A reappraisal of the effects of ACTH on the response of the central nervous system to injury.

ACTH possesses powerful immuno-suppressive properties and also retards healing by inhibiting fibroblast proliferation and the deposition of collagen. In the present work this hormone was used to test both the 'barrier' and 'immune' hypotheses relating to the failure of CNS regeneration after injury, dosages being administered which had been found to stimulate maximal adrenal production of glucocorticoids in control animals. Although the scarring in the CNS and the systemic humoral and cellular immune responses were significantly depressed, no increase in CNS axon growth was noted, even when large doses of thyroxine were added. We conclude that both 'immune' and 'barrier' hypotheses should be rejected, and that the beneficial effects of thyroxine treatment reported by some workers are not substantiated.


Adenosine-5'-O-(3-thiotriphosphate) as an affinity probe for studying leader RNA's transcribed by vesicular stomatitis virus.

The ATP required for in vitro transcription by vesicular stomatitis virus can be replaced by the analogue adenosine-5'-O-(3-thiotriphosphate) without affecting the rate of transcription or the nature of the mRNA products. RNA chains (less than 5 S) initiated with adenosine-5'-O-(3-thiotriphosphate) can be isolated by affinity chromatography on mercury-agarose columns used in these experiments. At least 80% of the leader RNA synthesized in vitro by infectious B virions and 70% of the leader RNA synthesized by defective-interfering particles of vesicular stomatitis virus contained a 5'-terminal triphosphate as judged by binding to mercury-agarose of leader RNA synthesized in the presence of the 5'-thiotriphosphate adenosine analogue.


Preparation of biologically active radioiodinated cholecystokinin for radioreceptor assay and radioimmunoassay.

A modified method is described for the preparation of stable, high specific activity radioiodinated cholecystokinin (CCK) by its conjugation to 125I-Bolton Hunter reagent (125I-BH). Purified radioiodinated CCK (125I-BH-CCK) stimulated amylase release from isolate rat pancreatic acini with a potency identical to that of native CCK. Further, 125I-BH-CCK was highly immunoreactive and reacted with antisera directed toward both the NH2- and COOH-terminal portions of the CCK molecule. Finally, 125I-BH-CCK bound to specific receptor sites on isolated pancreatic acini.


Mitochondria contain a distinct DNA topoisomerase.

A topoisomerase (nicking-closing enzyme) has been isolated from rat liver mitochondria. It has purified by double-stranded DNA-cellulose chromatography approximately 50,000-fold, based on the crude mitochondrial extract. It possesses a minimum specific activity of 1.9 x 10(5) units/mg. The enzyme has been shown to be distinctly mitochondrial, differentiated from the nuclear topoisomerase by its sensitivity to the intercalating drug, ethidium bromide, and to the non-intercalating trypanocidal drug, Berenil.


Coordinated modulation of albumin synthesis and mRNA levels in cultured hepatoma cells by hydrocortisone and cyclic AMP analogs.

The treatment of Hepa-2 cells, a permanent mouse hepatoma cell line, for 72 h with hydrocortisone (10(-6) M), N6,O2-dibutyryl cyclic AMP (10(-3) M), or 8-bromocyclic AMP (10(-3) M) results in a 2-,3- or 4-fold increase, respectively, in rates of synthesis and secretion of mouse serum albumin. Simultaneous treatment with hydrocortisone and N6,O2-dibutyryl cyclic AMP results in a 10-fold stimulation in these parameters, an effect that is significantly more than additive for the two compounds tested. The number of albumin mRNA sequences, determined by hybridization of total cell RNA to albumin complementary DNA, was increased in direct proportion to the increases in albumin synthesis in all experiments. The relative rate of albumin synthesis approaches in vivo levels in cells treated simultaneously with hydrocortisone and N6,O2-dibutyryl cyclic AMP. We propose that these factors may be necessary to maintain the maximal level of differentiated function in the continuous culture of Hepa-2 cells.


Nucleotide release from tubulin and nucleoside-5'-diphosphate kinase action in microtubule assembly.

ATP and UTP support microtubule assembly through the action of brain nucleoside-5'-diphosphate kinase on GDP. Penningroth and Kirschner (1977) J. Mol. Biol. 115, 643-673) have proposed that microtubule assembly may occur by either of two mechanisms: indirectly, through nucleoside-5'-diphosphate kinase-catalyzed phosphorylation of uncomplexed GDP and directly by nucleoside-5'-diphosphate kinase-mediated transphosphorylation of tubulin-bound GDP at low tubulin concentrations. We find the rates of GDP and GTP release (0.68 and 0.32 min-1, respectively) are sufficiently fast relative to assembly to permit GDP release, phosphorylation, and GTP binding as the sole mechanism of nucleoside-5'-diphosphate kinase action in microtubule assembly. Computer simulation studies accord with the conclusion that GDP release is rapid relative to microtubule assembly. The specific activity of the nucleoside-5'-diphosphate kinase is 1.7 nmol/min/mg of microtubular protein under the conditions studied. Pulse-chase experiments with tubulin . [14C]GDP complex and the rapidity of GDP phosphorylation by the kinase are in agreement with this scheme. Finally, it was observed that the extent and rate of microtubule assembly depends upon the [ATP]/[ADP] ratio.


Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins.

A flavokinase preparation from Bacillus subtilis is described which catalyzes the phosphorylation of reduced, but not oxidized, riboflavin. The enzyme is distinguished from other known flavokinases also in having an unusually low Km for the flavin substrate, 50 to 100 nM. ATP is the obligatory phosphate donor; one ATP is utilized for each FMNH2 formed. Mg2+ or Zn2+ is required for the reaction; Co2+ and Mn2+ will substitute, but less effectively. The same enzyme preparation catalyzes the synthesis of FADH2 from FMNH2 and ATP, but not the synthesis of FAD from FMN and ATP. FADH2 is also formed from reduced riboflavin, presumably by sequential flavokinase and FAD synthetase action. Zn2+ cannot replace Mg2+ in FADH2 formation. The reverse reaction, formation of FMN from FAD, occurs only with reduced FAD, giving rise to FMNH2, and is dependent on the presence of inorganic pyrophosphate. The enzyme thus appears to be an FADH2 pyrophosphorylase. The two enzymatic activities, flavokinase and FADH2 pyrophosphorylase, although not separated during the purification procedure, are distinguished by differences in metal ion specificity, in concentration dependence for ATP (apparent Km for ATP = 300 microM for FADH2 synthesis and 6.5 microM for flavokinase), and in the inhibitory effects of riboflavin analogues.


Contribution of alpha helix formation in human plasma apolipoproteins to their enthalpy of association with phospholipids.

The human plasma apolipoproteins, apo-A-II and apo-C-III, assocaite with phospholipid with a concurrent increase in alpha helical structure of the apoprotein and an exothermic enthalpy of association. A linear correlation of the increase in alpha helical structure and the exothermic enthalpy of association gave a value of -1.3 kcal/mol of amino acid residues converted from a random coil to an alpha helical structure. This value is very close to that of alpha helix formation by charged polyamino acids (-1.2 kcal/mol) and suggests that amino acid side chains contribute little or no heat to the random coil leads to alpha helix transition in the plasma apolipoproteins. In the absence of a change in alpha helix in the apolipoproteins studied here, the enthalpy of association is practically nil, suggesting that alpha helix formation is a major enthalpic contribution to the total free energy of lipid-apolipoprotein association.


Purification of the T4 gene 32 protein free from detectable deoxyribonuclease activities.

Detailed procedures are presented which allow reproducible preparation of T4 gene 32 protein, a helix-destabilizing protein essential for DNA replication and genetic recombination in T4 bacteriophage-infected Escherichia coli cells. Although 32 protein can be purified to better than 99% homogeneity by any one of several procedures, these methods have been developed to remove trace amounts of contaminating deoxyribonucleases, which are present in high levels in the original infected cells. Two alternative preparations are presented, each involving three chromatographic steps. Both 32 proteins obtained are essentially "nuclease-free," when tested at physiological salt concentrations. However, we show here that the phenyl-Sepharose chromatography step, which is necessary to remove an exonuclease activity active only at low salt concentrations, also removes a second protein present in trace amounts. In some cases, retention of this second protein is desirable, since it is essential for obtaining RNA primed, de novo DNA chain starts in an in vitro DNA replication system, when this system is constructed by mixing highly purified preparations of each of the six replication proteins coded for by T4 genes 32, 43, 44, 62, 45, and 41.


Azide binding to the cytochrome c ferric heme octapeptide. A model for anion binding to the active site of high spin ferric heme proteins.

Equilibrium constants for the binding of azide to the ferric heme c octapeptide in 50% ethylene glycol 50% buffer were measured spectrophotometrically. The equilibrium constant for azide binding at 20 degrees C and pH* 7.4 is 29.2, which is approximately 3 to 4 orders of magnitude lower than that observed for azide binding to various ferric hemeproteins. The equilibrium constant was indepent of pH* in the range from 7 to 8. Equilibrium constants at several temperatures exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta H0 = -26,100 J/mol (-6240 cal/mol) and delta S0 = -61.5 J/0K mol (-14.7 e.u.). Comparison of these values to the values for the heme proteins enables one to explain the differences in equiliberium constants in terms of differences in the polarity of the heme environments. The results are consistent with the concept that the oxygen affinity of heme complexes increases with the polarity of the heme environment. The data also suggest that an increase in the polarity of the heme environment should result in a corresponding increase in the susceptibility of ferrous heme dioxygen complexes toward oxidation by the dioxygen.


Adenylate cyclase activity and cyclic AMP levels during the development of Dictyostelium discoideum.

Adenylate cyclase activity and endogenous cyclic AMP levels were measured using a highly sensitive radioimmunoassay and protein binding assay during 24 h of development of Dictyostelium discoideum. Adenylate cyclase activity was not detected until the aggregation stage of development (10 h) when a sudden peak of activity was found. The enzyme was active at all subsequent stages, although a slow decline in activity was observed. Similarly, cyclic AMP levels were not detectable through the first 7 h of development and then showed a sudden peak at aggregation. Following aggregation the cyclic AMP levels decreased to approximately 1/2 the peak value and maintained that level throughout the remainder of the developmental cycle. Adenylate cyclase had a narrow range of substrate saturation with a maximum velocity at 1 to 4 mM ATP at both the aggregation stage (10 h) and the sorocarp stage (24 h). At levels of ATP higher than 6 mM the enzyme from both stages was strongly inhibited. No activity was observed in the absence of Mg2+ or dithiothreitol. The activity from 10-, 14-, and 20-h stages was found bound to a 25,000 x g pellet fraction. The sudden appearance of adenylate cyclase and its product cyclic AMP at aggregation provides additional evidence of a role for this nucleotide in chemotaxis, and the retention of enzyme activity and nucleotide level during the subsequent stages may reflect a further function of cyclic AMP during formation of the two cell types.


