties. The formation of 7-methylguanine in rat liver mitochondrial DNA following the administration of the powerful carcinogen, dimethylnitrosamine, and the weak carcinogen, methyl methanesulphonate was measured and compared to the alkylation of nuclear DNA by these agents. At all doses tested mitochondrial DNA was alkylated more extensively than nuclear DNA by dimethylnitrosamine but both types of cellular DNA were alkylated to about the same extent by methyl methanesulphonate. The physical structure of rat liver mitochondrial DNA isolated from animals treated with these agents was investigated by electrophoresis in agarose gels and by isopycnic centrifugation in CsCl gradients. These procedures carried out in the presence of ethidium bromide, an intercalating dye, separate closed circular forms of mitochondrial DNA from open circular molecules (containing a single-strand break) and linear molecules. Administration of dimethylnitrosamine produced a considerable decrease in the amount of mitochondrial DNA which could be isolated in the closed circular form and at higher doses of dimethylnitrosamine no closed circular mitochondrial DNA could be found. Methyl methanesulphonate was less effective at reducing the amount of closed circular mitochondrial DNA. One explantation of these results is that dimethylnitrosamine leads to strand breaks in mitochondrial DNA and the possible use of this system to investigate carcinogen-induced breaks in DNA is discussed.


Synthesis of threo-4,5-dihydroxy diastereomers of sphinganine;. The threo-4,5-dihydroxy diastereomers of sphinganine were prepared by the following sequence of reactions: (a) benzoylation of sphingenine to the tribenzoyl derivative; (b) osmylation followed by resolution of the mixture of threo-4,5-dihydroxy tribenzoyl (DHTBS) diastereomers; and (c) alkaline hydrolysis to yield the threo-4,5-dihydroxysphinganines (DHS). Carbon atoms 4 and 5 of the high and low melting threo-4,5-DHTBS diastereomers and the compounds derived from them were tentatively assigned 4R, 5S and 5R configurations, respectively;


Arrangement of lipid and protein in human serum high density lipoproteins: a proposed model. Based on size and compositional data, models for the two major density subclasses of human serum high density lipoproteins have been developed. Descriptions of both the surface structure of the high density lipoproteins and the structure of their apolar core regions are integral parts of these models. The surface is described as a mosaic of amphiphilic lipid and helical protein regions. The conformation and orientation of the various apolar lipid components as restricted by the surface organization is discussed.


An ESR Study of the mobility of the cholestane spin label in oriented lecithin-cholesterol multibilayers. The motion of the cholestane spin label in oriented lecithin-cholesterol multibilayers is described in terms of a rotational diffusion about the long molecular axis with diffusion coefficient D parrell and a restricted random librational motion about axes perpendicular to the long axis with diffusion coefficient D1. The diffusion coefficients have been determined from the angular dependence of the ESR line shape at various temperatures and cholesterol contents. The temperature dependence of D parrell and D1 clearly shows the transition from the gel to liquid crystalline phase. Increasing amounts of cholesterol reduce the transition temperature. A strong reduction is found from o to 10 mole % cholesterol. At 50 mole % no longer a sharp transition is observed. In the temperature range from 40 to 80 degrees C the range of D is about 10 times larger than the range of D parrell, indicating a high activation energy for the librational motion arising from a strong hindrance by interaction with surrounding molecules. Cholesterol contents up to 10-20 mole % give an increase of D parrell and D1, arising from strong decrease of the transition temperature in this range. Above 10-20 mole % a reduction of D parrell and D1 is found. However, the effect  of cholesterol is much stronger on D1 than on D parrell. In the liquid crystalline phase at about 60 degrees C the effect of cholesterol on D parrell is even negligible, while D1 strongly changes. This indicates that in the liquid crystalline phase only the librational motion is influenced by cholesterol, due to a denser packing of the molecules in the bilayer.


An ESR Spin label study of structural and dynamical properties of oriented lecithin-cholesterol multibilayers. Oriented dipalmitoyllecithin-cholesterol multibilayers with 11% water have been studied with the cholestane spin label. From the ESR spectra the order parameters and the mobility of the spin label about its long axis have been calculated. The results on pure lecithin multibilayers indicate a transition from gel to liquid crystalline phase at 52 plus or minus 2 degrees C. In the gel phase the lecithin alkyl chains are highly ordered, but tilted with respect to the normal to the bilayers by about 25 degrees. Above 52 degrees C the tilt disappears and the mobility of the cholestane spin label increases, indicating an increase of mobility of the lecithin alkyl chains. When cholesterol is added, below about 52 degrees C a decrease of order is found. Furthermore, already small cholesterol contents (smaller than or equal to 10 mole %) remove the tilt. Above about 52 degrees C cholesterol improves the order by decreasing the amplitude of the librational motions. Cholesterol lowers the transition temperature of the system and reduces the mobility of the lecithin alkyl chains in the liquid crystalline phase. However an increase in mobility is found at cholesterol contents up to 10 mole %. A very broad phase transition is observed at 50 mole % cholesterol. In all systems an increase in temperature results in a reduction of order through an increase of the amplitude of the librational motions of the molecules. The librational motions are to some extent cooperative. The asymmetry of the order matrix is found to be a measure for the lateral ordering. Cholesterol increases the lateral ordering, indicating that the flat cholesterol molecules orient parallel to each other.


Intervesicular phospholipid exchange. An NMR study. Phospholipid exchange between phosphatidylinositol and phosphatidylcholine ?vesicles has been studied by NMR spectroscopy with use of hydrophilic paramagnetic lanthanide probes (Pr-3+ and Eu-3+ ions). The dependence of the lanthanide induced shifts in the1-H and 31-P NMR spectra on the phospholipid composition of the vesicles could be used for its quantitative evaluation. The method has been proved to be applicable for studying phospholipid exchange stimulated by soluble proteins (postmicrosomal supernatant fraction) from rat liver. Furthermore it has been shown that the phospholipid molecules newly introduced by protein-stimulated exchange are predominantly incorporated into the outermonolayer of the vesicular bilayer membrane. This makes it possible to produce liposomes with asymmetric distribution of the phospholipids across the bilayer.


13-C NMR investigation of phospholipid membranes with the aid of shift reagents. 13-C NMR spectra of sonicated phospholipid dispersions have been investigated with the aid of paramagnetic ions (Pr-3+) as shift reagents. This method permits discrimination between the outer and the inner surfaces of the vesicular membrane and gives a larger variety of signals than 1-H NMR. The lanthanide induced shifts have been found to depend on other ions present and on the lipid composition of the membrane.


Phospholipid unsaturation and plasma membrane organization. A comparison has been made between the unsaturation of plasma-membrane phospholipids,present in the human erythrocyte, rat liver, mouse liver and a rapidly growing rat hepatoma. Of the double bonds present in the hydrocarbon chains of the membrane phospholipids,onethird is contributed by sphingomyelin plus phosphatidyl choline and the remainder by phosphatidyl serine, ethanolamine and inositol. Assuming that the phospholipids are asymmetrically distributed in the two leaflets of the bilayer in general, the consequences of this asymmetry in combination with cholesterol content and fatty acid distribution on plasma membrane organization and function are discussed. It is suggested, that the organizational disposition of plasma membrane components other than phospholipids is at least related if not dependent upon the latter's asymmetric distribution in the bilayer.


