Immune response of athymic nude mice to papovavirus SV40 tumor-associated antigens. The requirement of T cell functions in the induction of immune response to SV40-specific transplantation rejection antigen and intranuclear tumor antigen was studied using athymic nude mice. The results obtained indicate that virus-immunized athymic nude mice were unable to reject SV40 tumor cell challenge, and sensitized lymphocytes capable of inhibiting tumor growth in vivo could not be demonstrated in the spleens of virus-immunized mice. Athymic nude mice bearing tumor induced by virus-free SV40-transformed BALB/c cells failed to develop antibodies to intranuclear T antigen. Athymic nude mice also failed to respond to viral antigens. Thus it can be concluded that T cell functions are required in the induction of cellular immune response to SV40-specific transplantation rejection antigen and in humoral immune response to SV40-specific T antigen and virion antigen.


Hepatography and liver lymphography by retrograde biliary ethiodol infusion: an experimental study in the dog. Ethiodol in dosages of 0.5-3 ml/kg was retrogradely infused with hydrostatic pressure of 50--100 cm water into the biliary system of nine cholecystectomized dogs equipped with a Thomas cannula in the second portion of the duodenum, allowing cannulation of the common bile duct with a Foley catheter under direct vision. A total of 18 examinations were performed. A dense hepatogram and complete visualization of the hepatic lymph nodes were obtained when the infused Ethiodol dosage exceeded the capacity of the biliary system by 10 ml or more. The hepatogram lasted for three to four months and the lymph nodes were opacified for a year and longer. Contrary to angiographic contrast agents, Ethiodol retrogradely infused into the biliary system primarily refluxes from the biliary system to the lymph and not to the blood. Judged by serial chest radiographs and lung scans, pulmonary Ethiodol emboli did not occur. A significant temporary increase in serum bilirubin and liver enzymes followed the procedure but these returned to normal within a week to a month. Periodic needle biopsies of the liver showed Ethiodol droplets initially in the interstitium and lymphatics, and subsequently in hepatocytes, Kupffer's cells and the epithelium of small bile ductules. No hepatic damage was found histologically on long-time follow-up examinations.


Oxidative enzymes in the sebaceous glands of the domestic cat. The distribution and activities of several oxidative enzymes in various regions of the sebaceous glands of the domestic cat have been studied. The results obtained emphasize the outstanding importance of NADP-linked dehydrogenases for lipogenesis during sebum production. In particular, the reactions for glucose-6-phosphate dehydrogenase were very strong. Among the NAD-linked dehydrogenases investigated, lactate dehydrogenase showed strong activity in the peripheral cells of the sebaceous gland. The reactions for cytochrome oxidase and succinate dehydrogenase were weaker.


Supersaturation by counterperfusion and diffusion of gases. Gaseous supersaturation can be induced under steady-state conditions when two inert gases are transmitted in opposite directions across any system comprising a diffusion barrier adjacent to a zone of limited convective capacity. This has many implications for bubble formation in vivo and can explain the occurence of symptoms of decompression sickness without decompression.


In vivo aminoacylation of transfer ribonucleic acid in Bacillus subtilis and evidence for differential utilization of lysine-isoaccepting transfer ribonucleic acid species. The presence or absence of certain amino acids has different effects on the ability of Bacillus subtilis to sporulate, and the intracellular pool size of amino acids has been reported to vary during sporulation. The idea that these variations might exert a regulatory effect through aminoacylation of transfer ribonucleic acid (tRNA) was investigated by studying the levels of aminoacylation in vivo in the logarithmic or stationary phase of growth. Both the periodate oxidation method and the amino acid analyzer were used to evaluate in vivo aminoacylation. The results indicated that in general the level of aminoacylation of tRNA's remained constant through stage III of sporulation, although there were detectable variations for specific amino acid groups. Our studies also showed that periodate oxidation damaged certain tRNA's; therefore, the results obtained by such a method should be interpreted with caution. Because the damage can affect certain isoaccepting species specifically, the periodate oxidation method cannot be used to establish which isoaccepting species are acylated in vivo. We also investigated the possibility of preferential use of particular tRNA species by polyribosomes. These results demonstrated a preferential use of lysyl-tRNA's at different growth stages. Control mechanisms operating during the early stages of sporulation, therefore, do not affect the overall level of aminoacylation. However, there is an effect on the levels of aminoacylation of specific amino acids and on which isoaccepting species are utilized by the polyribosome system.


Levels of small molecules and enzymes in the mother cell compartment and the forespore of sporulating Bacillus megaterium. We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.


Selective incorporation of membrane proteins into proteoliposomes of different compositions. 1. Cytochrome oxidase was incorporated into preformed liposomes containing phosphatidylserine. When confronted with a mixture of liposomes, some containing phosphatidylserine and some without it, the enzyme was incorporated only into the phosphatidylserine-containing liposomes. 2. The hydrophobic proteins of the oligomycin-sensitive ATPase incubated in the presence of a mixture of liposomes with and without cytochrome oxidase were preferentially incorporated into cytochrome oxidase-containing liposomes. This selectivity was abolished by either cytochrome c or ascorbate. 3. Cytochrome oxidase incubated in the presence of a mixture of liposomes with and without the hydrophobic proteins of the ATPase was preferentially incorporated into liposomes that did not contain the hydrophobic proteins. 4. Cytochrome oxidase and the oligomycin-sensitive ATPase were preferentially incorporated into pure liposomes over bacteriorhodopsin-containing vesicles. 5. Reduced coenzyme Q (QH2)-cytochrome c reductase was incorporated randomly when incubated in the presence of a mixture of pure liposomes and liposomes containing the hydrophobic proteins of the ATPase complex. 6. The significance of the incorporation procedure as a model for membrane biogenesis is discussed.


Effects of estrogen, glucocorticoid, glucagon, and adenosine 3':5'-monophosphate on catalytic activity, amount, and rate of de novo synthesis of hepatic histidase. The mechanisms by which estrogen, glucocorticoid, glucagon, and adenosine 3':5'-monophosphate (cAMP), regulators which participate in the postnatal development of rat liver histidase, elevate the catalytic activity of this enzyme have been explored. A monospecific antibody against homogeneously purified preparations of rat liver histidase has been elaborated in the goat. Employing this antibody in immunotitration experiments, it has been demonstrated that the elevations of hepatic histidase activity elicited by administration in vivo of estradiol-17beta, cortisol acetate, glucagon, and N6,O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP) are paralleled, in each instance, by equivalent increments in immunoprecipitable histidase protein. Following administration of each of the three hormones and dibutyryl cAMP, rates of [14C]leucine incorporation in vivo into rat liver histidase, isolated by immunoprecipitation, relative to incorporation rates into total soluble hepatic protein, increase in magnitudes which are comparable to increases in enzyme amount and catalytic activity. It is thus inferred that estrogen, glucocorticoids, and glucagon, via cAMP, each regulate rat liver histidase development at specific postnatal stages by inducing increases in histidase biosynthetic rates.


