Steroid-receptor quantitation and characterization by electrophoresis in highly cross-linked polyacrylamide gels. Conditions for discontinuous polyacrylamide gel electrophoresis have been defined in which progesterone receptors of chick oviduct cytosol and a variety of steroid-binding proteins from other sources are stable and amenable to quantitative analysis. The essential modifications from standard procedures include the use of (1) separation gels in which the cross-linking agent/acrylamide monomer = 15:85, (2) glycerol (10% v/v) in all phases of the Trisglycine-HCl buffer system (pH 10.2 in the separation phase during electrophoresis at 0 degrees), and (3) a layer of a charged reducing agent, thioglycolate, beneath the sample layer. Electrophoresis of untreated oviduct cytosol labeled with [3H]progesterone +/- competing steroids revealed a heterodisperse slow peak and a sharp fast peak. Both peaks displayed the steroid-binding specificity and saturability that are characteristic of intracellular receptors. Recovery of steroid from both the slow and fast components increased linearly with sample load up to 60 mul of cytosol (1.2 mg of protein)/gel (6 mm diameter). The specific progesterone binding detected by this technique was comparable to that detected by charcoal-dextran treatment or ion exchange filtration. Relative electrophoretic mobilities (Rf) of globular protein standards and steroid-protein complexes in cytosol and chick serum were measured in separation gels with total gel concentrations (T) systematically varied from 5 to 15% (w/v). Data were processed by computer programs to obtain weighted linear regressions of log Rf on T (Ferguson plots) and the joint 95% confidence limits of the slopes (-KR) and intercepts of these plots. Molecular radii (R) of the binding components and apparent molecular weights (M) were calculated from the linear correlation of R with KR 1/2 for the standards. The value of M is approximately 158,000 obtained for the cytosol fast component was independent of the length of the separation gel, the presence of a stacking gel or prior exposure of the cytosol to KCl. It was higher than expected from the sedimentation coefficient of 4.2 S in the same pH 10.2 buffer. Electrophoresis in 170-mm separation gels without stacking gels revealed that KCl extracts of protamine-precipitated cytosol contain a different receptor form, of lower net negative charge than the cytosol fast form. The results demonstrate the utility of electrophoresis in highly cross-linked gels of several concentrations to discriminate between various receptor forms and steroid-binding components of serum. This method may lead to overestimates of M for highly asymmetric receptor forms.


Quantitative demonstration of the reciprocity of ligand effects in the ternary complex of chicken heart lactate dehydrogenase with nicotinamide adenine dinucleotide oxalate. The reciprocity of effects of two ligands simultaneously bound to a protein as a ternary complex may be proven by measurements of four standard free energies of binding. Two of these are for the binding of each ligand in the absence of the other, and the other two for the binding of each ligand in the presence of saturating amounts of the other (conditional free energies). These four quantities have been measured for the complexes of oxalate and nicotinamide adenine dinucleotide with chick heart lactate dehydrogenase. The differences between conditional and unconditional free energies are: oxalate, -1.1 +/- 0.3 kcal; NADH,-1.3 +/- 0.2 kcal, thus proving the reciprocity within experimental error.


Carbon-13 nuclear magnetic resonance spectroscopy of [2-13C]carboxymethylcytochrome c. Horse heart cytochrome c has been carboxymethylated under various reaction conditions using [2-13C]bromoacetate. Direct analysis of reaction products using 13C nuclear magnetic resonance spectroscopy shows that the protein can be much more extensively modified than has previously been assumed. The proximity of one carboxymethylmethionine residue to the paramagnetic center of the ferric protein allows it to be distinguished from a more constant carboxymethylmethionine residue on the basis of the chemical shift of its labeled methylene group. Refolding of cytochrome c after alkylation at low pH apparently gives a different configuration of modified methionine residues within the protein compared to that produced by alkylation at neutral pH in the presence of cyanide.


A protective function of superoxide dismutase during respiratory chain activity. (1) Aerobic incubation of heart muscle submitochondrial particles in phosphate buffer after treatment with NADH causes a progressive and substantial inhibition of the NADH oxidation system. Succinate oxidation remains almost unaffected by NADH treatment. (2) The loss of NADH oxidase activity is due to an inhibition of the respiratory chain-linked NADH dehydrogenase. This inhibition of the enzyme is very similar to that caused by combination of the organic mercurial mersalyl with NADH dehydrogenase. (3) The inhibition of NADH oxidation is largely prevented by compounds that are known to react with superoxide ions (02-.), including superoxide dismutase, cytochrome c, tiron and Mn2+. EDTA also has a protective effect, but a number of other metal chelating agents, and several proteins, including catalase, are without effect. (4) It is concluded that the inhibition of NADH oxidation of NADH oxidation by superoxide ions or by mersalyl is reversible and is therefore not due to the loss of oxidoreduction components from the respiratory chain or to an irreversible change in protein conformation. (6) The function of mitochondrial superxide dismutase is discussed in relation to the key role of NADH dehydrogenase in energy-conserving reactions and the formation of hydrogen peroxide during mitochondrial oxidations.


Chlorophyll radical cation in photosystem II of chloroplasts. Millisecond decay at low temperature. We compare the absorption changes, in the near infrared and in the green part of the spectrum, induced in spinach chloroplasts suspensions, at -- 170 degrees C, by continuous light and by flashes. (1) Following flash excitation, an absorption increase peaking at 825 nm which reverses rapidly (t 1/2 = 3.0 ms) is not affected by ferricyanide; it is suppressed when chloroplasts are preilluminated in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) and hydroxylamine. The reversion of that signal is simultaneous with a partial back reoxidation of C-550 (fully reduced by the flash) and with partial (about 25%) oxidation of cytochrome b559. The magnitude of the signal peaking at 825 nm (that we attribute to the radical cation of the trap chlorophyll of Photosystem II, acting as a primary electron donor) decreases progressively within a series of successive flashes. (2) An absorption increase (40% of which is slowly reversible) with a broad peak around 810 nm is induced by continuous light or by a flash. It is suppressed by pretreatment with ferricyanide, but it is little affected by the treatment with 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea and hydroxylamine. We attribute it to oxidized P700. (3) With chloroplasts pretreated with 10 mM ferricyanide, an absorption increase, whose magnitude is nearly independent of wavelength between 790 and 870 nm, can be induced by continuous light. One saturating flash produces only 20% of the signal. This absorption change (20% of which is reversible in 30 s) might be due to a secondary donor of Photosystem II.


Surface binding and interiorization of homologous and heterologous serum lipoproteins by rat aortic smooth muscle cells in culture. Rat aortic smooth muscle cells in culture were incubated with rat or human iodinated low and high density lipoprotein at 5-50 mug/ml for 3 h. With the homologous lipoproteins, 25-49% of total cellular protein radioactivity was trypsin releasable and was considered as surface-bound radioactivity, while the balance represented cellular uptake. The ratio of surface-bound to cellular label was higher when the cells were incubated with human lipoproteins and was about 9 : 1 with human high density lipoprotein. Cellular uptake of rat low density lipoprotein was about twice that of rat high density lipoprotein, while degradation of labeled protein, which had presumably followed protein uptake, was similar and ranged from 20 to 25% of protein uptake in 3 h. Experiments designed to test the effect of cell density on lipoprotein uptake have shown that the uptake was related inversely to cell density. Thus, the lower lipoprotein uptake encountered in the rat smooth muscle cells, compared to that described for human fibroblasts (Goldstein, J.L. and Brown, M.S. (1974) J. Biol. Chem. 249, 5153-5162), could be due in part to the much lower cell density used in the latter studies, as well as to cell type and species difference.


Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats. Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.


Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat. The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.


Effects of cholera toxin on cyclic adenosine 3',5'-monophosphate concentration and secretory processes in the exocrine pancreas. 1. The effect of purified cholera toxin on secretory processes of exocrine pancreas has been studied in the isolated, saline-perfused cat pancreas and in incubated pieces of rat pancreas. 2. The toxin evoked a biphasic secretory response from the perfused cat pancreas. An initial small phase, which began within minutes of toxin application, was an artefact due to the presence of NaN3 in the cholera toxin preparation as supplied; it could be entirely reproduced by NaN3 at the concentration expected during toxin stimulation. A second, sustained phase of secretion, due to the action of the toxin proper, began within 30-60 min, increasing in magnitude for many hours and persisting in the absence of toxin. It was accompanied by a parellel rise in tissue cyclic AMP concentration, and could be potentiated by theophylline. 3. The composition of the secretion stimulated by cholera toxin resembled that evoked by secretin; e.g. it contained a high concentration of bicarbonate and only basal amounts of digestive enzymes. 4. Similarly, cholera toxin did not stimulate enzyme secretion by incubated rat pancreas, despite large rises in tissue cyclic AMP concentration. 5. Because cholera toxin has thus far been shown to have no other effect than that of stimulating adenylate cyclase, these observations support the conclusion that cyclic AMP does mediate the electrolyte secretory response of the pancreas to secretin, but offers no evidence that cyclic AMP plays a similar role in the regulation of pancreatic enzyme secretion stimulated by cholecystokinin-pancreozymin or acetylcholine.


The effect of antiestrogens on egg yolk protein synthesis and estrogen-binding to chromatin in the rooster liver. Nafoxidine and CI-628, two well known antiestrogenic compounds, reduce the stimulating effect of estradiol on the estrogen-binding capacity of the liver chromatin from roosters. In vitro both antiestrogens compete with [3H]estradiol for the binding sites on the liver chromatin. They inhibit the estrogen-induced synthesis of egg yolk proteins (vitellogenin) and fail to induce this estrogen-specific protein synthesis by themselves. They show the ability, however, to increase the estrogen-binding sites on the liver chromatin to some extent.