Relationship between serum cholinesterase and low density lipoproteins in children with nephrotic syndrome. 1. A significant increase in the activity of serum cholinesterase and concentration of dextran sulphate precipitable lipoproteins, cholesterol and triglycerides was observed in sixteen children with a diagnosis of nephrotic syndrome when compared with the values obtained in 22 other normal children of comparable ages. 2. In 7 children with nephrotic syndrome serum concentrations of beta-lipoprotein and cholinesterase were markedly higher in the acute stage compared to the convalescent stage. A working hypothesis is proposed to explain the hyperlipoproteinemia in nephrotic syndrome.


Composition of low-density lipoprotein in children with hyperlipoproteinaemia. Low-density lipoprotein composition, studied in 7 pre-pubertal children with secondary hyperlipoproteinaemia, did not differ significantly from that found in 12 normal children. This contrasts with the findings in 12 children with the heterozygous form of familial hypercholesterolaemia in whom the low-density lipoprotein was significantly deficient in triglyceride (p smaller than 0.001) and protein (p smaller than 0.025), and had an increased proportion of cholesterol esters compared with normal (p greater than 0.01). Treatment which lowered serum cholesterol and low-density lipoprotein concentration in these children did not materially alter the abnormalities of composition of the low-density lipoprotein. The data support the hypothesis that an abnormality of composition of low-density lipoprotein is an integral feature of familial hypercholesterolaemia and does not simply result from an increased concentration of low-density lipoprotein of whatever cause.


Hexokinase isozyme pattern in CCl(4)-injured rat liver. Activities of hexokinase isozymes in carbon tetrachloride (CCl(4))-injured rat liver were determined quantitatively by DEAE-cellulose column chromatography and compared with those of regenerating liver, fetal liver and ascites hepatoma cells (AH 130). The CCl(4)-injured liver revealed an isozyme distribution with predominant Types I, II and III (3.2, 8.8 and 6.8 times higher than the control values, respectively) and with undetectable activity of Type IV hexokinase (glucokinase). Although the isozyme pattern generally resembled that of fetal liver or hepatoma cells, the relatively high activity of hexokinase Type III in CCl(4) treatment characterizes the pattern of hexokinase isozyme in acue liver damage.


Determination of human liver cytochrome P-450 by electron paramagnetic resonance of liver biopsies. Human liver cytochrome P-450 level has beenen determined by direct electron paramagnetic resonance spectroscopy at 77 degrees K of liver specimens obtained by surgical biopsy. A preliminary experiment with rat livers was performed in order to calibrate the intensity of the cytochrome P-450 EPR signal as a function of the cytochrome concentration obtained by the classical spectrophotometric method. 44 subjects have be examined. We found a cytochrome P-450 value of 21.5 plus or minus 11.2 nmoles per gram of liver in normal subjects. Variations of this level in some pathological liver states are discussed.


Human very low density lipoproteins: loss of electrophoretic mobility on enzymatic removal of sialic acid residues. Incubation of very low density lipoproteins (VLDL) from three hyperlipoproteinemic patients with neuraminidase resulted in a progressive liberation of sialic acid residues. After the original sialic content had been reduced by approx. 75% a lipoprotein fraction having zero electrophoretic mobility on paper appeared. This fraction increased until, after the removal of all the sialic acid residues, the mobility of the entire VLDL fraction was reduced to zero.


Viral hepatitis and cirrhosis.

Viral hepatitis and cirrhosis. Most of the knowledge of post-hepatitic cirrhosis comes from studies performed in the last five years on the hepatitis B antigen-related variety. The position of other types of hepatitis (particularly type A) as an aetiological factor in cirrhosis remains conjectural. In general, the post-hepatitic cirrhosis develops insidiously after a mild or unrecognised acute episode of hepatitis. General progress is slow. Early deaths are due to liver failure. Later, primary hepatocellular carcinoma assumes increasing importance. Needle biopsy of the liver is usually necessary to confirm the diagnosis of cirrhosis and to estimate the degree of activity. Sampling errors when such a small specimen of liver is obtained must be taken into account, when formulating a diagnosis and prognosis. Prednisolone therapy is usually given if the patient is symptomatic, biochemical tests are abnormal and the liver biopsy confirms active chronic hepatitis with or without cirrhosis. The evidence of benefit is not so strong as for other forms of active hepatitis and cirrhosis such as the lupoid type. The management of the cirrhosis is otherwise along orthodox lines.


Lp(a) lipoprotein/pre-beta1-lipoprotein in Swedish middle-aged males and in patients with coronary heart disease. A strong, positive association (P smaller than 0.0001) was found between the presence in serum of pre-beta1-lipoprotein upon electrophoresis in 0.5% agarose and the Lp(a+) phenotype in a series of 103 males aged 50-52 years participating in a health control study and in a series of 58 patients with sustained myocardial infarction. Both lipiprotein phenomena were more prevalent in the 58 patients than in the 103 presumably healthy males. The lack of any significant difference between total cholesterol values in the patients and the controls, despite the higher frequency of phenotype Lp(a+) in the former group, argues against the suggestion by Walton et al. (1974) that the increased Lp(a+) frequency in patients with coronary artery disease may be caused by a general increase in the concentration of low density lipoproteins (LDL).


Growth following renal transplantation in children and adolescents. Growth following renal transplantation in children and adolescents. Sixteen children who received a cadaveric renal transplant surviving 1 to 5 years, had their growth rate assessed while receiving 10 to 25 mg of prednisone every 48 hours. Six of 9 patients witha a bone age of 12 years or less grew at a "normal" rate. None of the patients whose bone age was greater than 12 years grew at a "normal" rate. Since low-dose alternate-day prednisone therapy allows for optimal growth but does not appear to compromise kidney survival, we feel such a regime should be instituted in growing children receiving a kidney transplant.


Dose-response to tibric acid: a new hypolipidemic drug in type IV hyperlipoproteinemia. This double-blind study compares the effects of tibric acid at four different doses to that of placebo in type IV hyperlipidemic patients. Fifty patients, divided at random in five groups of 10 patients each, received one of the following treatments: placebo, tibric acid 500 mg, 750 mg, 1,000 mg, or 1,250 mg per 24 hr. The data suggest that the 1,000 mg and the 1,250 mg doses are effective in lowering the serum triglyceride levels after 6 wk of treatment; the effect on total cholesterol is less pronouced. No effect is observed on the concentration of serum esterified cholesterol, phospholipids, and free fatty acids. It is also shown that in the dose range studied, the best fitting curve defining the four mean values of triglycerides and total cholesterol obtained with the four active treatments did not deviate significantly from linearity. Due to lack of toxicity, tibric acid may be useful in the treatment of type IV hyperlipoproteinemia.


Observations on embryonic chick-bone crystals by high resolution transmission electron microscopy. High resolution transmission electron microscopy of bone mineral at a vary early stage in its deposition in embryonic chick frontal bone demonstrates crystals of 2-2.5 nm thickness and a profile up to 40 nm long. They appear in groups which may show as few as one or two crystals. Associated with the crystals is a less dense amorphous material. Using specimen tilting, the change in crystal appearance indicates that crystals are tablet-shaped (rectangular) rather than rod-shaped. The amorphous material may be tablet-shaped crystals in orientations which are not dense enough to register as crystals in the image. Truly amorphous mineral does not show by bright-field electron microscopy, but preliminary results indicate that the dark-field technique may prove to be useful in imaging amorphous mineral provided that sections can be cut thinly enough and that precautions are taken during specimen preparation to avoid mineral loss. Crystals have been located in various parts of the osteoid zone and were not associated specifically with any one component. Rapid processing using anhydrous techniques must be used to avoid the possibility of artefactually altering the in vivo conditions of the mineral at the time of excision of the specimen.


