Lipid composition of serum lipoproteins in patients with primary type IIb and type IV hyperlipoproteinemia. Cholesterol, triglyceride and phospholipid concentrations of VLDL, LDL and HDL were studied in 20 patients with primary type IIb, 25 patients with primary type IV and in 18 controls. Both types are not only characterized by different concentrations of lipoprotein lipids, but also by their different lipid composition. Type IIb had more triglycerides in the LDL, type IV in the LDL and HDL. The HDL cholesterol content of type IV was decreased. The percent phospholipid concentration of HDL was identical in the 3 groups, demonstrating the constant role of this lipid fraction. The lipid relationships between the lipoproteins showed that the LDL/HDL lipid ratio of type IIb exceeded type IV ratio in spite of normal HDL lipid concentration in type IIb.


Relative increase in apolipoprotein CII content of VLDL and chylomicrons in a case with massive type V hyperlipoproteinaemia by nicotinic acid treatment. VLDL of fasting serum was fractionated into 4 fractions of decreasing particle size by preparative ultracentrifugation in a density gradient from a patient with massive type V hyperlipoproteinaemia before and after treatment with 12 g daily of nicotinic acid. Serum triglycerides fell from 58 to 9 mmol/1 in response to treatment due particularly to reduction of larger VLDL particles. Total, insoluble (apoprotein B) and soluble protein of all VLDL fractions also fell but the ratio of these 3 protein fractions to triglyceride rose particularly in the smaller VLDL fractions. The relative amount of apolipoprotein CII increased in all fractions and the ratio apo CII to triglycerides increased by 60--90% in all VLDL fractions. The relative amounts of apo CI, apo CII-2 and apo E appeared also to increase. The results are consistent with the hypothesis that low amounts of apo CII may play a role for the development of hypertriglyceridaemia.


HDL-cholesterol, apolipoproteins A1 and B. Age and index body weight. Some data of previous literature have emphasized a negative correlation between plasma apo A1, HDL cholesterol and coronary heart disease. The present paper stresses a high negative correlation existing both in females and males between values of index body weight (IBW) and plasma levels of HDL cholesterol and apo A1. No correlation has been found between age and, respectively, HDL cholesterol, apo A1 and apo B.


Further studies on serum lipoproteins in connective tissue diseases. Men with ankylosing spondylitis (AS) and females with systemic lupus erythematosus (SLE) were found to have low total serum lipid concentrations similar to results previously obtained in patients with rheumatoid arthritis (RA). In AS men total serum TG was about 50% of control values and in AS men and SLE women total serum cholesterol was 78% of control values but close to corresponding RA concentrations. This was explained mainly by low LDL concentrations. There was a marked difference between RA patients and AS and SLE patients in that the two latter groups had normal HDL cholesterol concentrations whereas in RA patients the HDL cholesterol concentration was only 70% of control values. Thus in spite of similar and low total serum lipid concentrations, differences in lipoprotein composition were found in the three different rheumatic diseases, underlining the importance of lipoprotein analyses in the study of dyslipoproteinaemia.


Neuromuscular refractoriness during blockage of transmission. A quantitative study. The effects of suxamethonium and tubocurarine on the refractoriness of neuromuscular transmission were studied in 21 anaesthetized adult subjects under various levels of neuromuscular block. The ulnar nerve was stimulated every 12 s with twin supramaximal stimuli 4 ms apart. At any level of block, the refractory fraction (the fractional decrement of the compound electromyographic response of the adductor pollicis to the second twin stimulus, relative to the response to the first) was used to quantify neuromuscular refractoriness. The magnitude of block was determined by the response to the first stimulus. Correlation between refractoriness and the degree of block was sought. Without block, neuromuscular transmission averaged 23% (SEM 4) refractory with this twin interval. The refractory fraction was increased markedly by suxamethonium, reaching 0.69 (SEM 0.1) at 50% block. Complete refractoriness occurred during 25--75% block in eight of 11 instances. Tubocurarine did not significantly alter refractoriness, paired responses to the twin stimuli decreasing proportionately during block.


Prolyl hydroxylase and collagen biosynthesis in rat liver following varying degrees of partial hepatectomy. Prolyl hydroxylase activity and collagen biosynthesis have been measured at intervals following 2 extents of hepatic resection in rats. Prolyl hydroxylase activity is validated as a measure of collagen biosynthesis. The levels rise to a peak at 72 h and thereafter decline. The implications for the process of liver regeneration are discussed.


Studies on the effect of quartz, bentonite and coal dust mixtures on macrophages in vitro. The effect of quartz, bentonite and coal dusts as well as the effect of the artificial mixture of these dusts on TTC reduction and extra-and intra-cellular lactate dehydrogenase activity in peritoneal rat macrophages was determined in vitro. The cell-membrane-damaging effect of quartz caused a significant extracellular release of lactate dehydrogenase. Bentonite caused no extracellular enzyme release, which leads us to believe that the biological effect of this dust is shown by decrease in intra-cellular lactate dehydrogenase activity. TTC reduction was inhibited equally by quartz and qentonite. In mixtures of quartz (60%)-bentonite (40%) dust the specific effect of quartz was inhibited by bentonite in vitro and also in vivo. We obtained the same results with coal-quartz-bentonite dust mixtures in vitro. Our experiments show that comparison of the biological effects of artificial dust mixtures and airborne dust samples is justified, and prove that performing various examinations simultaneously give fuller particulars on the probable biological effect of mineral dusts.


A new syndrome with pigmentation, scleroderma, gynaecomastia, Raynaud's phenomenon and peripheral neuropathy. A middle-aged man developed impotence, gynaecomastia, peripheral neuropathy, pigmentation lymphadenopathy, scleroderma, malabsorption and Raynaud's phenomenon following prolonged exposure to trichlorethylene. Somes, but not all, of these features have previously been reported in vinyl chloride disease, and trichlorethylene is related chemically to vinyl chloride. Seven patients with a similar but not identical syndrome have been described in Japan and U.S.A., apparently unrelated to exposure to industrial toxins.


Cell membrane enzymes. II. Alkaline phosphatase and alkaline phosphodiesterase I in normal and leukaemic lymphocytes. The distribution of two cell membrane enzymes, alkaline phosphatase and alkaline phosphodiesterase I has been studied in normal and leukaemic lymphocytes. No reduction in the level of activity of either enzyme was found in the chronic or acute B- and T-cell leukaemias. Alkaline phosphatase activity was elevated in the lymphocytes from T-CLL, cord blood and tonsils and the blast cells from Null-ALL. Alkaline phosphodiesterase was elevated in lymphocytes from cord blood and tonsils and the blast cells from Null-ALL. As findings in Null-ALL were based on only two cases, they need confirmation in a larger series. The significance of these results is discussed in relation to current theories of maturation and differentiation in the lymphoproliferative disorders.


Chemical modification studies on the Ca2+-dependent protein modulator: the role of methionine residues in the activation of cyclic nucleotide phosphodiesterase. Methionine residues have been implicated in the activation of cyclic nucleotide phosphodiesterase by the Ca2+-dependent protein modulator [Walsh, M., & Stevens, F.C. (1977) Biochemistry 16,2742-2749]. Treatment of the modulator with N-chlorosuccinimide in the presence of Ca2+ resulted in selective oxidation of methionine residues at positions 71,72, 76, and, possibly, 109 in the modulator sequence. These residues lie on the surface of the molecule exposed to solvent. This modification has several effects on the modulator protein: (1) the Ca2+-binding properties of the oxidized modulator are changed with apparent loss of high-affinity binding sites, (2) the oxidized protein no longer interacts with phosphodiesterase, and (3) troponin C like activities, viz., Ca2+-dependent change in mobility on urea-polyacrylamide gel electrophoresis and formation of a urea-stable complex with troponin I, are lost upon oxidation of the modulator. The phosphodiesterase binding domain of the modulator protein appears to be located between the second and third Ca2+-binding loops, a region of the molecule known from previous partial proteolysis studies [Walsh, M., Stevens, F.C., Kuznicki, J., & Drabikowski, W.(1977), J. Biol. Chem. 252, 7440-7443] to be exposed in the presence of Ca2+.


Fluorescence and chemical studies on the interaction of Escherichia coli DNA-binding protein with single-stranded DNA. Nanosecond and steady-state fluorescence spectoscopy were used to probe the environment of the tryptophan residues of Escherichia coli DNA-binding protein. A spectral shift and a change in quantum yield of the protein upon binding to DNA or oligonucleotides indicate that the tryptophan residues are near or at the DNA binding site. The observation of two excited-state lifetimes of the protein indicates that there is heterogeneity in the microenvironments of these tryptophan residues. The "short-lifetime" tryptophan residues are more sensitive to the interaction with DNA than the "long-lifetime" residues. The results of solute-perturbation studies with iodide or acrylamide indicate that there are tryptophan residues near the surface of the protein which are heterogeneous in their accessibility to these quenchers and that they become less accessible after DNA binding. Also, lysine residues of the protein have been shown to be essential to DNA binding by chemical-modification studies. Tyrosine, arginine, and cysteine residues appear not to be involved in this binding process. From studies of the decay of fluorescence anisotropy of the binding protein in the presence and absence of DNA, it has been concluded that (a) the tetrameric binding protein does not dissociate into subuniits upon binding to the oligonucleotide d(pT)16 and (b) the binding protein-fd DNA complex possesses "local flexibility" and, therefore, cannot be described as a continuous, rigid rod.