An analysis of the binding of the chick oviduct progesterone-receptor to chromatin. The binding of progesterone-receptor complexes to chromatin from target and nontarget tissues was studied in vitro. Chromatin from both target and nontarget tissues responds in a similar manner to saly and cofactors and has the same K(D) (approx. 3.10(-9) M) for the progesterone-receptor complex. The only observed difference in the binding of the progesterone-receptor complex to target and nontarget chromatins is the difference in total number of acceptor sites. oviduct chromatin has approx. 1300 sites/pg DNA, spleen chromatin has approx. 840 sites/pg DNA, and erythrocyte chromatin has about 330 sites/pg DNA. The K(D) and number of acceptor sites for progesterone-receptor complex binding to oviduct chromatin remains the same even after extensive purification of the progesterone-receptor complex. Activation of cytosol labeled with [3H]progesterone by preincubation at 25 degrees C, analogous to that required for maximal nuclear binding, occurs if the binding studies to chromatin are performed in 0.025 M salt. The absence of an observable temperature effect when the studies are performed at 0.15 M salt is due to the activation of the receptor by salt. The dissociation of the progesterone-receptor complex from chromatin exhibits a single dissociation rate and the initial event is the appearance of free progesterone rather than a progesterone-receptor complex. Lastly, the treatment of chromatin with an antibody prepared against either single-stranded DNA or double-stranded DNA does not alter the extent of binding of the progesterone-receptor complex. Similarly, pretreatment of chromatin with a single-stranded nuclease does not inhibit the capacity of chromatin to bind the hormone-receptor complex.


Stimulation of sugar transport in rat diaphragm by 8-bromoguanosine 3', 5'-monophosphate. The cyclic GMP derivative, 8-bromo cyclic GMP, increases the uptake of D-xylose and of 2-deoxy D-glucose into intact rat diaphragm incubated in vitro. 8-Bromo cyclic GMP does not stimulate the incorporation of [14C]glucose into glycogen in the diaphragm, or the uptake of alphalpha-amino isobutyric acid into this tissue. The effect of 8-bromo cyclic GMP on the diaphragm is consistent with the hypothesis that cyclic GMP plays a role in the regulation of sugar transport in muscle.


Measurement of particle sizes by elastic and quasi-elastic light scattering. A method of measuring an average particle radius in a highly polydisperse dispersion using the wavelength dependence of turbidity is described. For particles which are no larger than 0.3 of the wavelength of light used, a polynomial representation of the scattering cross-section can be used. For larger particles, more extensive numerical calculations are required. The use of the method is illustrated by determining the average particle radius of casein micelles by both elastic and quasi-elastic light scattering techniques. A polydisperse homogeneous sphere model is found to be a reasonably accurate representation of casein micelles. Several modifications of the model which would improve the agreement between the two techniques are mentioned briefly.


Tritiation of endotoxin.

Tritiation of endotoxin. Tritiated endotoxin was synthesized by three different methods: (1) sodium boro[3H]hydride reduction of native endotoxin; (2) sodium boro[3H]hydride reduction of endotoxin that had been oxidized previously with sodium metaperiodate; and (3) exposure of dry endotoxin to 3H2 gas. Sodium borohydride reduces aldehyde groups and sodium metaperiodate oxidizes vicinal glycol groups to aldehydes. Chromatographic analysis of the three tritiated endotoxins, using agarose, revealed that the biological activity associated with each labeled product appeared at the void volume, and in each case the biological activity coincided with a peak in radioactivity. The labeled product of the first method had a specific radioactivity of 0.18 mCi/g and a biological activity equal to that of native endotoxin. The labeled products of the second and third methods had specific activities of 2.1 mCi/g and 60.0 mCi/g, respectively, while their biological activities were one hundred-fold less than native endotoxin, as determined by the Limulus amebocyte lysate assay. These three labeled endotoxins are potentially ueled endotoxin.


Studies on the reconstitution of bovine erythrocyte superoxide dismutase. V. Preparation and properties of derivatives in which both zinc and copper sites contain copper. 1. We have developed a procedure for preparing derivatives of bovine superoxide dismutase in which primarily the Cu binding sites are occupied by Cu2+ (2 Cu2+-) and in which both the Zn and Cu binding sites are occupied by Cu2+ (4 Cu2+-). 2. The 2 Cu2+ protein shows approximately one-half the superoxide dismutase activity of an equivalent amount of native protein. A two-fold enhancement of the activity of 2 Cu2+-dismutase was observed upon occupation of the Zn sites either with Zn2+ or Cu2+. 3. The electron paramagnetic resonance spectrum of 4 Cu2+ protein was recorded over the temperature range 5-100 degrees K and the results suggest an antiferro-magnetic interaction between Cu2+ in the Zn site and Cu2+ in the Cu site having a coupling constant of approx. 52 cm-1. 4. The binuclear Cu2+ complex was found to accept only one electron from ferrocyanide. 5. One-half the total Cu+ of dithionite reduced 4 Cu+ protein was found to react rapidly with bathocupreine sulfonate whereas the other half reacted slowly. Reduced native protein did not react with bathocupreine sulfonate below 70 degrees C.


Regulation of GMP reductase in Salmonella typhimurium. The levels of guanosine 5'-phosphate reductase (EC 1.6.6.8) in Salmonella typhimurium appear to be modulated by changes in the ratio of the adenine and guanine nucleotide pools. Alterations of this ratio may be induced by high levels of guanosine in the culture medium or by genetic lesions in one of several purine interconversion enzymes, such as pur A or pur B mutants. The induction of the reductase requires transcription and translation processes and, in contrast to earlier observation with Escherichia coli, is not dependent on cyclic adenosine 3',5'-phosphate or the cyclic adenosine 3',5'-phosphate receptor protein.


Studies of a halophilic NADH dehydrogenase. II. Kinetic properties of the enzyme in relation to salt activation. 1. An NADH dehydrogenase, obtained from an extremely halophilic bacterium, was activated by various salts when enzyme activity was measured as the observed velocity, whereas the maximum velocity was unaffected by either the salt concentration or the nature of the salt. 2. Two ion effects were observed; a quantitative cation effect, reflected in changes in the apparent Michaelis constant for 2,6-dichlorophenolindophenol, and a qualitative anion effect, reflected in the apparent Michaelis and dissociation constants for NADH. 3. The data suggest that cations act by neutralizing electrostatic charges surrounding the 2,6-dichlorophenolindophenol-binding site, whereas the anions affect the conformation of the enzyme by altering the accessibility of the NADH-binding site to the bulk solvent. 4. Thus, the apparent activation of this enzyme, obtained from an extremely halophilic bacterium, is a reflection of measuring enzyme activity at non-saturating substrate concentrations.


Thyroidal autoregulation. Iodide-induced suppression of thyrotropin-stimulated cyclic AMP production and iodinating activity in thyroid cells. In continuing our study of the thyroidal autoregulation phenomenon, we have investigated the effects of iodide on several thyroidal responses to thyrotropin. Thus, we have found that the 2--4-fold thyrotropin stimulation of protein iodination in beef thyroid cells was reduced about 30% by 4 h of preincubation with 10 muM iodide, and virtually abolished with 50 muM iodide. Similarly the 8-fold thyrotropin stimulation of cyclic AMP accumulation in the cells was reduced about 30% by 3 h of preincubation with 50 muM iodide. It appears therefore that the so-called autoregulation of the thyroid gland does include influences of iodide on the thyrotropin stimulation of cyclic AMP production, iodide transport, and protein iodination which can be demonstrated in vitro in the dispersed thyroid cell system. Two other effects of thyrotropin, namely, the stimulation of [14C]leucine incorporation into protein and of iodide efflux were not at all affected by treatment with excess iodide, and hence may not be subject to the autoregulatory influence of iodide.


Intracellular formation of analogs of cyclic AMP. Studies with brain slices labeled with radioactive derivatives of adenine and adenosine. A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N6-etheno[14C] adenosine nor 1,N6-etheno[14C] adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-3H] adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chloro-cyclic AMP was not obtained. N6-Benzyl[14C] adenosine was converted only to intracellular monophosphates and significant formation of radioactive N6-benzylcyclic AMP was not detected during a subsequent incubation. 2'-Deoxy-[8-14C] adenosine was converted to both intracellular radioactive 2'-deoxy-adenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2'-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.


The effect of temperature on the spectroscopic properties of the heme c octapeptide from cytochrome c. The effect of temperature on the optical properties of the acetylated heme c octapeptide from cytochrome c was examined. At above ambient temperatures the observed optical spectrum with maxima at 549 and 424 nm was characteristic of high-spin ferrous hemeproteins. At below ambient temperatures the optical spectrum became characteristic of low-spin ferrous hemeproteins with maxima at 547, 518, and 410 nm. A thermodynamic characterization of this two component system yielded a deltaHO of -10.1 +/- 0.7 kcal/mol and a delta S0 of-37.6 +/- 2.5 e.u. for the temperature dependent process. Discussion of the spectroscopic and thermodynamic parameters was presented in terms of the consistent magnetic and structural properties of heme complexes.


Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases. 1. The action of eight purified phospholipases on intact human erythrocytes has been investigated. Four enzymes, e.g. phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells. On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50% of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80% of the sphingomyelin. 2. Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes. Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect. 3. With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved. 4. Due to its absolute requirement for Ca2+, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure. Subsequent addition of Ca2+ stimulates phospholipase A2 activity at the inside of the resealed cell, eventually leading to lysis. Before lysis occurs, however, 25% of the lecithin, half of the phosphatidylethanolamine and some 65% of the phosphatidylserine can be hydrolysed. This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes.


Further purification of "Triton subchloroplast fraction I" (TSF-I particles). Isolation of a cytochrome-free high-P-700 particle and a complex containing cytochromes f and b6, plastocyanin and iron-sulfur protein(s). The "Triton Subchloroplast Fraction I" or "TSF-I particles" can be further fractionated into a cytochrome fraction and a P-700-containing fraction essentially free of cytochromes. The cytochrome complex contains cytochromes f and b6 in approx. equimolar amounts, and, in addition, also plastocyanin and one iron-sulfur protein, all in the bound state. Bound plastocyanin was characterized by EPR spectroscopy. The EPR spectrum of the bound iron-sulfur protein resembles that previously detected in Phostosystem I particles under highly reducing conditions at lower than -560 mV. The redox potential of P-700 in the cytochrome-free high-P-700 particles was measured to be +468 mV; those of cytochromes f and b6 are +345 and -140 mV, respectively. Among the four components present in the complex, only cytochrome f can be coupled to a Photosystem I particle and undergoes photooxidation. This coupled photooxidation is totoally inhibited by KCN and only partially inhibited by HgCl2. The similarity of the complex containing cytochromes f and b6, plastocyanin, and an iron-sulfur protein to complexes III and IV of the mitochondrial respiratory redox chain and a possible involvement of the complex in cyclic photophosphorylation are noted and discussed.