The interferon-induced protein kinase PK-i from mouse L cells.

Interferon-treated L cells are characterized by an increased protein kinase activity that can selectively phosphorylate the small subunit of eukaryotic initiation factor 2. This protein kinase, PK-i, has been extensively purified and shown to be a potent inhibitor of mRNA translation. The purified PK-i contains the endogenously phosphorylated 67,000 Mr protein characteristic of interferon-treated cell extracts. PK-i can also phosphorylate arginine-rich histones. Purified PK-i can be activated by preincubation with ATP (but not adenylyl imidodiphosphate) and low concentrations of double-stranded RNA. The activation results in an increase in the first rate of eIF-2 phosphorylation. Activated PK-i becomes resistant to high concentrations of double-stranded RNA and more thermostable. A stimulator of PK-i activity, factor A, was isolated, as well as a specific phosphoprotein phosphatase that dephosphorylates the 67,000 Mr protein and eIF-2. These two factors, which are present in untreated L cells, may regulate the translation inhibitory activity of the interferon-induced and double-stranded RNA-activated protein kinase PK-i.


The effect of cytochrome P-450cam on the NMR relaxation rate of water protons.

Cytochrome P-450cam in the native, substrate-free state (Fe3+, S = 1/2) substantially reduces the NMR relaxation times, T1 and T2, of water protons. Temperature and frequency dependences of T1 and T2 were measured; they are consistent with a model of one or two protons exchanging between a binding site on a heme ligand and bulk water. The relevant parameters of this model have been deduced from the data. The spin relaxation time of the heme iron, tau S similar to 0.5 ns at 25 degrees C, is unusually long for a low spin ferric heme protein but is compatible with the line widths measured for paramagnetically shifted heme resonances. The proton residence time on the ligand, tau M similar to 1 microsecond at 25 degrees C, follows an Arrhenius law with activation energy EM similar to 15 kcal/mol. A scalar hyperfine interaction A/h = 2.2 MHz (3.1 MHz for one-proton exchange) of the found proton(s) with the heme iron is deduced from the difference between T1 and T2 observed in the fast exchange limit. The iron-proton distance is found to be 2.9 A (2.6 A for one-proton exchange). Variation of pH between pH 6.4 and 8.6 does not affect T1. The bearing of these results on the question of the axial heme ligand is discussed.


Human hypoxanthine-guanine phosphoribosyltransferase. IMP-GMP exchange stoichiometry and steady state kinetics of the reaction.

Steady state kinetics of hypoxanthine guanine phosphoribosyltransferase-catalyzed reactions are studied. The results obtained suggest that IMP, GMP, P-Rib-PP, and pyrophosphate bind to the same enzyme form, while hypoxanthine and guanine bind to a different form. Guanine activates IMP-pyrophosphorolysis. During the reaction guanine and IMP are consumed with formation of GM


Characterization of a calcium-modulated protein from transformed chicken fibroblasts.

A calcium-modulated protein has been isolated from secondary cultures of virus transformed chicken embryo fibroblasts and has been characterized in terms of its physical, chemical, and functional properties. These properties demonstrate that this protein is a calmodulin and distinguish it from other calcium-modulated proteins found in various muscle and non-muscle tissues. In addition, this transformed cell calmodulin has been shown to be indistinguishable in both structure and function from normal cell calmodulins isolated from chicken gizzard and brain. A novel change in the electrophoretic behavior of these calmodulin preparations that is dependent on sample history has been observed. These alterations may be the basis for previous reports of tissue specific differences in calmodulin and for some of the differences occasionally observed in peptide maps of calmodulins.


5'-Hydroxyl polyribonucleotide kinase from HeLa cell nuclei. Purification and properties.

An enzyme, 5'-hydroxyl polyribonucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of RNA in the presence of ATP, has been isolated from extracts of HeLa cell nuclei. The kinase requires a divalent cation (Mg2+ or Mn2+) for activity, has an alkaline pH optimum, and is sensitive to the sulfhydryl antagonist N-ethylmaleimide. 5'-hydroxyl terminated polydeoxyribonucleotides are phosphorylated much less efficiently than the 5'-hydroxyl terminated polyribonucleotides, and the kinase preparation is inactive on ribonucleoside 3'-monophosphates. Enzyme activity is inhibited by ADP and by pyrophosphate. The sedimentation coefficient of the kinase is estimated to be 5.6 S from glycerol gradient centrifugation.


Phosphorylation of 5-iodo-5'-amino-2',5',dideoxyuridine by herpes simplex virus type 1 encoded thymidine kinase.

Herpes simplex virus type 1 (HSV-1) encoded thymidine kinase converts 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd), a highly specific anti-herpes agent, into the 5'-diphosphate (AIdUDP) derivative in vitro. AIdUDP was identified by its acid lability, sensitivity to alkaline phosphatase hydrolysis, chromatographic behavior, and ratio of double isotope (125I, 32P) labeling. ATP, but not AMP, is a phosphate donor, and the direct transfer of the beta and gamma phosphate of ATP as pyrophosphate to AIdUrd was ruled out. The presence of a phosphoramidate bond was supported by the acid lability of AIdUDP which has a half life (t1/2) of 320 min at pH 3.0. At neutral pH, the hydrolysis products are AIdUrd and orthophosphate, with AIdUrd monophosphate being the probable hydrolytic intermediate at these pH values. However, at acidic pH, some pyrophosphate was detected in addition to AIdUrd and orthophosphate. AIdUrd competitively inhibited the phosphorylation of thymidine and deoxycytidine. Escherichia coli thymidine kinase, even though 100-fold higher in activity, was unable to phosphorylate AId-Urd under similar incubation conditions.


Dispersed mammary epithelial cells. Receptors of lactogenic hormones in virgin, pregnant, and lactating rabbits.

The number and affinity of binding sites for lactogenic hormones have been determined in dispersed mammary cells from virgin, pregnant, and lactating rabbits. Dispersed epithelial cells, prepared from mammary glands by enzyme digestion, calcium chelation, and gentle shearing, were separated from nonepithelial cells by density centrifugation. 125I-labeled ovine prolactin (oPRL) and 125I-labeled human growth hormone (/GH) were used as tracers. Association and dissociation of 125I-oPRL or 125I-hGH were time- and temperature-dependent. The rate of association followed a second order reversible reaction with a rate constant of approximately 0.5 at 4 degrees C, approximately 2.0 at 23 degrees C, and approximately 9 x 10(7) M-1 min-1 at 37 degrees C. Maximum binding was achieved after 120 h at 4 degrees C, 48 h at 23 degrees C, and 2 to 4 h at 37 degrees C. Dissociation of 125I-oPRL or hGH from cells by unlabeled oPRL was complete at 4 degrees C after 160 h, following a first order reaction (5-1 = 9.9 x 10(-5) min) and incomplete at 23 degrees C and 37 degrees C even after prolonged time. Internalization of receptor-bound 125I-oPRL was studied by quantitative electron microscope autoradiography. Grain distribution over- and volume densities of cellular organelles was analyzed as a function of time and temperature. At 37 degrees C, there was a rapid and specific translocation of lactogenic hormones to intracellular organelles. Autoradiographic grains were found associated with vesicles, Golgi elements, lysosome-like structures, and the nucleus. One class of high affinity binding sites was estimated from Scatchard plot and direct kinetic analyses at 4 degrees C. Whereas the apparent affinity constant (approximately 10(10) M-1) did not change significantly throughout pregnancy and early lactation, the number of receptors extrapolated from Scatchard plots at 4 degrees C varied in an inverse relation to serum progesterone concentration. Thus, approximately 1900 sites were detected in virgin rabbits (progesterone, approximately 200 pg/ml), and midpregnancy (progesterone, approximately 15,000 pg/ml), and approximately 1800 during early lactation (progesterone, approximately 500 pg/ml). The binding properties of lactogenic hormones to dispersed cells was compared with those to Triton X-100 solubilized microsomal membrane preparations. Good correlation between the two systems was found indicating that cell dispersion did not alter binding properties. Our results indicate that dispersed mammary cells bind lactogenic hormones in a saturable and reversible process, that the number of exposed receptors varies throughout gestation and lactation, and finally that lactogenic hormones are internalized following interaction with their membrane receptors.


Requirements for synthesis of ribonucleic acid primers during lagging strand synthesis by the DNA polymerase and gene 4 protein of bacteriophage T7.

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze DNA synthesis on duplex DNA templates. Synthesis is initiated at nicks in the DNA template, and this leading strand synthesis results in displacement of one of the parental strands. In the presence of ribonucleoside 5'-triphosphates the gene 4 protein catalyzes the synthesis of oligoribonucleotide primers on the displaced single strand, and their extension by T7 dna polymerase accounts for lagging strand synthesis. Since all the oligoribonucleotide primers bear adenosine 5'-triphosphate residues at their 5' termini, [gamma 32P]ATP is incorporated specifically into the product molecule, thus providing a rapid and sensitive assay for the synthesis of the RNA primers. Both primer synthesis and DNA synthesis are stimulated 3- to 5-fold by the presence of either Escherichia coli or T7 helix-destabilizing protein (DNA binding protein). ATP and CTP together fully satisfy the requirement for rNTPs and provide maximum synthesis of primers and DNA. Provided that T7 DNA polymerase is present, RNA-primed DNA synthesis occurs on either duplex or single-stranded DNA templates and to equal extents on either strand of T7 DNA. No primer-directed DNA synthesis occurs on poly(dT) or poly(dG) templates, indicating that synthesis of primers is template-directed.


Characterization of the ribonucleic acid primers and the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 4 protein of bacteriophage T7.

The DNA polymerase and gene 4 protein of phage T7, in the presence of helix-destabilizing protein (DNA binding protein), catalyze DNA synthesis on duplex templates. As has been previously shown (Kolodner, R. D., and Richardson, C. C. (1978) 4. Biol. Chem. 253, 574-584), in the absence of ribonucleoside 5'-triphosphates DNA synthesis is initiated at nicks, and all of the newly synthesized DNA is covalently attached to the template. In this paper we characterize the DNA synthesized in the presence of ribonucleoside 5'-triphophates and show that, in contrast, the major portion of the newly synthesized DNA is not attached to the template, having an average chain length of 5000 to 6000 nucleotides. In addition, each chain of newly synthesized DNA is terminated at its 5'-end by a covalently attached tetranucleotide RNA primer whose sequence is predominantly pppApCpCpC and pppApCpCpA. The results of isotope transfer experiments are in agreement with the number of initiation events determined by the incorporation of [gamma-32P]ATP and indicate that each of the four deoxyribonucleotides is present at the RNA-DNA junction.