Qualitative aspects of hydrogen-deuterium exchange in the 1H, 13C, and 15N nuclear magnetic resonance spectra of viomycin in aqueous solution. The 1H, 13C, and 15N high field nuclear magnetic resonance spectra of the cyclic peptide viomycin have been fully assigned using homo- and heteronuclear double resonance experiments and pH effects. In addition it is shown how the two- and three-bond H-D isotope effects upon carbonyl resonances may assist in their assignment. The resistance to exchange with solvent water of the amide proton involved in the transannular hydrogen bond is observed directly in the 1H spectra, via the isotope effect on a carbonyl resonance in the 13C spectra, and via the one-bond 1H couppling in the 15N spectra.


Transmembrane movement and distribution of cholesterol in the membrane of vesicular stomatitis virus. The transmembrane movement and distribution of cholesterol in the vesicular stomatitis virus membrane were studied by following the depletion of cholesterol from virions to interacting phospholipid vesicles and by exchange of radiolabeled cholesterol between virions and phospholipid-cholesterol vesicles. The kinetics of the cholesterol exchange or depletion reactions revealed the presence of two exponential rates: a rapid rate, dependent on the vesicle to virus ratio, and a slower rate, independent of the vesicle to virus ratio. The kinetics of cholesterol movement could be best interpreted by a model of the virion membrane considered as a two pool system in which approximately 30% of the cholesterol resides in the outer monolayer and approximately 70% in the inner monolayer. The half-time for equilibration of the two pools was calculated to be 4--6 h and was assumed to represent the time required for transmembrane movement of cholesterol across the bilayer. The initial rate of transfer of cholesterol from virus into vesicles increased when vesicle phospholipids contained more unsaturated and shorter chain fatty acids. Furthermore, the transfer of cholesterol appeared to occur by a collisional mechanism requiring membrane-membrane contact. Interaction with lipid vesicles did not significantly affect the integrity of the virion membrane as assessed by the relative inaccessibility of internal proteins to lactoperoxidase-catalyzed iodination and by the small loss of [3H]amino acid labeled protein from the virus.


Magnetic cross-relaxation among protons in protein solutions. The magnetic spin-lattice relaxation rates of solvent water nuclei are known to increase upon addition of diamagnetic solute protein. This enhancement of the relaxation rate is a function of magnetic field, and the orientational relaxation time of the protein molecules can be deduced from analysis of the field-dependent relaxation rates. Although the nature of the interactions that convey information about the dynamics of protein motion to the solvent molecules is not established, it is known that there is a contribution to the relaxation rates of solvent protons that plays no role in the relaxation of solvent deuterons and 17O nuclei. We show here that the additional interaction arises from a cross-relaxation process between solvent and solute protons. We introduce a heuristic three-parameter model in which protein protons and solvent protons are considered as two separate thermodynamic systems that interact across the protein-solvent interface. The three parameters are the intrinsic relaxation rates of each system and a cross-relaxation term. The sign of the latter term must always be positive, for all values of magnetic field, in order for magnetization energy to flow from the hotter to the cooler system. We find that the magnetic field-dependence of the cross-relaxation contribution is much like that of the remaining solvent proton relaxation, i.e., about the same as the deuteron relaxation field dependence. This finding is not compatible with the predictions of expressions for the cross-relaxation that have been used by other authors, but not applied to data over a wide range of magnetic field strength. The model predicts that the relaxation behavior of both the protein protons and the solvent protons is the sum of two exponentials, the relative contributions of which would vary with protein concentration and solvent isotopic composition in a fashion suggestive of the presence of two classes of protein protons, when there is in reality only one. This finding has immediate implications for the interpretation of published proton relaxation rates in complex systems such as tissues; these data should be reexamined with cross-relaxation taken into account.


The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria. The phospholipid depletion of rat liver mitochondria, induced by acetoneextraction or by digestion with phospholipase A2 or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A2. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipid-deficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes. These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.


Variable chlorophyll a fluorescence from P-700 enriched photosystem I particles dependent on the redox state of the reaction centre. 1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence. 2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs. 3 EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite. 4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles. 5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P+X are more effective quenchers of chlorophyll fluorescence than PX-, where P is P-700.


Flash-induced photophosphorylation in Rhodospirillum rubrum chromatophores. I. The relationship between cytochrome c-420 content and photophosphorylation. The content of cytochrome c-420 in Rhodospirillum rubrum chromatophores prepared by grinding with alumina is 5--10% of that in whole cells, and 20--40% in chromatophores by 'French' pressing. Flash-induced phosphorylation of various chromatophores which varied in cytochrome content from 7 to 40% is proportional to the cytochrome content. Extrapolating the cytochrome c-420 content to that observed in whole cells, a ratio ATP/P+X- near 1 is calculated. At low flash intensity the phosphorylation per flash is proportional to flash energy. Photophosphorylation in flashes given after a time of several minutes is only slightly dependent on the number of flashes. If the flashes are spaced from 0.1 to 10 s, relative phosphorylation in the first flash is about 70% and in the second 90+ of that observed in the following flashes. Proton binding is not affected by the cytochrome c-420 content and a ratio of H+/P+x- of 2.3 was found. These results can be explained by a working hypothesis in which charge separation occurring at one reaction centre and the resulting electron transport mediated amongst others by c-420, results in the injection of two protons into an ATPase, this in contrast to a chemiosmotic mechanism, where the protons are released in the chromatophore inner space.


Pseudomonas cytochrome c peroxidase. Initial delay of the peroxidatic reaction. Electron transfer properties. A delay of some seconds is observed in the reaction of Pseudomonas cytochrome c peroxidase if the reaction is initiated by adding the enzyme to the reaction mixture containing reduced electron donor and hydrogen peroxide. This lag phase is avoided if the enzyme is incubated with the reduced electron donor and the reaction is started by adding hydrogen peroxide. The nature of the initial delay has been studied and it is shown that the peroxidase is reduced before a steady-state rate in the peroxidatic reaction is reached. The ability of the peroxidase to accept electrons from various electron donors emphasizes its cytochrome-like properties.


Effects of inhibitors of lipid synthesis on the replication of Rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins. The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholestetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1(-14C)]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [35S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examinatin of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.


The influence of cellular amino acids and the Na+ : K+ pump on the membrane potential of the Ehrlich ascites tumor cell. The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.


Effects of adenosine 3' : 5'-monophosphate and guanosine 3' : 5'-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle. The effects of adenosine 3' : 5'-monophosphate (cyclic AMP), guanosine 3' : 5'-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P). While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10(-5) M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP. Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10(-8) M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10(-8) M, while with cyclic AMP a concentration of 10(-5) M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P. These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.


Plasma membranes from rat intestinal epithelial cells at different stages of maturation. I. Preparation and characterization of plasma membrane subfractions originating from crypt cells and from villous cells. To determine the mechanism of the maturation of the brush border membrane in intestinal epithelial cells, purification of the plasma membrane from undifferentiated rat crypt cells and of the basal-lateral membrane from villous cells has been performed. The method is based on density perturbation of the mitochondria to selectively disrupt their association with the membrane. With both cell populations, two membrane subfractions displaying the same respective density on sucrose gradient have been obtained with an overall yield of 15--20% and a 10-fold enrichment of the plasma membrane markers 5'-nucleotidase and (Na+ + K+)-dependent, ouabain-sensitive ATPase chosen to follow their purification. The four fractions were constituted by sheets and apparently closed vesicles of various sizes. Each fraction was characterized by a distinct protein composition and different levels of enzyme activities. The cells, used for the preparation of the membranes, were isolated as a villus to crypt gradient. This separation and that of the membranes, led to the conclusion that the (Na+ + K+)-dependent ATPase is localized principally in the plasma membrane of all cells whatever their state of maturation, while 5'-nucleotidase is predominantly located in the basal-lateral membrane of the villous cells and may serve as a specific marker for the purification of this membrane. Finally it has been shown that aminopeptidase, dissacharidases and alkaline phosphatase do not appear simultaneously in the maturation process of the cells, alkaline phosphatase being absent from the crypt cells and aminopeptidase being the first to be synthesized. This enzyme seems to appear in the crypt cells membrane before being integrated into the mature brush border membrane.


Specific association of two homologous DNA-binding proteins to the native 30-S ribosomal subunits of Escherichia coli. The native 30-S ribosomal subunits from Escherichia coli are shown to be associated with two proteins which are different from the known ribosome-associated and ribosomal proteins. Neither protein is foune on native 50-S subunits or on intact ribosomes in the cell extract. The purified proteins re-bind in vitro to free 30-S subunits, but do not bind to either free 50-S subunits or intact ribosomes. The proteins, denoted NS1 and NS2, have been purified and characterized. Both proteins showed the same molecular weight of 9500 by sodium dodecyl sulfate gel electrophoresis but 34 000 by gel filtration. Upon treatment with cross-linking reagents the purified proteins gave higher molecular weight species up to the tetrameric ones showing that they exist in solution as tetramers. The amino acid compositions, tryptic fingerprint patterns and N-terminal sequences of the two proteins have been determined. These data show that NS1 and NS2 possess distinct primary structures but with extensive sequence homology. Antibodies raised against the purified proteins cross-reacted in double immuno-diffusion tests confirming further the homology. Because of the similarity in properties a sample of the DNA-binding protein HD (Berthold, V. and Geider, K. (1976) Eur. J. Biochem. 71, 443--449) was compared to NS1 and NS2. In terms of several criteria, the protein HD is found to be a mixture of two proteins, namely NS1 and NS2. The present report is the first instance of an association of DNA-binding proteins to the ribosome.