EEG and neurophysiological studies of early infantile autism. My aims in this presentation are to review EEG findings that have been published recently in children variously labeled as autistic, schizophrenic, or psychotic. I will also report new results from EEG investigations of an expanded series of Dr. DeMyer's subjects. Sleep research and studies of cerebral evoked responses and the contingent negative variation will be reviewed, and preliminary results of wuantitative analyses of EEG from autistic and normal children will be reported.


Serotonin inhibition and sleep.

Serotonin inhibition and sleep. Methysergide a clinically used blocker of serotonin receptors, was given for 48 hr to 11 normal adults, at a dose of 8 mg per 24 hr. Total REM sleep time was decreased, although total sleep time was unchanged. Stage 4 decreased and stage 3 increased, while total slow wave sleep remained constant. There was a tendency toward a decrease in the number of intact sleep cycles. The relationship of these data to published reports on p-chlorophenylalanine is discussed.


Hypnosis and eye movements.

Hypnosis and eye movements. Eye movements (with closed lids) were studied in a group of highly hypnotizable experimental subjects experienced in self-hypnosis, and compared with a random sample of control subjects that had never been hypnotized and were low in waking suggestibility. Approximately half the experimental subjects rolled their eyes upwards to a greater extent when hypnosis was induced than during eye closure while awake. In some subjects eye flutter occurred during hypnosis, but not in the awake condition. During passive hypnosis the mean rates of rapid eye movements were lower, but those of slow eye movements were higher than during the resting awake condition of the same subjects or the random control subjects. The mean rates of horizontal eye movements during suggestions about begin in a train and watching passing telephone poles were higher for the experimental subjects in the hypnosis and 'imagination' sessions than that of the random control group in the imagination session. A proportion of the experimental subjects made more lateral eye movements during hypnosis than during the imagination session, but an equal proportion did not differ between the two conditions. The mean rates and durations of horizontal eye movements during dreaming about a tennis match were greater during hypnosis ('hypnotic' dream), than during the awake condition a few minutes later ('natural' dream), or the awake condition in the imagination session ('imagination' dream) of the same subjects or random controls. The performance and subjective involvement of the experimental subjects during the Barber suggestibility scale, 'nystagmus' suggestions and 'dreaming' did not differ significantly between the two hypnosis sessions, but in most cases were significantly greater during hypnosis than during the imagination session of the same group or the random control group.


The hepatic chalone. II. Chemical and biological properties of the rabbit liver chalone. A factor has been purified from rabbit liver which decreases the incorporation of 3H-thymidine in DNA of regenerating rat liver slices. This effect is due mostly to an inhibition of DNA synthesis from the deoxynucleoside triphosphates. The purified rabbit liver factor thus interferes with the liver cell division cycle. This inhibitor of DNA synthesis is specific for liver cells; it is not toxic for cultured hepatocytes and its action on DNA synthesis is reversible: at low dose, the inhibition of DNA synthesis in regenerating liver slices is transitory. The purified rabbit liver inhibitor is thus a chalone. The purified rabbit liver chalone ultrafiltrates through 1.2 nm pores, is destroyed by trypsine and pronase, carries a negative charge at pH 8.8 and a positive charge at pH 4.6; it is thermostable and likely a small peptide. It also inhibits RNA and protein synthesis in regenerating liver slices. The inhibition of protein synthesis is immediately maximal then decreases with time, while the maximum inhibition of DNA and RNA synthesis appears after a delay. When a low dose of chalone is used (0.2 unit per 5 ml), the inhibition of DNA and RNA synthesis disappears after some time: this is not due to a destruction of the chalone, but to a loss of sensitivity of the slices incubated in Hanks solution. The inhibitor content of liver cells, normal or malignant, seems inversely correlated with their state of growth. It is much lower in the liver of a young animal or in regenerating liver than in adult liver. Hepatomas produced by feeding DAB contain three times less inhibitor than the normal liver. The purified liver chalone is 5-10 times less active on the incorporation of 3H-thymidine in DNA of DAB hepatoma slices than in DNA of regenerating liver slices. It has no apparent action on adult liver slices; this might be due to the fact that 3H-thymidine incorporation into DNA of the adult organ depends, for the greater part, on other processes than DNA replication in hepatocytes.


Lp (a) blood types. Comparison of immunological testing with results of polyacrylamide gel electrophoresis. The results of serum beta-lipoproteins typing of 100 random human sera for Lp (a) antigen by the standard immunological method were compared with those of polyacrylamide gel electrophoresis which may detect an Lp (a) band migrating more slowly than the bulk of LDL. This band was found in all the 33 Lp (a+) but also in 9 of the 67 Lp (a-) sera examined.


Axon voltage-clamp simulations. III. Postsynaptic region. This is the third in a series of four papers in which we present the numerical simulations of the application of the voltage clamp technique to excitable cells. In this paper we discuss the problem of voltage clamping a region of a cylindrical cell using microelectrodes for current injection and voltage recording. A recently developed technique (Llinás et al., 1974) of internal application of oil drops to electrically insulate a short length of the postsynaptic region of the squid giant synapse is evaluated by simulation of the voltage clamp of an excitable cylindrical cell of finite length with variable placement of the current and voltage electrodes. Our results show that ENa can be determined quite accurately with feasible oil gap lengths but that the determination of the reversal potential for the synaptic conductance, ES, can be considerably in error. The error in the determination of ES dependp, and especially the membrane resistance at the time the synaptic conductance occurs. It is shown that the application of tetraethylammonium chloride to block the active potassium conductance very significantly reduces the error in the determination of ES. In addition we discuss the effects of cable length and electrode position on the apparent amplitude and time course of the syn aptic conductance change. These results are particularly relevant to the application of the voltage clamp technique to cells with nonsomatic synapses. The method of simulation presented here provides a tool for evaluation of voltage clamp analysis of synaptic transmission for any cell with known membrane parameters and geometry.


A proton-relaxation enhancement study of the interaction of manganous ions with phospholipids in aqueous dispersions. An interaction of dipalmitoylphosphatidylcholine (PC) and phosphatidylserine (PS) with manganous ions has been investigated by measuring the effect of bound manganese upon the longitudinal relaxation rate, 1/T1, of the solvent water protons and evaluating the enhancement factor epsilon b. The observed enhancement values were used to determine the number of interacting sites per polar head group, n, and the values of association constants, KA, of manganese to PC and PS. Changes in epsilon b correlate with structural changes at the interacting site. By increasing the temperature one can see an abrupt decrease in epsilon b within the temperature interval from 40 to 50 degrees C indicating the thermal phase transition of PC as established by calorimetry, fluorescence and high-resolution NMR measurements. That an enhancement of 1/T1 of the solvent-water protons occurs at all is explained by assuming a restricted rotation of the Mn2+-aquo complex in the bound state. In addition we suppose that the rotation of the Mn2+-aquo complex is the mechanism which dominates the relaxation of the water protons in teh bulk solvent when phospholipids are present.


Effect of DNA base composition on the intercalation of proflavine. A kinetic study. The effect of DNA base composition on the kinetics of the association between DNA and proflavine has been investigated using the temperature jump relaxation method. It is found that, regardless of the G + C base composition the results fit a two step mechanism, the second of which exhibits characteristics of intercalation of proflavine into DNA. However, they two equilibrium constants corresponding to these steps, KI and KII, depend on the nature of the DNAs. The constant KI is found to be an order of magnitude greater for M. lysodeikticus DNA (72% G + C) than for calf thymus DNA (48% G + C). Increasing G-C content thus appears to favor the intermediate non-intercalated complex of proflavine with DNA. Methylation of M. lysodeikticus DNA with dimethyl sulfate, preferentially yielding N7 methyl guanine as the modified base, again leads to an apparent two step mechanism, with the value of KI unchanged with respect to untreated DNA, while the affinity of proflavine for the intercalated complex measured by the value of KII increases for methylated DNA.


Acute monoblastic leukemia: diagnosis and treatment of ten cases. During a large clinicopathologic study of acute nonlymphocytic leukemia (ANLL), ten patients were identified in whom the leukemic blasts demonstrated striking morphologic and cytochemical similarities and who seemed to form a specific subgroup of ANLL. The patients' leukemic blasts were studied in routine blood and bone marrow preparations and by cytochemical and ultrastructural techniques. In routine smears, the blasts showed no clear evidence of differentiation. Cytochemically, the blasts exhibited strongly positive nonspecific esterase activity, which was completely inhibited by incubation with sodium fluoride, and were myeloperoxidase and sudan black B negative. Ultrastructural features of the blasts were similar to those described for monocytic leukemias. Striking clinical features included the occurrence primarily in young patients, the high frequency of lymphadenopathy at presentation, and the high incidence of post-treatment disseminated intravascular coagulation. Complete remissions were frequently initially obtained with duanorubicin in combination with various other agents and later in the disease with VP16-213. Based on the cytochemical and ultrastructural features, we concluded that this form of ANLL was a variety of acute monocytic leukemia. Recognition of the entity is important for optimal therapy.


The effects of tetraphenylboron on neuromuscular transmission in the frog. 1 The effect of tetraphenylboron (TPB) were studied on the frog sciatic nerve-sartorius muscle preparation. 2 TPB (0.01-1 mM) blocked indirectly elicited twitches of the preparation. 3 TPB (0.01-0.1 mM) produced no depolarization but lowered membrane resistance. TPB increased miniature endplate potential (m.e.p.p.) frequency, the rate of rise of the endplate potential (e.p.p.), and slowed the rate of rise and rate of fall of the muscle action potential. 4 In Mg2+ solutions the quantal content of e.p.ps was initially increased by TPB (0.01 mM). This was followed by a decrease of e.p.p. and m.e.p.p. amplitudes, accompanied by a lack of e.p.p. failures. 5 Larger concentrations of TPB (0.1 mM) produced an increase in e.p.p. amplitude followed by the sudden abolition of e.p.ps. This effect was associated with abolition of the nerve terminal spike. 6 TPB (0.1 mM) exhibited no postjunctional blocking action. 7 The results indicate that TPB acts prejunctionally, initially causing an increased release of acetylcholine. Subsequently, transmitter output is reduced by a reduction of quantal size.