Calcium accumulation by chondrocyte mitochondria. Chick epiphyseal plate mitochondria observed in vitro suggest that energy dependent Ca++ uptake was maximal in the presence of ATP and a respiratory substrate. However, nucleotides other than ATP had no effect on this type of cation uptake. The observation that Ca++ accumulation was sensitive to the presence of 2,4-DNP and a number of respiratory inhibitors suggested that the mechanism of cation accumulation was similar to that described in tissues that do not undergo biological mineralization. Non-energy supported Ca++ acumulation was studied in the presence of rotenone and antimycin A. Under these conditions, the amount of Ca++ bound by skeletal tissue mitochondria was greater than bound by mitochondria obtained from noncalcifying tissues. Following isopycnic centrifugation, the Ca++ loaded mitochondria banded at different sucrose densties but the Ca++ affinity of mitochondria at each density band was similar. Hence, no particular mitochondrial species seems to be responsible for cation transport.


Enzyme activities and properties of lysosomes and brush borders in jejunal biopsies from control subjects and patients with coeliac disease. 1. Reduced activities of four enzymes from brush borders were found in intestianl biopsies from patients with untreated coeliac disease. The activities returned towards control values after treatment by gluten withdrawal. Parallel changes were noted for the cytosol enzyme lactate dehydrogenase. 2. Measurement of brush-border integrity by differential centrifugation of biopsy extracts indicated increased fragility of the brush borders in biopsies from untreated patients. Normal values were obtained for biopsies from treated patients. 3. Increased activites of six acid hydrolases (lysosomal enzymes) were found in biopsies from untreated coeliac patients. Normal values were obtained for biopsies from treated patients. 4. Assement of lysosomal integrity by assay of latent N-acetyl-beta-glucosaminidase and of sedimentable activity of four acid hydrolases demonstrated increased lysosomal fragility in untreated coeliac mucosa. These lysosomal changes return to within the normal range after treatment by gluten withdrawal. 5. The lysosomal changes are consistent with their having a pathogenic role in the enterocyte damage of coeliac disease. Possible mechanisms for the lysosomal changes are discussed.


Vitamin D metabolism and parathyroid function in man. 1. The metabolism of an intravenous pulse dose of double-isotope-labelled cholecalciferol has been studied in control subjects with widely differing states of vitamin D nutrition and in patients with primary disorders of parathyroid function. 2. The formation of labelled 1,25-dihydroxy-cholecalciferol [1,25-(OH)2D3] and labelled 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] has been related to the prevailing concentrations in serum of 25-hydroxycholecalciferol [25-(OH)D3], immunoreactive parathyroid hormonel, calcium and orthophosphate (Pi). 3. In control subjects with relative vitamin D deficiency [serum 25-(OH)2D3 was related inversely to the serum 25-(OH)D3 and serum calcium, and directly to serum immunoreactive parathyroid hormone. No formation of 1,25-(OH)2D3 was detectable to form labelled 24,25(OH)2D3 preferentially. 4. No control subject produced significant amounts of both labelled 1,25-(OH)2D3 and labelled 24,25-(OH)2D3 simultaneously. 5. All subjects with primary hyperparathyroidism produced significant amounts of labelled 1,25-(OH)2D3 and labelled 24,25-(OH)2D3 simultaneously; the renal turnover of 25-(OH)D3 was apparently greater than in nutritionally matched controls. Serum labelled 1,25-(OH)2D3 in this disease was not correlated with serum 25-(OH)D3, immunoreactive parathyroid hormone, calcium or Pi. Production of labelled 24,25-(OH)2D3 was inappropriately high for the prevailing nutritional state. 6. The indirectly estimated their concentration of 1,25-(OH)2D3 showed only a fourfold variation in control subjects (45-180 pmol/l), compatible with its having a regulated hormonal function. 7. The data suggest that the production of 1,25-(OH)2D3 from a pulse dose of cholecalciferol is normally regulated, directly or indirectly, by the parathyroid hormone.


Persistent papillomatosis associated with immunodeficiency. A case report of a young bull with persistent papillomatosis associated with immunodeficiency is presented. Humoral immune responses were normal but cell mediated immunity was deficient. The possible significance of the findings to pathogenesis and therapy of the disease is discussed.


Transbronchial forceps lung biopsy through the fiberoptic bronchoscope. tdiagnosis of diffuse pulmonary disease. Transbronchial forceps biopsy through the fiberoptic bronchoscope was performed in 37 patients. Tissue was technically inadequate for examination in two cases. A pathologic diagnosis was obtained in 26 (72 percent) and in five additional critically ill patients, direct histologic examination of lung tissue allowed exclusion of certain entities, thus significantly influencing therapy. Overall, the procedure was of value in 31 of 35 patients (90 percent). There was no significant hemorrhage and there was one 15 percent pneumothorax. The safety and high diagnostic yield of this technique in the diagnosis of diffuse lung disease is proved.


Cyclophosphamide, vincristine and sequential split-course radiotherapy in the treatment of small cell lung cancer. Thirty-nine patients with small cell lung cancer were treated with large dose intravenous cyclophosphamide combined with vincristine. Sequential split-course radiotherapy was added when the gross disease was limited to one hemithorax and draining scalene nodes. Fifteen of 16 patients in the limited disease category showed objective response, eight of which were complete. Fourteen of 23 patients in the extensive disease category yielded an objective response, six of which were complete. The median survival for complete responders was 48 weeks, 38 weeks for partial responders and 14 weeks for non-responders. The difference between responders and non-responders was statistically significant. The major toxicity was myelosuppression with a median leukocyte nadir of 500/mm-3 noted on treatment day no. 15. Prompt recovery was the rule. Toxicity appeared to be cumulative for patients receiving radiotherapy. These results are superior to those evolving from treatment with cyclophosphamide as a solitary agent.


Colorectal cancer in children: epidemiologic aspects. Despite many similarities to colorectal cancer in adults, the rare childhood form has some peculiarities. Childhood mortality is greater among Negroes than Caucasians, particularly in boys, reflecting the rising incidence of this tumor in the young Negro population. In addition, the percentage of childhood cases with precancerous diseases (polyposis, colitis) appears greater than in adults. Most striking is the high percentage of mucin-producing tumors in young people with colorectal cancer. The mucoid tumors tend to occur after the age of 10 years, whereas younger children are more likely to develop non-mucoid carcinoma in an adenomatous polyp.


Adenocarcinoma arising in a diverticular abscess of the colon: report of a case. A case of a patient with adenocarcinoma in an abscess cavity that resulted from perforated diverticulitis of the descending colon is described. Serial barium-enema studies over a nine-year period show the diverticulosis, perforation, abscess cavity, and finally the adenocarcinoma in the abscess cavity. Although it is infrequent, the possibility of carcinoma must be considered in cases of patients who have unhealed pericolonic abscess cavities.


Metastasis from a pedunculated adenomatous colonic polyp with focally invasive carcinoma: report of a case. A patient who had metastasis from a focus of invasive carcinoma confined to the head of an otherwise benign pedunculated adenomatous polyp of the sigmoid colon is described. That only 20 such cases have been reported previously attests to the rarity of this phenomenon. Because the morbidity and mortality of radical surgery far outweigh the liklihood of metastasis from such foci of invasive carcinoma in pedunculated adenomatous colonic polyps, local removal is recommended.