The phosphohydrolase activity in lamellar bodies and its relationship to phosphatidylglycerol and lung surfactant formation. Phosphohydrolase activity of a lamellar body-enriched preparation from pig lung was examined to ascertain if two separate enzymes catalyze the hydrolysis of phosphatidic acid and phosphatidylglycerol 3-phosphate. From sulfhydryl inhibition, heat inactivation and substrate specificity studies, we suggest that one phosphohydrolase may account for the hydrolysis of both substrates. The relationship of the reported experiments to the biosynthesis of the glycerophospholipids present in surfactant is discussed.


Electron spin resonance studies of radicals obtained by the reaction of alpha-tocopherol and its model compound with superoxide ion. alpha-Tocopherol (vitamin E) and its model compound, 6-hydroxy-2,2,5,7,8-pentamethylchroman, were found to be oxidized by O2- to yield free radicals which were detected at room temperature by ESR spectroscopy. The ESR spectra of these radicals showed seven main lines with additional hyperfine structure and have the same g-values at 2.0046. Assignments of the ESR spectra were done on the basis of the spectra of the free radicals of deuterated hydroxypentamethylchroman obtained from the same reaction with O2-. The radicals observed are chromanoxyls generated by the abstraction of hydrogen from the 6-hydroxy group of tocopherols.


Nitrogenase from Bacillus polymyxa. Purification and properties of the component proteins. A purification procedure is described for the components of Bacillus polymyxa nitrogenase. The procedure requires the removal of interfering mucopolysaccharides before the two nitrogenase proteins can be purified by the methods used with other nitrogenase components. The highest specific activities obtained were 2750 nmol C2H4 formed . min-1 . mg-1 MoFe protein and 2521 nmol C2H4 formed . min-1 . mg-1 Fe protein. The MoFe protein has a molecular weight of 215 000 and contains 2 molybdenum atoms, 33 iron atoms and 21 atoms of acid-labile sulfur per protein molecule. The Fe protein contains 3.2 iron atoms and 3.6 acid-labile sulfur atoms per molecule of 55 500 molecular weight. Each Fe protein binds two ATP molecules. The EPR spectra are similar to those of other nitrogenase proteins. MgATP changes the EPR of the Fe protein from a rhombic to an axial-type signal.


Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae). Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.


Conformation of corticotropin. An infrared spectrometry study of hydrogen exchange kinetics. 1H--2H exchange kinetics of the peptide hydrogens in corticotropin have been examined in 2H2O and CF3C2H2O2H solutions by means of infrared absorption measurements. In aqueous solution, around pH 3, the experimental data suggest a partially ordered structure, since in the two corticotropins 1--24 and 1--32 about 6 slowly exchanging peptide protons are numbered. These might belong to the N-terminal part of the molecule. The C-terminal 25--32 octapeptide segment appears to be unordered and slightly destabilizes the overall hormone conformation. For corticotropin1--24 in CF3C2H2O2H, the qualitative interpretation of infrared spectra and the quantitative analysis of exchange data give evidence of a strong stabilization: a predominantly alpha-helical structure is induced by trifluoroethanol.


Comparison of polypeptides from cultured human fibroblasts and sarcoma cells. The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.


The interaction of nitric oxide with soybean lipoxygenase-1. The interaction of nitric oxide with the non-heme iron dioxygenase lipoxygenase is reported. This apparently resulted in a novel type of complex where an electron is donated to the NO molecule. In addition a new position for an EPR transition from iron was discovered which, it is suggested results from high spin ferric iron in a field of axial symmetry characterised by a very low value for D.


Factors regulating the induction of ornithine decarboxylase in fetal rat liver cells in culture. Various hormonal and non-hormonal agents were tested for their ability to induce ornithine decarboxylase (EC 4.1.1.17) in primary cultures of fetal rat liver cells that retain many of the differentiated functions of hepatocytes. The only agents to induce ornithine decarboxylase in this cell type were fetal calf serum, prostaglandin E1 and cyclic AMP derivatives. Also, the amino acid arginine would induce ornithine decarboxylase in this cell type following arginine starvation for 24 h. These observations are in contrast to the wide range of hormones, e.g. insulin, hydrocortisone, glucagon and growth hormone, than can induce ornithine decarboxylase in vivo in the adult rat liver but which are all without effect on fetal rat liver cells.


The formation of hydroxyproline in collagen by cells grown in the absence of serum. L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but  ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggests that reductants other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.


Effects of CuCl2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart. CuCl2 non-competitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg2+, 10 muM Ca2+ and phosphodiesterase activator with Ki values of approximately 2 muM for both substrates. CuCl2 inhibition was also non-competitive with Mg2+, Ca2+ and phosphodiesterase activator. Dialysis demonstrated that CuCl2 inhibition is reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl2 was not additive with that of p-hydroxymercuribenzoate, therefore CuCl2 may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl2 also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I50 value of 18 muM. Several effects of Cu2+ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.


Topical epinephrine causes a decrease in density of beta-adrenergic receptors and cathecholamine-stimulated chloride transport in the rabbit cornea. A single administration, or twice daily administration for 4.5 days, of topical 2% epinephrine to the rabbit eye in vivo causes a 30-40% decrease in the density of beta-adrenergic receptors on membranes prepared from the cornea. Such treatment also causes complete loss of the ability of excised corneas to respond to epinephrine in vitro with enhanced active chloride transport. These findings indicate that stimulation with a high concentration of catecholamine depresses the entire pathway from receptor to physiological response.


REM sleep and memory consolidation.

REM sleep and memory consolidation. One proposal referring to a specific function of REM sleep has been that it is necessary for, or at least conductive to, the progress of memory consolidation. This hypothesis was tested by comparing the effects on story retention of REM deprivation against an S4 deprived control. It was found that recall accuracy following REM deprivation was significantly poorer than following S4 deprivation. Furthermore, the degree of deterioration in recall accuracy during REM recovery sleep was less than during S4 recovery sleep. These findings were interpreted as evidence for active REM facilitation of memory consolidation. However, alternative explanations based upon proactive influences need to be investigated.


A time-resolved electron spin resonance study of the oxidation of ascorbic acid by hydroxyl radical. Time-resolved electron spin resonance (ESR) spectroscopy for the study of radicals produced by pulse radiolysis is illustrated by a study of the oxidation of ascorbic acid by OH radical in aqueous solution. In basic solution, the direct oxidation product, the ascorbate mono-anion radical, is formed within less than 2 mus of the radiolysis pulse. In acid solutions (pH 3(-4.5), N(2)O:saturated) three radicals are initially formed, the ascorbate mono-anion radical, an OH adduct seen also in steady-state ESR experiments, and an OH adduct at C2 with the main spin density at C3 of the ring. The first OH adduct decays with an initial half-life of about 100 mus, probably by biomolecular reaction. The second OH adduct, which shows one hyperfine splitting about a(H) = 24.4 +/- 0.3 G and g = 2.0031 +/- 0.0002, decays with a half-life of about 10 mus. On this same time scale the concentration of the ascorbate radical approximately doubles. It is concluded that the adduct at C2, but not the other adduct, loses water rapidly to form the ascorbate radical.


Fast electron transfer processes in cytochrome C and related metalloproteins. Various free radicals formed on pulse radiolysis of aqueous solutions have been used to investigate the mechanisms of reduction of cytochrome(III) c by inter- and intramolecular electron transfer. The rapid formation of free radicals (t less than 1 mus) and their high reactivity with cytochrome (k approximately 10(8)(-5) x 10(10)M(-1)s(-1)) make such studies feasible. Reduction of cytochrome by free radicls is monitored by optical methods. Fast optical changes in the 1(-500)-mus region correspond to reduction of the iron center; whereas the slower changes in the 10(-500)-ms region are attributed to postreduction conformational changes. It has been concluded that the reduction path is mediated through the crevice and that no reduction intermediates are being formed.


Fast reactions in carbon monoxide binding to heme proteins. Using fast flash photolysis, we have measured the binding of CO to carboxymethylated cytochrome c and to heme c octapeptide as a function of temperature (5 degrees-350 degreesK) over an extended time range (100 ns(-1) ks). Experiments used a microsecond dye laser (lambda = 540 nm), and a mode-locked frequency-doubled Nd-glass laser (lambda = 530 nm). At low temperatures (5 degrees-120 degreesK) the rebinding exhibits two components. The slower component (I) is nonexponential in time and has an optical spectrum corresponding to rebiding from an S = 2, CO-free deoxy state. The fast component (I*) is exponential in time with a lifetime shorter than 10 mus and an optical spectrum different from the slow component. In myoglobin and the separated alpha and beta chains of hemoglobin, only process I is visible. The optical absorption spectrum of I* and its time dependence suggest that it may correspond to recombination from an excited state in which the iron has not yet moved out of the heme plane. The temperature dependences of both processes have been measured. Both occur via quantum mechanical tunneling at the lowest temperatures and via over-the-barrier motion at higher temperatures.