Isolation and characterization of the transferrin receptor from human placenta.

The transferrin receptor has been isolated from human placenta using immunochromatography and affinity chromatography. The receptor is a glycoprotein and has a Mr = 90,000 on sodium dodecyl sulfate-gel electrophoresis in the presence of 2-mercaptoethanol. The isolated receptor is immunologically related to the transferrin receptor on the reticulocyte cell surface.


Adenosine 3':5'-monophosphate-mediated inhibition of myosin light chain phosphorylation in bovine aortic actomyosin.

Ca2+-dependent phosphorylation of the myosin light chains in bovine aortic native actomyosin is markedly depressed in the presence of cyclic AMP and its dependent protein kinase. This inhibition occurs with either cardiac, skeletal, or aortic protein kinase plus cyclic AMP, while little or no inhibition occurs with either cyclic AMP or protein kinase alone. The extent of inhibition is related to the concentration of protein kinase and approaches a maximum of approximately 50%. Concomitant with the inhibition of myosin light chain phosphorylation is (a) an increased phosphorylation of a 100,000-dalton moiety which possibly corresponds to the myosin light chain kinase present in the native actomyosin preparation and (b) a decrease in the actomyosin Mg2+-ATPase activity. These findings suggest that modulation of actin-myosin interactions by the cAMP system directly at the level of the contractile proteins may represent a mechanism by which beta adrenergic relaxation occurs in mammalian vascular smooth muscle.


Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.


Cytochrome c oxidase subunits in contact with phospholipids. Hydrophobic photolabeling with azidophospholipids.

The technique of photolabeling of membrane proteins with arylazidophospholipids was applied to cytochrome c oxidase. The "deep" and "shallow" labels employed reacted with all subunits of cytochrome c oxidase except V and VI: Subunits I, III, and VII were heavily labeled, Subunit II was labeled to a lesser extent, and Subunit IV was poorly labeled. Subunit I was labeled more by the deep label and Subunit VII by the shallow one. The other subunits were equally labeled by the two probes. This technique has revealed what subunits of cytochrome c oxidase interact with the lipid and their approximate position in the membrane.


Inhibition of low density lipoprotein uptake in confluent endothelial cell monolayers correlates with a restricted surface receptor redistribution.

Binding of either low density lipoprotein (LDL) or Concanavalin A (ConA) to actively growing vascular endothelial cells is associated with a redistrubution of the appropriate cell surface receptor sites which form patches and caps. This receptor lateral mobility is greatly restricted when endothelial cells reach confluence and adopt the configuration of a cell monolayer composed of closely apposed and non-overlapping cells. In this case, although the cells still exhibit specific LDL binding to the appropriate cell surface receptor sites, neither the binding of LDL nor of ConA induces a receptor redistribution. The lack of LDL receptor redistribution correlates with a marked decrease in the rate of LDL internalization. In contrast, no such density-dependent changes are observed in cell types which grow on top of each other and form multiple cell layers at confluence. Thus, neither LDL nor ConA induced cap formation in either sparse or confluent smooth muscle cell cultures and the same rate of LDL internalization is observed at both cell densities. Similarly, adsorptive endocytosis of cationized LDL (which enters the cells independently of the LDL receptor sites) was not correlated with a detectable receptor redistribution, nor was it significantly affected by changes in cell density and spatial organization. The formation of a confluent cell monolayer resting on an underlying basement membrane might therefore provide, via a change in membrane dynamics, a mechanism whereby the endothelium of large blood vessels can function as a protective barrier against the high circulating levels of LDL in plasma.


Regulation of cyclic nucleotide phosphodiesterase forms by serum and insulin in cultured fibroblasts.

A rapid reduction of cyclic nucleotide phosphodiesterase activity occurs after the replating of confluent cultures of BHK 21 c/13 fibroblasts into fresh medium. This reduction in activity depends on the density to which the cultures are reseeded and the concentration of serum in the medium. Enzyme activity in BHK cells is restored after 24 to 48 hours if cells are diluted into medium containing 10% fetal calf serum or 0.5% fetal calf serum supplemented with insulin (10(-6)M), but not into 0.5% serum alone. The restoration in enzyme activity is blocked by cycloheximide or Actinomycin D. When BHK cells become quiescent by maintanance in 0.5% serum conditions for 48 hours, a rapid (15--60 minutes) increase in cyclic AMP phosphodiesterase activity occurs when 10% serum is added to the cultures. Enzyme activity is increased even further after 24 to 48 hours in the 10% serum. Cycloheximide or Actinomycin D do not affect the rapid increase in enzyme activity in response to serum, but completely inhibit the long term increase. In contrast to serum, insulin (10(-8) to 10(-6)M) has no short term effect, but does increase enzyme activity after 24 to 48 hours to levels comparable to those seen with addition of 10% serum. As is the case with serum, this long term effect of insulin on enzyme activity is prevented by inhibitors of protein and RNA synthesis. Kinetic analyses of cyclic AMP phosphodiesterase activity in homogenates of quiescent BHK cells indicate the presence of only high Km (congruent to 20 muM) enzyme activity. Addition of serum or insulin to quiescent cells results in the appearance of apparent low Km enzyme activity in homogenates. Sucrose gradient analysis of BHK cells displays two forms of cyclic AMP phosphodiesterase enzyme activity: a 3--4 S form and 5--6 S form. In quiescent cells, the 5--6 S form greatly predominates relative to the 3--4 S form. Addition of serum to quiescent cells results in a rapid appearance of increased 3--4 S form enzyme activity. Insulin also increases the activity of this higher affinity 3--4 S enzyme form after 24 to 48 hours in culture. The functional significance of short and long term regulation of cyclic nucleotide phosphodiesterase(s) in cells is discussed.


A variant of S49 mouse lymphoma cells with enhanced secretion of cyclic AMP.

A novel variant of S49 mouse lymphoma cells is described which is resistant to growth arrest and cytolysis by dibutyryl cyclic AMP but, in contrast to previously described variants, has normal cyclic AMP-dependent protein kinase. The variant is also resistant to N6-monobutyryl cAMP but is sensitive to killing by 8-bromo cAMP and cholera toxin. Extracts of the variant appear to contain wild type levels of both O2'-butyrylesterase and cyclic AMP phosphodiesterase activities. Accumulation of exogenous [3H]dibutyryl cyclic AMP is reduced in the variant suggesting a defect in either uptake or secretion of the analog or its metabolic products. Accumulation of cyclic AMP in variant cells after stimulation of adenylate cyclase with either isoproterenol or cholera toxin is also reduced compared with wild type cells, although cyclase activity of membranes prepared from the variant cells is normal. Extracellular accumulation of cyclic AMP after stimulation of variant cells with isoproterenol is greater than that found with wild type cells. It is concluded that the variant has an alteration in its cyclic AMP secretion mechanism resulting in more efficient extrusion of cyclic AMP than in wild type cells.


Quantitative analysis of vitamin D3 in a feed using normal phase high pressure liquid chromatography.

A procedure is described for the separation and quantification of Vitamin D3 from different feeds and premixes. The study was conducted, first using a liquid partition step as a preliminary clean-up after extraction, then chromatography on activated Silica gel 60 before final analysis on a high pressure liquid chromatograph (HPLC) using a LiChrosorb NH2 (10 mu) column and a variable wavelength UV detector set at 264 nm. Total analysis on the HPLC was achieved in fifteen minutes. The detector response curve for an authentic D3 standard was linear using peak areas with a minimum detectable amount being 5 ng. The overall percent recovery of D3 in feeds was 94.4 +/- 2.4%. The minimum detectable amount of D3 in animal feeds was found to be in the region of 2,000 I.U./kg.


Interaction between thyroid-stimulating immunoglobulins and thyrotropin receptors in fat cell membranes.

In the TSH radioreceptor assay to study the interaction between Graves' immunoglobulins (Ig) and TSH receptors in guinea pig fat cell membranes, Graves' Ig were found to inhibit [125I]TSH binding to fat cell membranes in a dose-dependent manner. Scatchard analysis of [125I]TSH displacement curves by Graves' Ig indicated a single population of the binding sites in fat cell membranes, in contrast to two populations of TSH-binding sites in the membranes. Displacement of [125I]TSH bound to fat cell membranes by both Graves' Ig and unlabeled TSH were time and temperature dependent, with similar dissociation curves, suggesting a specific binding of Graves' Ig to the membrane sites related to the TSH receptor in the fat cells. Such Ig are referred to as fat cell-binding Ig, to be distinguished from the thyroid-stimulating Ig (TSI) detected by TSH radioreceptor assay using human thyroid membranes. Both fat cell-binding Ig and TSI were detected in the sera of a great majority of untreated patients with Graves' disease. A significant correlation was found between both values (r = 0.80; n = 19; P less than 0.001). According to these results, TSI might represent an autoantibody to the membranes associated with the TSH receptor of the target tissues without a strict organ specificity.


Plasma high density lipoprotein cholesterol in thyroid disease.

The plasma levels of high density lipoprotein cholesterol (HDL-C) were reduced in 16 hyperthyroid female patients compared to 37 euthyroid women (33.5 +/- 8 vs. 51.5 +/- 13 mg/dl (mean +/- SD); P less than 0.001). When 5 patients were restudied after restoration of the euthyroid state, plasma HDL-C increased from 29 +/- 5 to 43 +/- 11.5 mg/dl (P less than 0.05). In addition, in 22 hypothyroid women, HDL-C levels were also diminished compared to the euthyroid group (43.4 +/- 15.5 vs. 51.5 +/- 13 mg/dl; P less than 0.05). Nine patients were restudied after L-T4 replacement therapy; their levels of HDL-C increased but not to a statistically significant degree. The daily administration of 0.3 mg L-T4 to eight normal male volunteers for 1 month did not significantly affect HDL-C levels.


Decreased high density lipoprotein cholesterol in hypopituitarism.

Serum triglyceride, cholesterol, and high density lipoprotein cholesterol (HDL-C) levels were measured in 10 men and 3 women with hypopituitarism. The mean total cholesterol and triglyceride levels were significantly increased but were within the range of controls. The mean HDL-C concentration and the HDL-C to total cholesterol ratio were significantly decreased. Similar findings were present in a patient with isolated GH deficiency. Two men with Kallman's syndrome and 3 patients with active acromegaly had values that were either within or just below the normal range. Replacement therapy with thyroid hormone and prednisone resulted in a normalization of HDL-C concentrations in four of the five patients studied. This improvement suggests a role for thyroid hormone and/or glucocorticoids in the maintenance of normal HDL-C concentrations. These observations also suggest that the distribution of plasma lipoprotein fractions is dependent in part, either directly or indirectly, on pituitary hormones.