[Paroxysmal ACTH secretion from an adrenal medullary tumour (author's transl)]. A secondary Cushing's syndrome developed in a 38-year-old woman from an ACTH secreting phaeochromocytoma. Large amounts of ACTH were demonstrated in the tumour by radioimmunological tests. A remarkable feature was that clinical and biochemical signs of increased catecholamine secretion completely regressed with time and the adrenals were stimulated at intervals by the ectopic ACTH secretion, steroid excretion falling to normal in between.


Investigations upon adenohypophysial cell proliferation in vitro: influence of oestradiol, thyrotropin releasing hormone, dibutyryl cyclic AMP and theophylline. Mitotic indices were counted in adenohypophysial fragments cultured in vitromthe cultures exposed to 17-beta oestradiol, synthetic thyrotropin releasing hormone (TRH) and dibutyryl cyclic AMP showed a higher number of mitoses as compared with respective controls. However, theophylline decreased the mitotic index in cultured adenohypophysis.


Lipolysis and cyclic-3', 5'-AMP content in rat adipose tissue at different age. The content of cAMP, the release of glycerol and nonesterified fatty acids (NEFA) of adipose tissue with and without the stimulation by ACTH and norepinephrine were determined in young and old rats. After the lipid mobilizing hormones lowered concentration of cAMP and impaired stimulation of lipolytic activity were found in old rats. Furthermore, less stimulation of glycerol and NEFA release by cAMP was observed in these animals. These results suggested that in old animals a lower lipolytic activity per tissue and lipid mass is available in intact and hormone stimulated fat tissue and therefore the accumulation of lipids in fat depots is facilitated.


Triiodothyronine binding in rat anterior pituitary, posterior pituitary, median eminence and brain. In vivo studies of the exchange of tracer [125-I]-L-triiodothyronine (T3) between plasma (P), and the anterior pituitary (AP), posterior pituitary (PP), median eminence (ME) and the frontal lobes of the brain (B), in the rat show that from 2.5 h onwards the concentration of new [125I]T3 in AP, PP and B were parellel to that of the plasma, with a t1/2  of 7.4 h; the t1/2 for ME was 10.3 h. The extrapolation of these curves to zero time was assumed to indicate the relative concentration of T3 per unit weight in terms of total body T3. T3 content of these tissues was determined by radioimmunoassay. The values obtained validated the steady state parameters derived from the radio-isotopic measurements. As an indicator of the concentration gradient between tissue and plasma the organ/plasma (O/P) ratio was calculated; these data indicate that under steady state conditions, the order of T3 concentration is AP greater than PP greater than ME greater than B. Binding studies have shown that AP and PP contain "specific," saturable binders while ME and B do not. Evaluation of the binding parameters of the high affinity binders in both AP and PP gave similar association constants. These associations constants, when corrected for the binding strength of T3 to plasma proteins, resulted in values similar to those of neuclear T3 binders.


Effect of insulin and lipolytic hormones on cyclic AMP phosphodieterase activity in normal and diabetic rat adipose tissue. Insulin has been shown to lower cyclic AMP (cAMP) levels in hormonally sensitive tissue. The mechanism by which this lowering occurs has not yet been fully defined. We studied the effects of insulin on rat adipose tissue cyclic nucleotide phosphodiestrase (PDE) in an incubation system. The adipose tissue used was from both normal animals and animals rendered diabetic by intravenous injections of streptozotocin. Rat epididymal fat pads were incubated in a Krebs-Ringer bicarbonate-4% albumin system with O, 100, 1,000 or 10,000 PU/ml insulin (INS); epinephrine (EPI) or glucagon (GLU) at several different concentrations. After 15 min of incubation, each tissue was homogenized, centrifugated, and the supernatant assayed for cAMP PDE activity using the breakdown of (3-H)cAMP. The data was used to characterize cAMP PDE into apparent high and low K-m PDE components. In the normal animals, INS increased Vmax of the low Km PDE components; 100 pU/ml INS, 30%, 1000 p1/ML INS, 40; and 10,000 pU/ml INS, 20%. In contrast, streptoxotocin diabetes lowered this Vmax by 30%. In the diabetic animals, INS also increased Vmax by 30%. In the diabetic animals, INS also increased Vmax of the low Km PDE component; 100 pU/ml INS, 30%; 1000 pU/ml INS, 50% and 10,000 pU/ml INS, 100%. Epinephrine at 1, 10, and 100 pg/ml stimulated low Km cAMP PDE activity by 67%, 73% and 44% respectively. The stimulatory effect of EPI on both the low and high Km cAMP PDE activity was neutralized by propranolol or adenosine. In comparison to EPI, GLU at very low concentrations, 10-9M, stimulated low Km cAMP PDE. These studies suggest that some of the biologic actions of insulin, an antilipolytic substance, are mediated through activation of low Km PDE. Furthermore, this enzymatic activity is lower in experimental diabetes. The stimulation of low Km PDE by lipolytic hormones may reflect a long-range protective action of these agents.


Regulation of thyroid ornithine ornithine decarboxylase (ODC) by thyrotropin. I. The rat. We studied the effects of TSH on rat thyroid ornithine decarboxylase (ODC) activity. After 1 day of goitrogen treatment, there was an abrupt fall in serum triiodothyronine (T3) a rise in circulating TSH, and a dramatic increase in thyroid ODC activity. Despite the continued rise in TSH and progressive increase in thyroid gland size with further treatment, thyroid ODC activity declined on the third day and remained at submaximal levels. Thyroid ODC activity was also stimulated in a dose-related manner by administration of exogenous TSH. Little TSH effect was noted before 3 h. Maximal ODC activity occurred between 4 and 5 h. The TSH stimulation of ODC could be inhibited by pretreatment with actinomycin D or cycloheximide, suggesting that the increase in ODC activity requires new RNA and protein synthesis. Although pretreatment with agents that alter microtubule structure (e.g., colchicine and vinblastine) prevent stimulation of ODC activity by TSH, additional data suggest, but do not confirm, that hrmone secretion and ODC activation may be dissociable. Further studies were undertaken to determine whether cyclic AMP (cAMP) or prostaglandins played any role in the regulation of thyroidal ODC activity. Dibutyryl cAMP, alone, or together with aminophylline, did not stimulate thyroidal ODC activity in dosages which concomitantly stimulated adrenal enzyme activity. Likewise, prostaglandin E2 (PGE2) did not stimulate thyroidal ODC activity, but did stimulate adrenal enzyme activity in a dose-related manner. However, pre-treatment of rats with inhibitors of prostaglandin synthesis prevented the activation of thyroidal ODC BY TSH. One inhibitor, indomethacin, attenuated the TSH stimulation of enzyme activity in a dose-related manner. Indomethacin pretreatment also resulted in approximately a 10-fold decrease in thyroidal prostaglandin levels. Exogenous PGE9, in dosages as high as 500 pg, did not overcome the inhibitory effect of indomethacin on ODC activation. Although the precise role for endogenous prostaglandins remains to be defined, it does appear that a reduction in thyroidal prostaglandins prevents activation of the enzyme by TSH.