Modeling the role of cyclic AMP in catabolite repression of inducible enzyme biosynthesis in microbial cells. Modeling the role of cyclic AMP (cAMP) in catabolite repression of inducible enzyme production in microbial cells was studied. A catabolite repression index, F, was defined based on the postulation that complex formation occurs between RNA polymerase (RNAP) and DNA, and shifting from the inert form to the open form of this complex (the latter form is required for transcription) is accelerated by the cAMP.CRP complex. The catabolite repression index, F, was incorporated into model equations of mRNA production. Empirical relationships between intracellular cAMP level and medium glucose concentration were established based on experimental data and introduced into the model. Computer simulation results were obtained for a number of interesting cases. The practical utility of the proposed model was demonstrated by comparing it with the experimental results on glucose isomerase biosynthesis.


Sex steroid production by the human fetus: its role in morphogenesis and control by gonadotropins. Normal male genital differentiation requires fetal testicular secretion of both testosterone and the nonsteroidal müllerian-inhibitory factor. It appears that secretion of testosterone, at least during the critical period of differentiation, occurs in response to stimulation by CG, although pituitary LG may influence Leydig cell function in later fetal life. To date, there is no evidence to support a similar endocrine function for the fetal ovary in female genital differentiation. In both sexes normal fetal pituitary gonadotropin secretion appears to be required for maturation of the germ cells and related elements. The pattern of FSH and LH secretion in the fetus reflects gradual maturation of a functional hypothalamo-pituitary unit responsive to feedback inhibition by sex steroids. The higher levels of gonadotropins in female fetuses from from 12--20 weeks suggest either that this maturation occurs earlier in males than in females, or, more probably, that feedback recognition of androgens is established before gestation reflects development of an estrogen-mediated feedback mechanism, and in addition the possible influence that hormones, such as prolactin or placental estradiol, may have on testicular steroidogenesis.


[Electrophysiologic, morphometric and histocytochemical characteristics of frog sartorius muscle fibers]. Three groups of muscle fibers (dark, light, and intermediate) were revealed in the fibers of the frog sartorius muscle in examination of the succinate dehydrogenase (SDH) activity. There was revealed a reverse relationship between the diameter of the muscle fibers an the SDH activity in them. The external surface of sartorius muscle is chiefly represented by dark muscle fibers, whereas the internal one--by light ones. Microelectrode study demonstrated that the fibers of the external surface were characterized, in comparison with those of the internal one, by lesser action potentials, prolonged trace negative potential, low quant composition of the end plate potentials, high amplitude and low frequency of the end plate miniature potentials. Analysis of the data obtained demonstrated definite interrelationship between the histochemical profile of the muscle fibers of the frog sartorius muscle and their electrophysiological characteristics.


[Biochemical characteristics of the synaptosomes and mitochondria of the motor area of the cerebral cortex following switching off of sensory impulsation]. Changes in the specific activity of cytochrome oxidase, monoamine oxidase, acetylcholinesterase and Na,K-ATPase in the light and heavy synaptosomes and mitochondria of neurone bodies fractions in the motor cerebral cortex of rabbits were demonstrated under conditions of light (visual) deprivation. These changes were specific of different metabolic cycles and differed in individual cell ultrastructures. The influence of sensory impulsation on functional neuron activity in different projection regions of the cerebral cortex is discussed.


[Enzymatic lipid peroxidation and oxidative metabolism of aminazine in brain microsomal fractions]. NAD (P) H-dependent enzymic systems, both of lipid peroxidation and chlorpromazine oxidative metabolism are shown to be localized in the microsomal fractions from human and rat brain. Hydroxy-derivatives of chlorpromazine (e.g. 7-OH-chlorpromazine) formed in the course of enzymic NADPH-dependent metabolism possess antioxidant activity and inhibit lipid peroxidation in the brain microsomes. The properties of enzymic NAD (P) H-dependent oxigenase systems in the membranes of the microsomal reticulum of the liver and brain are compared.


[Effect of bradykinin and morphine on rat sensomotor cortex neurons]. As revealed in acute experiments on rats bradykinin applied microiontophoretically produced neuronal activation in the sensory-motor region of the rat brain cortex. Morphine applied iontophoretically prevented bradykinin effect. It is suggested that bradykinin interacts with opiate receptors in the cortical neurones.


[Functional activity of rat adenohypophysis cells in a primary monolayer culture and the effect of several regulators on it]. Some peculiarities of labeled growth hormone (GH) and prolactin (PL) secretion in the 5-day monolayer culture of the rat adenohypophysis was studied. The hormones from the culture medium were obtained by electrophoresis on polyacrylamide gel. Natrium-dibutyril of cyclic adenosine-3',5'-monophosphate and theophylline, stimulated the GH and PL secretion. Thyrotropin-releasing hormone (TRH) increased the incorporation of 14C-1-leucine into the cell protein, stimulated PL secretion, but did not act on the GH release. Somatostatin completely abolished the GH secretion mediated by theophyllin, but not that of PL. Some peculiarities in the formation of labeled GH and PL pool in the cells and secretion of these hormones into the culture medium are discussed.


[Enzymatic micromethod for determining Na, K-ATPase activity in rat cerebral cortex membranes]. A method for the assay of Na,K-ATPase activity of unpurified synaptosomal fraction obtained from the microquantities (2--3 mg of fresh tissue) of the rat cerebral cortex is described. This method is based on the fluorimetric determination of ADP formed in the course of ATPase reaction. The method is highly sensitive and may be used to determine the membrane preparations Na,K-ATPase activity with the protein content of 0.05--10.0 microgram per sample.


The effect of dbcAMP on the lysolecithin induced hemolysis of calf and adult cattle erythrocytes. The calf erythrocytes have an increased sensitivity against lysolecithin as compared to their adult counterparts. 10(-3)M dbcAMP increases the hemolysis induced by 5 microgram of lysolecithin in 0.15 M NaCl containing 10 mM phosphate buffer (pH 7.4). By increasing the level of phosphate buffer (75 mM) in the incubation mixture, 10(-3)M dbcAMP decreases the hemolysis induced by 5 microgram of lysolecithin. These data suggest a dual effect exerted by dcbAMP: the relatively labilizing or stabilizing effect prevails as a function of exogenous inorganic phosphate level. 10(-6)M dcbAMP also has a relative protective effect against lysolecithin. The combined addition of cAMP (10(-3)) and theophyllin (10(-4)M) does not stabilize the membrane. By increasing the level of lysolecithin to 20 microgram/ml the stabilizing effect of dcbAMP disappears. DbcAMP (10(-3)) as well as cAMP (10(-3)M) and theophyllin (10(-4)M) have a minimum increasing effect on hemolysis in the absence of lysolecithin, too.


Effect of mercuric chloride on the digestive system of a teleost fish, Channa punctatus. The effect of 1.8 mg/liter (LC50) of mercuric chloride exposure on the activities of alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, amylase, pepsin, trypsin, tripeptidase glycyl-glycine dipeptidase and carnosinase has been examined in Channa punctatus. The three phosphatases have been inhibited in the liver but showed an increase in activity in the intestine and pyloric caeca. Amylase, pepsin and trypsin have also shown a slight increase in activity. There has been no significant alteration in the activites of the peptidases. The results show that mercury inhibits the activites of phosphatases in the liver but has no significant effect on the digestive enzymes within the experimental period of 96 hours.


The neuropathology of malignant external otitis. A case report of malignant external otitis is presented with particular emphasis on the neuropathological sequelae. The most striking finding was a mycotic aneurysm of the basilar artery with resultant thrombosis and brain stem infarction. Diffuse pachymeningitis was also present. The severity of the changes described in this case further point out the need for early recognition and adequate treatment of this potentially fatal disease complex.


Adjuvant chemotherapy: an approach to the management of malignant disease. The definition of the roles of surgery and radiation therapy in controlling local malignant disease has become much clearer in the past two decades. Nonetheless, most patients who die of their malignancy do so from metastatic disease. With the advent of effective combination chemotherapy for many patients with advanced malignant disease, the employment of systemic treatment at an earlier stage has become a clinical possibility. Important advances in the systemic control of a number of malignancies have been described. These are examples of a few of the presently ongoing trials (Salmon and Jones, 1977). For the merits and demerits of prophylactic systemic treatments to be accurately determined in malignant disease, it is important that the future role of adjuvant chemotherapy in man be evaluated only in the context of carefully controlled clinical trials. The fact that adjuvant therapy is effective in prolonging survival in some malignant diseases is established; its role in many of the commoner malignant diseases awaits more complete evaluation.


Adrenocorticotrophic hormone fragments mimic the effect of morphine in vitro. 1 Fragments of the N terminal part of adrenocorticotrophic hormone (ACTH) inhibited the electrically evoked contractions of the mouse vas deferens. This inhibition could be antagonized by naloxone. 2 The same fragments displaced radiolabelled morphine from morphine antiserum. 3 Structure-activity relationship studies showed that in both assay systems the active core is located within the sequence ACTH 7--10. 4 It is postulated that the Trp9 residue and the peptide bond between Trp9 and Gly10 are particularly important for interaction of ACTH fragments with morphine receptors.


Genital herpesvirus infection in women attending a venereal diseases clinic. Routine cervical viral cultures (and cultures from lesions if present) were performed on 2630 female patients attending a venereal diseases clinic over a period of four years. Of these patients 96 (3.7%) had genital herpetic infection; of these 23 (24%) were asymptomatic. The association of herpesvirus with other genital infections is considered, but no increase in the incidence of Neisseria gonorrhoeae, Trichomonas vaginalis, and genital warts was found; there was an increased incidence of Candida albicans. A significantly higher percentage of the patients with herpesvirus took oral contraceptives. The findings are discussed and compared with previous reports.