A syndrome of apparent mineralocorticoid excess associated with defects in the peripheral metabolism of cortisol.

A syndrome is described whose features, suggestive of primary mineralcorticoid excess, included hypertension, hypokalemia, low PRA, and responsiveness to spironolactone. Aldosterone levels were subnormal but as yet there has been no evidence of overproduction of other mineralocorticoids by chemical analysis or by bioassay of plasma and urinary extracts. The steroidal abnormalities that were observed involved peripheral matabolism rather than secretion. One patient exhibited a transient delay in reduction of the 3-keto group in the A ring, and both patients exhibited a decrease in the metabolism of cortisol to biologically inactive cortisone. This was shown by the marked decrease in the excretion of urinary metabolites bearing an 11-keto group and a decrease in the oxidation of 11 alpha-[3H]cortisol to tritiated water. The defect appeared not to be a deficiency of the 11 beta-oxidoreductase system itself, since the reverse reaction of conversion of cortisone to cortisol proceeded normally, but, rater, an alteration in the equilibrium position of 11 beta-oxidoreduction in favor of the reduced form. This was also expressed by a prolongation of the half-time of disappearance of cortisol. The decrease in the MCR permitted the maintenance of normal cortisol plasma levels and normal glucocorticoid function at a diminished rate of secretion. The decreased rate of conversion of cortisol to cortisone serves as a biochemical marker of this hypertensive syndrome.


Albright's osteodystrophy in a patient with renal hypercalciuria.

We have investigated an 18-yr-old hypercalciuric female with features of both renal hypercalciuria and pseudohypoparathyroidism. She had increased circulating parathyroid hormone levels, which are common to both diseases. She also had a modest hypocalcemia and low normal basal cAMP excretion, both of which are more likely to occur in pseudohypoparathyroidism. She also had Albright's osteodystrophy, which is frequent in patients with pseudohypoparathyroidism and has never been reported in patients with renal hypercalciuria. In contrast to patients with pseudohypoparathyroidism, her serum 1,25-dihydroxycholecalciferol level was increased and her renal responses to parathyroid hormone infusion, including renal calcium reabsorption, were normal. This patient, therefore, raises the possibility that some patients with renal hyperalciuria may have a forme fruste of pseudohypoparathyroidism.


Cell-mediated and humoral immune responses to herpes simplex virus and cytomegalovirus in renal transplant patients.

Cell-mediated immunity to herpes simplex virus and cytomegalovirus, using the lymphocyte transformation test and interferon induction in lymphocytes, was studied in 59 patients from 1 day to 7 years after allotransplantation and compared with the results in normal subjects. Both parameters were permanently depressed with regard to cytomegalovirus. With herpes simplex virus, interferon production was also permanently depressed, whereas the transformation reaction was normal during the first year after transplantation and only slightly depressed in patients more than 1 year after transplantation. In 6 patients the above-mentioned assays and the complement fixation reaction were performed serially and related to the clinical signs of herpes simplex virus and cytomegalovirus infection. The relationship between depression of the transformation reaction and interferon production in lymphocytes and the occurrence of clinically evident herpes simplex virus and cytomegalovirus infections was, however, equivocal. The humoral immune response to herpes simplex virus was measured by the complement fixation test and the more sensitive antibody-dependent, cell-mediated cytotoxicity reaction, and a good correlation was found between these two tests, although only a few persons were found to be negative in the antibody-dependent, cell-mediated cytotoxicity reaction. The suggestion is made that only a few adults are "true" herpes simplex virus seronegative.


Enzyme-linked fluorescence assay: Ultrasensitive solid-phase assay for detection of human rotavirus.

Enzyme-linked immunosorbent assay (ELISA) has proven to be a useful assay system for the direct detection of infectious agents. However, when the usual color-producing substrates are employed, relatively large amounts of substrate must be hydrolyzed by the bound enzyme before detection can be achieved. We attempted to improve the sensitivity of ELISA by utilizing a substrate that yields a fluorescent product on enzyme action. The enzyme-linked fluorescence assay (ELFA) based on this principle was approximately 100 times more sensitive than the corresponding ELISA or radioimmunoassay for the detection of human rotavirus in a standard stool suspension. In addition, the ELFA for human rotavirus was capable of detecting antigen in six specimens that were negative by ELISA. Five of these specimens were obtained late in the course of confirmed rotavirus infections. ELFA provides a simple, reliable, ultrasensitive method for the rapid detection of viral antigen.


Efferent connections of the habenular nuclei in the rat.

The efferent connections of the medial (MHb) and lateral (LHb) habenular nuclei in the rat were demonstrated autoradiographically following small injections of tritiated amino acids localized within various parts of the habenular complex. Comparison of individual cases led to the following conclusions. MHb efferents form the core portion of the fasciculus retroflexus and pass to the interpeduncular nucleus (IP) in which they terminate in a topographic pattern that refects 90 degrees rotations such that dorsal MHb projects to lateral IP, medial MHb to ventral, and lateral MHb to dorsal IP. Most MHb fibers cross in the interpeduncular necleus in the "figure 8" pattern described by Cajal, and terminate throughout the width of IP with only moderate preference for the ipsilateral side. However, the most dorsal part of MHb projects almost exclusively to the most lateral IP zone in a cluster pattern that is particularly dense on the ipsilateral side. The MHb appears to have no other significant projections, but very sparse MHb fibers may pass to the supracommissural septum and to the median raphe nucleus. Except for some fibers passing ventrally into the mediodorsal nucleus, all of the LHb efferents enter the fasciculus retroflexus and compose the mantle portion of the bundle. No LHb projections follow the stria medullaris. In the ventral tegmental area LHb efferents become organized into groups that disperse in several directions: (a) Rostrally directed fibers follow the medial forebrain bundle to the lateral, posterior and dorsomedial hypothalamic nuclei, ventromedial thalamic nucleus, lateral preoptic area, substantia innominata and ventrolateral septum. (b) Fibers turning laterally distribute to the substantia nigra, pars compacta (SNC); a small number continue through SNC to adjacent tegmentum. (c) The largest contingent of LHb efferents passes dorsocaudally into paramedian midbrain regions including median and dorsal raphe nuclei, and to adjacent tegmental reticular formation. Sparse addition LHb projections pass to the pretectal area, superior colliculus, nucleus reticularis tegmenti pontis, parabrachial nuclei and locus coeruleus. No LHb projections appear to involve the interpeduncular nucleus. All of these connections are in varying degree bilateral, with decussations in the supramammillary region, ventral tegmental area and median raphe nucleus. On the basis of differential afferent and efferent connections, the LHb can be divided into a medial (M-LHb) and a lateral (L-LHb) portion. The M-LHb, receiving most of its afferents from limbic regions and only few from globus pallidus, projects mainly to the raphe nuclei, while L-LHb, afferented mainly by globus pallidus and in lesser degree by the limbic forebrain, projects predominantly to a large region of reticular formation alongside the median raphe nucleus. Both M-LHb and L-LHb, however, project to SNC. The reported data are discussed in correlation with recent histochemical findings.


The GABA neurons and their axon terminals in rat corpus striatum as demonstrated by GAD immunocytochemistry.

Glutamic acid decarboxylase (GAD, EC 4.1.1.15), the enzyme which catalyzes the alpha-decarboxylation of L-glutamate to form the neurotransmitter gamma-aminobutyric acid (GABA), was localized immunocytochemically in rat neostriatum, pallidum and entopeduncular nucleus. A large amount of GAD-positive reaction product was observed in both the pallidum and entopeduncular nucleus in light microscopic preparations and was localized ultrastructurally to axon terminalis that surrounded dendrites and large somata. In the neostriatum the relative numbers of GAD-positive axons terminals per unit area were substantially less than in the pallidum. GAD-positive terminals predominantly formed symmetric synapses with somata, dendrites and spines, but a small number of them formed asymmetric synapses with either dendrites or spines. The presence of GAD within these terminals is consistent with results of other investigations which have indicated that the striatopallidal and striatoentopeduncular pathways as well as neostriatal local circuit neurons and/or collaterals from neostriatal projection neurons, use GABA as a neurotransmitter. GAD-positive reaction product was also localized within the somata and dendrites of neostriatal and pallidal neurons in colchicine-injected preparations. The GAD-positive somata in the pallidum were medium-sized neurons and since such cells project to the substantia nigra, our results are in agreement with those from other studies which demonstrate a GABAergic, pallidonigral pathway. In the neostriatum, GAD-positive somata were identified light microscopically as medium-sized neurons with either round or fusiform shapes. Electron microscopic examinations also showed GAD-positive reaction product within the perikaryal and dendritic cytoplasm of these neurons, as well as in dendritic spines. These findings are in accord with the results of studies which have indicated that medium-sized, spinous neurons of the neostriatum give rise to a GABAergic, striatonigral pathway. The significance of GAD localization within these neostriatal neurons is discussed in relation to recent findings which show that substance P is contained within this same class of striatonigral projection neuron.


Effects of the beta-adrenoceptor blocking agent sotalol on CNS: sleep, EEG, and psychophysiological parameters.

Sotalol (Sotalex), 320 or 960 mg, was administered to 12 healthy subjects daily for a period of four days in a double-blind trial over 11 days. The effects of sotalol on heart rate, blood pressure, EEG, subjective quality of sleep, polygraphically determined sleep pattern, and psychophysiological parameters such as psychomotor performance, memory, perception, vigilance, and general condition were studied and were related to dosage and plasma levels. Steady-state plasma levels of sotalol were reached within 24 hours after a single dose; 960 mg resulted in plasma levels three times higher than those reached with 320 mg, which indicates first-order linear absorption. The effects of sotalol on EEG, sleep, and performance in psychological tests were equivocal and do not yield evidence for CNS activity of sotalol.


Fluoride inhibition of polymorphonuclear leukocytes.

Significant inhibition of PMN metabolic activity by F- occurred at 0.1, 0.5 and 0.0 mM F- for O2- generation, 1-C14 CO2 release labeled glucose and NBT-reduction respectively. This inhibition resulted primarily from F- suppression of nonoxidative glucose metabolism. As the concentration of F- in the O2- generating system increased beyond 10 mM, a rise in production of O2- anion occurred, peaking at 20 mM F-, followed by a rapid decline.


Isolation, identification and characterization of camelpox virus in Iraq.

A virus was isolated from pox-like lesions in camels during an outbreak of camelpox disease which occurred in December 1977 in an area near the Iraqi-Iranian border. It was identified serologically as a virus of the Orthopoxvirus group. The biological properties of the isolate indicated that it was probably identical with strains of camelpox virus isolated from Iran, Egypt, Kenya and the U.S.S.R.