Effects of chaotropic agents versus detergents on dihydroorotate dehydrogenase. As chaotropic salts are generally believed to affect water structure in a manner which increases lipophilicity of water, they may seem to be capable of substituting for detergents in the solubilization of particulate enzyme. Although solubilization either by detergents or by chaotropic salts has been demonstrated with several membrane proteins, the effects these agents have on the properties and activity of an enzyme may be quite different. This is illustrated by the effects on mammalian mitochondrial dihydroorotate dehydrogenase. Stability of the solubilized enzymic activity is dependnet on the presence of a detergent and maximum enzymic activity is observed at the critical micelle concentration of the detergent. Addition of low concentrations of various anions of the chaotropic series further enhances activity while higher concentrations of these anions, although increasing solubility of the enzyme, irreversibly inhibit catalysis.


Determination of the nucleotide sequence of part of the regulatory region for the galactose operon from Escherichia coli. We have determined the sequence of 59 base pairs in the DNA preceding the site for initiation of transcription in the galactose operon. DNA from a lambdagal transducing phage was digested with restriction endonucleases to obtain a DNA fragment from the gal regulatory region. This fragment extends from 59 base pairs prior to the transcription initiation site through the 45 base pairs which specify the 5'-terminal sequence of gal mRNA. Analyses of RNA transcripts derived from this fragment and a variety of direct DNA sequence analyses allowed us to deduce the following sequence for the DNA in this fragment: (formula: see text) Position +1 corresponds to the site for initiation of gal mRNA synthesized in the presence of cyclic AMP and its receptor protein, CRP. Transcription experiments indicate, however, that this fragment lacks some element of the gal promoter required for the stimulation of transcription by CRP-cAMP. There are, nonetheless, some similarities between the gal sequence preceding the transcription start site and the sequences of other promoter regions. These include the heptamer sequence T-A-T-G-G-T-T (--12 to --8) and the sequence A-C-A-C-T-T-T (--36 to --30).


Acquisition of increased hormone sensitivity during in vitro adipocyte development. Murine 3T3-L1 fibroblasts enter a differentiation program subsequent to prolonged maintenance in the confluent state and develop into adipocytes. The hormone sensitivity of adenylate cyclase and the physiological responsiveness to insulin were compared in 3T3-L1 preadipocytes and adipocytes. The following observations, comprising several distinct categories of hormone responsiveness, were made. (a) (2.5 micronM) isoproterenol stimulated adenylate cyclase 15-fold in adipocyte homogenates, but only 2.5-fold in preadipocyte preparations, suggesting a considerable magnification in beta-adrenergic responsiveness during development. (b) A totally new control element, adrenocorticotropic hormone responsiveness, was incorporated into the adenylate cyclase system of the adipocytes. (c) Sensitivity to prostaglandin E1 was observed in both preadipocytes and adipocytes, but no change in responsiveness could be detected in the differentiated cells. (d) Glucagon-sensitive adenylate cyclase could not be detected in either preadipocytes or adipocytes. (e) Both preadipocytes and adipocytes possess considerable insulin binding activity, but near physiological levels of insulin stimulate the conversion of glucose to CO2 and lipid only in the differentiated cells.


Inhibition of carbamyl phosphate synthetase by P1, P5-di(adenosine 5')-pentaphosphate: evidence for two ATP binding sites. Studies on the effect of a series of alpha, omega-diadenosine 5'-polyphosphate (ApnA; n = 2 to 6) on carbamyl phosphate synthetase showed that only Ap5A is an effective inhibitor. Ap5A also inhibits two partial reactions catalyzed by the enzyme: bicarbonate-dependent ATPase and ATP synthesis from carbamyl phosphate and ADP. The data indicate that Ap5A binds to the enzyme sites that interact with ATP. Of a variety of ATP-utilizing enzymes (kinases, hydrolases, synthetases), only adenylate kinase (Leinhard, G. E., and Secemski, I. I. (1973) J. Biol. Chem. 248, 1121--1123) and carbamyl phosphate synthetase are inhibited by Ap5A. The present findings provide strong evidence that carbamyl phosphate synthetase has two separate binding sites for ATP in which the gamma-phosphate moeities of ATP are bound in close proximity to the bicarbonate binding site of the enzyme.


Reduced concentrations of Z protein in Morris hepatomas. Possible role in abnormal regulation of lipid metabolism. The cytoplasmic concentration of Z protein (Mr approximately 12,000) was significantly reduced in a series of implanted Morris hepatomas with varying degrees of differentiation. Approximately half of the [14C]palmitoyl-CoA added to cytosol fractions from control or host livers was bound to the Z protein region whereas a much smaller proportion was bound to this region in the cytosol of nine Morris hepatomas studied. The possible implications of these findings are discussed in relation to the abnormal regulation of lipid metabolism in hepatomas.


Calcium fluxes in isolated acinar cells from rat parotid. Effect of adrenergic and cholinergic stimulation. Rat parotid acinar cells dispersed by a combination of enzymatic treatments remain sensitive to adrenergic and cholinergic agonists. Previous studies have implicated Ca2+ in both adrenergic and cholinergic responses. This paper describes the effects of adrenergic and cholinergic stimulation upon 45Ca2+ fluxes in isolated parotid acinar cells. Suspensions of dispersed cells took up 45Ca2+ from the medium. The net rate of isotope influx was increased by the adrenergic agonists epinephrine, norepinephrine, isoproterenol, and phenylephrine, and by the cholinergic agonists acetylcholine and carbamylcholine. In 1 mM Ca2+, epinephrine was capable of increasing the 45Ca2+ influx in 40 min to three times that of resting cells. Isoproterenol, a beta-adrenergic agonist, was only half as effective as epinephrine in stimulating maximal calcium uptake although it was equally effective in stimulating maximal amylase release in the same cells. Experiments with the alpha-adrenergic antagonist phentolamine, the beta-adrenergic antagonist propranolol, and the cholinergic antagonist atropine confirmed that alpha- and beta-adrenergic and cholinergic stimulation each had a direct stimulatory effect on 45Ca2+ uptake. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate also caused some stimulation of net calcium uptake. Direct measurement of Ca2+ efflux indicated that the increased calcium uptake in the presence of epinephrine was not the indirect result of a decrease in efflux. The rates of both basal and epinephrine-stimulated calcium uptake increased with increasing calcium concentration in the medium. Epinephrine had little effect on the rate of calcium uptake at 0.15 mM Ca2+. Although the energy poison NaCN had little effect on the basal rate of calcium uptake, the stimulable component of calcium uptake was inhibited by NaCN at all calcium concentrations tested (0.2 to 4.1 mM).


Imidazole, the ligand trans to mercaptide in ferric cytochrome P-450. An EPR study of proteins and model compounds. A crystal field analysis of EPR data for various low spin ferric cytochromes P-450 suggests that in all of them, regardless of source or method of induction, the heme ligands are a sulfur atom, presumably from cysteine, and an imidazole from histidine. The imidazole can be displaced in the ferric protein by cyanide, guanidine, or by an amine, analogous to its displacement by CO or NO in the ferrous protein. The resulting changes in the EPR parameters for the ferric protein are consistent with similar substitutions in heme thiol model compounds. The analysis of the latter can be understood on the basis of alterations of the electronic structure of the ligands to the heme iron.