Sensory action potentials and biopsy of the sural nerve in neuropathy. In 167 consecutive patients with various types of neuropathy, the amplitude of the sensory potential and the maximum conduction velocity along the sural nerve were compared with conduction in other sensory nerves, and were related to structural changes revealed by nerve biopsy. Electrophysiological findings in the sural nerve were similar to those in the superficial peroneal and the median nerve, though the distal segment of the median nerve was normal in 20 per cent of the patients when it was abnormal in the sural nerve. Quantitation of histological findings was a more sensitive method than the electrophysiological study in that two-thirds of 33 patients with normal electrophysiology in the sural nerve showed mild loss of fibres or signs of remyelination in teased fibres. The amplitude of the sensory potential was grossly related to the number of large myelinated fibres (more than 7 micrometer in diameter). Considering the 95 nerves from which teased fibres were obtained, maximum conduction velocity was abnormal in half. In 18 of these nerves, slowing in conduction was due to axonal degeneration: the velocity was as to be expected from the diameter of the largest fibres in the biopsy ("proportionate slowing"). In 9 nerves slowing was severe and more marked than to be expected from loss of the largest fibres ("disproportionate slowing"); these nerves showed paranodal or segmental demyelination in more than 30 per cent of the fibres. In 16 nerves from patients with neuropathy of different aetiology neither loss of fibres nor demyelination could explain the moderate slowing. The cause of slowing in these nerves is unknown; other conditions are referred to in which slowing in conduction cannot be attributed to morphological changes. Finally, electrophysiological and histological findings are reported in some patients with neuropathy associated with malignant neoplasm, with rheumatoid arthritis, with polyarteritis nodosa, with acute intermittent porphyria and with cirrhosis of the liver.


Hypersensitivity to histamine in the guinea-pig brain: microiontophoretic and biochemical studies. Electrolytic lesions of the medial forebrain bundle induce a fall in histidine decarboxylase activity (the specific synthetic enzyme of brain histamine) in the ipsilateral cerebral cortex and hippocampus of the guinea pig brain; these results suggest the presence of an ascending histaminergic pathway in the guinea pig brain similar to that described in the rat. Possible alterations in the sensitivity of histaminergic receptors present in the target areas were studied following this type of lesion by combining electrophysiological and biochemical approaches. Microiontophoretic applications of histamine or noradrenaline reveal a hypersensitivity (lower ejecting currents for threshold and maximal responses) in cortical neurons ipsilateral but not contralateral to the lesion, whereas responses to iontophoretically applied GABA are not modified. In contrast the responsiveness of histamine-sensitive cyclic AMP generating systems is not modified, neither in the cerebral cortex nor in the hippocampus after this type of lesion. Similar conclusions are reached from the data obtained with specific agonists of the two classes of histaminergic receptors and measurements in the presence of a phosphodiesterase inhibitor. Several hypotheses are discussed in order to reconcile the finding of a denervation hypersensitivity revealed by iontophoresis contrasting with an unaltered responsiveness of the histaminergic receptors linked to the adenylate cyclase.


Role of the locus coeruleus in transmission onto anterior colliculus neurons. The present study was an attempt to elucidate the role of locus coeruleus (LC) on neuronal activities in the anterior colliculus of rats anesthetized with alpha-chloralose. The spike latency of neurons in the deep grey and white layers of anterior colliculus elicited by stimulation of the optic chiasm (OC), lateral geniculate body (LGB) and visual cortex (VC) was longer than that of neurons in the more superficial layers: optic and intermediate grey layers. When conditioning stimuli were applied to LC, a significant inhibition of spike generation upon OC, LGB and VC stimulation was observed on neurons of deep grey and white layers, but not on those of optic and intermediate grey layers. The conditioning stimulation did not alter spike generation, either in the neurons of deep grey  and white layers or those of optic and intermediate grey layers, following stimulation of the superficial grey layer. These results strongly suggest that noradrenaline originating in the LC could produce an inhibition of neuronal activities in the deep grey and white layers, and such is probably the result of inhibition of neurons located in the superficial layer of the anterior colliculus.


Reflex activity of regenerating frog spinal motoneurons. The reflex activity of frog spinal motoneurons whose axons had been sectioned 7 days to 10 months previously was examined. Before the severed axons reinnervated muscle, reflex latency was prolonged, and reflex amplitude was depressed. Examination of input-output relations revealed the presence of a subset of axotomized motoneurons having supranormal excitability. This subset was not present in normal preparations or in preparations tested after reinnervation took place. Following reinnervation of muscle, reflex latency returned to preoperative levels, while amplitude recovery was typically more variable. Decreased conduction velocity of the severed ventral root fibers accounted for the alterations in reflex latency. These and other data form the basis for concluding that motoneurons, even within a particular preparation, show a wide range of responses to section of their axons and will provide a framework for interpretation of more selective intracellular experiments.


Regional distribution of angiotensinogen in rat brain. The regional distribution of angiotensinogen, the prohormone of angiotensin I, was examined in rat brain. Quantification of brain angiotensinogen concentration was difficult because of the presence of an endogenous angiotensin I degrading (i.e. angiotensinase) activity which was active at the pH of the renin-angiotensinogen incubation. This degrading activity was unequally distributed throughout the brain, and its presence in homogenates invalidated measured levels of angiotensinogen. Only following removal of the angiotensinase activity by ammonium sulfate precipitation of the prohormone could the distribution of the prohormone be determined. Angiotensinogen was widely distributed throughout 31 brain regions; however there was an approximate 12-fold variation in concentration. Highest levels of the prohormone were found in the dorsal and ventral periventricular hypothalamus, area postrema, organum vasculosum lamina terminalis, periventricular thalamus, dorsal raphe and lateral reticular formation. Significantly lower amounts were found in the parietal cortex, cerebellum, septum and pituitaries. While the majority of regions examined exhibited similar concentrations of angiotensinogen, the demonstration of regions containing either significnatly low or high amounts of prohormone is consistent with a topographical distribution of angiotensinogen in rat brain.


Effects of acetylcholine and cyclic GMP on input resistance of cortical neurons in awake cats. The effects on input resistance (Rm) of extracellular iontophoresis of acetylcholine (ACh) and of intracellular iontophoresis of cyclic GMP (cGMP) were studied in neurons of the coronal-pericruciate cortex of awake cats. Control studies were also conducted including iontophoresis of saline extracellularly and 5'-GMP intracellularly. (1) Substantial increases in Rm occurred in approximately half the neurons given ACh or cGMP. (2) In the absence of associated repetitive spike discharge induced by intracellular injection of depolarizing current pulses during iontophoretic applications, the increases in Rm were transient occurring in less than 30 sec and lasting 4--5 min. (3) With associated current-induced spike discharge, the increases in Rm persisted for as long as the neurons could be held -- up to 1.5 h maximally. (4) Rm was not increased if saline was substituted for ACh, if 5'-GMP was substituted for cGMP, or if the neuron was only discharged repeatedly. (5) The magnitude and time course of both transient and persistent increases in Rm were comparable between cells given ACh or cGMP and whether action potentials and resting potentials were greater than or equal to 40 mV (average 47 mV)* or were less.


Ligand binding studies in the mouse olfactory bulb: identification and characterization of a L-[3H]carnosine binding site. Binding sites for the dipeptide L-carnosine (beta-alanyl-L-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[3H]-carnosine was saturable, reversible and stereospecific and had a Kd of about 770 nM. The stereospecific binding of L-carnosine represented about 30% of the total binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibited by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interferred with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. Binding sites for 6 other radiolabeled receptor ligands were also detected in bulb membranes. Peripheral deafferentation of the olfactory bulbs by intranasal irrigation with ZnSO4 led to a loss greater than 90% of the L-[3H]carnosine binding in 4--5 days with much smaller losses in binding of the other 6 ligands over a 180-day observation period. This initial loss of carnosine binding after denervation was due to a loss of binding site stereo-specificity followed by a loss of binding sites. The characteristics of the carnosine binding site in olfactory bulb fulfil 6 of the 7 criteria considered relevant for a functional receptor.


Effect of 1,25-dihydroxycholecalciferol on adenosine 3',5'-cyclic monophosphate production in calvaria of mice. 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), added at doses which stimulate bone resorption in the system used, did not increase adenosine 3',5'-cyclic monophosphate (cAMP) production in vitro in mouse calvaria incubated for up to 48 h. Thus 1,25-(OH)2D3 does not appear to stimulate bone resorption through involvement of an increased cAMP production.


Direct lymphocytotoxicity against herpes simplex virus infected cells. This study was undertaken to determine if direct cytotoxicity (DC) against herpes simplex virus infected cells, perhaps mediated by T cells, could be demonstrated in individuals subject to recurrent herpes labialis. The mononuclear cells from 7 out of 17 individuals with recurrent herpes expressed DC whereas no DC was ever exhibited by 7 individuals without a previous history of herpes infections. Several approaches were used to show that the cytotoxicity being detected was predominately of the direct type rather than antibody-dependent cell cytotoxicity (ADCC). Since the effector cells of the DC were sensitive to trypsin treatment and behaved as do natural killer (NK) cells upon cell fractionation, the results were taken to imply that the DC was attributable to a NK-effector cell type rather than a classical T lymphocyte.