Bioactive and immunoactive ACTH in the rat pituitary: influence of stress and adrenalectomy. Tissue levels of bioactive and immunoactive ACTH were measured in both the anterior and neuro-intermediate lobes of the rat pituitary. Similar concentrations of bioactive (65 ng/mg) and immunoactive (83 ng/mg) ACTH were found in the anterior lobes control rats. A 2-min ether stress had no effect on either bioactive or immunoactive ACTH levels in the anterior lobe. Twenty-four h after adrenalectomy the anterior lobe content of both bioactive and immunoactive ACTH decreased only to return to supranormal levels 21 days after the operation. A 30-min neurogenic stress had no effect on anterior lobe bioactive ACTH content but reduced the immunoactive ACTH level to 50 ng/mg. Synthetic alphah-17-39 ACTH was used in our radio-immunoassay in order to measure the C-terminal ACTH activity of the neuro-intermediate lobe. The concentration of such C-terminal activity in control rats (890 ng alphah-17-39 ACTH/mg) considerably exceeded the amount of bioactive ACTH (15 ng/mg). This is presumably due primarily to the presence of the so-called corticotropin-like intermediate lobe-peptide (CLIP). The amounts of bioactive or C-terminal immunoactive ACTH in the neuro-intermediate lobe were not affected by ether stress nor short term (24-h) or long term (21-day) adrenalectomy. Neuro-intermediate lobe bioactive ACTH decreased (to 8 ng/mg) only with the introduction of a 30-min neurogenic stress. Neurogenic stress had no effect on the concentration of CLIP, but when the stress was imposed 24 h after adrenalectomy, a significant reduction was observed. The data support the presence of bioactive ACTH in the intermediate lobe of the rat pituitary and suggest that  such ACTH is preferentially released by neurogenic stress and not appreciably regulated by circulating levels of glucocorticoids. Until the biological function and/or target organ of CLIP is identified, the significance of the changes in tissue levels of C-terminal immunoactive ACTH will remain unknown.


The effect of stress on the cytology and immunocytochemistry of pars intermedia cells in the rat pituitary. The purpose of this study was to determine if there were ultrastructural and immunocytochemical changes in intermediate lobe cells following stress. We found marked cytological changes in intermediate lobe cells after 30 min of stress with a buzzer and stroboscopic light (neurogenic). Following such treatment, the cytoplasm was filled with pale graules or empty vesicles. There was cytological evidence of increased secretory and synthetic activity. Innunocytochemical staining (for 17-39-ATCH) revelaed that there were fewer stained granules per cell in such  rats although the individual granules stained as strongly as those in control rats. No cytological changes were observed following ether stress. These results correlated well with our bioassay and radioimmunoassay data which showed that only neurogenic stress resulted in depletion of ACTH from neuro-intermediate lobes (1). In agreement with the work of others, we found no obvious cytological changes in intermediate lobes from adrenalectomized or cortisol treated rats wheras anterior lobe ACTH cells hypertrophied following adrenalectomy and involuted following cortisol treatment. Anterior lobe ACTH cells from intact rats showed no degranulation following 30 min of buzzer stress or after 2 min of ether stress.


Autoradiographic localization of radioactivity from [3-H]oxytocin in the rat mammary gland and oviduct. [3-H]Oxytocin was incubated in vitro with pieces of oviduct and mammary gland and the tissue were subjected to autoradiography. Radioactivity was localized only in smooth muscle cells of the oviduct and in regions of mammary tissue where myoepithelial cells are found. In contrast, radioactivity was not concentrated in any region of skeletal muscle, a nontarget tissue for oxytocin. The localization of radioactivity in oviduct and mammary cells was absent when the tissues were incubated with [3-H]oxytocin and an excess of unlabeled oxytocin. These findings provide evidence for the presence of specific and high affinity receptors for oxytocin in its target tissues.


Effects of age and obesity on insulin binding to isolated adipocytes. We have measured insulin binding to isolated adipocytes prepared from rats of varying ages and body weights. The ability of adipocytes to bind insulin progressively decreases as animals get older and fatter until about 70 days of age and 300 g body weight are reached. From this point on further decreases in insulin binding to adipocytes were not seen as rats got older and fatter. Analysis of the data indicated that this decrease in insulin binding could be accounted for by decreased numbers of insulin receptor sites per cell. Further studies were conducted in which animals were allowed to age, but obesity was prevented or reversed by hypocaloric diets. In these experiments decreased insulin binding was either prevented or restored to normal by the negative caloric state, indicating that age had no appreciable effect on the ability of adipocytes to bind insulin. The influence of the obesity associated variables-hyperinsulinemia and increased fat cell size-on insulin binding was also examined. These latter studies are consistent with the concept that elevated in vivo plasma insulin levels lead to decreased insulin receptors, and further suggest a role for additonal factors in regulating the adipocyte insulin receptor. 1. Decreased insulin binding to adipocytes is closely related to the obese state. 2. This decrease in insulin binding can be accounted for by decreased numbers of receptor sites per cell, and 3. The mecahnism(s) underlying this decreased insulin binding are complex and probably represents an effect of more than one variable.


Specific binding sites for natural glucocorticoids in plasma membranes of rat liver. The presence of sites specifically binding natural glucocortocoids in plasma membrane (PM) preparations (PM0, density=1.13-1.16; PM1, density=1.16-1.18) from rat liver was elucidated by equilibrium dialysis as well as by centrifugal methods. Equilibrium dialysis showed the presence of binding sites having a higher affinity for [3-H]cortisol (Kd=1.4 times 10- minus 9M at 4C) in PM0, and that of the binding sites having a lower affinity for [3H] cortisol (Kd=1.3 times 10- minus 8M at 4C) in PM1, while centrifugal analysis showed the presence of higher affinity binding sites (Kd=1.5-1.9 times 10- minus 97 at 0 C) in both PM0 and PM1, and also of intermediate affinity binding sites (Kd=4.1 times 10- minus 9M at 0 C) in PM1. The discrepancy in the cortisol binding parameters obtained by the two different methods seems to be due mainly to the lability of some binding sites, especially the PM1. The glucocorticoid-binding sites in the plasma membranes of rat liver appear to have the highest affinity of corticosterone, followed by cortisol and cortisone. A synthetic glucocorticoid [3H]-dexamethasone, did not show any specific binding to the liver plasma membranes. Neither dexamethasone nor nonglucocorticoids such as estradiol given simultaneously affected [3H] cortisol binding to the plasma membranes.


Studies on inhibition of TSH stimulation of adenyl cyclase activity in thyroid plasma membrane preparations by propranolol. Propranolol (1 mM) was found to inhibit TSH stimulation of adenyl cyclase activity in a subcellular fraction from bovine thyroid enriched in plasma membranes. However, stimulation due to PGE1 or NaF was not similarly inhibited. Since (i) and inhibition was observed at concentrations of propranolol between 10-minus 4 and 10- minus 3M, and appeared to be noncompetitive (ii) the optical isomers of propranolol were equipotent, (iii) inhibition was specific for propranolol since it was not observed with the closely related drug practolol (1 mM), and (iv) quinidine (1 mM) and the local anaesthetics lignocaine and aptocaine also proved inhibitory, we concluded that propranolol inhibition of TSH stimulation was due to its "quinidine-like" properties (i.e., relatively specific and characteristic membrane-active properties) and not to its action as a beta-adrenergic antagonist.