L-Arginine kinase from tobacco hornworm, Manduca sexta (L.). Purification, properties, and interaction with L-canavanine. Arginine kinase (adenosine 5'-triphosphate: L-arginine phosphotransferase, EC 2.7.3.3) was purified from the larvae of the tobacco hornworm, Manduca sexta (L). This enzyme catalyzes the production of L-phosphoarginine, which is the principal reserve of high energy phosphate compounds in insect muscle. The enzyme also phosphorylates L-canavanine, a guanidinooxy analogue of arginine which severely disrupts all developmental stages of this insect. Evaluations of certain kinetic and thermodynamic parameters of the reactions with arginine and canavanine suggest that reactions known to be much more sensitive to canavanine, such as protein synthesis or genome expression, rather than phosphagen formation and function account for the pronounced toxicity of canavanine in this insect. Sedimentation equilibrium and electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate indicate that this insect enzyme has a molecular weight of about 40,000. This value is consistent with molecular weights of arginine kinases of non-insect arthropods. Its amino acid composition is also very similar to that of other arthropod arginine kinases. Km values for the enzyme are: L-arginine, 0.5 mM; Mg-ATP, 2.5 mM; L-canavanine, 22 mM; L-phosphoarginine, 0.7 mM; Mg-ADP, 0.45 mM; and L-phosphocanavanine, 27 mM. Turnover numbers (expressed as moles of product per min per mol of enzyme) are: L-arginine, 8,320; L-canavanine, 1,635; L-phosphoarginine, 25,875; and L-phosphocanavanine, 3,040. The apparent equilibrium constants at 37 degrees for phosphagen formation are 0.44 with arginine and 0.1 with canavanine. A procedure for L-phosphocanavanine synthesis is also presented.


Characterization of triton X-100-solubilized prostaglandin E binding protein of rat liver plasma membranes. Rat liver plasma membranes bind prostaglandins E1 and E2 (PGE) with high affinity and specificity. We have solubilized plasma membranes, prelabeled with radioactive PGE1, in water solutions of Triton X-100. We sedimented this material into sucrose density gradient containing H2O and D2O. From numerical integration of the sedimentation equation, taking explicitly into account the density and viscosity gradients present during the centrifugation, we have determined a value of s20,w = 5.6 to 5.7 X 10(-13) s and a partial specific volume, v = 0.80 to 0.81 cm3/g, for the PGE binding protein-Triton X-100 composed of 60% (w/w) protein and 40% (w/w) detergent. Gel filtration in water solutions of Triton X-100 gives a Stokes radius of 53 A for the complex. These data imply a molecular weight of 105,000 for the detergent-free binding protein and a frictional ratio of 1.3 for the complex. If the detergent is bound to the protein in a monolayer, about 40% of the PGE binding protein's surface would be covered with detergent. The procedures used in the analysis of the sedimentation behavior of the PGE binding protein-detergent complex, when coupled with a gel filtration measurement of the Stokes radius, allow valid determination of the size, shape, and extent of detergent binding of a wide variety of membrane proteins, even when they are present as minor components of complex mixtures.


3(17)beta-Hydroxysteroid Dehydrogenase of Pseudomonas testosteroni. Ligand binding properties. The binding of NAD and NADH to electrophoretically pure 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni was determined by Fluorescence spectroscopy and gel filtration. Four moles of cofactor are bound/mol of tetrameric enzyme; the binding sites are equivalent and independent. The dissociation constants for NAD and NADH are 16 and 0.25 micronM, respectively. As measured by gel filtration in the absence of cofactor, 0.4 mol of estradiol-17 beta is bound/mol of tetrameric enzyme. Data obtained from isotope exchange at equilibrium indicate that the binding of the cofactor to the enzyme is favored over the binding of steroid, although each may bind in the absence of the other. The rates of cofactor dissociation from the ternary complexes are slower than the rates of steroid dissociation; cofactor dissociation is probably the rate-limiting step. Cofactor analogs modified in the pyridine moiety are cosubstrates, whereas modified adenine derivatives are not. The enzyme also utilized as substrate a number of potential steroid affinity labels; no enzyme inactivation by these compounds was observed.


Endogenous protein kinase inhibitors. Purification, characterization, and distribution in different tissues. A thermostable inhibition of ATP-protein phosphotransferase (EC 2.7.1.37) (protein kinase) which is present in crude tissue extracts has been resolved by gel chromatography (Sephadex G-100) into two molecular forms. These two forms will be referred to as type I and type II inhibitor. The type I inhibitor (Mr approximately or equal to 24,000) is specific for cAMP-dependent protein kinase and corresponds to the inhibitor described earlier (Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fisher, E. H., and Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985). The type II inhibitor (Mr approximately or equal to 15,000) competes for the enzyme with various substrate proteins (histone, alpha-casein, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). The type II inhibitor blocks protein phosphorylation catalyzed by several types of protein kinases (cAMP- and cGMP-dependent or cyclic nucleotide-independent protein kinases). The type II inhibitor from rat brain has been purified 1500-fold; this protein is thermostable, has acidic characteristics, and does not require Ca2+ ions for its activity. Different ratios and concentrations of type I and type II inhibitors of protein kinase are found in rat skeletal muscle, pancreas, cerebellum and corpus striatum, and in lobster tail muscle.


Interaction of canine and swine lipoproteins with the low density lipoprotein receptor of fibroblasts as correlated with heparin/manganese precipitability. Canine HDL1 and canine and swine HDLc were fractionated into several lipoprotein subpopulations by heparin/manganese precipitation. The ability of the various subfractions of HDL1 or HDLc to compete with 125I-labeled low density lipoproteins (LDL) for binding and degradation by human fibroblasts was compared. The HDL1 or HDLc which precipitated at the lowest concentration of heparin (a concentration which precipitates LDL) were the most effective in competing with 125I-LDL for binding, internalization, and degradation. A striking characteristic of these lipoproteins was the occurrence of a prominence of the arginine-rich apoprotein. The HDL1 or HDLc subfractions which were not precipitated by heparin/managanese lacked detectable arginine-rich apoprotein and did not compete significantly with the 125I-LDL for binding and degradation. Furthermore, the lipid to protein ratio differed in the precipitable and nonprecipitable lipoproteins, with those which were most efficiently bound and degraded containing more cholesterol. Specific lipoprotein interaction with heparin and with the cell surface receptors may occur by a common mechanism; namely, through a positively charged region on the lipoprotein surface which may reside with the B and arginine-rich apoproteins.


Fatty acid enhancement of human serum albumin binding properties. A spin label study. The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.


Cyclic AMP gradient in migrating pseudoplasmodia of the cellular slime mold Dictyostelium discoideum. Several lines of experimental evidence suggest that an anterior-posterior gradient of cyclic AMP exists in migrating pseudoplasmodia of the cellular slime mold Dictyostelium discoideum, and that this gradient may be responsible for control of the proportions of stalk and spore cells that form during culmination. In experiments reported here, the amounts of cyclic AMP present in the anterior and posterior portions of pseudoplasmodia were measured using a protein binding assay, and the levels obtained normalized to protein and DNA. For a variety of pseudoplasmodia migration conditions examined, the anterior portion was found to contain cyclic AMP concentrations 40 to 70% higher than the posterior portions.