Studies on the stability of rat testis membrane receptors for hLH, hCG and hFSH.

Receptor preparations from rat testes were stored at 4 C with various protease inhibitors and chemicals, at -20 C and -80 C, in freeze-dried form as well as at 4 C and 37 C after reconstitution in various media. Each preparation was tested for stability in terms of binding capacity of human luteinizing hormone (hLH), human chorionic gonadotropin (hCG) and human follicle-stimulating hormone (hFSH). The freeze-dried samples were stable for at least six months, although once reconstituted and stored at 4 C, they were less stable than fresh ones. Furthermore, Na azide and Na azide with diisopropylfluoro-phosphate (DIFP) prolonged the stability of the fresh samples. The samples stored at -20 C or -80 C were stable for at least 90 days and repeated freezing and thawing (15 times) did not change the binding capacity of either the hCG/hLH or hFSH receptors.


Tanapox. A serological survey of the lower Tana River Valley.

Sera collected from the indigenous population of the Tana River valley during the Tana River Expedition in 1976 were examined for neutralizing antibody to Tanapox virus. Antibody was found in 9.2% of the population in infected areas. This result and the presence of antibody in four children indicated that infection had continued to occur in the area since the 1962 outbreak.


Trypan Blue as a marker of plasma membrane permeability in alloxan-treated mouse islet cells.

Suspensions of pancreatic islet cells from noninbred ob/ob-mice were incubated with Trypan Blue. Microscope photometry showed that apparently viable cells excluded the dye completely, whereas the nuclei of nonviable cells accumulated Trypan Blue by a saturable process. The nucleus-to-medium dye gradient was more then 30:1 in media containing 0.1% or less Trypan Blue. The apparent affinity constant for nuclear binding of the dye was 3.1 X 10(4)l/mol. Albumin partially inhibited the nuclear staining. More than 0.5% Trypan Blue in the medium was toxic per se. In the absence of albumin, 0.5 or 20 mmol/l alloxan, 1 mmol/l N-ethylmaleimide, or 0.1 mmol/l chloromercuribenzene-p-sulphonic acid, but not 20 mmol/l streptozotocin, increased the frequency of islet cells stained with 0.1% Trypan Blue. The absorbance of nuceli was also increased in cells treated with alloxan or N-ethylmaleimide, but not in those treated with chloromercuribenze-p-sulphonic acid. It is concluded that alloxan rapidly increases the permeability of the plasma membrane in mouse beta-cells. This action of alloxan appears to be more acute than any such effect of streptozotocin.


In vivo and in vitro studies on temperature-sensitive mutants of swine vesicular disease virus.

Two temperature-sensitive mutants of the Ukg 27/72 strain of swine vesicular disease virus were isolated in tissue culture and a third was derived following adaptation in mice. All three were found to have similar growth restrictive temperatures, but varied considerably in their virulence when administered to pigs. The route of inoculation appeared to exert a considerable influence on the apparent degree of attenuation, the antibody titre engendered and the transmission of disease to pigs held in contact with inoculated animals. One strain appeared almost totally attenuated when inoculated animals. One strain appeared almost totally attenuated when inoculated into pigs but spread to animals in contact causing severe disease. Virus re-isolated from one such animal was found to have retained its temperature sensitive phenotype, suggesting that virulence in this case was not directly related to temperature sensitivity. Pigs with high antibody titres were found to be susceptible when placed in contact with challenge animals, although the lesions which developed were mild.


Inhibition of the in vitro outgrowth of Epstein-Barr virus-infected lymphocytes by TG lymphocytes.

Human T lymphocytes subpopulations from subjects not acutely infected with EBV have been selected and examined for ability to inhibit the outgrowth of autologous B lymphocytes that have undergone in vitro infection with EB virus. The results show that only TG lymphocytes are inhibitory. TG lymphocytes from subjects who are EBV antibody-negative inhibit as well as those from subjects who have EBV antibody. TG lymphocyte populations, as well as other T cell fractions obtained from neonatal subjects, fail to inhibit the outgrowth of infected, autologous lymphocytes under the conditions tested. We propose that NK cells are responsible for the inhibitory effects described in this report.


Chemotactic factor-induced generation of superoxide radicals by human neutrophils: evidence for the role of sodium.

The role of sodium ion in superoxide (O2-) generation by human peripheral neutrophils was investigated. Cells were activated by exposure to the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and O2- release was assessed by ferricytochrome c reduction after 5 min of incubation at 37 degrees C in the presence of FMLP 4 X 10(-8) M. In the absence of monovalent cations (isotonic glucose), negligible O2- generation occurred. There was a progressive increase in the magnitude of FMLP-induced O2- generation with increasing Na+ concentration up to 90 mM, where the response was noted to plateau. Varying the K+ concentration (1 to 10 mM) had no effect on the amount of O2- produced in the presence of Na+ 140 mM. FMLP also stimulated 22Na+ and 48Ca2+ uptake by the cells in a dose- and time-dependent fashion. FMLP-induced 22Na+ uptake appeared to be independent of the external Ca2+ concentration ( to 4 mM). In contrast, there was a progressive decrease in themagnitude of the FMLP-induced increase in 45Ca2+ uptake as the Na+ concentration was reduced by replacement with choline+ or glucose. These studies support a requirement for Na+ in FMLP-induced O2- generation and suggest that a Na+ influx may underlie the nature of this requirement. The data are also consistent with the hypothesis that a Na+ influx may precede the Ca2+ influx in the FMLP-induced activation sequence.


Purification of human T and B cells by a discontinuous density gradient of percoll.

A simple technique for isolating human peripheral B and T cells is described. This method is based on the different mobility of cells in a discontinuous density gradient of Percoll. A population with a mean of 72.8% of surface stable Ig (B cells) is found in the top layer (fraction I) whereas T cells with less than 3% of Ig are distributed in the two lower layers (fractions II and III). B cells obtained by this method were highly viable and reacted strongly with anti-DRw specific sera, whereas T cells did not respond. The clear-cut difference between positive and negative reactions makes this technique very suitable for DRw typing. The proliferative response to PHA is decreased in fractions I and III while greatly increased in fraction II. The different mitogenic responses in the two fractions containing T cells probably represent two functionally distinct T cell subsets. Cells from all fractions are less activated than unseparated cells when Con A and PWM are used as mitogen. The technique is very rapid, avoids the interaction of cells with their specific receptors and gives both T and B cells in one step, all advantages over other methods commonly used.


Primary oat cell carcinoma of the larynx.

A case of primary oat cell carcinoma of the larynx in a forty-year-old female has been described. The tumour is rare and extremely aggressive. Surgical treatment and irradiation appear to halt the disease for a short time.


Apolipoprotein B transports cholesterol in plasma as low density lipoprotein (LDL) and targets its delivery to cells by binding to a specific plasma membrane receptor. The cellular consequences of apoB binding to its receptor were investigated to determine whether it suppresses cholesterol biosynthesis and reduces the number of cellular receptors for the apoprotein. Upon preincubation of fibroblasts with lipoprotein-deficient medium alone or supplemented with either LDL or apoB complexed to BSA (apoB-BSA), LDL suppressed cholesterol biosynthesis, but apoB enhanced it. Similarly, fibroblasts preincubated in medium supplemented with LDL bound decreased amounts of either (125)I-labeled LDL or (125)I-labeled apoB-BSA to their receptors, while preincubation with apoB-BSA increased the binding relative to the controls. These latter results occurred in association with a decrease in cellular cholesterol content, indicating that apoB in the medium bound cholesterol and removed it from the cells, thus stimulating both cholesterol synthesis and cellular binding of apoB. Accordingly, fibroblast cholesterol synthesis and the number of functional LDL receptors are not suppressed by the binding of the apoprotein to the receptor, and the known role of apoB remains that of transporting cholesterol in plasma and delivering it to the cell. A possible physiologic role for apoB in depleting cells of cholesterol is presently unknown since apoB is not known to exist free in plasma; however, these findings demonstrate such a functional capability for this apoprotein.-Shireman, R. B., and W. R. Fisher. Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.


In this study we have determined the fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis of rat plasma very low density lipoprotein (rat VLDL). The experiment was carried out in vitro with lipoprotein lipase purified from bovine milk, VLDL labeled with [(14)C]palmitate, [(3)H]cholesterol, [(32)P]phospholipids, and (125)I-labeled apolipoprotein C and in plasma-devoid systems. Triglyceride hydrolysis ranged between 0 and 98.6%. [(32)P]Phospholipids, unesterified [(3)H]cholesterol, and (125)I-labeled apolipoprotein C were removed from the VLDL (d < 1.019 g/ml) during lipolysis. About one-third of the [(32)P]phosphatidylcholine was hydrolyzed to lysolecithin, and was transferred to the fraction d > 1.21 g/ml. The other two-thirds of the phospholipids were removed unhydrolyzed, mainly to the fraction d 1.04-1.21 g/ml. With the progression of the lipolysis, unesterified [(3)H]cholesterol was removed from VLDL at increasing rates, predominantly to the fraction d 1.04-1.21 g/ml. (125)I-Labeled apolipoprotein C removed from the VLDL partitioned between the fraction of d 1.04-1.21 g/ml and d > 1.21 g/ml. Negative-staining electron microscopy of the fraction d 1.04-1.21 g/ml (containing phospholipids, unesterified cholesterol, and apolipoprotein C) revealed many discoidal lipoproteins. [(3)H]Cholesteryl esters remained associated with the VLDL even when 70-80% of the triglycerides were hydrolyzed. These observations suggest that during in vitro lipolysis of VLDL, surface constituents leave the lipoprotein concomitantly with the hydrolysis of core triglycerides. The process of removal of surface constituents is independent of the presence of an acceptor lipoprotein and may occur in the form of a surface-fragment particle. -Eisenberg, S., and T. Olivecrona. Very low density lipoprotein. Fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis in vitro.


Preparation and properties of soluble, immunoreactive apoLDL.

Immunoreactive apo-low density lipoprotein (LDL), soluble in mildly alkaline buffers of low ionic strength, was prepared by attaching LDL to a DEAE-Sepharose column and eluting the lipids with a 0--2% (w/v) gradient of nonionic detergents. Brij-36T, Nonidet P-40, and Triton X-100 gave similar results. After washing the detergent from the column, the bound apoLDL was eluted with 1 M NaCl, pH 7.4, with recoveries up to 85%. This apoLDL could be dialyzed extensively against low ionic strength solutions, and remained soluble as long as the pH was above 7. Spectrophotometric analysis showed that less than 0.1% %w/v) of cholesterol or phospholipids and less than 1% (w/v) of detergent remained associated with the protein. The apoLDL cross-reacted with LDL against antisera prepared vs. intact LDL. Pore-gradient polyacrylamide gel electrophoresis, with SDS and urea, showed that this preparation was less aggregated than organic solvent extracted apolLDL and appeared to be made of oligomers of two monomeric subunits, one with molecular weight around 22,700 and a smaller one of approximately 8000. Isoelectric focusing showed that there also was charge heterogeneity in the soluble apoLDL.