Hydrolysis of estrogen conjugates by the lungs of the dog in vivo. Although hydrolysis of certain estrogen conjugates released into the systemic circulation is now known to occur in the human and in the dog, the site or sites of such hydrolysis remained unidentified. In an attempt to identify such sites, radioactive estrone was infused intravenously into a series of male dogs and serial paired samples of blood were obtained from the right ventricle (RV) and a peripheral artery (A). The samples were analysed for total radioactivity, unconjugated radioactive estrogens, radioactive estrone, estradiol-17beta, estrone sulfate, estrone glucosiduronate and estradiol-17beta glucosiduronate(s). The mean percent extraction across the lungs (see article) was calculated for each radioactive moiety analyzed. There was no significant percent extration of total radioactivity or of estrone sulfate. The mean percent extractions for unconjugated estrogens and for estrone and estradiol-17beta were -24.7, -21 and -34.3 respectively (P less than 0.01), indicating net production by the lungs. The mean percent extractions for estrone glucosiduronate and estradiol-17beta glucosiduronate(s) were +19.8 and +10.0 respectively (P less then 0.01), indicating net uptake of these conjugates by the lungs. Statistical analysis showed that the two processes were related. Our findings support the hypothesis that the lungs are one site, possibly the main site, at which hydrolysis of estrogen glucosiduronates occurs in the dog.


Anterior pituitary hormone release in vitro inversely related to extracellular osmolarity. Release of hormones from bovine anterior pituitary tissue in vitro was inveresly related to the osmolarity of the incubation medium. Addition of each of several ionic or non-ionic solutes to Krebs-Ringer-bicarbonate (KRB) or to beef serum inhibited hormone release. If the value of 286 mOsm/liter of KRB is considered as 100%, the osmolarity of the medium was altered from -15% to +10% of control. Over this range, an increase in osmolarity of 10% resulted in the following percentage inhibition: ACTH, 47; PRL, 43; TSH, 36; LH, 23; GH, 18; maximal percentage inhibition over this range was as follows: ACTH, 80; PRL, 60; TSH, 60; LH, 45; GH, 40. The inverse relationship between extracellular oxmolarity and secretion of ACTH and PRL would appear to be appropriate in view of the salt and water retaining actions of these hormones. The sensitivity of the response to osmotic changes suggests a possible role of body osmolarity in the regulation of adenohypophysial secretion.


Effect of dexamethasone on the secretion of thyrotropin in the rat: dose and time relations. Serum TSH and corticosterone concentrations were measured in intact rats and in rats given TRH or exposed to short-term cold 3 h and 12 h after pretreatment with dexamethasone in various doses. Dexamethasone given 3 h before experiemtns significantly depressed both TRH- and cold-induced TSH responses at all dose levels. Dose of 25 pg/100 g body weight significantly depressed serum TSH concentration when given 3 h before the experiment. However, when given 12 h before the experiment the drug augmented TRH-induced TSH secretion, although the cold response was unaltered. In intact rats dexamethasone significantly depressed serum TSH concentration in doses of 250 and 500 mug/100 g body weight. In all experiments the steroid blocked ACTH secretion. These results support the view that the effect of dexamethasone on thyroid function is highly dependent on the time relations. A single large dose of dexamethasone has first an inhibitory effect at the pituitary level and then facilitates pituitary to TRH and at the same time inhibits secretion of TRH in response to cold.


Evidence for persistent binding of biologically active thyrotropin to thyroid in vitro. Differences exist in the rates at which hormones are inactivated by, or dissociate from, their target tissues. The present studies examined the binding of biologically active TSH to thyroid slices and compared its characteristics to those of PGE. Canine thyroid slices were initally incubated with 5 mU/ML OF BOVINE TSH (TSH-Inital) for 15 min, washed and incubated in media free of hormone for 3 hr. At the conclusion of this second incubation period all slices were again washed. Some were then transferred to media containing 10-2M theophylline for a final 10 min incubation and subsequent measurement of cAMP and protein kinase, while others were transferred to media containing (l-14C)glucose without theophylline for a final 45 min incubation to assess glucose oxidation. Identically treated slices never exposed to TSH served as controls, while others were exposed to TSH only during the final 10 or 45 min incubation periods (TSH-Final). cAMP content determined after significantly increased in TSH-Initial (mean 2.98 plus or minus 0.36 (se) pmol/mg wet wt) compared to control (0.35 plus or minus 0.04), but was less than that in TSH-Final (5.76 plus or minus 0.51). This phenomenon was not unique to canine thyroid, since comparable results were noted in studies of human, bovine or porcine thyroid slices. The protein kinase activity ratio (-cAMP/+cAMP) and glucose oxidation of TSH-Initial were also significantly increased above control following the final 10 min or 45 min incubations respectively. Addition of trypsin to the 3 h incubation abolished the subsequent increase in cAMP in TSH-Initial, while addition of TSH antiserum appreciably reduced this increase. These results are consistent with the persistent binding of biologically active TSH to thyroid. By contrast, evidence of similar persistent binding of PGE1 to thyroid, glucagon to liver, or parathyroid hormone to renal cortex was lacking when assessed by an identical experimental procedure. Differences between the duration of interaction of TSH and PGE1 with thyroid may be dependent or a more gradual dissociation to tissue bound TSH, a more rapid inactivation of bound-PGE1, or both.


Effect of temperature during preparation of rat adrenal quarters and isolated cell suspension of their ACTH responsiveness. The effect of temperature during preparation of rat adrenal quarters or isolated adrenal cell suspension on their response to ACTH was examined through a comparison of amounts of corticosterone produced after their incubation. The response to ACTH added in vitro was considerably higher when adrenal quarters and isolated adrenal cell suspension were prepared at room temperature (25 degree C) than when prepared at ice-cold. Endogenous steroidogenesis was not affected by the temperature. It seemed unlikely that this higher response to ACTH of adrenal quarters or isolated adrenal cell suspension prepared at room temperature was due to an activation of the cells. A possibility was discussed that cooling adrenal quarters or isolated adrenal cell suspension during the preparation may create an unphysiological state in some place to related the cell membrane.


Inhibitory effect of ouabain on epinephrine-induced stimulation of adrenal corticosterone secretion in rats. Chronic administration of ouabain (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) definitely inhibited epinephrine-induced increase of adrenal corticosterone secretion. The inhibition rate increased along with frequency of ouabain administration. Increase in adrenal corticosterone synthesis and secretion by ACTH (20-50 mU/rat) administration was partially suppressed by pretreatment with chronic ouabain administration. A slight but significant increase of adrenal corticosterone secretion caused by epinephrine administration in hypophysectomized rats was also inhibited by pretreatment with ouabain administration. Chronic administration of neither phentolamine (1 mg/rat, intraperitoneally, once daily for consecutive 15 days) nor propranolol (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) caused significant changes in adrenal corticosterone secretion in response to ACTH as well as to epinephrine. Chronic administration of ouabain in rats causes not only elevated secretion of ACTH from anterior pituitary but also functional change in adrenals leading to suppression of corticosterone secretion in response to ACTH or epinephrine administration.


Effect of triiodothyronine or iodide on the thyroidal secretion in vitro: Inhibition of TSH- and dibutyryl-cyclic-AMP induced endocytosis. T3 or iodide, at the concentration of 10- minus 3M, inhibits TSH- or dibutyryl-cyclic AMP-induced thyroidal intracellular colloid droplet formation, while leaving the TSH-induced increase in cyclic AMP levels intact. The effect of T3 appeared not to be mediated through the iodide derived from deiodination of T3. Those results clearly demonstrate that T3 or iodide acts at the point beyond cyclic AMP generation and prior tothe formation of intracellular colloid droplets. This is also the site at which microtubule active reagents exert their influence. Whether T3 or iodide fits this category or acts in as yet undetermined manner remains to be established.