Purification and subunit composition of guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. The guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung was purified to apparent homogeneity by affinity chromography using 8-2-aminoethylthio-cGMP coupled to Sepharose 4B. The kinase activity was purified approximately 6000-fold with an overall recovery of approximately 20%. The product isolated by affinity chromatography contained both cGMP-binding and cGMP-dependent histone kinase activity, indicating that the enzyme was not dissociated into regulatory and catalytic components by the immobilized cGMP derivative. The enzyme had a molecular weight of approximately 165,000 and a sedimentation coefficient of 7.8 S. The purified kinase displayed several characteristics similar to that of the partially purified enzyme including specificity for cGMP and stimulation by high concentrations of magnesium. On sodium dodecyl sulfate gels, only one major polypeptide chain was present having a molecular weight of approximately 81,000. This subunit bound 1 mol of cGMP and exhibited cGMP-dependent protein kinase activity. It is proposed that the native enzyme consists of two identical subunits (Mr=81,000), each of which binds cGMP and catalyzes protein phosphorylation.


Preparation and properties of N6-monobutyryl adenosine 5'-monophosphate. A major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate. The synthesis and properties of N6-monobutyryl adenosine 5'-monophosphate are described. The properties of synthesized monobutyryl nucleotide have been compared to those of a metabolite recognized in previous studies (Castagna, M. C., Palmer, W.K., and Walsh, D.A. (1977) Arch. Biochem. Biophys. 181, 46-60) as the major radioactive product produced in the liver upon perfusion with N6,O2'-dibutyryl cyclic [3H]adenosine 3':5'-monophosphate. By the criteria of cochromatography on DEAE-cellulose and in three thin layer chromatographic systems and from equivalent rates of alkaline hydrolysis, N6-monobutyryl adenosine 5'-monophosphate has been identified as a major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.


The dissociation of the surface architecture described by enhanced lectin agglutinability and the transformed phenotype expressed as anchorage independence. Using a series of cold-sensitive variants of chemically transformed BHK-21 cells, revertants to the normal phenotype derived from a dimethyl-nitrosamine transformed clone of BHK-21 as well as revertants to the normal phenotype derived from polyoma transformed BHK-21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed phenotype.


Regulation of intracellular calcium in chick embryo fibroblast: calcium uptake by the microsomal fraction. The total membrane fraction of a chick embryo fibroblast (CEF) homogenate accumulates calcium in an energy-dependent manner. This activity can be dissociated into azide-sensitive and azide-insensitive components. The azide-sensitive component of calcium uptake is believed to represent mitochondrial calcium uptake. The azide-insensitive component of calcium uptake is enhanced by the presence of a calcium trapping agent such as oxalate, and cannot utilize, ADP, inorganic phosphate and a Krebs cycle substrate to support uptake. The distribution of the azide-insensitive calcium uptake in subcellular fractions suggests that this uptake occurs in other than mitochondrial membranes. The membranes most likely to contribute to the azide-insensitive component of calcium uptake are the endoplasmic reticulum and plasma membrane. A microsomal preparation from CEF cells is essentially devoid of the azide-sensitive calcium uptake activity. This microsomal activity is similar in characteristics to the sarcoplasmic reticulum of skeletal muscle. However the specific activity of CEF microsomal calcium uptake system is much less than that found in the skeletal muscle system. The transport of calcium by these membranes provide a mechanism for the regulation of cytosol calcium levels and may play a role in the control of movement and growth of cultured cells.


Colloidal iron hydroxide-binding to the surfaces of chick embryo fibroblasts transformed by wild-type and a temperature-sensitive mutant of Rous sarcoma virus. The densities of colloidal iron hydroxide (CIH) particles binding to the surfaces of chick embryo fibroblasts were determined before and after transformation with wild type Rous sarcoma virus and a temperature sensitive (ts) mutant of this virus. On the basis of in vitro behavior, cells transformed by the ts virus manifest a malignant phenotype at 36 degrees C (permissive temperature) and appear normal at 41 degrees C (non-permissive temperature). At the permissive temperatures there is a significant increase in CIH particle-binding to spaces of cell surface between microvilli on the wild type and ts transformed cells. At the non-permissive temperature this significant increase in binding is only observed on the wild type transformant, while the density found on the ts transformant is not significantly different from the untransformed state. Therefore, in vitro characteristics of normalcy and malignancy are reflected in changes in the CIH binding properties of the cell surface spaces between microvilli. The CIH densities observed on the microvilli are significantly different from the density on the spaces between them for each of the classes of cells studied at either temperature. The microvilli are found to bind a lower density of particles in five of the six cases. No correlations between microvilli particle density and transformation to in vitro malignant characteristics were observed.


The storage lipids in Tangier disease. A physical chemical study. The physical states and phase behavior of the lipids of the spleen, liver, and splenic artery from a 38-yr-old man with Tangier disease were studied. Many intracellular lipid droplets in the smectic liquid crystalline state were identified by polarizing microscopy in macrophages in both the spleen and liver, but not in the splenic artery. The droplets within individual cells melted sharply over a narrow temperature range, indicating a uniform lipid composition of the droplets of each cell. However different cells melted over a wide range, 20-53 degrees C indicating heterogeneity of lipid droplet composition between cells. Furthermore, most of the cells (81%) had droplets in the liquid crystalline state at 37 degrees C. X-ray diffraction studies of splenic tissue at 37 degrees C revealed a diffraction pattern typical of cholesterol esters in the smectic liquid crystalline state. Differential scanning calorimetry of spleen showed a broad reversible transition from 29-52 degrees C, with a maximum mean transition temperature at 42 degrees C, correlating closely with the polarizing microscopy observations. The enthalpy of the transition, 0.86+/-0.07 cal/g of cholesterol ester, was quantitatively similar to that of the liquid crystalline to liquid transition of pure cholesterol esters indicating that nearly all of the cholesterol esters in the tissue were free to undergo the smectic-isotropic phase transition. Lipid compositions of spleen and liver were determined, and when plotted on the cholesterol-phospholipid-cholesterol ester phase diagram, fell within the two phase zone. The two phases, cholesterol ester droplets and phospholipid bilayers were isolated by ultracentrifugation of tissue homogenates. Lipid compositions of the separated phases approximated those predicted by the phase diagram. Extracted lipids from the spleen, when dispersed in water and ultracentrifuged, underwent phase separation in a similar way. Thus (a) most of the storage lipids in the liver and spleen of this patient were in the liquid crystalline state at body temperature, (b) the phase behavior of the storage lipids conformed to that predicted by lipid model systems indicating lipid-lipid interactions predominate in affected cells, (c) lipid droplets within individual cells have similar compositions, whereas droplet composition varies from cell to cell, and (d) cholesterol ester does not accumulate in the splenic artery. Since Tangier patients lack high density lipoprotein, we conclude that high density lipoprotein-mediated cholesterol removal from cells is essential only for those cells which have an obligate intake of cholesterol (macrophages).