Determination of mevalonate in blood plasma in man and rat. Mevalonate "tolerance" tests in man.

A method is described for the determination of mevalonate in ultrafiltrates of blood plasma. The method depends on the phosphorylation of mevalonate with [gamma-32P]ATP and mevalonate kinase to 5-[32P]phosphomevalonate, and the subsequent isolation of the 5-[32P]phosphomevalonate together with known amounts of added 5-phospho[14C]mevalonate by ion-exchange chromatography. The 32P/14C ratio in the isolated 5-phosphomevalonate is a linear function of the mevalonate content of the samples. The smallest amount that can be determined is 1--2 pmol. The fasting level in human plasma varied between 20 and 75 pmol/ml. Human red blood cells absorb mevalonate from plasma relatively slowly; their maximum storage capacity is about 1.3 pmol/10(6) red cells. An oral and intravenous "mevalonate tolerance test" in man is described that can be carried out with 200 and 30 mumol. respectively, of the unlabeled (RS)-mevalonate in a 70-kg man. Beer and wine contain mevalonate at a concentration of  3--8 microns, too low to provide a significant amount of mevalonate even for heavy drinkers. The mevalonate content of the plasma from the blood of the vena cava inferior of male rats varied between 81 and 502 pmol/ml and is positively related to the levels of liver 3-hydroxy-3-methylgultaryl-CoA reductase, suggesting that the liver is probably the main source of mevalonate circulating in blood. The plasma of renal venous blood contained only 33--85% as much mevalonate as the arterial plasma.


Lipids of whole cells and plasma membrane fractions from Balb/c3T3, SV3T3, and concanavalin A-selected revertant cells.

The lipid composition of Balb/c3T3, SV3T3, and the concanavalin A-selected SV3T3 revertant cells has been analyzed at the whole cell and plasma membrane levels. In comparison to untransformed 3T3 whole cells, SV3T3 cells showed an unchanged content of triacylglycerols, free fatty acids, and glycerylether diesters but a lower concentration of total phospholipids, while no significant difference was found in the phospholipid composition. Whole SV3T3 revertant cells exhibited a lipid composition similar to that in untransformed 3T3 cells with the exception of a higher proportion of sphingomyelin. Analysis of isolated plasma membranes did not reveal any significant differences in the cholesterol to phospholipid molar ratio between 3T3 and SV3T3 or SV3T3 revertant cells. The major changes in the acyl chain pattern SV3T3 compared with whole 3T3 cells consisted of an increase of oleic and palmitoleic acids coupled with a decrease of C20 and C22 polyunsaturated acids in phosphatidylethanolamine and phosphatidylcholine; an increase of oleic acid was also evident in SV3T3 phosphatidylinositol plus phosphatidylserine. An increase of palmitoleic and oleic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine of SV3T3 plasma membranes; the only change in SV3T3 plasma membrane phosphatidylcholine was an increase of oleic acid. An increase of monoenoic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol plus phosphatidylserine of SV3T3 revertant cells at the level of both whole cells and plasma membranes.


Apoproteins of high density lipoproteins in the urine of normal subjects.

Apoproteins of high density lipoproteins were detected in the urine of normal subjects after the urinary proteins were highly concentrated. By immunoelectrophoresis, all of the urinary apoproteins gave precipitin lines with similar electrophoretic mobility. This suggests that the various apoproteins are present in the same particle. The apoproteins were present only in the ultracentrifugal fraction of density greater than 1.24 g/ml. Neither apoprotein B nor apoprotein E were detected in the urine, suggesting that very low density and low density lipoproteins are not excreted in the urine of normal subjects.


Interaction of bovine serum high density lipoprotein with mixed vesicles of phosphatidylcholine and cholesterol.

The interaction of sonicated, small vesicles of egg phosphatidylcholine and cholesterol (2:1, mol/mol) with bovine high density serum lipoproteins was examined in terms of lipid transfer between both types of particles and the resulting changes in lipoprotein structure. Saturation of high density lipoprotein preparations with vesicle lipids gave final lipoprotein particles with essentially unchanged protein content and composition, unchanged cholesterylester and nonpolar lipid content, but with markedly increased phospholipid content (59% increas by weight) and moderately increased cholesterol content (20% increase by weight). The lipoproteins enriched in lipid were relatively uniform, spherical particles, 110 +/- 3.6 A in diameter (6 A larger than the original lipoproteins); they had a markedly decreased intrinsic protein fluorescence, a red-shifted fluorescence wavelength maximum, and more fluid lipid domains. These results indicate that the direct addition of excess lipids from membranes or other lipoproteins is a possible mechanism for lipid transfer to high density lipoproteins. Also they suggest a structural flexibility of high density lipoproteins that allows the addition of significant amounts of surface components.


Aminopropyl silica gel as a solid support for preparation of glycolipid immunoadsorbent and purification of antibodies.

Aminopropyl silica gel was prepared from porous silica gel and was used as a solid support for immunoadsorbent in the purification of anti-glycolipid antibodies. For neutral glycosphingolipids, a carboxyl function was generated by oxidation of the olefinic double bond of the sphingosine moiety, whereas for gangliosides the carboxyl group of sialic acid was used to couple with aminopropyl silica gel in the presence of a carbodiimide. These compounds were used for purifying anti-glycolipid antibodies from serum of immunized rabbits. The antibodies bound to the su-strate were released by 2 M potassium thiocyanate and their immunological properties were studied. Aminopropyl silica gel may be preferred over conventional organic solid supports for the following reasons: 1) faster flow rate; 2) higher capacity; 3) easier handling; 4) more economical; and 5) lower susceptibility to microbial attack.


Evaluation of the bovine testicular radioreceptor assay for pituitary follicle-stimulating hormone.

A modified bovine testicular receptor was used to evaluate highly purified follicle-stimulating hormone (FSH) from a number of species. The particulate receptor obtained from adult bovine testes could be stored frozen or lyophilized for long periods without appreciable decrease in the binding of the ligand facility or in loss of specificity. The bovine testis receptor binds twice as much 125I-labelled ovine FSH as 125I-labelled human hormone. When FSH from different species was compared against NIH-FSH-S10, using various FSH ligands, the ovine hormone was clearly the most active, although many had comparable in-vivo biological potencies. The results suggest that there is probably some species specificity in the hormone-receptor interactions. As the ovine hormone is structurally closer to the bovine, from which the receptor was derived, it appears to have the highest activity in vitro. Marked differences in the biological activities of the different preparations between the human chorionic gonadotrophin-augmentation test and the in-vitro assays have been observed. In the in-vitro assays, all preparations, with the exception of the porcine hormone preparation, were less active and the ratio of bioassay/radioreceptor assay varied widely. In the radioreceptor assays, all FSH preparations except pregnant mare serum gonadotrophin (PMSG) showed parallel inhibition curves. The three different PMSG preparations examined gave inhibition lines that were parallel to each other.


Effects of prostaglandin E2 and dibutyryl cyclic AMP on the histamine-induced production of cortisol and corticosterone in isolated canine adrenal cells.

The steroidogenic effect of histamine in isolated adrenocortical cells of the dog was investigated in the presence of prostaglandin E2 (PGE2) and/or dibutyryl cyclic AMP (dbcAMP) in the medium. The effect of histamine, in combination with PGE2 was less than their total individual effects in the production of cortisol, but not ofcorticosterone. With dbcAMP the effect was just equal to them. However, the combination of histamine, PGE2 and dbcAMP showed an increase twice that of their total individual effects in the production of both steroids. These results indicate that, in the dog, histamine, PGE2 and dbcAMP may act synergistically in the adrenocortical cells.


Intestinal parasites in Laotian refugees.

Laotian refugees relocated in the Columbus, Ohio, area were screened for intestinal parasites. Eight different pathogens were isolated. The prevalence of the various organisms ranged from 4 to 61 percent of persons affected and 18 to 100 percent of families. These data show a higher prevalence of parasitic disease than that revealed by previous observations by the Center for Disease Control among Southeast Asian refugees. It was also apparent that neither federal nor local programs were adequate to meet the special health care needs of these refugees nor to safeguard the health of communities they entered.


Cell-mediated immunity to cytomegalovirus in patients receiving immunosuppressive therapy.

Employing the techniques of complement-fixation (CF), immunofluorescence (IF), and in vitro lymphocyte transformation (LTF), the humoral antibody response and cell-mediated immune (CMI) response to cytomegalovirus (CMV) were studied in the serum and peripheral blood lymphocytes in 19 normal children (controls) and 23 patients with renal disease who were receiving immunosuppressive therapy or undergoing hemodialysis. The LTF activity was determined by the whole blood microassay using two strains of CMV (AD-169 and Davis) and phytohemagglutinin (PHA). The antibody level responses to CMV in different groups of subjects were generally similar. The LTF response to PHA as evidenced by delta cpm activity was moderately depressed in immunosuppressed and hemodialyzed subjects compared to the response observed in the controls. The mean delta cpm activities in response to AD-169 and Davis strains of CMV in seropositive immunosuppressed patients were about one-fifth and one-third lower respectively than those of seropositive normal controls. These observations suggest that an impairment of CMV specific cellular immunity may be an important mechanism underlying the increased susceptibility to CMV infections in patients with chronic renal disease who receive immunosuppressive therapy.


Human rotavirus in an adult population with travelers' diarrhea and its relationship to the location of food consumption.

The role of human rotavirus in adult diarrhea was evaluated in 164 newly arrived US students attending summer school at an urban Mexican university. Rotavirus was identified in stool samples by electron microscopy. Rotavirus was found in 26 of 109 students with diarrhea (24%) and in 8 of 55 asymptomatic control students (15%). Although bacterial pathogens were recovered from virus positive students with diarrhea, viral shedding also occurred independently of other agents. Clinical disease in students excreting only rotavirus tended to be mild and was accompanied by a low density of viral shedding. Food consumption in the home and at public eating establishments was examined the week before illness. While the location of food consumption was found to be important in the acquisition of diarrhea, there was no apparent relationship of the site where meals were eaten and the acquisition of rotavirus by students newly arrived in Mexico. These data support our previous study in a US student population residing in a rural setting in Mexico and implicate rotavirus as a cause of diarrhea among students traveling to Mexico from the United States. The present study offers additional evidence that rotavirus infection in this population might be spread by a nonfood vehicle of transmission which differs from spread of enterotoxigenic E coli, Shigella, or Salmonella strains in the same population.