Involvement of ACTH and MSH in active and passive avoidance behavior. Intracerebroventricular injection of antiserum to alpha--MSH induces a weak reduction of passive avoidance latencies after administration prior to retention testing. Administration of antiserum to ACTH 1--24 induces a more marked effect in this respect, whereas injection of a combination of these antisera results in the strongest reduction of passive avoidance retention. No effect of this treatment is observed when these antisera are injected immediately after the learning trial. In active avoidance behavior a facilitation of extinction of the response is observed after intracerebroventricular administration of the antisera prior to each extinction session. This effect is comparable with the one observed in passive avoidance behavior. From these data it is suggested that ACTH and alpha-MSH play an important role in processes related to the retrieval of information stored in the brain.


Possible serotonin-agonist action of veratramine. Veratramine produces a characteristic excitatory action on the central nervous system, producing both tremor and a characteristic "struggling" behavior. This behavioral excitation is accompanied by changes in serotonin content in hypothalamus. The central serotonin agonist methysergide in doses of 5--15 mg per kg produces a dose-dependent inhibition of veratramine's action, while parachlorophenylalanine has no effect. These results are consistent with a serotonin agonist mechanism to explain veratramine's action.


Abnormalities in pulmonary function after brief exposure to toxic metal fumes. A 26-year-old welder became ill after exposure to zinc and cadmium fumes at work. His initial clinical course was consistent with that of metal fume fever, but persistence of symptoms and signs beyond the usual duration in this condition led to suspicion of a toxic pulmonary reaction to cadmium. The finding of high percentages of both metals in the urine confirmed this diagnosis. Pulmonary function tests showed restriction of lung volumes, with increased elastic recoil and reduced diffusion, but no evidence of airways obstruction. Chest roentgenograms indicated central pulmonary edema, which cleared in 6 days. Follow-up assessment 2 years later showed incomplete improvement of the restrictive ventilatory defect.


Bordetella pertussis serotypes in Canada. A study was done to determine the major antigenic factors of Bordetella pertussis strains isolated throughout Canada and whether these isolates have the same antigenic structure as the bacilli in the currently used vaccines. Testing for the major pertussis antigens, factors 1, 2 and 3, was conducted with 440 freshly isolated strains of B. pertussis received from seven canadian provinces between August 1976 and February 1978 and six batches of pertussis vaccine or immunizing agents containing pertussis vaccine. With the aid of specific antisera prepared in rabbits, the antigenic factors were detected by a slide agglutination technique. Almost all (98.9%) of the pertussis strains examined were serotype 1,3. All six batches of pertussis vaccine or immunizing agents containing pertussis vaccine proved to be rich in each of the three main pertussis agglutinogens.


Effects of decapitation-stress on the phosphorylation of cortical membrane proteins. The endogenous phosphorylation of membrane-bound proteins was studied in preparations from the cerebral cortex of rats sacrificed by immersion in liquid nitrogen or by decapitation. Compared to quick-frozen rats, samples from decapitated animals demonstrated a two-fold increase in 32P-phosphate incorporation into specific protein bands with apparent molecular weights of 56K, and 52K (designated E1 and E2) and a decreased incorporation into a phosphoprotein of 47K (designated F). The phosphorylation of two proteins (78K and 34K) in membranes from decapitated rats was found to be highly stimulated by exogenously added cyclic AMP. On the other hand, the phosphorylation of specific protein bands in preparations from quick frozen rats was minimally affected by addition of cyclic AMP. The results indicate that conditions which lead to increases in cyclic AMP levels in the brain in situ induce specific changes in phosphorylative activity, and these can be detected by assaying isolated membrane fragments in vitro.


The disparity of indirect and direct zirconyl-gel assays for carcinoembryonic antigen. Experience with the zirconyl phosphate gel (Z-gel) radioimmunoassays for plasma CEA levels below 20 ng/ml (the indirect method) and for levels greater than 20 ng/ml (the direct method) has shown that a disparity of values exists, caused by shifting from one assay to the other. This disparity is at least partially due to PCA-labile proteins reacting in the direct assay. It may be constant for individual patients but varies among patients. The magnitude of this disparity is independent of the CEA level (above 15 ng/ml).


Scintigraphic evaluation of sarcomata in children and adults by Ga67 citrate. Gallium-67 scans were performed on 32 patients with adult and childhood sarcomata as part of clinical staging studies. Gallium-67 scans proved to be highly accurate in assessing sites of disease in patients with malignant schwannoma, Ewing's sarcoma, and rhabdomyosarcoma. Gallium-67 scans were inaccurate in determining sites of disease in patients with other types of sarcomata. It is concluded that Gallium-67 scanning is a useful adjunct to clinical staging in selected patients with sarcomata.


A stratified randomized study of intradermal BCG in patients with carcinoma of the bronchus: prolongation of quality but not length of life in inoperable patients. This paper reports the results of a stratified, randomized trial of monthly intradermal injections of Glaxo BCG in addition to conventional therapy (surgery, radiotherapy or no treatment) in a consecutive series of 75 men with confirmed bronchial carcinoma. BCG treatment did not significantly prolong survival but had consistenly more effect in prolonging the period in good general condition and in "acceptable" clinical condition. These were significantly prolonged among the BCG patients (all histopathologies) treated with a full course of radiotherapy (p = 0.01, p = 0.005) and among the 43 patients with squamous carcinoma after adjustment for treatment and general prognostic factors (ratio of observed to expected deaths (O/E) for BCG 0.65, P = 0.025). There was a tendency for BCG patients with oat cell carcinoma to survive less well than controls (O/E for BCG 1.40 not significant). Within comparable groups of patients with squamous carcinoma the delay in decline of general condition was accompanied by reduced weight loss.


Undifferentiated sarcoma of the kidney: a tumor of childhood with histopathologic and clinical characteristics distinct from Wilms' tumor. The pathological and clinical features of 123 patients with Wilms' tumor diagnosed between 1961 and 1977 were analyzed. A subgroup of nine patients with distinctive histology characterized by the presence of exclusively sarcomatous elements was identified. These patients were indistinguishable clinically from the other patients with Wilms' tumor and were treated as Wilms' tumor. The rate of recurrence in this group of sarcomatous Wilms' tumor was very high (7/9) and long-term disease-free survival correspondingly low )3/9 free of disease from 15 months to nine years) when compared to the patients with classical Wilms' tumor (50% recurrence rate and 71% currently free of disease). In addition, patients with metastases (seven of seven patients with recurrence had skeletal involvement as contrasted to one of 50 patients with metastatic classical Wilms' tumor). We conclude that the "sarcomatous Wilms' tumor" represents a distinct clinicopathological entity identifiable morphologically at diagnosis, best described as renal sarcoma and that in the future optimal treatment of this disease may vary somewhat from the treatment of classical Wilms' tumor.


Bone metastasizing renal tumor of childhood: morphological and clinical features, and differences from Wilms' tumor. A bone metastasizing primary renal tumor of childhood is described, and five cases are presented. Although all five cases were originally diagnosed as nephroblastomata, there appear to be sufficient histological, ultrastructural and clinical differences to suggest that these tumors should comprise a separate and distinct entity. Using the data from the Manchester Children's Tumor Registry, the incidence of this tumor was found to be 2.3% of all primary renal neoplasms in childhood.


Association of breast cancer with meningioma: report of two cases and review of the literature. We report two cases of breast cancer in whom meningioma subsequently developed and provide a brief review of the literature. A hormonal interrelationship is suggested. The knowledge of this potential association is important in the interpretation and treatment of patients with breast cancer who develop central nervous system manifestations.


Transbronchial lung biopsy in the diagnosis of lymphangitic carcinomatosis. The clinical diagnosis of lymphangitic carcinomatosis of the lung has generally been confirmed by open lung or transthoracic needle biopsy, precedures with significant morbidity and mortality, particularly in patients with respiratory insufficiency. We present six cases of lymphangitic carcinomatosis diagnosed by transbronchial biopsy. Autopsy confirmation was obtained in 4 patients and ultrastructural confirmation of intralymphatic location of tumor in one. The diffuse bronchial and peribronchial lymphatic involvement demonstrated here suggests that this relatively noninvasive technique should be the procedure of choice in the diagnosis of lymphangitic carcinomatosis of the lung.


Detection and significance of occult axillary node metastases in patients with invasive breast cancer. Blocks of axillary lymph nodes from 78 patients with invasive breast cancer, which after "routine" pathological examination were regarded as negative for metastases, were step-sectioned at 20 u intervals. Occult metastases were detected in 24% of the cases. A significant association between such metastases and a lack of or slight degree of an intraductal carcinomatous component of the dominant cancer was noted. There was no relationship between occult metastases and 15 other histopathological and 3 clinical features investigated, including an average 5 year survival rate. Similarly there was no correlation between any of the possible discriminants investigated in the 24% of patients who have died of their diseases or are living with recurrence regardless of the presence of occult metastases. Factors accounting for the lack of universal survival in patients with Stage I (and those with occult metastases that might be designated as stage 11/2) remain enigmatic. It is concluded that attempts to detect occult metastases by extending histopathological methods may be more academic than practical or therapeutically significant.


Pancytopenia induced by aminoglutethimide in the treatment of breast cancer. Aminoglutethimide is an investigational agent of proven benefit in the treatment of metastatic breast carcinoma. We report herein a case of aminoglutethimide-induced pancytopenia complicated by bleeding and gram-negative septicemia. Severe pancytopenia is a rare but important side effect of this new drug and is rapidly reversible when the agent is withdrawn.