An inhibitory effect of atropine on responses of the vas deferens of the mouse to field stimulation. 1 Atropine is shown to impair responses of the vas deferens of the mouse to field stimulation by acting at a site proximal to the smooth muscle cells. 2 The inhibitory effect of atropine is prevented by desmethylimipramine and reversed by dexamphetamine, and appears similar to the adrenergic-neurone blockade of guanethidine. 3 Electronmicroscopical studies show the presence in vas of presumptive noradrenergic axons which have acetylcholinesterase reaction product associated with their axolemmae. 4 These results are discussed in relation to the controversial hypothesis of a 'cholinergic link' in noradrenergic transmission.


The effects of cyclic N-2-O-dibutyryl- adenosine 3',5'-monophosphate, adrenaline and aminophylline on the isometric contractility of the isolated hemidiaphragm of the rat. 1 N-2-O-dibutyryl adenosine 3',5'-monophosphate (db cyclic AMP), adrenaline and aminophylline produce a potentiation of the tension developed (Td) and the maximum rate of rise of tension (dT/dt max) in the rat isolated diaphragm during indirect electrical stimulation. Aminophylline and db cyclic AMP also produce the same effect during direct stimulation. 2 Propranolol produced a depression of the action of adrenaline on Td and dT/dt max during indirect stimulation of the diaphragm. On the other hand, the potentiating actions of db cyclic AMP and of aminophylline on Td and dT/dt max during indirect stimulation were unaffected by propranolol. 3 The results support the idea that cyclic AMP may be involved not only in regulating the processes associated with synthesis, mobilization and storage of transmitter in the motor nerve terminal, but also in modifying some metabolic processes which regulate the function of the contractile elements.


Some characteristics of pre- and post-synaptic inhibitory receptors at the hermit crab neuromuscular junction. 1 The effects of gamma-aminobutyric acid (GABA), beta-guanidinopropionic acid (betaGP) and picrotoxin on the pre- and post-synaptic receptors of the hermit crab neuromuscular junction were studied quantitatively usine electrophysiological techniques. Reductions in excitatory junction potential (e.j.p.) amplitude and membrane resistance were measured simultaneously from the same cells. 2 The pre- and post-synaptic receptors were activated by the same order of concentration of GABA, whereas betaGP stimulated the pre-synaptic receptors at a concentration ten times lower than was required to affect the post-synaptic membrane. 3 Picrotoxin appeared to antagonize the pre-synaptic action of betaGP in a competitive manner. The affinity constants (+/- s.e. mean) for picrotoxin 5 times 10(-6)M and 2 times 10(-4)M were 6.80(+/-0.46) times 10(5)M-1 and 6.42(+/-1.8) times 10(5)M-1 respectively. 4 The effect of GABA on e.j.p. amplitude also appeared to be antagonized competitively by picrotoxin whereas the post-synaptic effect was antagonized in a non-competitive manner. 5 Possible differences in the nature of the pre- and post-synaptic receptors are discussed.


Extraction from ox retractor penis of an inhibitory substance which mimics its atropine-resistant neurogenic relaxation. The inhibitory post-ganglionic transmission in the retractor penis of the ox resembles that of the dog and is not cholinergic or adrenergic. Acid extracts of this tissue have yielded an unidentified, labile, inhibitory substance which mimics the effect of its inhibitory nerves.


The effects of hypercholesterolaemic plasma on vascular sensitivity to noradrenaline. 1. The pressor responses to injected noradrenaline (NA) of 42 isolated perfused femoral arteries of the rabbit were studied. 2. Potentiation of the responses was found when hypercholesterolaemic plasma was perfused through the arteries. No change was found with normal plasma. 3. Potentiation of the responses was found when isolated beta-lipoprotein in Krebs solution was perfused. No change was found with similar amounts of bovine-albumen. 4. Pure cholesterol dissolved directly into normal plasma, and dissolved via propanol into Krebs solution or plasma caused no potentiation. Propanol alone in Krebs or plasma had no effect. 5. Potentiation was caused by a decreased equilibrium coefficient (Keq) for the NA-adrenoceptor interaction and an increased maximal pressor response (Rmax). 6. It is concluded that cholesterol carried on its apoprotein is capable of potentiating the pressor effects of noradrenaline.


Dopamine-induced amylase secretion from guinea-pig submandibular gland. 1. The effects of dopamine, 5-hydroxydopamine (5-OHDA) and noradrenaline on amylase secretion from the guinea-pig submandibular gland were investigated under in vitro conditions. 2. All three amines greatly enhanced amylase secretion. Blockade of dopamine beta-hydroxylase did not inhibit the response to dopamine. 3. Noradrenaline and dopamine stimulated amylase release from salivary glands of reserpine-treated animals, whereas 5-OHDA had no stimulatory effect on secretion in guinea-pigs pretreated with reserpine. 4. Haloperidol completely inhibited dopamine-induced enzyme discharge, but did not affect noradrenaline-initiated secretion. 5. Apomorphine caused a slight enzyme release by itself; it diminished the dopamine secretory effect, but did not modify that of noradrenaline. 6. Pimozide and fluspirilene attenuated the dopamine-induced enzyme discharge, but compared with haloperidol they were less effective. 7. It is concluded that dopamine exerts a secretagogic action different from that of noradrenaline. The possible presence of a specific dopamine receptor in salivary glands is discussed.


Fatal Clostridium welchii septicaemia following acute cholecystitis. A case of Clostridium welchii septicaemia following acute cholecystitis is described. The onset was acute and a rapidly fatal outcome ensued. Radiological findings were negative. An approach to the antibiotic treatment and general management is discussed.


Value of infarct-specific isotope (99mTc-labelled stannous pyrophosphate) in myocardial scanning. With the use of 99mTc-labelled stannous pyrophosphate scans positive for myocardial infarction were obtained from 28 patients in the acute stage of the disease. In some cases the scan was positive when the initial electrocardiogram gave equivocal results. Negative scans were obtained from a control group of patients and from eight patients in hospital with chest pain but with no other evidence of recent myocardial infarction.


Arsine toxicity aboard the Asiafreighter. Eight sailors on board the Asiafreighter were exposed to arsine that had escaped from a cylinder in the cargo hold. Four suffered severe toxicity and within a few hours had developed fever, weakness, nausea, vomiting, diarrhoea, abdominal pain, and haemoglobinuria. These patients had pronounced intravascular haemolysis, which in one patient was complete. This patient was also stuporose and anoxic, a condition attributed to failure of oxygen transport and sludging of red cell debris in the cerebral and pulmonary circulations, but he regained a normal level of consciousness after exchange transfusion. Evidence of marrow depression was present: the reticulocyte response to the haemolysis was poor and there was a thrombocytopenia. All four patients developed renal failure, one being totally anuric for five weeks. Two patients developed peripheral neuropathy, and one was still severely disabled six months after the incident. The other four patients had a similar, though less severe, illness.


Treatment of wounds from animals suspected of carrying neurotropic viruses. The methods used at the Clinical Research Centre, Northwick Park Hospital, to treat wounds potentially infected with neurotropic viruses are outlined. Emphasis is laid on first aid and on surgical attention. Patients should remain under medical supervision for at least four weeks after the injury, the need for specific post-exposure treatment depending on the nature of the exposure.


Test for left-right chemospecificity in frog cutaneous nerves. Studies of the effects of competitive reinnervation by left and right cutaneous nerves were carried out in Rana pipiens in an effort to test for the existence of left-right biochemical differentiation of neural tissue. The dorsal cutaneous nerves were cut, transposed and allowed to regenerate under different conditions of denervation and competition and the resultant reinnervation and reflex patterns were determined by behavioral and electrophysiological mapping techniques. Contrary to earlier findings, there was no indication in the present investigation that the growth patterns were influenced by the laterality of the fibers. Other factors such as availability of terminal sites created by complete or partial denervation and the tendency of fibers to seek terminal connections seemed to override any qualifications imposed by the existence of lateral specificity on the formation of terminal contacts.


Endogenous phosphorylation of four cerebral cortical membrane proteins: role of cyclic nucleotides, ATP and divalent cations. Endogenous phosphorylation of 4 membrane-bound protein components of cerebral cortex was studied in vitro. Cyclic AMP was shown to stimulate phosphorylation to a major extent in protein components designated D, E and G, but to a minor extent in protein component F. The time course of phosphorylation of each of these components is affected differently by (a) the presence of cyclic nucleotides or zinc ions and (b) the concentration of ATP and magnesium ions in the reaction mixture. Although a different membrane preparation was used here than by Ueda et al., molecular weight determination and response to divalent cations, magnesium and zinc, suggest that components D and E may be the same as their proteins I and II. The newly described phosphoproteins, F and G, are respectively the least and most stimulated by the addition of cyclic AMP. Under certain conditions phosphate content in protein E can be increased by cyclic AMP and decreased by cyclic GMP.23Author


Thyrotropin releasing hormone (TRH): apparent receptor binding in rat brain membranes. Thyrotropin releasing hormone (TRH) binds to membranes of rat brain tissue via high- and low-affinity binding components. The high-affinity binding of TRH to brain membranes resembles binding to pituitary membranes in terms of its affinity and specificity for most peptides. In equilibrium studies, the affinity and specificity for most peptides. In equilibrium studies, the dissociation constant for high-affinity binding to brain membranes is about 50 dissociation constant for high-affinity binding to brain membranes is about 50nM, which is about the same as for aat pituitary membranes, while low-affinity binding to brain membranes has a dissociation constant of about 5 muM. Liver membranes display low-affinity binding for TRH with a dissociation constant similar to the low-affinity binding component of brain membranes. No high-affinity binding can be detected with liver membranes. Negligible saturable binding to TRH can be detected with membranes of any tissues examined other than liver, pituitary and brain...