Decreased H2 histamine response of granulocytes of asthmatic patients. Increased bronchial sensitivity to inhaled histamine in asthma is well known. The mechanism of this increased bronchial sensitivity is not known nor has it been demonstrated that isolated cells respond abnormally to histamine. Polymorpho-nuclear leukocytes (PMNs) provide a homogeneous cell population to study agonist response. Release of granulocyte lysosomal enzymes is inhibited by agonists increasing the PMN cyclic AMP concentration. The release of the lysosomal enzyme beta glucuronidase by serum-activated particles of zymosan was similar in PMNs isolated from normal and asthma subjects. Histamine (100-0.01 muM) inhibited enzyme release. Except at the maximal concentration of histamine (100 muM), the response to histamine was decreased in asthma. The inhibition of enzyme release paralleled an increase in intracellular PMN cyclic AMP. In asthma, the cyclic AMP response to histamine was reduced. The H2 antihistamine metiamide blocked histamine inhibition of lysosomal enzyme release and the increase in cyclic AMP. The effect was maximal at concentrations equimolar to those of histamine. The H1 antihistamine chlorpheniramine had no effect on histamine inhibition of granulocyte lysosomal enzyme release. A decrease in the inhibition of the release of the inflammatory lysosomal enzymes from granulocytes in asthma may contribute to an enhanced bronchial inflammatory reaction.


Hydrogen peroxide production in chronic granulomatous disease. A cytochemical study of reduced pyridine nucleotide oxidases. The ability of polymorphonuclear leukocytes (PMN) to produce H(2)O(2) in response to phagocytic stimulation was examined cytochemically using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Normal PMN oxidized diaminobenzidine within the phagocytic vacuole by a reaction dependent upon endogenous H(2)O(2) and myeloperoxidase. CGD PMN failed to oxidize diaminobenzidine, which is consistent with the biochemical data showing a lack of H(2)O(2)-generating capacity. A plasma membrane enzyme (oxidase) activated by phagocytosis is capable of H(2)O(2) production in PMN. The localization of this oxidase activity was explored in CGD PMN using a cytochemical technique specific for H(2)O(2). The enzyme activity is stimulated by exogenous NADH, but not NADPH. Reaction product formation, indicative of activity of the oxidase, is dependent upon precipitation of cerium ions by the enzymatically generated H(2)O(2). The advantage of this approach is that enzyme activity of individual cells can be assessed, allowing determination of numbers of reactive cells in the population and their relative degrees of reactivity. NADH oxidase was found to be active both on the plasma membrane and within the phagocytic vacuoles of control PMN, whereas those cells from three CGD patients showed greatly reduced activity in both these sites. Assessment of the reactivity of individual cells showed the number of cells with oxidase activity in CGD to be significantly reduced when compared to control values. Additionally, of those cells that do react, a higher percentage of them are only weakly reactive. Omission of NADH from the incubation medium reduced the percentage of control cells showing enzyme activity but had no effect on CGD PMN, implying that the enzyme is not saturated with substrate in control cells, but in CGD the diminished enzyme is fully saturated. The defect may lie in the fact that in CGD patients there are fewer cells capable of peroxide generation, and a majority of these reactive cells produce only reduced amounts of this bactericidal agent.


Structural features of Salmonella typhimurium lipopolysaccharide required for activation of tissue factor in human mononuclear cells. Activation of mononuclear cell tissue factor was examined utilizing lipopolysaccharides obtained from wild-type and both Rc and Re mutants of Salmonella typhimurium. Wild-type (smooth) lipopolysaccharide, galactose-deficient (Rc) lipopolysaccharide, heptose-deficient (Re) lipopolysaccharide, and lipid A preparations were all active in their ability to generate tissue factor activity in human mononuclear cells grown in tissue culture. Polymyxin B has been reported to prevent some of the lethal effects of endotoxin in vivo, and the drug reportedly binds to the 2-keto-3-deoxyoctulosonate-lipid A region of the lipopolysaccharide molecule. Polymyxin B was effective in inhibiting the tissue factor generating activity of wild-type lipopolysaccharide, Re lipopolysaccharide, and lipid A in a dose-dependent fashion. Treatment of lipid A preparations with mild alkali abolished the ability of these preparations to activate tissue factor in cells. Analogous to many of the other biologic properties of lipopolysaccharide, tissue factor activation in human mononuclear cells appears to depend upon the integrity of the lipid A portion of the molecule.


Overloading human aortic smooth muscle cells with low density lipoprotein-cholesteryl esters reproduces features of atherosclerosis in vitro. Human aortic smooth muscle cells accumulate only small amounts of cholesteryl esters in tissue culture, even when incubated for prolonged periods with high levels of plasma low density lipoprotein (LDL). This failure to overaccumulate LDL-cholesteryl esters is due to an LDL-mediated feedback suppression of the activity of the cell surface LDL receptor, a regulatory action that limits the rate at which the cells take up LDL. This regulatory system can be bypassed by incubating smooth muscle cells with LDL that has been given a strong positive charge by covalent linkage with N,N-dimethyl-1,3-propanediamine (DMPA-LDL). The unregulated uptake of DMPA-LDL produces a massive deposition of cholesteryl esters in the form of inclusions within the cell. These inclusions take up lipid stains and exhibit positive birefringence with formée crosses that are typical of liquid crystals of cholesteryl esters. By electron microscopy, the cholesteryl ester inclusions appear as homogeneous gray cytoplasmic lipid droplets. The current studies demonstrate that the unregulated uptake of LDL-cholesteryl esters by human aortic smooth muscle cells can reproduce in vitro the major biochemical and morphological alterations that occur within smooth muscle cells in vivo during the process of atherosclerosis.


Polybrominated biphenyls in raw milk and processed dairy products. Milk from four dairy herds identified by the Michigan Department of Agriculture as containing less than .3 ppm (fat basis) physiologically incorporated polybrominated biphenyls was processed individually into cream, skim milk, butter, and stirred curd cheese. Pasteurized and freeze-dried whole milk, skim milk, and cream, spray-dried whole milk and skim milk, and condensed whole milk were made also. Polybrominated biphenyls were concentrated in the high-fat products. Pasteurized skim milk, buttermilk, and whey had slightly more polybrominated biphenyls than pasteurized whole milk on a fat basis. Spray-drying reduced the polybrominated biphenyls in whole milk and skim milk while pasteurization, freeze-drying, aging of cheese, and condensation were not effective.


Shock and the kidney: pathophysiology and pharmacological support. This article reviews current knowledge as to the physiological mechanisms that control renal vascular resistence. The contribution of both extrinsic and intrinsic neuro-humoral regulation of both blood flow and glomerular filtration rate are described. The changes that occur both to the renal blood flow and glomerular filtration rate in the pathophysiological situation of "pre-renal uraemia" as well as "acute tubular necrosis" are described. Within this setting pharmacological manoeuvres that may improve both renal blood flow and glomerular filtration rate are discussed. In addition, the indications for and general principles of haemo-and peritoneal dialysis are described.


Target cells for interferon production in human leukocytes stimulated by sendai virus. Whole leukocytes, mononuclear cells, polymorphonuclear cells (PMN), MONOCYTES, PURIFIED LYMPHOCYTES, AND T (rosette-forming cells, RFC) and non-T (nonrosette-forming cells, nonRFC) lymphocytes isolated from the human peripheral blood were stimulated by Sendai virus, respectively, and examined for interferon production in their culture fluids. High levels of interferon were produced by mononuclear cells, but not by PMN. Removal of monocytes from the mononuclear cell population did not affect at all the levels of interferon produced, although it strongly suppressed interferon induction by polyinosinic-polycytidylic acid (poly IC) and mitogenic response to phytohemagglutinin (PHA) of the lymphocytes. Purified monocytes and T lymphocytes were unresponsive to the virus. In contrast, a population of purified non-T lymphocytes produced high levels of interferon. Addition of monocytes to the interferon-producing non-T lymphocytes did not affect the levels of interferon produced. No detectable levels of interferon were produced in the mixture of T lymphocytes and monocytes. It is concluded that non-T lymphocytes may be a major target for interferon induction of human leukocytes by Sendai virus.