Epidemiology of cytomegalovirus infection in end stage renal disease.

Since primary cytomegalovirus (CMV) infection is a cause of morbidity among renal transplant recipients, we undertook a prospective study of our maintenance hemodialysis patients and personnel to determine whether these sources posed a risk for transmission of CMV. Our study of 85 patients and 49 personnel showed that CMV was detected in eight nontransplanted, older dialysis patients and 13 patients who had lost their allograft. In spite of the presence of CMV on the unit, no patient or staff member developed primary infection from interpersonal transmission or from transfused frozen red blood cells. All primary infections in renal transplant patients could be accounted for by acquisition from the transplanted kidney, thus eliminating the dialysis unit or frozen blood as a risk to either patients or personnel.


Profile of Mood States: the factors and their physiological correlates.

Spontaneously occurring mood states and various physiological indices were evaluated on three occasions in a group of eight healthy males. Results reveal that significant positive correlations exist among five of the six factors comprising the Profile of Mood States (POMS). This finding is related to previous research on the POMS and varied methodologies utilized in those reports. Reliable positive correlations of the POMS factors Depression and Tension with heart rate and diastolic blood pressure were obtained. The Anger factor of the POMS also correlated positively and significantly with heart rate. These findings are discussed in terms of the relationship between POMS factors and anxiety.


Dopamine-sensitive adenylate cyclase activity in the caudate nucleus and adrenal medulla in Parkinson's disease and in liver cirrhosis.

In human caudate nucleus and adrenal medulla dopamine-stimulated adenylate cyclase was measured. At a concentration of 100 microM dopamine, liver cirrhosis, carcinoma and therapy resistent parkinsonian patients showed decreased stimulation or even a reduction compared to controls. Therapy responding parkinsonian patients showed a decreased stimulation compared to controls, but this stimulation was significantly higher than in drug resistent patients.


Correlated effects of acetylcholine and cyclic guanosine monophosphate on membrane properties of mammalian neocortical neurons.

The effects of successive extracellular iontophoresis of acetylcholine (ACh) and atropine, and intracellular hyperpolarizing iontophoresis of cyclic GMP (cGMP) were studied in single neurons of the coronal-pericruciate cortex of awake cats. (a) Fifty-seven percent of the neurons that were tested responded to ACh with an increase in neuronal input resistance (Rm) and 50% responded to ACh with an increase in firing rate; 65% responded to cGMP with an increase in Rm and 60% responded to cGMP with an increase in firing rate. (b) After application of atropine, increases in Rm and firing rate associated with iontophoresis of ACh failed to recur. (c) Persistent increases in Rm following application of ACh accompanied by current-induced neuronal discharge were not diminished by subsequent application of atropine. (d) Atropine did not prevent increases in Rm and firing rate associated with intracellular iontophoresis of cGMP. (e) All cells tested with both ACh and cGMP that were shown initially to respond to extracellular ACh with increases in Rm were later shown to have comparable responses to cGMP.


Electrophysiological study of the peripheral nervous system in children. Changes in proximal and distal conduction velocities from birth to age 5 years.

Hoffman's reflex was evoked in the soleus muscle of 105 normal children from birth to age 5 years. This technique made it possible to determine conduction time and to estimate conduction velocity over the reflex arc. For 59 children, proprioceptive and motor distal nerve conduction velocities were calculated for the tibial nerve trunk. These measurements enabled the common changes for these three velocities to be described in terms of an exponential curve. Proximal conduction velocity has similar values to those of proprioceptive distal nerve fibres: it is always higher than motor nerve conduction velocity, but the difference gradually diminishes, remaining constant after the eighteenth month. Conduction time diminishes between birth and one year of age, whereas height increases. Then conduction time increases slowly, reaching at about age 5 years, when average height is 1050 mm, the value it had just after birth. For the child born at term, maturation of the nervous system is thus especially rapid during the first year of life.


Postnatal rat sympathetic neurons in culture. II. Synaptic transmission by postnatal neurons.

1. It was shown in the preceding paper that postnatally derived rat superior cervical ganglion neurons (SCGN) will grow in dissociated cell culture and form functional synaptic connections with each other. In this report, synaptic transmission by the postnatal SCGN is detailed. 2. Synaptic interactions between SCGN were blocked by the nicotinic cholinergic antagonist hexamathonium (C-6), indicating that acetylcholine was the transmitter substance used by these neurons. This was found to be the case even for neurons taken from 12.5-wk-old animals. 3. In a few cases, the beta-adrenergic blocking agent, propranolol, was found to block synaptic potentials, suggesting that a catecholamine might be involved in the transmission process. The possible mechanisms of this involvement are discussed. 4. SCGN taken from up to 10-wk-old rats were able to form functional synaptic contacts with cocultured skeletal muscle cells. These interactions were sensitive to low external Ca2+ and to 1--2 microM d-tubocurarine (d-TC). 5. It is concluded that even adult SCGN retain a certain amount of neurotransmitter "plasticity" when grown under appropriate culture conditions. From the data on the neuron-neuron and SCGN-skeletal muscle interactions, it is suggested that a matching of presynaptic transmitter with postsynaptic receptor is a sufficient condition for the formation of functional nerve-target interactions.


Simultaneously occurring salivary gland tumors of different types.

Two cases of simultaneously occurring salivary gland tumors of the parotid gland are reported. Both patients were women in the sixth decade of life. In the first patient, a carcinoma in pleomorphic adenoma and an oncocytoma were found in the right parotid gland. The second patient had an adenoid cystic carcinoma associated with a sebaceous cell adenoma. The addition of these cases to the previously published data brings the total number of simultaneously occurring salivary gland tumors of different types to ten.


Bilateral nephroblastoma in a horseshoe kidney.

A case of bilateral nephroblastoma in a horseshoe kidney is presented. It is the second to be reported in the international literature.


Reduction of opiate binding to brainstem slices associated with the development of tolerance to morphine in rats.

Previous studies revealed that the characteristics of opiate binding sites are different in brainstem slices and homogenates. In the present study, the binding of opiate agonists, morphine and etorphine, and that of the antagonist, naloxone, to thin slices of rat brain stem was studied. Sodium ion inhibits agonist binding and enhances antagonist binding in brainstem slices. Development of the analgesic tolerance to morphine is accompanied by a reduction of opiate binding. The opiate binding recovers partially toward control values during morphine withdrawal. Kinetic analyses indicate that sodium-induced changes in opiate binding sites are different from those caused by chronic morphine treatment. These results provide evidence that analgesic tolerance is associated with changes in opiate receptors.


Adrenergic innervation and cyclic adenosine 3':5'-monophosphate levels in response to norepinephrine in stomach of postnatal rats.

Changes in receptor function, innervation and cyclic adenosine 3':5'-monophosphate (cAMP) levels in response to norepinephrine were investigated in the stomach of postnatal developing rats. We found that although the beta adrenergic receptor function and responsiveness of cAMP-generating system to norepinephrine are present in the early postnatal stage (3-day-old), these systems are not fully developed at birth and that increase in sensitivity comes with age. Adrenergic innervation is present at birth in Auerbach's plexus but not in smooth muscle, during the 1st postnatal week. Direct adrenergic innervation to smooth muscle appears 1 week after birth and matures during the first 4 weeks. This maturation of direct adrenergic innervation runs in parallel with the increased sensitivity of beta receptors and is accompanied by an elevated responsiveness of the cAMP-generating system to norepinephrine.


Nicotinic, muscarinic and adrenergic receptors in a parasympathetic ganglion.

Transmission in submandibular ganglia of hamsters was blocked by hexamethonium and dimethylphenylpiperazinium. Dimethylphenylpiperazinium caused depolarization and decreased membrane resistance (Rm). The muscarinic agonist, bethanechol (BCh) caused depolarization of some cells and hyperpolarization of others. Regardless of the change in membrane potential, BCh always increased Rm. Since the responses to BCh persisted in the absence of [Ca++]0, it was concluded that BCh acted directly on the ganglion cells and did not depend upon a transsynaptic process. All responses to BCh were prevented by atropine. The evidence suggests that the ganglion cells possess muscarinic receptors. Like BCh, norepinephrine (NE) either depolarized or hyperpolarized the ganglion cells. There was no relationship between the blockade of transmission by NE and the effect of NE on the membrane potential. The responses to NE were prevented by dihydroergotamine, suggesting the presence of alpha adrenergic receptors on the ganglion cells.


Catecholaminergic alpha-receptors and shivering in the rat.

1. A rise or a fall in systemic blood pressure brought about by the I.V. infusion of peripherally acting drugs (adenosine, noradrenaline or methoxamine) inhibited shivering in the cold-exposed rat. 2. Since the injection of commonly used doses of noradrenaline (0.05-0.10 mumole) into a lateral cerebral ventricle of a rat was usually accompanied by a rise in blood pressure special precautious were required to determine whether noradrenaline had a specific central effect on shivering. 3. Small doses of noradrenaline (0.02-0.03 mumole) or clonidine (0.01 mumole) which had no effect on blood pressure when injected into a lateral cerebral ventricle still inhibited shivering in the cold-exposed rat and this effect was prevented by phentolamine. 4. It is concluded that noradrenaline can inhibit the cold sensor-shivering pathway in its central course by an action on alpha-receptors.


Supportive nursing: a critical review of the literature.

Nurses have long thought that providing support was a part, if not a central part, of their nursing practice. In the literature, nurses have described their ability to provide support and have discussed their inability as well as their reasons why they have been unable to give support. Support for nurses has at least three parameters: physical, social, and emotional. The emotional parameter has received the most attention by nurses. The meaning of support in nursing needs to be clearer and more precise. Although several nurses, either through theoretical discussions or exploratory research studies, have begun to further define support, this construct warrants more thought and investigation by nurses. Once an operational definition is determined and supportive behaviors identified, nurses can further investigate the influence of support to the promotion of health.


Microscopic observations on the filopodia of Entamoeba histolytica.

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter micrometer thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 micrometers in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called "surface-active lysosomes with trigger," "dendritic plasmalemmal extensions," and "extra-amebic vesicles" by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.


Trichomonas vaginalis resistance to Mengo virus infection.

We have investigated the susceptibility of Trichomonas vaginalis to Mengo virus infection by comparing the outcome of Mengo virus or purified Mengo virus RNA infection in T. vaginalis and in CCL-1 mouse fibroblasts. While the adsorption and entry of Mengo virus into T. vaginalis occurred in the same manner as in fibroblasts, the uncoating was much slower. In addition, Mengo virus infection of T. vaginalis displayed no eclipse nor any subsequent production of infectious virus. Purified RNA failed to initiate productive infection in T. vaginalis, whereas it provoked viral replication in the fibroblast controls. It was shown by assessment of protein synthesis in T. vaginalis and mouse fibroblasts cell-free systems that the protozoan ribosomes were able to translate endogenous mRNA and poly-U, but not viral RNA.