Metabolic responses of the awake cerebral cortex to anoxia hypoxia spreading depression and epileptiform activity. Using the time sharing fluorometer/reflectometer the measurement of NADH fluorescence as well as the reflected light was obtained from the surface of the awake rat brain cortex. The light was transferred to and from the brain via a flexible light pipe (made of quartz fibers) connected to a cannula implanted permanently above the brain. Exposing the rat to pure nitrogen atmosphere increased the fluorescence (reduction of NADH) by 32.3 +/- 6.1% in comparison to the normoxic fluorescence level. During cortical spreading depression (SD) the NADH fluorescence decreased (oxidation of NADH) by 17.3 +/- 2.8%. Exposing the rat to nitrogen after SD was elicited blocked the oxidation cycle observed during SD. Exposing the awake ras to 10, 7.5 or 5% O2 did not block the response of the brain to spreading depression or to Metrazol applied locally to the cortex. Under hypoxic conditions the brain showed a typical response to SD, namely, an oxidation cycle of NADH except that the duration of the cycle was longer and the decrease in the NADH level was smaller. The EEG activity recovered to normal even under 5%. The same effect of hypoxia was found when Metrazol was applied and epileptic activity was developed.


Recruitment and firing rate modulation of motor unit tension in a small muscle of the cat's foot. Maintained contractions were elicited in the first deep lumbrical muscle of the cat's foot by electrical stimulation of the contralateral motor cortex or, reflexly, by pinching of the foot pad. The discharges of all significant motor units of the muscle were monitored by electromyography, and contractions of the various motor units were observed in isometric recordings of muscle tension. Over a wide range, muscle tension could be enhanced by an increased intensity of pad pinching or cortical stimulation. This increase in muscle tension was caused by a recruitment of new motor units as well as by an increase in the firing rate of already active motor units. The latter mechanism was clearly of great importance. Pad pinching or cortical stimulation could sometimes cause the muscle to produce a tension close to that of a maximum tetanic contraction. This was several times greater than the mean tension that would have been caused by motor unit recruitment alone (i.e. by the motor units firing at their minimum steady rate). Cortical stimulation as well as pad pinching commonly recruited weak units more easily than stronger ones of the same muscle. The recruitment order obtained in response to pad pinching often differed, however, in various details from the recruitment caused by cortical stimulation.


Evaluation of inactivated infectious bovine rhinotracheitis vaccines. Sixty-five calves of approximately three months of age and of mixed sex were vaccinated twice at four week intervals with either attenuated or inactivated infectious bovine rhinotracheitis vaccines. Following initial vaccination there was no demonstrable serum infectious bovine rhinotracheitis titer in any of the calves receiving the inactivated vaccine with 20.7% of the calves receiving the attenuated vaccines having demonstrable titers. Following a second administration of vaccine at eight weeks post-initial vaccination 63.9% of the calves receiving the inactivated vaccine had no demonstrable titer with 72.4% of the calves receiving the attenuated vaccine exhibiting a blood titer of four or greater.


Identification of multiple equine infectious anemia antigens by immunodiffusion reactions. Equine infectious anemia (EIA) cell antigens prepared from infected equine spleen, equine leukocyte cultures or a persistently infected equine dermis cell line contained at least two serologically reacting components. For convenience one component was designated as soluble antigen (SA) and the other as cell-associated antigen (CAA). The SA appeared as a single component when it was prepared from EIA virus precipitated from infectious tissue culture fluid with polyethylene glycol and ether treated but it was mixed with CAA when the source was infected cells. Cytolytic or mechanical disruption of infected cells appeared to accelerate the release of CAA. Reaction to each component could be identified in double and radial immunodiffusion tests by increasing the concentrations of SA in a two-component antigenic mixture. The CAA component does not appear to affect the value of the immunodiffusion test as a diagnostic aid.


In vitro antimicrobial sensitivity of Mycoplasmas isolated from the bovine genital tract. The in vitro antimicrobial sensitivity of 14 mycoplasma and 13 ureaplasma strains isolated from the genital tracts of bulls was examined. It was found that at relatively low concentrations, tetracycline, declomycin and tylosin were lethal to both types of organisms. Lincospectin, berenil, streptomycin and erythromycin were lethal to mycoplasmas but were only inhibitory to the ureaplasma strains at the same concentrations. Polymyxin B and novobiocin were ineffective at the levels tested.


Viral susceptibility of a cell line derived from the pig oviduct. Seventeen of 24 RNA viruses and eight of nine DNA viruses replicated in a cell line derived from a pig fallopian tube. The following RNA viruses grew poorly in it: the virus of transmissible gastroenteritis of pig and the swine-influenza, Sendai and bovine para-influenza type 3 viruses. Among other RNA viruses an untyped swine para-myxovirus and some picornaviruses, rhabdoviruses and togaviruses attained high titers and produced an extensive cytopathic effect. Among the DNA viruses a porcine adeno, equine rhinopneumonitis, infectious bovine rhinotraceheitis, pseudorabies and porcine cytomegalo viruses replicated in pig fallopian tube cells as well as in other cells generally used to grow them.


Transmissible gastroenteritis (TGE) of swine: effect of age of swine testes cells culture monolayers on plaque assays of TGE virus. A continuous line of swine testes cell culture monolayers was infected at various ages with both cell culture-adapted transmissible gastroenteritis (TGE) virus and tissue infected with TGE virus. Both produced increasing numbers of plaques as the cell monolayers aged from two to five days. Therefore, allowing the swine testes cell monolayer to age five to six days before inoculation should increase the likelihood of detecting TGE virus by plaque assay.


An outbreak of avian encephalomyelitis in turkeys in Canada. An outbreak of avian encephalomyelitis in two week old turkey poults is described. The disease was diagnosed by the fluorescent antibody technique and was confirmed by neutralization tests conducted in chicks.


An adrenal cell system suitable for long term study of factors affecting steroidogenesis. Cell suspensions of normal adult rad adrenals have been prepared by trypsinization and incubated in Ham's nutrient mixture F10 containing horse serum, fetal calf serum, and lima bean trypsin inhibitor. In most experiments culture medium was not changed during incubation. In this system the number of cells fell to 50% after 2 days, then slowly declined to 20% after 1 month of incubation. A corticosterone (B) response was seen to as little as 5 muU of ACTH per millilitre, a concentration which is within the range found in normal rat serum. With maximal stimulation (100 mU ACTH/ml) the rate of accumulation was highest during the first 24 h then slowly decreased over the following 9 days. When in separate experiments ACTH was added after various times of incubation up to 3 weeks, there was a B response which continued for as long as 1 week after the ACTH was added; the later the time at which ACTH was added the lesser was the initial B response and the longer the lag period before a substantial response occurred. In cell suspensions in medium containing 5.0 mequiv. of K+ per litre, aldosterone content increased for approximately 24 h, then showed little or no change over the next 9 days. With increased K+ concentration, aldosterone was found in greater amounts and accumulation continued for longer periods, both without and with ACTH. This adrenal cell system appears suitable for long term study of factors affecting steroidogenesis.


Adrenocorticotropin secretion rates following histamine injection in adult and newborn dogs. The metabolic clearance rate (MCR) for ACTH in adult dogs was previously shown not to vary significantly with varying plasma ACTH concentrations or among dogs. This is confirmed here for pups aged 1-7 days. Hence, ACTH secretion rates can be continuously calculated from a continuous function of plasma ACTH vs. time. Each of seven adult dogs under Nembutal anesthesia received two or three intravenous (i.v.) injections of histamine with increasing doses. The first injections in each dog ranged from 7 to 50 mug/kg, while the last dose was 62-108 mug/kg. A total of 16 injections were given. Twelve pups (two litters of six) aged 1-7 days each received one injection of histamine of 76-116 mug/kg (i.v.). ACTH concentrations in plasma were determined by an adrenal cell suspension bioassay before, and 6 times after each injection. Nine pups also underwent determinations of their MCR for ACTH, with plateau concentrations determined at three times during an ACTH infusion. Continuous curves of ACTH secretion rates were calculated for all 28 histamine injections, showing that all except the 1-day-old pups secrete considerable ACTH when stressed. Compared to adult dogs, the pups show lower secretion rate peaks and shorter periods of rapid secretion. Changes in plasma glucocorticoids also suggest that the adrenal cortex of newborn dogs can respond to ACTH by increased glucocorticoid secretion.


Uncoupling of lipolysis from cyclic AMP by procaine: a tool for studying the mechanism of action of antilipolytic agents. The initial rate of net glycerol release in norepinephrine-stimulated adipose tissue fragments was inhibited (40-78%) by procaine-HCl (1-5mM), whereas basal (unstimulated) lipolysis was unaffected. A dose-related inhibition of norepinephrine-induced lipolysis by procaine-HCl (0.1-1 mM) also occurred in adipocytes. Procaine-induced antilipolysis was associated with an augmented rather than a reduced hormone-stimulated increment in intracellular cyclic AMP. The dissociation of lipolysis from cyclic AMP accumulation has been termed the uncoupling effect of procaine. This effect of procaine was employed to define the precise mechanism of action of the antilipolytic drug clofibrate (Atromid-S) which inhibits lipolysis by reducing cyclic AMP. A reduction in cyclic AMP by clofibrate was demonstrated in norepinephrine-stimulated cells exposed to procaine (uncoupled system). Thus, the inhibitory effect of clofibrate on cyclic AMP could not be attributed to accumulation of products of lipolysis. Because neither procaine-HCl nor clofibrate had any effect on the low Km 3':5'-cyclic-AMP phosphodiesterase (EC 3.1.4.17) activity in hormone stimulated cells, the clofibrate-induced reduction in cyclic AMP was attributed to its direct action on adipocyte adenylate cyclase.