Somatic cell hybrids between mouse peritoneal macrophages and SV40-transformed human cells. III. Identification of surface antigens coded for by human chromosomes 7 and 17. Somatic cell hybrids between SV40-transformed human cell lines and mouse peritoneal macrophages (MPM) containing either human chromosome 7 or 17 carrying the SV40 genome were injected into mice syngeneic to the mouse parental cells. Since either chromosome 7 or 17 was the only human chromosome present in the hybrids used as immunogens, the humoral immune response to gene products coded for by either chromosome was assayed. Using a sensitive radioimmunoassay, we were able to identify noncross-reactive cell-surface antigen(s) specifically coded for by either human chromosome 7 or 17, and present in normal, tumor-derived and virus-transformed human cells. However, no reactivity against SV40 tumor-specific surface antigen (TSSA) could be detected in the antisera.


The role of neutrophils in antiviral defense--in vitro studies on the mechanism of antiviral inhibition. Highly enriched populations of bovine neutrophils were added, in the presence of antiviral antibody, to herpesvirus-infected bovine cell cultures. A cell dose-dependent reduction in virus-induced cytopathology was observed. The mechanism of inhibition was presumed to be mediated by a subcellular neutrophil product and not the result of either direct cytotoxicity or antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, inhibition of comparable magnitude was observed when neutrophils and virus-infected cells were separated by cell impermeable membranes. In addition, plaque reduction occurred when antiviral immunoglobulins or fragments unable to mediate ADCC were used in the assays. Killed neutrophils and sonicates were unable to mediate plaque inhibition. Speculations were made as to the origin of the virus-inhibitory substances and the role that neutrophils might assume in mediating recovery from virus infection.


Effect of LDL-In, a normal immunoregulatory human serum low density lipoprotein, on the interaction of macrophages with lymphocytes proliferating in response to mitogen and allogeneic stimulation. The immunoregulatory properties of LDL-In, a normal species of human serum low density lipoprotein which suppresses indictive events involved in triggering of lymphoid cells by lectins and antigens, were analyzed in order to distinguish between a primary effect on macrophages and lymphocytes. LDL-In was found to be equally effective in suppression of the response of human lymphocytes to tpha at concentrations of lectin demonstrated to impart apparent macrophage-independence or macrophage-dependence to the culture system. Exposure of only the macrophages to LDL-In was shown to be without effect on subsequent in vitro lymphocyte responses to either PHA or allogeneic cells, whereas exposure of only the lymphocytes to LDL-In was fully effective. The cellular locus was further identified by demonstrating that the responder lymphocytes, but not the stimulator lymphocytes, were the target of the suppressive activity in mixed lymphocyte reactions. These data considered in conjunction with previous studies suggest that the primary untriggered lymphocyte is the most probable cellular target for the bioregulatory effects of LDL-In.


The elimination of herpes simplex plaques by antibody and the emergence of resistant strains. Established type 1 HSV plaques were eliminated by antibody. Antibody had to remain in contact with infected cells for several days to have a maxi;mum effect. It did not prevent the production of viral induced antigens in a cell when applied after the cell was infected but did prevent the transmission of infection to contiguous cells. Strains which were resistant to elimination by antibody formed syncytia, did not grow to significantly higher titers than nonresistant strains, and were as easily neutralized by antiserum as nonresistant strains.


Antibody-dependent, eosinophil-mediated damage to 51Cr-labeled schistosomula of Schistosoma mansoni: effect of metabolic inhibitors and other agents which alter cell function. The effect of drugs known to inhibit different metabolic pathways or cell functions on antibody-dependent, eosinophil-mediated damage to schistosomula was determined. Eosinophil-mediated damage was completely inhibited by cytochalasin B, inhibitors of glycolysis, aminophylline, and treatment of cells with diazotized sulfanilic acid and tosyl-lysyl-chloromethyl ketone. The effects of cytochalasin and 2-deoxyglucose were reversible. On the other hand, eosinophil-mediated damage was unaffected by agents which inhibit DNA replication, protein synthesis, oxidative respiration, prostaglandin synthesis, and microtubule aggregation. The findings suggest that alteration of the cell surface membrane and microfilaments prevents damage by interfering with cell-target interactions, that energy derived from glycolysis is required for cytotoxicity, that cell-associated esterases are probably involved, and that cytotoxicity may be modulated by cyclic nucleotides. Some of the attributes of eosinophils that allow it and not other cells to mediate this reaction are discussed.


Development of cellular anti-tumor immunity in chickens bearing tumors induced by Rous sarcoma virus. The technique of peripheral lymphocyte stimulation in response to antigen was used to demonstrate the existence of cell-mediated anti-tumor immunity in chickens bearing tumors induced by avian sarcoma viruses. The expression of this anti-tumor response against virus-containing transformed cell culture supernatant fluids and cell extracts varied among animals in terms of time after inoculation with the oncogenic agent. Animals whose tumors had completely regressed rapidly lost the ability to mount such continuing immunity. In addition, we found evidence that some normal animals express endogenous levels of natural immunity against neoplasms induced by these viruses and/or against the viruses themselves.


In vitro primary induction of cytotoxic T cells against virus-infected syngeneic cells. An in vitro method is described for primary induction of murine cytotoxic T cells against syngeneic cells infected with ectromelia or lymphocytic choriomeningitis (LCM) virus. Cytotoxicity was assayed by 51Cr release from macrophage or L929 target cells. Cytotoxic activity was sensitive to anti-theta and complement and was expressed only against target cells infected with the same virus and sharing H-2K or H-2D genes with the infected stimulator cells. The crucial factors in generating responses were mouse strain, responder: stimulator ratio, nature of infected stimulator cells, and presence of sufficient macrophages. C57BL/6 cells were less demanding than CBA/H and BALB/c cells. Under optimal conditions defined here, the in vitro response had similar kinetics and potency to the primary response in the spleen in vivo.


Immune adherence hemagglutination: further observations on demonstration of antibody to varicella-zoster virus. The immune adherence hemagglutination test for measurement of antibody to varicella-zoster (V-Z) virus is well suited to performance in a routine serology laboratory. This test is useful in assessing susceptibility to varicella, measuring antibody to V-Z virus in  globulin preparations, and evaluating some effects of passive immunization. Since the complement-fixation test for antibody to V-Z virus lacks sensitivity and the fluorescent antibody to membrane antigen test is not readily available, the immune adherence hemagglutination test may be of practical value in patient management.


Synergistic infection with murine cytomegalovirus and Candida albicans in mice. A previous report from this laboratory demonstrated that mice infected intraperitoneally with a 20% lethal inoculation of murine cytomegalovirus (CMV) exhibited markedly enhanced mortality rates (80%-100%) after an intravenous injection of a 0-20% lethal inoculum of Candida albicans. The current study revealed that mice infected with murine CMV alone had high titers of virus in spleen, liver, lung, and kidney from days 3 through 20, whereas those inoculated with C. albicans alone had a self-limited fungal infection involving only the kidney. In the combined murine CMV-C. albicans infection, the titers of murine CMV in tissues were changed very little. In contrast, C. albicans was recovered from multiple organs, and a progressive renal infection developed. This altered pathogenesis of the candida infection in murine CMV-infected mice resembled that produced by a 100% lethal inoculum of C. albicans alone. These results indicate that the murine CMV infection enhanced the susceptibility of mice to infection with C. albicans and suggest that death was due to progressive fungal infection of the kidney.