Changes in enzyme activity in human trichinosis. Changes in enzyme activity in human trichinosis have been studied. The activities of 19 enzymes and some isoenzymes were investigated in 12 patients from the 3rd to the 20th week. The activities of muscle-specific enzymes (CK, ALD and MK) were greatly increased with peaks during the 4th week; activities returned to normal ranges during the 7th and 8th weeks. Liver-specific enzymes (GTP, OCT, guanase, GLDH, SDH and MDH) and AP, ChE, cerulosplasmin remained within normal limits in all patients with the exception of a child who died. An explanation of the origin of hyperenzymemia is proposed on the basis of these investigations. The data obtained support the proposition that hyperenzymemia is of muscular origin and that the liver is seldom affected in case of trichinosis.


Properties of enzymes. V. Catalase inhibition of glucose oxidase reaction. The radical produced aerobically in the glucose oxidase reaction reduces the oxidated cytochrome c. The extent of reduction depends on the concentrations of substrate (glucose) and enzyme. Superoxide dismutase purified from various sources does not inhibit the cytochrome c reduction, but catalase does, in proportion to its concentration. This inhibition, which might be utilized for quantitative catalase determinations, provides further evidence for the formation of H2O2 in the glucose oxidase reaction.


Cyclic AMP phosphodiesterase activity in three Morris hepatomas. Rat liver cAMP phosphodiesterase has been fractionated into four peaks of activity with isoelectrofocusing column chromatography. The major two liver peaks (high Km enzymes) decreased with increasing growth rate while the minor two liver peaks (low Km enzymes) increased in one fast growing Morris hepatoma. There was also less total phosphodiesterase activity in the fast growing hepatoma.


Differential effects of metal-binding agents on the D-glucose-6-phosphate phosphohydrolase activity of the rat liver. The metal-binding agents (citrate, oxalate, bicarbonate, EDTA) exert dual effects on D-glucose-6-phosphate phosphohydrolase activity in the homogenate as well as in the subcellular fractions. The important differences of the effects are associated with the concentration of the chelator and with time of its addition. The small (appropriate) concentrations of the metal-binding agents stimulate and stabilize the enzyme activity. However, chelators used in higher concentrations exert the inhibitory influence on the activity of glucose-6-phosphatase. Stimulation of the reaction was observed only if the chelator was added before the enzyme-substrate complex formation has been started. The formation of the ternary complex: the enzyme(metal)-chelator substrate exerting a protective influence on the active centre has been suggested. The hypothesis of a similar action of the metal-binding agents and Pi on the glucose-6-phosphatase as a metaloproteid has been proposed.


Carbohydrate content and regulation following injection of different glycogenolytic enzymes. The effect of injection of glycogenolytic enzymes on tissue glycogen, blood glucose and plasma insulin was studied in mice. No effects were observed following phosphorylase, whereas the hydrolytic enzymes, alpha-amylase and acid amyloglucosidase depressed liver glycogen. In addition acid amyloglucosidase induced a decrease in blood glucose, a slight elevation of plasma insulin and a marked increase in tolbutamide-stimulated insulin release. At the doses given none of the enzymes affected muscle glycogen. Amyloglucosidase pretreatment markedly enhanced insulin release induced by glibenclamide, leucine, isoleucine, lysine and glucose whereas insulin release stimulated by IPNA, ACTH, glucagon and "CCK-PZ" was unaffected. Injection of acid amyloglucosidase has a profound influence on carbohydrate content and regulation in mice. It is suggested that the dependence or independence of amyloglucosidase activity among the insulin secretagogues tested might reflect different or partially different mechanisms in the process of insulin secretion.


Conformational studies of equilibrium structures in fragments of horse heart cytochrome c. Ultraviolet absorption and circular dichroism studies have been carried out on horse heart apo-cytochrome c and heme-free peptide fragments obtained by cyanogen bromide cleavage of the native protein. It was noted that the various peptides assume predominantly an unordered conformation in water solution. Increasing ionic strength and addition of 2-chloroethanol increase the right-handed helical content. Guanidinium hydrochloride favors the coil state. It was also demonstrated that two non-interacting helical regions of different stability are present in the apo-protein in 2-chloroethanol.


Action of rheumatoid synovial collagenase on cartilage collagen. Different susceptibilities of cartilage and tendon collagen to collagenase attack. The action of purified rheumatoid synovial collagenase on purified cartilage collagen, alpha-1(II)-3, in solution at 25 degrees C has been characterised. The enzyme attacked cartilage collagen in solution producing a 58% reduction in specific viscosity and resulting in the appearance of two reaction products which represented approximately three-quarter and one-quarter fragments of the intact molecule as shown by disc electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The alpha-chain fragments which comprised each of these components corresponded to molecular weights of approximately 74000 and 21000. Electron microscopy of segment-long-spacing crystallites of the reaction products revealed three-quarter (TC-a) and one-quarter (TC-b) length fragments, and permitted accurate localization of the cleavage locus between bands 41 and 42 (I-41). This cleavage site and the formation of TC-a and TC-b reaction products are very similar to those found for type-I collagen substrates. Cartilage collagen in solution was found to be more resistant to collagenase attack than tendon collagen, the rate of cartilage collagen degradation being six times slower than that for tendon collagen, as judged by viscometry. The mid-point melting temperatures (T-m) for lathyritic cartilage and tendon collagen were 40.5 and 41.5 degrees C, and for the collagenase-produced reaction products 38.5 and 37.5 degrees C, respectively. The significance of these findings is discussed in relation to the structure of type I and II collagens.


Drainage of thoracic duct lymph in twelve patients with myasthenia gravis. The effect of thoracic duct lymph drainage (5-34 days) in 12 patients with myasthenia gravis on muscular function has been followed for 5-43 months. Among the results obtained were: (1) During the drainage the myasthenic symptoms decreased markedly after 1-4 days and remained so during the drainage. (2) The doses of cholinesterase inhibitors had to be markedly reduced during the lymph drainage in eight patients. (3) Discontinuation of the lymph drainage increased the myasthenic symptoms within a few days. However, after a median observation time of 14 months with conventional treatment all but one of the patients had improved. (4) Retransfusion of the patients own cell-free lymph caused a worsening of the myasthenic symptoms. This effect could also be obtained following infusion of IgG preparations from the patients lymph. Three retransfusions of cell suspensions obtained from the thoracic duct lymph from two patients had no effects on their myasthenic symptoms. (5) It is suggested that thoracic duct lymph drainage can be combined with other forms of treatment in severe cases of myasthenia gravis.


The absence of stimulation of lipolysis by papaverine, a strong inhibitor of phosphodiesterase. Quantitative studies of the action of theophylline and papaverine were performed in rat epididymal fat pads, both on the lipolytic effect and on the activity of phosphodiesterase, adenylate cyclase and protein kinase. Papaverine, a stronger inhibitor of phosphodiesterase than theophylline, did not produce lipolysis. The maximum lipolytic effect (glycerol release) of theophylline was much higher than that of epinephrine and nearly approached the effect exerted by dibutyryl cyclic AMP. While theophylline potentiated or was without any effect on lipolysis produced by epinephrine and dibutyryl cyclic AMP, papaverine at concentration 10- minus 3 M reduced the effect of both drugs as well as of theophylline by 90 per cent. These concentrations of papaverine also strongly inhibited the activity of adenylate cyclase. Neither papaverine nor theophylline prevented the activation of protein kinase by cyclic AMP. The data suggest that the lack of a lipolytic effect of papaverine migth be caused by a combination of its inhibitory effect on adenylate cyclase and direct inhibition of activation of triglyceride lipase.