Growth response of axenic Entamoeba histolytica to hydrogen, carbon dioxide, and oxygen.

Entamoeba histolytica required CO2 for growth in axenic culture while growth was inhibited by H2. The organism was tolerant to 5% O2 in the gas phase and it was able to detoxify products of O2 reduction in the medium. The ameba did not require a negative oxidation-reduction potential for axenic growth. However, little or no free O2 was present in media exposed to 5% O2 in the gas phase. Growth was improved by adding yeast extract to the medium.


Antitumor immunity in breast cancer patients. Biologic and therapeutic implications.

As judged by in vivo and in vitro indices of cellular immunity, mammary carcinogenesis appears to be associated with immunogenic changes in the transformed cells and prognostically favorable antitumor cell-mediated immunity in the host. The prognostically favorable cell-mediated immunity appears to be directed against a component(s) which is shared by most stage 0 in situ breast cancers and the major envelope glycoprotein (gp55) of RIII murine mammary tumor virus. Progressive disease is associated with a loss of gp55-like tumor immunogenicity and/or anti-gp55 cell-mediated immunity of the host. It is appropriate that measurements of tumor immunogenicity and tumor-specific cell-mediated immunity of the host be included in the prognostic assessment and therapeutic decisions regarding individual patients. Such data are particularly pernitent to the development and evaluation of immunotherapeutic and immunoprophylactic protocols.


Malignant trophoblastic disease in paraguay.

The incidence of malignant trophoblastic diseases from 1960 to 1969 and 1970 to 1974 in the population of Parguay was low, as shown by the analysis of unselected hospital admissions as compared to the total population. Paraguay showed disease characteristics comparable to those of developing countries where malignant diseases of the trophoblast have elevated frequencies. The apparently high frequencies in lower socioeconomic groups appear to be due to the fact that these groups make up a large proportion of the hospital population. The hardship of poverty predisposes to the hospital concentration of pathologic pregnancies from around the country and to the home delivery of grossly normal gestations. Thus, a biased incidence of diseases results.


Cellular antiproliferative action exerted by auranofin.

Auranofin (AF) is a new orally absorbed coordinated gold compound currently undergoing Phase I studies for its use in the treatment of rheumatoid arthritis. Our investigations with RAJI lymphoma, HeLa carcinoma, and EBV-transformed cells indicate AF exerts an inhibitory effect on DNA, RNA, and protein synthesis as assessed by 3H-thymidine, 3H-uridine, and 3H-leucine uptake, respectively. A rapid and persistent dose dependent inhibition of 3H-thymidine uptake was observed at gold concentrations of 50-100 microgram/dl while all parameters were inhibited after a 24 hr exposure to 100 microgram/dl. Reductions in viability and surface morphological changes were also observed. These results suggest AF exerts a significant inhibitory effect on essential biological processes and functions.


Synthesis of 11 beta,13 beta- and 13 beta,16 beta-propano steroids: probes of hormonal activity.

Syntheses of 11 beta,13 beta- and 13 beta,16 beta-propano derivatives of 17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one are described. The 13 beta,16 beta bridge was constructed by intramolecular alkylation of the C-16 enolate anion from 3-methoxy-13 beta-[3'-(tosyloxy)propyl]gona-3,5-dien-17-one, the latter being obtained via Birch reduction of both aryl groups of 17 beta-hydroxy-3-methoxy-13 beta-(3'-phenoxypropyl)gona-1,3,5(10),8-tetraene (1). The 11 beta,13 beta bridge was constructed by Prins cyclization of 17 beta-acetoxy-3-methoxy-13 beta-(3'-oxopropyl)gona-1,3,5(10),9(11)-tetraene, itself obtained via Birch reduction of only the side-chain aryl group of 1. Binding affinities of certain of these compounds and substituted 13 beta-propyl derivatives of 17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one for the uterine cytosol receptor of progesterone are reported, and the origin of the high progestational activity of norgestrel and 11 beta-substituted progestins is discussed.


Photoaffinity labeling of the angiotensin II receptor. 3. Receptor inactivation with photolabile hormone analogues.

It has been shown that the receptor of angiotensin II (AT) in rabbit aorta strips, rat aorta, and rat stomach can be blocked specifically and irreversibly by several photolabile analogues of Sar-Arg-Val-Tyr-Val-His-Pro-Phe ([Sar1]AT) with irradiation. The effectiveness of a photolabel with light of wavelength 365 nm depends on the labeling amino acid (L-4'-nitrophenylalanine, L-4'-diazoniumphenylalanine, or L-4'-azidophenylalanine) and on its position in the peptide (replacing Tyr4 and/or Phe8). The 4'-azido)Phe-containing analogues are all good to fair photoinactivators. Their decreasing order of effectiveness is as follows: [Sar1,(4'-azido)Phe8]AT, [Sar1,(4'-azido)Phe4,8]AT, and [Sar1,(4'-azidoPhe4]AT. The (4'-nitro)Phe analogues show the opposite relation: the good ligand [Sar1,(4'-nitro)Phe8]AT is almost ineffective, but the nonligand [Sar1,(4-nitro)Phe4]AT exhibits good, specific photoinactivation. This can be explained by the existence of a different photolysis pathway for (4'-nitro)Phe: this analogue probably undergoes a multiphoton decay with a long-lived first excited state. A peptide in this state may differ in its pharmacological properties from the ground state and become a ligand.


Synthesis and biological properties of N2-substituted spin-labeled analogues of actinomycin D.

We have synthesized N2-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)]actinomycin D And the related 1,2-diaminoethane and 1,3-diaminopropane derivatives and evaluated their biological properties. Binding studies with the spin-labeled actinomycin D analogues and DNA were carried out by using circular dichroism, electron spin resonance, and thermal denaturation. These studies have suggested that the derivatives bind to DNA and that their DNA-binding modes are similar but not identical. Spin-labeled actinomycin D derivatives were less potent in inhibiting Escherichia coli DNA-dependent RNA polymerase reaction than actinomycin D and were less toxic to L1210 cells in vitro than the parent compound. Spin-labeled actinomycin D derivatives were more common than the parent compounds against P-388 leukemia cells in vitro with little or no toxicity.


Crystal-induced inflammation, enzyme release and the effects of drugs in the rat pleural space.

The pleural space of the rat has been used to study the acute inflammatory response to crystals of monosodium urate monohydrate, calcium pyrophosphate dihydrate and hydroxyapatite. Each crystal caused a brisk inflammatory response accompanied by crystal phagocytosis and release of lysosomal enzymes. These responses were modified by the anti-inflammatory drugs indomethacin, dexamethasone, colchicine and cyclophosphamide. The results of these experiments suggesting that the resting cell population of the pleural space contributed to the response, and that lysosomal enzyme release may play only a minor role in the mediation of increased vascular permeability.


Frequency sweep analysis of neuromuscular junction continuity.

A new technique for measuring neuromuscular junction continuity is described. Junctional transmission is evaluated by integration of the electromyographic (EMG) response induced by intramuscular electrical stimulation. The unique characteristics of this monitoring technique are the use of intramuscular stimulation and a varying stimulation rate. The stimulus frequency is made to increase exponentially from 1 to 100 pulses per second over a period of 10 or 15 seconds. Thin coiled wire electrodes are used for stimulation and recording of the EMG response. The frequency sweep electromyogram (FS-EMB) response was measured in uncurarized and curarized tibialis anterior muscle of cat. In the uncurarized preparation, the FS-EMG response was only slightly decreased with an increasing stimulus frequency. During neuromuscular blockade with d-tubocurarine the FS-EMG response diminished with increasing frequency. The response failed at successively lower frequencies during deeper levels of neuromuscular block. Recovery proceeded in the reverse order. The frequency sweep technique provides quantitative assessment of neuromuscular junction continuity over a broad spectrum of driving frequencies without inducing fatigue. This method does not require force measurements nor voluntary effort. Intramuscular stimulation minimises painful stimuli thus making the frequency sweep technique suitable for evaluating neuromuscular transmission in either conscious or unconscious individuals.


Conformational changes of membrane-bound (Na+--K+)-ATPase as revealed by trypsin digestion.

To distinguish ligand-induced structural states of the (Na+--K+)-ATPase, the purified membrane-bound enzyme isolated from rat kidneys was digested with trypsin in the presence of various combinations of Na+, K+, Mg++ and ATP. It was found that first the large and then the small polypeptide chain of the (Na+--K+)-ATPase was degraded, indicating that the lysine and arginine residues of the large chain are more exposed than are those of the small one. The (Na+--K+)-ATPase activity was inactivated in parallel with the degradation of the large polypeptide chain. After the degradation of the large polypeptide chain, about 75% of the (Na+--K+)-ATPase protein remained bound to the membrane, demonstrating that the split protein segments were only partially released. It was found that the combinations of ATP, Mg++, Na+ and K+ present during trypsin digestion influenced the time course and degree of degradation of the (Na+--K+)-ATPase protein. The degradations of the large and the small polypeptide chain were affected in parallel. Thus, certain ATP and ligand combinations influenced neither the degradation of the large nor the degradation of the small polypeptide chain, whereas by other combinations of ATP and ligands the degree of susceptibility of both polypeptide chains to trypsin was equally increased or reduced. In the absence of ATP the time course of trypsin digestion of the (Na+--K+)-ATPase was the same, whether Na+ or K+ was present. With low ATP concentrations (e.g., 0.1 mM), however, binding of Na+ or K+ led to different degradation patterns of the enzyme. If a high concentration of ATP (e.g. 10 mM) was present, Na+ and K+ also influenced the degradation pattern of the (Na+--K+)-ATPase, but differentially compared to that at low ATP concentrations, since the effects of Na+ and K+ were reversed. Furthermore, it was found that the degradation of the small chain was only influenced by certain combinations of ATP, Mg++, Na+ and K+ if the large chain was intact when the ligands were added to the enzyme. The described results demonstrate structural alterations of the (Na+--K+)-ATPase complex which are supposed to include a synchronous protrusion or retraction of both (Na+--K+)-ATPase subunits. The data further suggest that ATP and other ligands primarily alter the structure of the large (Na+--K+)-ATPase subunit. This structural alteration is presumed to lead to a synchronous movement of the small subunit of the enzyme. The structural state of the (Na+--K+)-ATPase is regulated by binding of Na+ or K+ to the enzyme-ATP complex. The effects of Na+ and K+ on the (Na+--K+)-ATPase structure are modulated by the ATP binding to "high affinity" and to "low affinity" ATP binding sites.