Fecal excretion of hepatitis A virus in humans. To define more completely the period of fecal excretion of virus during hepatitis A virus infection, we studied 24 fecal samples from six children with clinical illness during an epidemic of type A hepatitis. As determined by immune electron microscopy, the six patients had detectable viral excretion before or by the time of the first abnormality in serum glutamic-pyruvic transaminase (alanine aminotransferase). Viral excretion reached a peak early and declined to undetectable levels before levels of serum enzyme reached a peak. These data accord with epidemiologic evidence that the person who already has symptoms and signs of type A hepatitis is unlikely to transmit the infection to others. Immune electron microscopy, therefore, may be a better index to the period of communicability than studies of experimental infection in human subjects. This conclusion would imply that precautions against fecal contamination are not usually necessary for patients hospitalized with type A hepatitis.


Inhibition of simian virus 40-induced tumors by antisera to fetal hamster tissue. Growth of tumors induced in hamsters with simian virus 40 (SV40) or SV40-transformed cells was inhibited by antisera to fetal hamster thymocytes or liver. This result was in contrast to the enhancement of tumor growth by antisera to adult hamster thymocytes.


The effect of sodium azide on the chemiluminescence of granulocytes--evidence for the generation of multiple oxygen radicals. It has been established that granulocytes generate superoxide (O-2) as well as hydrogen peroxide (H2O2) during phagocytosis. The chemiluminescence (CL) generated by phagocytes appears dependent on these oxygen radicals (or). however, recent studies suggest that oxygen molecules, including singlet oxygen (1O2) or hydroxyl radicals (OH-), may also be generated during phagocytosis and contribute to CL. We have tested this possibility by studying human granulocyte CL in the presence of 0.1 mM sodium azide, a known inhibitor of myeloperoxidase and catalase and a scavenger of 1O2. The effects of azide on CL were correlated with the effects of this compound on hexose monophosphate shunt (hmps) activity, nitroblue tetrazolium (NBT) dye reduction, formate oxidation, and cytochrome c reduction. CL generated by granulocytes during the phagocytosis of zymosan particles was markedly impaired by azide (24% to 47% of control values). On the other hand, phenomena dependent in part on the presence of O2 radicals, i.e., reduction of NBT dye and cytochrome c, were not impaired by the presence of azide. As would be expected, inhibition of catalase by azide virtually abolished the oxidation of formate, but the burst in HMPS activity associated with phagocytosis was augmented further. The latter observation indicated that azide did not impair generation of H2O2 but increased the relative amount detoxified via the HMPS. The experiment provides evidence that radicals other than O-2 and H2O2 are generated during phagocytosis and that these radicals are major contributors to the CL phenomenon.


Automated microdensitometry and quantification of lipoproteins by agarose gel electrophoresis. A major obstacle in the application of quantitative microelectrophoresis has been tedious manipulations and calculations. To overcome these difficulties, we have developed an automatic system for the microdensitometry and calculations as part of a quantitative agarose gel electrophoresis facility. Results are internally standardized by serum cholesterol and/or triglyceride measurements. The hardware consists of a densitometer, an analog to digital converter, a cathode ray tube terminal, a teleprinter, and a small computer. A program in 4K words allows sample coding, electrophoretic scan display, indexing, and systematic identification of each peak. Data are acquired from scans of electrophoretic patterns of serum alone or in combination with the 1.006 gm/ml VLDL top and/or bottom preparative lipoprotein fractions. As many as 30 scans can be stored in 4K words of memory and then sent via high-speed telephone line to a larger computer for remote processing. The analysis corrects for baseline drifts and pre-beta asymmetry and will properly identify and quantify the amount of VLDL, LDL, and HDL with corrections for "sinking pre-beta" and "floating beta" in LDL and VLDL, respectively. Results are given in milligrams per 100 milliliter as well as percentile rank and standard deviation score ranking of each lipoprotein class as compared to an appropriate normal reference population. The latter data are in a form more meaningful to the physician and patient and provide a quantitative dimension to lipoprotein phenotyping.


The lecithin-cholesterol acyl transferase activity of rat intestinal lymph. The lecithin-cholesterol acyl transferase (LCAT) activity in rat mesenteric lymph was examined as a possible source of chylomicron cholesteryl ester. Lymph activity was only 2-3% of rat serum activity. Removal of d less than 1.006 lipoproteins increased lymph LCAT activity, but only to 6-8% of that of serum. Relative to total cholesterol in the d greater than 1.08 g/ml fractions, lymph LCAT activity in lymph from fasting rats was less than serum, but in lymph from nonfasting rats the ratio LCAT/HDL-cholesterol reached levels greater than serum, suggesting a contribution of enzyme from the gut. Both LCAT activity and HDL concentration in mesenteric lymph increased during feeding. Subfractions of lymph that inhibited serum LCAT were: chylomicrons, VLDL, chylomicron lipid, VLDL apoprotein, and HDL apoprotein. In the rat, the low LCAT activity of mesenteric lymph was in part due to the low enzyme concentration present, and the activity was apparently lowered further by lipid-rich lipoproteins that inhibited the reaction. Enzyme inhibition due to the apoprotein fractions of lipoproteins is probably minor in the rat in vivo.


Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat. The feeding of cholesterol to rats resulted in marked alterations in the type and distribution of the plasma lipoproteins and their apoproteins. The hyperlipoproteinemia was characterized by an increase in the d < 1.006 lipoproteins (B-VLDL and VLDL), an increase in the intermediate and low density lipoproteins (LDL), and the appearance of HDL(c). Associated with these lipoproteins was a prominence of the arginine-rich apoprotein. The high density lipoproteins (HDL) were decreased. A two-dimensional immunoelectrophoretic procedure was adapted to quantitate the changes in distribution of the arginine-rich apoprotein in the plasma and various ultracentrifugal fractions obtained from control and cholesterol-fed rats. In rats fed the cholesterol diet, the total plasma arginine-rich apoprotein increased from a control value of approximately 29 mg/dl to 47 mg/dl. The method of ultracentrifugation, however, was found to markedly alter the quantitative results. When the 60 Ti rotor was used at maximum speed to isolate the ultracentrifugal fractions, less than 50% of the total plasma arginine-rich apoprotein was associated with the lipoproteins in the d < 1.006 or the d 1.006-1.02, 1.02-1.063, or 1.063-1.21 ultracentrifugal fractions. By contrast, after limited ultracentrifugation with the 40 rotor, much less arginine-rich apoprotein was lost, with approximately 20% of the arginine-rich apoprotein in control rats and 10% in cholesterol-fed rats found in the d > 1.21 fraction. Significant alterations in the arginine-rich apoprotein quantitation notwithstanding, the observations of increased arginine-rich apoprotein in the B-VLDL, intermediate fraction, and HDL(c) following cholesterol feeding remained valid. However, precise quantitation awaits refinements in lipoprotein isolation techniques.