[Nitrosoguanidine-induced attenuated Clostridium perfringens type A mutant in gas gangrene]. A fully virulent classical type A strain of Clostridium perfringens was treated during its logarithmic growth phase with 100 mug/ml of N-méthyl-N'-nitro-N-nitrosoguanidine, the bacteria being exposed to the mutagen for 30 min at 37 degrees C in a phosphate buffer adjusted to pH 6.2; after treatment the suspension was streaked on sheep blood agar plates, and colonies that showed an alteration in the theta-hemolysis pattern were selected for isolation. The virulence of two mutants, thus altered in their theta-hemolysis, was studied. One, designated LNG 5, was still capable of killing most of the inoculated guinea pigs in less than 24 h with all the clinical, macroscopic, and bacteriological signs of gas gangrene; however, histological sections showed that tissue damage was not as marked as with the wild strain. On the contrary, the second mutant, labelled LNG 11, was completely avirulent as far as gas gangrene was concerned; indeed, the injection of fluid cultures containing 1 times 10(8) - 10(9)/ml viable bacteria, was not followed by any clinical, bacteriological, or histological signs of gas gangrene. However, strain LNG 11 did give rise to a firm swelling of the inoculated thigh with a corresponding acute inflammatory response of the connective tissue, although the muscle fiber was unaltered. Eventually, this local reaction was followed by necrosis of the skin accompanied by an acute or subacute inflammation with fibroblastic proliferation. These superficial lesions healed spontaneously. They could not be reproduced with crude filtrate alone or with washed bacilli. Strain LNG 11 was therefore considered to be soletly an attenuated strain since, although avirulent as far as gas gangrene is concerned. it is still capable of producing low levels of toxic material. This appears to be the first time that such a strain of C. perfringens type A has been obtained by nitrosoguanidine treatment.


Supersensitivity of central neurons--a brief review of an emerging concept. The concept that "denervation" or "pharmacological disuse" supersensitivity develops in central neuronal systems subsequent to sustained attenuation of normal neurohumoral mechanisms is reviewed. Particular emphasis is placed on biochemical and electrophysiological parameters of supersensitivity in dopaminergic (striatal) neuronal systems. The possible applicability of theories invoking changes in receptor sensitivity to the phenomenon of narcotic tolerance and physical dependence and to psychoactive drug therapy is discussed.


Pituitary melanocorticotrophinoma with amyloid deposition. The light and electron microscopic features of a pituitary adenoma composed of adrenocorticotrophic hormone (ACTH) and melanocyte stimulating hormone (MSH) cells with perivascular amyloid deposition is reported. Histochemical and fine structural data indicate that this material is APUDamyloid and is present in the extra-cellular perivascular spaces. It is suggested that the differences in fine structure and of distribution of the amyloid in pituitary adenomas is dependent upon the cell of origin.


Why transcortical reflexes?

Why transcortical reflexes? Experiments in humans and in monkeys have indicated that load perturbations, occurring during voluntary movements and postural activity, may be automatically compensated for. Overall muscle stiffness opposing load changes is determined by the visco-elastic properties of the muscle, by segmental reflex actions and finally by long-loop reflexes. Under certain circumstances, for instance when the subject or the experimental monkey is "prepared" to counteract perturbations which are unpredictable in time, the long-loop "reflexes" appear to be responsible for most of the corrective muscle tension. Experiments in anaesthetized monkeys revealed that signals from stretch afferents reach neurons of the motor cortex, possibly via a relay in the cortical area 3a. The latencies of these responses to well controlled muscle stretches were in the same range as motor cortical cell discharges recorded in alert monkeys subjected to load perturbations. Furthermore, these responses of cells in the motor cortex also had the appropriate timing to indicate a causal relationship with the long-latency electromyographic responses to load changes referred to above. These experimental results therefore strongly support the hypothesis, first proposed by Phillips (1969), of a transcortical servo-loop adjusting motor cortical output according to the load conditions in which  movements are performed. The major advantage of transcortical regulations as opposed to segmental regulations, seems to be a powerful gain control acting at the cortical level; it was repeatedly shown that the long-loop reflexes are strongly modifiable and under voluntary control. It is suggested that an adaptive gain control at the cortical level is a prerequisite to preserve the complex capabilities of the motor cortex as the chief "executive" for skilled, preprogrammed movements. A loss of this adaptive gain control may be, at least partly, the cause of motor disorders such as rigidity in Parkinsonian patients, as reported by Tatton and Lee (1975). It is suggested that further investigations of the control of transcortical reflexes may aid in the understanding of the pathophysiology of motor disabilities.


Prognosis and treatment of Wilms' tumor at Great Ormond Street Hospital for Sick Children--1960-1972. Eighty-one children with a diagnosis of Wilms' tumor who were treated at the Great Ormond Street Hospital for Sick Children between 1960 and 1972 are reviewed. The National Wilms' Tumor Study group staging system is used. Distribution by age and sex in each stage together with survival rate is given. Treatment and prognostic factors are discussed. Wilms' tumor is most common in children below the age of 3 years. There was a survival rate of 87% in Stage I, 36% in Stage II, 8% in Stage III, and 33% in Stage IV. The survival was worse in patients with locally advanced tumor than in patients who had pulmonary metastases in association with a locally resectable tumor. Whole abdominal irradiation is necessary in all patients in whom the tumor has breached the renal capsule. Single agent chemotherapy has not proved effective in preventing relapse in patients with advanced tumors. More intensive therapy is needed in this group to prevent both local recurrence and distant metastases. The addition of multiple drug chemotherapy to nephrectomy and whole abdominal irradiation is already improving the disease-free survival.


Treatment of hepatocellular carcinoma with adriamycin. Preliminary communication. In a Phase II clinical trial, 14 patients with histologically proven primary hepatocellular carcinoma were treated with adriamycin administered intravenously at a dose of 75 mg/m2 every 3 weeks. All 11 evaluable patients responded with 3 exhibiting complete tumor regression after two, three, and five courses of adriamycin respectively. The remission durations for these 3 were 3, 6, and 7 months, and their survivals were 8, 9, and 13 months, respectively. The median survival of the evaluable patients is 8 months (range 1-13 months). The side effects encountered included myelosuppression, anorexia, nausea, vomiting, and alopecia. Adriamycin seems to be an effective agent in hepatocellular carcinoma. Further trials are underway to test its true efficacy both singly and in combination with other drugs in the management of this tumor.


Treatment of grade III and IV astrocytoma with dimethyl triazeno imidazole carboxamide (DTIC, NSC-45388) alone and in combination with CCNU (NSC-79037) or methyl CCNU (MeCCNU, NSC-95441). The effectiveness of DTIC in the treatment of Grade III and IV astrocytomas was analyzed in two phases. In the first phase, 14 patients (Group A) with progressive neurologic dysfunction following primary treatment were treated with DTIC alone (8 patients) or in combination with CCNU or methyl CCNU (6 patients) and evaluated for change in neurologic status. Five of the 8 treated with DTIC responded symptomatically for a median duration of 18 weeks, and 3 of 6 treated with the combination of drugs responded for a median duration of 12 weeks. In the second phase, 15 patients (Group B) were treated within 4 weeks of surgical resection with radiation therapy and adjuvant chemotherapy with DTIC and/or MeCCNU. These patients were followed for survival and compared to a historical control group of 15 patients (Group C) treated with surgery and radiation only. The drug-treated group had a median survival of 55 weeks, compared to 35 weeks for the control group. Hematologic toxicity was life threatening in 2 of 14 patients treated with combination drugs, but mild with DTIC alone. DTIC appears to be active against malignant astrocytomas. Survival may be lengthened by combining chemotherapy with surgery and radiation therapy.


Ectopic placental proteins in nontrophoblastic tumors. Serial measurements following chemotherapy. Four men with metastatic carcinoma (two bronchus, one stomach, one primary unknown) and ectopic placental protein production (one chorionic gonadotropin (hCG and its alpha subunit; one placental lactogen (hPL); one hCG alone; one hCG and hPL) were studied with serial retrospective measurements of the ectopic protein concentrations in serum, correlated with objective assessment of clinical status. The biochemical marker and clinical response were documented during treatment with a variety of agents and during periods without therapy. Of nine assessable periods, seven showed concordance of biochemical marker and clinical responses.


Herpes simplex encephalitis in Hodgkins disease. Isolation of drug-sensitive virus from brain following unsuccessful treatment with idoxuridine. Herpes simplex encephalitis developed in a patient with Hodgkin's disease under therapy. Despite treatment with idoxuridine in a total dose of 280 mg/kg intravenously, he died without showing any clinical response. At autopsy, there was no gross or microscopic evidence of Hodgkin's disease, and virus isolated from the brain postmortem was inhibited in vitro by idoxuridine 0.5 mug/ml. Failure of idoxuridine to affect the course of infection by a drug-sensitive virus may be due to poor tissue penetration, although the role of the Hodgkin's disease cannot be discounted.


Ultrastructure of human bronchiolo-alveolar cell carcinoma. Ultrastructural features were correlated with a series of special staining reactions in eight cases of bronchiolo-alveolar carcinoma. Ultrastructural, all tumors were similarly composed of large cells with abundant cytoplasm and small nuclei in close contact with each other. Straight membranes or complex interdigitations occurred within adjacent tumor cells, attached to each other by scattered desmosomes. Microvilli or cilia abutted from free surfaces of the cells, and were noted in different stages of evolution. Numerous organelles were seen in the cytoplasm, including prominent mitochondria and single or coalescent secretory vacuoles with granular matrix resembling mucin. Other cytosomes less commonly found were irregular, partially lamellated inclusions and dark, homogeneous structures without limiting membranes. The stroma of the tumors was rich in elastin and collagen. Both the number of secretory vacuoles in the cytoplasm of tumor cells and the amount of connective tissue fibrils in the stroma of the tumors correlated well with the findings in the series of special staining reactions. No definite ultrastructural feature was present to identify the tumors as orginating from Type II alveolar epithelial cells, but the possibility exists that they arose in the bronchiole, from undifferentiated basal cells or mucinous cells per se. Our impression in these eight cases studied is consistent with the view that bronchiolo-alveolar carcinomas are indistinguishable at the ultrastructural level from other bronchogenic adenocarcinomas.