A simple assay procedure for beta-D-mannanase. A simple assay procedure for beta-D-mannanase enzyme has been developed which employs carob D-galacto-D-mannan dyed with Remazolbrilliant Blue. Additionally, the procedure is quantitative, relatively sensitive, and highly specific for beta-D-mannanase enzyme. It can be readily used for the determination of beta-D-mannanase activity in crude enzyme preparations and column-chromatography eluates.


Comparison of transcription stimulating, phenol-soluble non-histone chromosomal proteins in normal rat liver and transplantable hepatocellular carcinomas. The non-histone chromosomal proteins (NHCP) of a rapidly and slowly proliferating transplantable hepatocellular carcinoma (THC) were compared to those of normal and regenerating rat liver. The total quantity of NHCP is approximately threefold higher in the THCs than in either normal rat liver at 4 h and 44 h regenerating rat liver. Only those NHCP that can be extracted from chromatin by 0.35 M NaCl were further examined and it was observed that the proteins of this highly complex fraction could be further fractionated by their differential phenol-solubility. The phenol-soluble 0.35 NHCP contained protein(s) capable of stimulating the level of DNA-directed RNA synthesis in vitro. The total amount of this stimulatory activity was 5 times higher in the rapidly growing THC and 1.6 times higher in the slowly growing THC than in normal rat liver. In order to assess the contribution of cell-cycle dependent alterations on the increase in the amount of stimulatory activity in the THCs, 44 h regenerating rat livers were examined. This tissue represents a mix of cells in various stages of the cell cycle which is similar to that found in the THCs. It was found that the total quantity of NHCP in the 44 h regenerating rat liver was the same as in normal rat liver. The total amount of the stimulatory activity also was similar in both the normal and 44 h regenerating rat liver. The amount of the stimulatory activity was found to double in 4 h regenerating rat liver, however. These data suggest that the alterations observed in the NHCP of the THCs are not due solely to cell cycle dependent changes, but may represent malignancy dependent alterations.


Gestational changes in pulmonary converting enzyme activity in the fetal rabbit. Changes in angiotensin-converting enzyme were measured in the lungs of fetal rabbits isolated and perfused in situ at varying ages from 22 days gestation to 7 days of age under controlled conditions of flow, pH, and temperature. Enzyme activity was assessed by infusing bradykinin or angiotensin I in Krebs-Henseleit solution and measuring residual peptide in the effluent by radioimmunoassay. The levels of substrate studied were below those required for enzyme saturation. Lungs of 22 day gestation fetuses removed only one-third of either peptide. The activity at term and in neonatal life resulted in more than 80% peptide removal. The time of the greatest rise in the percent substrate cleared occurs earlier than the time of the greatest increase in lung and body weight. The lower percentage of substrate cleared in early gestation appears to result in part from a limited surface area for enzyme activity in the primitive fetal pulmonary microvascular bed, since morphological studies with fluorescein-tagged anticonverting enzyme antibody demonstrated the presence of enzyme in the lung as early as 17 days of gestation. Electron micrographs of the pulmonary endothelial cell surface reveal that the degree of surface infolding and hence surface area increases with gestation. The higher percentage of substrate cleared in later gestation closely parallels the structural and ultrastructural development of the vascular bed. The presence of converting enzyme in the placenta by the second third of gestation and the large size of the placenta suggest that this organ may be a major locus of converting enzyme activity in the fetus.


Alpha-adrenergic receptors in rat myocardium. Identification by binding of [3H]dihydroergocryptine. [3H]Dihydroergocryptine ([3H]DHE) binds to sites in membranes derived from rat myocardium that have the characteristics expected of alpha-adrenergic receptors. The binding is saturable with 41 fmol [3H]DHE bound per mg of protein and of high affinity with KD = 2.9 nM. The binding is rapid and readily reversible. Adrenergic agonists compete with [3H]DHE for binding in the order: epinephrine greater than norepinephrine greater than isoproterenol; and adrenergic antagonists compete for binding in the order: phentolamine greater than propranolol. For comparison, (-)[3H]dihydroalprenolol [(-)[3h]dha] was used to bind to sites in the same membrane preparations having characteristics of beta-receptors. The number and affinity of beta-receptors were quite similar to those of the alpha-receptors with 46 fmol (-)[EH]DHA per mg protein bound at saturation and KD = 2.5 nM. These techniques allowed identification of both beta- and alpha-adrenergic receptors in membranes derived from isolated atria, right ventricular free walls, and left ventricles including interventricular septa. This is the first report documenting direct identification of myocardial alpha-receptors by radioligand-binding techniques and complements the literature previously reporting myocardial inotopic and electrophysiological responses to alpha-adrenergic stimulation.


Effects of well-defined ischemia on myocardial lysosomal and microsomal enzymes in a canine model. We have used a new technique for extraction of myocardial membranes (0.25 M sucrose, 0.6 M KCl) to isolate particulate and soluble proteins and enzymatic activities in an effort to quantify changes characteristic of progressive ischemia. Myocardial blood flow (MBF) was measured with microspheres (15 micrometer diameter) in all samples of tissue used for assay of proteins and enzymatic activities; MBF to the moderately ischemic areas (M-ischemia) was 53% of control (H-control); MBF to the severely ischemic areas (L-ischemia) was 9% of control. Significant decreases (P less than 0.001) in content of protein were seen in all post 1,000 g pellets and supernatant fluids in the L-ischemia zones; particulate lysosomal enzymatic activity was significantly decreased (P less than 0.001) in all four post 1,000 g pellets (2,500 g to 140,000 g) of the L-ischemic areas (for N-acetyl-beta-glucosaminidase and beta-glucuronidase). The increase in percent free activity of lysosomal enzymes (index of loss of latency) also was highly significant (P less than 0.001) in all particulate fractions of the L-ischemic areas. In addition, about 45% of the total activity of the microsomal marker enzyme, rotenone-insensitive NADH cytochrome C reductase (RINCR), was found in the 140,000 g pellet of H-control tissue (9.9 micronmol/min per g); this activity fell to 8.1 micronmol/min per g in M-ischemic areas (P less than 0.001) and to 5.3 micronmol/min per g in L-ischemic areas (P less than 0.001). This study demonstrates that changes in myocardial proteins, lysosomes, and other membrane-bound enzymes (RINCR) may provide reproducible bichemical parameters for assessing ischemic myocardial injury.


Lack of effect of methylprednisolone on lysosomal and microsomal enzymes after two hours of well-defined canine myocardial ischemia. Myocardial ischemia was produced for 2 hours by coronary ligation in 11 dogs pretreated with methylprednisolone (MP, 30 mg/kg). Myocardial blood flow (MBF) was measured with microspheres (15 micrometer) in each tissue sample used for enzymatic analysis. Homogenates of these tissue samples were separated by ultracentrifugation into lysosome-rich and microsomal fractions and were analyzed for N-acetyl-beta-glusosaminidase (NAGA), beta-glucuronidase (beta-gluc), rotenone-insensitive-NADH-cytochrome c reductase (RINCR), and cytochrome oxidase. The enzymatic data from centrifugal fractions were grouped according to MBF values for statistical analysis of inter-group effects of ischemia. Significant losses (P less than 0.001) of NAGA and beta-gluc were seen in all MP-treated lysosome-rich particulate fractions that were isolated from zones demonstrating MBF values less than 25% of control (L-ischemia). Similar significant losses (P less than 0.001) of RINCR were seen in microsomal fractions from L-ischemia zones. Samples with MBF values greater than 25% but less than 75% of control (M-ischemia) also demonstrated significant decreases of lysosomal and microsomal enzymatic activity in specific fractions. When the data of the above MP-treated group were compared with the untreated control group, no significant intergroup effects of treatment with MP were observed. In addition, enzymatic data (NAGA, RINCR) were normalized prior to performing linear regression analyses; percent loss of particulate enzymatic activity was plotted against percent decrease in MBF. The effects of 2 hours of ischemia on the above biochemical parameters were comparable between untreated and MP-treated groups. Finally, when myocardial samples were grouped according to similar levels of MBF, statistical analysis using the general linear models procedure revealed no beneficial effect of MP treatment on changes in lysosomal hydrolases, microsomal RINCR, or latency of lysosomes.


Segmental alpha-receptor-mediated vasoconstriction in the canine coronary circulation. Recent studies from this laboratory have indicated that sympathetic alpha-receptor-mediated coronary vasoconstriction can compete with local metabolic vasodilation to reduce the oxygen supply to the myocardium. In vitro studies from other laboratories on isolated coronary vessel strips suggest that large epicardial vessels are the dominant site of adrenergic alpha-receptor activity. In this study we used anesthetized, open-chest dogs to test the hypothesis that alpha-receptor-mediated vasoconstriction occurs predominantly in epicardial vessels, which are partially removed from the metabolic milieu in the myocardium. Adrenergic beta-receptor blockade was achieved by propranolol (3 mg/kg, iv). The circumflex coronary artery was pump-perfused at constant pressure to minimize passive changes in large vessel resistance. Pressure was measured at the tip of the perfusion cannula sealed in the circumflex artery, and in an apical branch of the circumflex artery. Large vessel resistance was calculated as the pressure gradient along the vessel segment divided by the coronary flow. Intracoronary injections of nitroglycerin were used as an independent measure of the vasomotor responsiveness of the large vessel segment. Adrenergic alpha-receptor activation was produced by intracoronary bolus injections of norepinephrine and by electrical stimulation of the left stellate ganglion. Alpha-receptor stimulation caused an increase in total coronary vascular resistance; however, the relative increase in the resistance of the large vessel was only about 60% of that seen for the entire coronary bed. These data suggest that, contrary to the proposed hypothesis, adrenergic alpha-receptor-mediated vasoconstriction in the large coronary vessels is not proportionally greater than that observed in the total coronary vascular bed.


Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues. Aspartate aminotransferase (EC 2.6.1.1) activity and the distribution of its isoenzymes in human liver were examined. Rabbit antiserum against porcin soluble (i.e., non-mitochondrial) enzyme cross-reacted with the soluble enzyme of human origin and was used in an immunoprecipitation assay to quantitate the soluble and mitochondrial isoenzymes. These were separated by rapid, semiquantitative electrophoresis on cellulose acetate and by three other quantitative techniques: isoelectric focusing and anion-and cation-exchange chromatography. The mitochrondrial enzyme averaged 81% of the total activity in normal adult human liver (n = 4). Its contribution was dramatically reduced in single specimens of human fetal liver (56% of total activity) and hepatoblastoma tissue  (38%). Total enzyme activities (mumol min-1 per gram of tissue) were: adult, 150; fetal, 38; tumor, 6. Total enzyme concentrations (micromoles of enzyme per kilogram of tissue) found were: adult, 10.8; fetal, 2.7; tumor, 0.4. The concentrations and isoenzyme distribution in human liver are compared to those in various animal model systems. Other methods for quantitative estimation of the isoenzymes and their adaptability for use in estimating concentrations in serum are reviewed.


Thyroxine binding and deiodination of thyroxine by human lymphocytes. Binding and deiodination of T4 by lymphocytes from euthyroid, hypothyroid and hyperthyroid subjects were studied. Two sets of binding sites were found on normal lymphocytes. One set with equilibrium association constant of 6.9 X 10(9) l/mol and a maximal binding capacity of 3000 mol T4 per lymphocyte, and another set with equilibrium association constant of 4.1 X 10(6) l/mol and a maximal binding capacity of 7.8 X 10(6) mol T4 per lymphocyte. The major part of the binding sites were non specific and the binding reversible. Debinding assays revealed also two types of binding site and indicated that deiodination took place predominately from one type of binding site. Deiodination assay showed that the deiodination process had a maximal deiodination capacity of 6 X 10(-13) mol T4/10(6) lymphocytes. Paper chromatography revealed only I and T4 after incubation of lymphocytes with T4. In comparing the patient groups with the control group, lymphocytes from hyperthyroids and hypothyroids exerted a higher T4 turnover (deiodinated T4/bound T4) although the difference was not statistically significant. Lymphocytes exerted a high T4 turnover at low temperature (3 degrees C) and propylthiouracil inhibited deiodination of T4 but not binding.


An assay method for the measurement of urinary inhibitors of calcium phosphate formation. An assay method has been developed for studying quantitatively the formation of calcium phosphate. The method will detect both substances which affect the solubility of calcium phosphate and substances which affect the crystal growth of the material. It is sensitive as or more sensitive than other methods described in the literature for detecting such compounds.


Isolation and identification of some urinary inhibitors of calcium phosphate formation. Normal urine has been examined for substances which inhibit formation of calcium phosphate. A separation scheme involving ultrafiltration, precipitation, electrophoresis and paper chromatography was devised to isolate these substances. Contrary to what has been suggested in the literature for many years, the urines examined did not contain a potent unidentified inhibitor. The major anionic inhibitors were citric acid, pyrophosphate and isocitric acid. These substances together with a small contribution from the cations appeared to account for most, if not all, of the inhibitory activity of urine.


Kasahara-variant alkaline phosphatase in a renal cell carcinoma. Electrophoretically Kasahara-variant alkaline phosphatase we found in a renal cell carcinoma tissue. This enzyme electrophoresed more quickly than liver alkaline phosphatase but more slowly than Kasahara isoenzyme. Neuraminidase treatment of the enzyme caused retardation of electrophoretic mobility which was the same as that of neuraminidase-treated Kasahara isoenzyme. The enzymic properties of this variant enzyme such as inhibition by L-phenylalanine, L-homoarginine, L-leucine, EDTA and urea are consistent with those of Kasahara isoenzyme. On Ouchterlony double diffusion, the precipitin lines of Kasahara and Kasahara-variant enzymes produced by antibody to Kasahara isoenzyme fused completely. These facts may mean that Kasahara-variant isoenzyme is different from the Kasahara one in terminal sialic acid content.


Acid glycosidase and arylsulfatase activities of human cerebrospinal fluid as measured by concanavalin A-sepharose affinity chromatography. An affinity chromatographic method using concanavalin A-Sepharose is described for the determination of N-acetyl-beta-D-glucosaminidase, arylsulfatase. alpha-L-Fucosidase and alpha-D-mannosidase activities in the human cerebrospinal fluid. By this method (starting with 12 to 20 ml samples of cerebrospinal fluid) the above enzymes could be obtained in a concentrated form and their activities could be determined within incubation periods of 30 min to 1 h under the assay conditions described. The pH optima of the enzymes were in the range of pH 4 to 5. About 80% of the total cerebrospinal fluid N-acetyl-beta-D-glucosaminidase was found to be the A form by DEAE-Sephadex A-50 chromatography. About 60% of the total arylsulfatase was also found to be the A form. Determination of these enzyme activities in a few samples of human cerebrospinal fluid indicated a rough proportionality between the enzyme activities and the protein concentration in the cerebrospinal fluid.


Unilateral adrenalectomy and pituitary irradiation in the treatment of ACTH-dependent Cushing's disease in children and adolescents. In four juvenile patients with Cushing's disease, the therapeutic approach used was unilateral adrenalectomy followed by irradiation of the pituitary. The follow-up time of these patients has ranged from 1.5 to 10 years. All four are clinically well. Concentrations of adrenal steroids are within normal limits and they require no additional medication. It is felt that this method gives young patients the chance for normal growth and pubertal development as well as a normal social life, so important in the stressful adolescent years, thus avoiding the handicap incurred by bilateral adrenalectomy and the consequent requirement for continuous substitution therapy.


Augmentation of the in vitro mitogenic response of owl monkey peripheral blood lymphocytes by levamisole and loss of this effect with the development of herpesvirus saimiri-induced lymphoma. Levamisole (LMS) has been shown to be capable of enhancing the proliferative response of normal owl monkey peripheral blood lymphocytes (PBL) to PHA, and, to a lesser degree, of increasing the level of spontaneous DNA synthesis. With the development of herpesvirus saimiri-induced lymphoma, these stimulatory responses were lost. LMS was not capable of stimulating tumour cells to normal functions, or of reversing the disease-induced suppressed functions on normal cells.


Localization of 99m Tc-pyrophosphate in an islet cell tumor of the pancreas. A case of 99m Tc-pyrophosphate uptake in an islet cell tumor of the pancreas is presented. A possible relationship of pyrophosphate uptake in this tumor with uptake in other neuroectodermal tumors which are of neural crest origin is considered.


Role of tissue culture in prediction of malignancy. It is now clear that tissue culture has a role to play in the prediction of the degree of malignancy of human brain tumors. The fact that virtually all human brain tumors grow at least for some interval in culture allows application of tissue culture to the study of all tumors. This almost universal culturability of tumors is truly a singular virtue of intracranial neoplasms. No other human solid tumor group has been so amenable to in vitro growth or study. Up to the present time, despite our experience with over 1100 human brain tumor cultures, we have been extremely conservative in altering patient management on the basis of in vitro data. As is now apparent, the basis for direct input into the clinical milieu exists and it is necessary to work on a patient by patient basis to see how well the existing criteria can be applied to help guide management. Indeed the emphasis given to tissue culture by Rubinstein already confirms the current interest in the applicability of tissue culture data to neuropathological study of tumors (48). Clearly for certain tumors which tend to be benign, tissue culture can serve to alert the clinician to the perhaps unexpected malignant potential of the lesion. For the malignant tumors, the degree of malignancy, the probable biological behavior, the role of host defense factors and therapeutic agents can all be more quantitatively defined for the individual patient by the detailed study of the patient's cultured cells. With improvement in the surgical treatment of benign tumors and with better chemotherapeutic and radiotherapeutic measures for malignant neoplasms this more detailed and precise characterization of tumor behavior has become increasingly relevant to the optimization of the clinical management of the brain tumor patient.


A study in vitro of the sodium pump in fulminant hepatic failure. 1. The mechanism underlying the raised leucocyte sodium content in fulminant hepatic failure was studied by measurement of sodium fluxes, (Na+ + K+)-dependent adenosine triphosphatase activity, and leucocyte ATP content. 2. The rate constant for sodium efflux in the leucocytes was significantly reduced, and attributable to reduced activity of the enzyme (Na+ + K+)-ATPase. Leucocyte ATP content was not significantly different from that of control cells. 3. Incubation of cells from patients in the sera of normal subjects resulted in a reversal of these changes. Inhibition of the leucocyte sodium efflux rate constants and (Na+ +K+)-ATPase of normal cells was achieved by incubation in sera from patients. 4. We suggest that the raised sodium content of leucocytes in fulminant hepatic failure is attributable to a defective sodium pumping mechanism, possibly due to a circulating toxin.