Restoration of potassium-stimulated respiration of glycerol-treated muscle. Agent that produced contracture in skeletal muscle, such as caffeine or K-depolarization, also caused an increased rate of oxygen consumption. Both of these functions are calcium dependent. In this study the respiratory response to K-depolarization and to caffeine was monitored in glycerol-treated and normal frog sartorius muscles. Although glycerol-treated muscle does not contract in response to K-depolarization, it does develop normal caffeine contractures. The respiratory response to both potassium and caffeine is greatly inhibited in glycerol-treated muscles. Pretreatment with dibutyryl cyclic AMP restored the respiratory response to normal levels in glycerol-treated muscle. Pretreatment with low levels of caffeine that had no effect on oxygen uptake markedly enhanced oxygen uptake with higher concentrations of caffeine and resulted in a normal respiratory response to K-depolarization even though there was no tension development. Caffeine had no effect on adenyl cyclase activity even at concentrations that markedly stimulated oxygen uptake. The data suggest that potassium stimulation of oxygen uptake in glycerol treated muscle is uncoupled by a defect in the formation of a cyclic nucleotide cofactor, rather than a defect in calcium influx.


Fluorocarbon emulsions and cerebral microcirculation. Mice were exchange transfused with a fluorocarbon emulsion (FC-80). Their hemotocrits were reduced to levels of 10-15. They were examined within 1 hour of completion of normal or slightly accelerated in the majority of the mice. Acceleration may have been due to reduced "blood" viscosity. All mice with normal or reduced transit times displayed reversible increases in brain NADH levels when they were exposed to room air or 100% N-2 for brief periods, rather than to the atmosphere of 100%O-2 used to support life in these mice. A small group of mice with prolonged fluorescein transit tended to display NADH levels that failed to rise when O-2 was reduced in the inspired air. In this small subgroup, microcirculatory failure had apparently produced anoxia and maximal NADH levels even before the supply of inhaled O-2 was reduced. Many FC-80 mice displayed reversible decreases in brain pO-2 levels when inspired O-2 was reduced. Pentylenetetrazol activated the EEG of FC-80 mice. The EEG activity was reversibly reduced when the animals were briefly exposed to room air or N-2. These data suggest that fluorocarbon emulsions can support brain microcirculation, oxygenation, and electrical activity in vivo, a result which is consonant with the observations of others who have performed in vitro experiments, or observed long-term survival.


[Evoked activity of cat association cortex neurons in different sleep stages]. Evoked activity of neurons in the parietal associative cortex in response to adequate and electric stimulations of the thalamic radiation fibers was studied during different phases of sleep. The number of responding neurons decreased during slow--wave and, particularly, during paradoxical sleep. With transition from phase to phase, responsiveness changed mainly in polysensory neurons which gave variable responses with long latencies. Mechanisms of change of responsiveness during the two phases appear to be different. Reliable increase of the inhibitory pause in responses during slow-wave sleep and its decrease during paradoxical sleep may indicate a strengthening of mechanisms of reciprocal inhibition during the first phase, and their weakening during the second phase.


Phosphorylation of purified thyroid plasma membranes incubated with [32-P]ATP. Plasma membranes have been prepared from porcine thyroid glands using sucrose gradients. The fractions having a density in sucrose of 1.18 g/ml mainly contained plasma membranes and were moderately contaminated with other subcellular components as shown by marker enzyme data. Purified plasma membranes incubated in the presence of [32-P]gamma ATP incorporated 32-P. Kinetics of incorporation of 32-P into endogenous substrates studied in various buffers and with increasing ATP concentration suggest a phosphodephosphorylating system related to cAMP-dependent protein kinase and phosphoprotein phosphatase activities. The two enzymatic activities associated with plasma membranes have been demonstrated using exogenous substrates. cAMP increases and fluoride ions decrease the extent of membrane phosphorylation. The specific activity of protein kinase was 10-12 times higher than in the initial homogenate and was only slightly enhanced in the presence of 0.5% Nonidet as compared to microsomal fraction. cAMP binding to membrane proteins was 3 times higher than to the other particulate fractions. TSH present in the incubating medium or added after 5 min of 32-P labelling induced a rapid stimulation of endogenous phosphorylation followed by a rapid decrease. Phosphorylated membrane substrates were analyzed: high voltage paper electrophoresis after partial hydrolysis indicated that [32-P]phosphate is incorporated into serine and threonine residues as o-phosphate derivatives. SDS-polyacrylamide gel electrophoresis showed several 32--labelled fractions. When enhanced by cAMP, no specific phosphorylation of protein components was observed.


Circulating antibodies and respiratory virus infections. Most respiratory viruses infect only the respiratory mucosa and mainly the surface epithelium, and it is therefore plausible that antibody within the circulation is unable to influence them, either by preventing infection or by terminating it once it has started. However there is experimental and observational evidence that some infections are prevented by circulating antibody. This may be because circulating immunoglobulins are released onto the surface possibly because of virus cytopathic effects or hypersensitivity to a viral antigen.


Meaning of circulating antibody titers in the infections of parainfluenza 3 virus and RS virus. It is generally accepted that secretory antibodies play a very important role in the prevention of respiratory infections, but it is also considered that the circulating antibody level is a very significant indication of the establishment of infections. In parainfluenza 3 and RS virus infections, reinfections are very common but seem to depend more or less upon circulating antibody levels. There is a correlation between circulating antibody level and infection rate in parainfluenza 3 infection in some groups of school children in our observations of about one year. In the case of RS virus infection, observations were made in some nursery institutions and an experience in school children, in which also a circulating antibody level of some degree presents indications of infection rates.


Contribution to the study of the parainfluenza antigens. From epidemiological data it appeared that an epidemic of parainfluenza type 1 occurred in Lyons in October-November 1967. Thereafter a few sporadic cases were detected. Then in 1970 strains were isolated which showed a change in the hemagglutinin antigen. The persistence of antibodies in the population is shorter than that of the other parainfluenza viruses (i. e. parainfluenza 3); it is not known if reinfections with parainfluenza 1 occur since there are cross-reactions by CF test between parainfluenza 1 and parainfluenza 3 which are known to cause reinfections. The study of antigens of parainfluenza strains was made by HI, Nt, CF and immunodiffusion tests with various antisera prepared in various animal species. This study showed antigenic differences and similarities between parainfluenza 1 and parainfluenza 3.


The anamnestic response of the secretory immune system: A review. Following topical immunization of the respiratory mucosa in man, persistence of antibody in the respiratory secretions can be demonstrated in some individuals for more than one year. The secretory response to replicating agents is generally more prolonged than the response to less persistent antigens. Repeat topical antigenic stimulation provokes a response which differs very little from the primary response in magnitude, duration or latency. Nevertheless, if more than 1 year following adequate primary intranasal immunization, at a time when secretory antibodies are no longer detectable, men are again vaccinated intranasally with a dose of formalin-inactivated rhinovirus vaccine which will not provoke a primary secretory immune response, some vaccinees will produce nasal antibody. Similarly in mice, a second topical immunization of the respiratory tract with a dose of tetanus toxoid which does not produce a detectable primary secretory immune response will cause 11S IgA antibodies to appear in bronchial and nasal washings. Thus, it is possible that the secretory immune system may possess certain attributes of immunologic memory as classifically defined by the responses of the internal immune system to repeated antigenic stimulation.