An ultrastructural study of mixed hepatoblastoma with osteoid elements. A case of mixed hepatoblastoma with osteoid elements was studied by light and electron microscopy. The ultrastructure of the epithelial elements showed a lack of differentiation and simple cytoplasmic organelles. However, an occasional cytoplasmic crystalloid structure were seen. The ultrastructure of the osteoid foci showed fibroblast-like cells capable of collagen formation, clearly distinguishable from the epithelial elements. These cells had neoplastic characteristics of nuclear pleomorphism and high nucleo-cytoplasmic ratio. These findings support the belief that hepatoblastoma arises from a multipotential blastema capable of both epithelial and mesenchymal differentiation, with the osteoid elements being an intrinsic neoplastic component of the tumor.


Primary liver cancer coincident with Schistosomiasis japonica. A study of 24 necropsies. The etiologic relationship of parasitic liver disease to primary liver cancer has long been debated. For this reason, a review of 4611 necropsies was carried out to determine the frequency with which hepatocellular carcinoma occurred in association with schistosomiasis. Of 227 cases of hepatocellular carcinoma, 24 (10.6%) were associated with schistosomiasis japonica. This was significantly higher than the incidence of this carcinoma without schistosomiasis (2.78%). The majority of the 24 cases exhibited the features of a mixed macronodular and micronodular cirrhosis (Gall's posthepatitic cirrhosis); this was super-imposed upon and caused a masking of schistosomiasis fibrosis. By radioimmunoassay hepatitis B antigen was positive in 27% of these cases. A review of the literature indicated that chronic schistosomiasis, on its own, is unlikely to be the cause of primary liver cell carcinoma. Histologic features resembling post-hepatitic cirrhosis combined with a high frequency of hepatitis B antigen suggest that viral hepatitis rather than S. japonicum is the more likely etiologic factor involved, or has a synergistic effect on carcinogenesis.


Radioautographic study of the rat brain and pituitary after injection of 3H dexamethasone. The central nervous system and the pituitary of adrenalectomized male rats injected with 3H-dexamethasone were examined by radioautography. At 1 hr after the injection, radioactivity concentration was high in the medial basal hypothalamus, the pituitary and the pineal gland. In the hypothalamus, radioactive material was found to be selectively concentrated in neurons in the ventral part of nucleus arcuatus and in the infundibular region. In the anterior pituitary, a large proportion of cells showed silver grains both in the cytoplasm and over the cell nuclei. However, in a small number of cells, the radioactive material was associated with the cell nuclei. Less radioactivity was present in the intermediate and posterior lobes. The pineal gland contained more silver grains than did other regions of the brain. The results obtained in the present study suggest essentially an action of dexamethasone in the medial basal hypothalamus and at the level of the pituitary.


Inhibitor induced alterations of chromatoid bodies in male germ line cells of Xenopus laevis. The action of inhibitors of protein synthesis on the structure of cytoplasmic inclusions found in the male germ cell line of the anuran, Xenopus laevis, has been studied by light and electron microscopy. Results indicate that one such inclusion, the chromatoid body, is sensitive to treatment with either chloramphenicol or puromycin. These drugs administered in vivo or in vitro cause up to a thirty-fold increase in the volume of the chromatoid body in all stages where it is normally present. Maximum size increase obtainable is the same for either drug, but is different and characteristic for each germ cell stage. Drug action is dose dependent, with "chromatoid body syndrome" occurring over a relatively narrow concentration range. Cyclohexamide, in contrast to chloramphenicol or puromycin, does not produce a clear increase in the size of chromatoid bodies, and is capable of blocking the action of the other drugs at normally effective concentrations. Results obtained in this investigation suggest that primary spermatogonia contain enough chromatoid body material to account for the total amount present in all subsequent germ cell stages. This fact, coupled with other studies where chromatoid-like bodies have been observed, suggests the hypothesis that the chromatoid body represents at least in part an aggregation stage of materials associated with the microtubule population of the germ cell line. Alternately, or in addition, ribonucleoprotein may contribute to the structure of the chromatoid body.


Ultrastructure of the adenohypophysis in the teleost Poecilia latipinna. In the sailfin molly, Poecilia latipinna, seven morphological endocrine cell-types could be distinguished with the electron microscope. Each of these was identified with one of the seven cell-types distinguished with the light microscope, to most of which endocrine functions have previously been allocated. Corticotrophs and prolactin cells form the rostral pars distalis, and the proximal pars distalis consists of an outer layer of gonadotrophs and an inner zone containing growth hormone cells and thyrotrophs. The pars intermedia contains two cell-types, of uncertain function. Stellate cells (interstitial cells) occur throughout the adenohypophysis, but are most numerous and prominent in the rostral pars distalis. The inner proximal pars distalis contains a cell-type not previously distinguished in this species with the light microscope, the Z-cell, which could be aminergic. The ultrastructural features of each cell-type are described in detail, and discussed in comparisons with the homologous cells described in other teleosts. There is good agreement for different teleosts in the ultrastructural details of each cell type.


Corticoid uptake by the paraventricular nucleus in the hypothalamus of the duck, Anas platyrhynchos. 3H-corticoids were localized by autoradiography in small neurons in the area of the magnocellular paraventricular nucleus of mallard ducks. Correlative data show that: (1) the label is principally unmetabolized steroid, (2) the hypothalamus competitively binds corticosterone, (3) the paraventricular nucleus contains immunoreactive neurophysin, is richly innervated by boutons of monoaminergic nerves and is involved in the adaptive response to osmotic stress.


Disposition of cytosine arabinoside (NSC-63878) and its metabolites: a pharmacokinetic simulation. A computer-based model of the pharmacokinetics of cytosine arabinoside (ara-C) and its active metabolite, ara-CTP, has been developed. This model, which is an intracellular extension of the Bischoff-Dedrick model of multi-organ pharmcokinetics gives predictions which are in agreement with the recent measurements of Chou et al on tissue concentrations of ara-CTP and Borsa et al on blood levels of ara-C. It is shown that the ara-CTP halving time in tissue is much greater than the ara-C halving time in blood because of low tissue levels of phosphatase. For a single dose at the LD10 level in mice, significant splenic DNA inhibition is calculated to occur for 26 hours, while the ara-C levels are negligible in 6 hours. The calculated duration of cytostatic effect at lower dosages (2.5 mg/kg) is 10 or 12 hours, while ara-C blood levels are negligible within 3 hours. Implications for cell kinetics and scheduling studies are also briefly described.


Ontogenesis of the alpha-MSH, beta-MSH and ACTH cells in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity. Pituitary sections from 15 to 21 day-old rat foetuses have been studied with the immunofluorescence technique, using antibodies anti alpha-MSH, anti beta-MSH and anti beta (1-24) ACTH. The first ACTH cells appear on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia. beta-msh cells have been observed on day 16 in the pars anterior and on day 17 in the pars intermedia, while alpha-MSH cells appear only on day 18 and exclusively in the pars intermedia. The cytodifferentiation of the beta-MSH and ACTH cells occurs in the pars intermedia with about a 24 hours delay in comparison to that of the pars distalis. The first revealed cells are always located in the posterior half of the pituitary gland. The corticostimulating activity of the hypophysis has been tested with the fluorescence intensity of the corticotrophs, the adrenal weight, the adrenal content in corticosterone and the plasma corticosterone level. The fluorescence of the corticotrophs increases on day 18, shows a maximum on day 19 and decreases until term. The adrenal weight rises regularly between day 16 to day 20, thereafer the increase subsides. Adrenal and plasma corticosterone concentrations reach a peak on day 19 of pregnancy. These data suggest that hypophyseal corticostimulating activity is very high between days 18 and 19 and decreases between days 19 and 21.


Trophic influences of sympathetic nerves and cyclic AMP on differentiation and proliferation of isolated smooth muscle cells in culture. Smooth muscle cells of the newborn guinea-pig vas deferens dispersed into single cells and grown in culture maintain their differentiation for approximately 5 days before undergoing dedifferentiation and mitosis. The presence of sympathetic nerve fibres in contact with the isolated cells delays this process by 3-7 days (Chamley et al., 1974). A similar delay in dedifferentiation of vas deferens smooth muscle cells in tissue culture in the presence of sympathetic ganglion extract is described in the present report, demonstrating that the trophic effect is elicited by a chemical substance. This effect is mimicked by the presence of either a confluent layer of RKA epithelial cells, dibutyryl cyclic AMP or theophylline. A similar, but considerably weaker, effect is also obtained with spinal cord and liver extracts and noradrenaline. Acetylcholine does not show an effect. It is suggested that a trophic substance (probably not noradrenaline) from sympathetic neurons activates the adenyl cyclase system of smooth muscle cells to increase the intracellular level of cyclic AMP which in turn promotes and maintains the differentiation of the cultured smooth muscle cells.


The retinal pigment epithelium and photoreceptor cells. Light- and electron microscopic studies on monkey eyes. In the perifoveal retina of the monkey, Cercopithecus aethiops, the melanin granules are accumulated in apical cytoplasmatic protrusions of the pigment epithelial cells, facing the end of the cones. The rods are inserted deeper into the pigment epithelium than the cones; they reach the bottom of the infoldings of the apical surface membrane of the pigment epithelial cells. No melanin granules or other inclusions are situated at the end of the rods. The outer extremity of the rods is considerably inclined and in sections often appears as groups of rod discs which are incompletely or completely separated from the main part of the outer segments. This separation is regarded as an artifact caused by the inclination of the rods, and it is therefore not considered to represent phagocytosis of the outer segments by the pigment epithelium. The inclusions of the pigment epithelial cells are classified in five categories which seem to be related to each other owing to their shared structural characteristics. It is suggested that melanin granules are produced, modified and destroyed by the pigment epithelial cells of the adult. Because of the relations between the photoreceptors and the melanin granules it is suggested that light scattered by the melanin granules may pass backwards through the outer segments of the cones, but not of the rods.


Ultrastructural identification of umbilical cord vein endothelium in situ and in culture. The ultrastructure of human umbilical cord vein endothelium in situ, after isolation by collagenase treatment, and in primary culture is described. The cultured cells formed a monolayer with typical "butt" and interdigitated junctions with specialized areas, and contained Weibel-Palade bodies, rod-shaped tubular organelles considered specific of endothelial cells. These morphological features were not present in cultures of human skin fibroblasts and fibroblast-like cells derived from umbilical cords. It is thus concluded that endothelial cells retain their characteristic fine structure in primary culture. Simple ultrastructural studies can thus be used to identify endothelial cells in culture.


Immunofluorescent localization of sites binding anti-synthetic LHRH serum in the median eminence of the greenfinch (Chloris chloris L.). With an indirect immunofluorescence technique and an antiserum raised against synthetic LHRH, immunofluorescent granules were observed in fine radially oriented fibres in the palisade layer of the zoma externa in both the anterior and the posterior divisions of the median eminence in the greenfinch. There was no specific immunofluorescence in the tractus supraoptico-hypophyseus. It is concluded that the greenfinch median eminence contains material which is similar to mammalian LHRH.


Studies on the function of polyadenylic acid on poliovirus RNA. RNAase H was used to remove selectively over 83% of 3' terminal polyadenylic acid [poly(A)] from poliovirus RNA. In a single cycle infection with this poly(A)-deficient RNA, no evidence for multiplication of the RNA was found. To elucidate further the function of poliovirus poly (A), the translational capacity of the RNAase H-treated and untreated RNA was studied in a cell-free protein synthesizing system prepared from HeLa cells. Removal of most of the poly(A) did not affect the pattern or amount of polypeptides synthesized. We conclude that the poly(A) on poliovirus RNA is probably necessary to the replication of the molecule.


Creatine kinase, myokinase, and acetylcholinesterase activities in muscle-forming primary cultures of mouse teratocarcinoma cells. Multipotential mouse teratocarcinoma cells in embryoid bodies were explanted on plastic or collagen substrates. Various modes of cell determination, including myogenesis, occurred. The predominant avenue of differentiation soon became myogenesis: many multinucleated myotubes formed and yielded an extensive network of skeletal muscle fibers. The process does not proceed to normal completion, as the fibers have a paucity of striations and are not contractile. Activities of several enzymes ordinarily associated with muscle differentiation were examined. Acetylcholinesterase activity increases, especially during myotube formation, as in normal myogenesis. However, creatine kinase activity rises during myotube formation and then drops abnormally, and myokinase activity fails to increase appreciably. The fetal isozymic form of creatine kinase is expressed in the cultures, although well differentiated solid tumors taken from mice show attainment of the adult muscle isozyme type if skeletal muscle is demonstrably present. The results are consistent with the interpretation that coordinately regulated changes in gene expressions controlling these functions may be required for later stages of myogenesis.


Genetic control of chlorophyll biosynthesis in chlamydomonas: analysis of a mutant affecting synthesis of delta-aminolevulinic acid. A Mendelian mutation, r-1, in Chlamydomonas reinhardtii has been isolated which elevates protoporphyrin accumulation of the Mendelian protoporphyrin mutants brS-1 and brC-1 more than 20 fold. This increased protoporphyrin accumulation is shown to result from increased delta-aminolevulinic acid synthesis in the double mutants brS-1 r-1 and brC-1 r-1 over that of brS-1 and brC-1 alone. By itself, the r-1 mutation has no detectable protoporphyrin accumulation and has reduced levels of delta-aminolevulinic acid synthesizing activity, chlorophyll, protoheme, and cytochrome oxidase activity. The low levels of chlorophyll and protoheme in r-1 can be increased by feeding delta-aminolevulinic acid. We hypothesize that r-1 may be a mutation of the gene coding for the delta-aminolevulinic acid synthesizing enzyme which reduces the sensitivity of this enzyme to feedback inhibition by protoporphyrin or heme as well as reducing the overall activity of the enzyme. Evidence is also presented for a single delta-aminolevulinic acid synthesizing enzyme serving both chlorophyll and heme biosynthesis.


[Effects of theophylline on chemically induced liver tumorogenesis and on bladder membranes in rats]. The addition of theophylline to the diet of rats given the carcinogen 4-dimethylaminoazobenzene did not significantly impair the development of hepatomas. In contrast, the feeding of theophylline has been associated to some histopathological changes in bladder membrane leading to the occurrence of malignant papillomas.


[Interconvertibility of molecular forms of 3'5' cyclic AMP phosphodiesterase of human platelets]. Three forms of cyclic AMP phosphodiesterase from human platelets are resolved upon sucrose gradient centrifugation : 2.5S (monomer), 4.8S (dimer) and 7S (tetramer). They are interconvertible and form an association-dissociation equilibrium depending on the concentration of enzyme. The dissociated form has a high Km for cyclic AMP (Km : 3-5.10(-4) M) whereas the associated form has a low Km (Km : 3-5.10(-5) M).


[Coprologic survey in Bilharzia infected children of the Adzope region (Ivory Coast)]. During a stool examination survey on 174 bilharzia attained children living in a rural area of Ivory Coast, 93% were found to have intestinal parasites mainly Entamoeba coli, Necator americanus and Ascaris lumbricoides. The prevalence of the cysts of Entamoeba histolytica (4,7%) is lower than the one found in France (7%). Concerning the other intestinal parasites, their respective occurrence have been compared with those noted in other regions of Ivory Coast and of Africa.


[Androgen receptors in the cytosol of normal mucosa and of pharyngo-laryngeal epitheliomas in humans]. Specific binding for 5 alpha dihydrotestosterone has been characterized in cytosol fraction from human pharyngeal and laryngeal mucosae. The high binding affinity of the protein receptor (Kd 2 x 10(-10)M) and its low capacity are similar in both sexes, and similar in both normal mucosa and epithelioma.


[Pralidoxime prevents certain teratogenic effects induced by bidrin in quail embryos]. Bidrin treatment of quail embryos results in axial anomalies as well as malformations of the beak and the limbs. Whereas the administration of pralidoxime to teratogen-treated embryos prevents the appearance of the axial anomalies, the morphogenesis of the beak and limbs remains profoundly altered.


Noninvasive quantitation of myocardial infarction with technetium 99m pyrophosphate. We sought to quantitate infarct size using radioactive imaging techniques. Infarcts were created in closed chest dogs. Using a scintillation camera interfaced to a computer, infarct images were made in the anterior, left lateral, LAO, and RAO projections, 48 hours after infarction and 75 to 90 min following the intravenous injection of 15 mCi of Technetium 99m pyrophosphate (Tc-PYP). Images were computer enhanced and area was calibrated with a radioactive grid source of known dimensions. Image radioactivity was normalized for decay and dose corrected for body weight. Animals were sacrificed two hours following the injection Tc-PYP. Postmortem images were also computer enhanced and calibrated. Gross infarct area and weight were estimated and transmural biopsies were evaluated for Tc-PYP activity and analyzed for creatine phosphokinase (CPK) content. Contiguous biopsies were pathologically analyzed and graded. There was a negative correlation between tissue Tc-PYP activity and CPK content (r=0.89). Pathologic severity worsened with increased Tc-PYP activity and diminished CPK content. There was a good correlation between gross infarct area and image infarct area, both in vivo (r


Biochemical studies on tissues from a patient with Lafora disease. Tissues from the cerebral cortex, liver and myocardium of a patient with Lafora disease were obtained at autopsy and were studied biochemically. 1. Glucose content in the myocardium and liver was almost nil while that in the controls was 0.66 mg/g wet weight in the former and 8.80 mg/g wet weight in the latter. Glycogen content in the cerebral cortex and myocardium was about 10 and 3 times more than in controls. 2. Polyglucosan extracted from the cerebral cortex, liver and myocardium had a longer exterior glucose chain than that in the liver of the control but a normal, alpha or beta 1,4-glucosidic linkage was observed. 3. The activities of glucose-6-phosphatase and amylo-1,6-glucosidase in the cerebral cortex, liver and myocardium were well preserved. The activities of acid maltase in the three organs mentioned above and of neutral maltase in  the myocardium were elevated twice and one and half times more than the control. Phosphorylase levels in the myocardium were extremely small, while in the cerebral cortex and liver normal activities were observed. In light of these findings, glycogen metabolism in Lafora disease is discussed.


[Ultrastructural observations of the velamentous astrocyte impregnated by Golgi's method]. Electron microscopy of the velamentous astrocyte in the cat inferior colliculus was performed on serial semi-thin and ultra-thin Golgi preparations. The glial origin of the velum has been considered and its topographical relationships with the other structures of the neuropile have been described.


Effects of apolipoproteins on lipoprotein lipase activity of human adipose tissue. The effect of apolipoproteins isolated from HDL and VLDL on the activity of lipoprotein lipase (LPL) of adipose tissue was studied. The CII apoprotein was found to activate LPL. This activation was strongly inhibited by CI, AI (apo-Lp-Gln I), and the arginine-rich apoprotein, whereas AII and CIII exhibited a considerably lower inhibitor effect.


Biochemical typing of hyperlipoproteinemia. The development of a nomogram. The accurate biochemical typing of hyperlipoproteinemia would require quantification of lipoprotein fractions. At present, typing is frequently based on the lipoprotein electrophoresis pattern together with serum lipid analyses. Lack of facilities for lipoprotein electrophoresis has however focused the clinical interest for classification of hyperlipoproteinemia into other means of a simple biochemical typing. In the present study a nomogram was developed to allow biochemical typing of hyperlipoproteinemia from serum cholesterol and triglyceride values. Serum cholesterol was determined according to a Liebermann-Burchard reaction by the method of Cramer and Isaksson [1], serum triglycerides by the determination of glyceride glycerol according to Carlson [2], and serum lipoprotein electrophoresis was performed on agarose gel [3]. Cholesterol content [4] of alpha-lipoproteins ("alpha-LP cholesterol") was obtained in serum after the precipitation of very-low-density lipoproteins(VLDL) and low-density lipoproteins(LDL) by manganese chloride and heparin [5]. Preparative ultracentrifugation was performed in a one-step, gradient procedure [6] isolating VLDL, d less than 1.006, LDL, d 1.006--1.063 g/ml and high-density lipoprotein (HDL) including very-high-density lipoproteins, d greater than 1.063 g/ml, or alternatively at d 1.006 g/ml according to the procedure by Gustafson et al. [7].