Parturition in goats: studies on the interactions between the foetus, placenta, prostaglandin F and progesterone before parturition, at term or at parturition induced prematurely by corticotrophin infusion of the foetus. Relationships between foetal corticosteroid concentrations, utero-ovarian prostaglandin F (PGF) and maternal peripheral progesterone have been examined in detail in goats shortly before spontaneous parturition at term. Foetal corticosteroids increased during the last 13-11 days of gestation and particularly sharply during the last 3 days and even during advanced labour. About 24 h before parturition, acute releases of PGF were evident in the vein draining the pregnant uterine horn, and these corresponded closely to the time of luteal regression. Further release of PGF occured when progesterone declined to low levels, probably reflecting in the course of labour. The changes observed before premature parturition, induced by infusing ACTH into foetal goats, were similar except for the more rapid increase in foetal corticosteoid concentrations. Immature neonates born after ACTH treatment were viable, placental delivery was normal and lactogenesis occurred in the mothers indicating that the treatment promoted full expression of the critical perinatal events. The early, acute releases of PGF were ipsilateral to the ACTH-infused foetus and were luteolytic provided the corpora lutea were also on that side. Luteolysis failed or was abnormally delayed if the corpora lutea were contralateral and prolonged ACTH treatment of the foetuses in such cases caused foetal death probably because of premature failure of the placenta. Similar findings were noted if ACTH infusion of the foetus was accompanied by simultaneous progesterone treatment of the mothers in order block the induction of labour. It was suggested that placental changes occurring during foetal hypercortisolism might be caused by increased placental oestrogen synthesis and the effect of this on the foeto-maternal junction along with a stimulatory action on PG synthesis in the maternal placenta. Experimental disruption of the normal sequence of events, when labour was blocked by progesterone, proved to be lethal to the foetus if the loss of placental integrity progressed sufficiently. The chain of regulatory signals linking increased activity of the foetal adrenal with parturition thus appears to involve stimulation of oestrogen biosynthesis, PGF release from the maternal placenta and the start of physical changes at the placental junction. Provided the foetus and corpora lutea are ipsilateral, the early releases of PGF effect luteolysis and a withdrawal of progesterone from the maternal circulation. When progesterone concentrations are sufficiently low, labour is initiated and its progress reflected by further release of PGF. The control mechanisms, which also provide for the final maturation of the foetus, clearly enable a close synchronization of the various perinatal events which are essential for the transition from foetal to postnatal life.


Urinary steroid excretion by the squirrel monkey (Saimuri sciureus). Urinary steroids and steroid conjugates were measured in the squirrel monkey (Saimuri sciureus). The principal steroids excreted were cortisol, 11beta,17alpha,20beta,21-tetrahydroxy-4-pregnen-3-one (20beta-dihydrocortisol), alpha- and beta-cortol and alpha- and beta-cortolone. The majority of the steroids were excreted unconjugated and a conspicuous feature of the pattern was the large amount of urinary free cortisol. Unlike man there was an insignificant excretion of 3alpha,17alpha,21-trihydroxy-5beta-pregnane-11,20-dione (tetrahydrocortisone) and 3alpha,11beta,17alpha,21-tetrahydroxy-5beta-pregnan-20-one (tetrahydrocortisol). A steroid not previously identified in urine from any species was one of the major glucuronide conjugates; it was characterized as having the structure 3beta,17alpha,20xi,21-tetrahydroxy-5beta-pregnan-11-one. Administration of dexamethosone resulted in complete suppression of steroid output, whilst the response to adrenocorticotrophic hormone was inconstant.


Regulation of cholesterol synthesis by low density lipoprotein in isolated human lymphocytes. Comparison of cells from normal subjects and patients with homozygous familial hypercholesterolemia and abetalipoproteinemia. The rate of cholesterol synthesis from [14C]acetate was low in circulating blood lymphocytes freshly isolated from 17 normal subjects and 4 subjects with homozygous FH. On the other hand, the rate of cholesterol synthesis was two to fourfold above normal in freshly isolated lymphocytes from two subjects with abetalipoproteinemia. When the lymphocytes from subjects with all three genotypes were incubated for 48-72 h in the absence of lipoproteins, the rate of cholesterol synthesis increased by 5-15-fold. The subsequent addition of plasma LDL, but not HDL, rapidly suppressed cholesterol synthesis in the lymphocytes from normal subjects. In contrast, lymphocytes from the FH homozygotes, which have been shown previously to be deficient in cell surface LDL receptors, were resistant to LDL-mediated suppression of cholesterol synthesis. In addition to its ability to suppress cholesterol synthesis after it had been elevated by incubation of the cells in the absence of lipoproteins, LDL was able to suppress the induction of the enhanced rate of sterol synthesis when added to normal lymphocytes immediately after their isolation from the bloodstream. In contrast to the former action of LDL, the latter action of LDL-i.e., the suppression of induction of sterol synthesis-also occurred to a limited extent in lymphocytes from FH homozygotes. However, the FH lymphocytes, but not the normal cells, could be made resistant to this action of LDL by inclusion in the incubation medium of lipoprotein-deficient serum (30 percent, vol/vol) plus HDL (1 mg protein/ml). Considered together with previous data demonstrating a deficiency of LDL receptors in freshly isolated lymphocytes from FH homozygotes, the current studies provide evidence in support of the hypothesis that the interaction of plasma LDL with its cell surface receptor serves to regulate cholesterol synthesis in human lymphocytes.


On the occurrence of cytochrome P-450 and aryl hydrocarbon hydroxylase activity in rat brain. The difference spectra of the carbon monoxide-complex of dithionite-reduced rat brain microsomes, compared with both reduced microsomes, alone, and the carbon monoxide-complex of oxidized microsomes, indicate the presence of small amounts of cytochrome P-450 in brain. As in liver, cytochrome P-450 in brain is degraded in vitro to its inactive form, cytochrome P-420 by methylmercury chloride. Aryl hydrocarbon hydroxylase activity is also present in rat brain microsomes and, at lower specific activity, in brain homogenates. This carcinogen metabolizing activity is increased four-fold in rats pretreated with 3-methylcholanthrene.


Guanosine 3',5'-cyclic monophosphate and the in vitro physiology of frog photoreceptor membranes. Frog rod outer segments freshly detached from dark-adapted retinas contain approximately 1-2 molecules of guanosine 3',5'-cyclic monophosphate (cyclic GMP) for every 100 molecules of visual pigment present. This cyclic GMP decays to 5'-GMP, and the conversion is accelerated upon illumination of the outer segments. Bleaching one rhodopsin molecule can lead to the hydrolysis of 1,000-2,000 molecules of cyclic GMP within 100-300 ms. The decline in cyclic GMP concentration becomes larger as illumination increases, and varies with the logarithm of light intensity at levels which bleach between 5 X 10(2) and 5 X 10(5) rhodopsin molecules per outer segment-second. Light suppression of plasma membrane permeability, assayed in vitro as light suppression of outer segment swelling in a modified Ringer's solution, occurs over this same range of light intensity. The correlation between cyclic GMP and permeability or swelling is maintained in the presence of two pharmacological perturbations: papaverine, a phosphodiesterase inhibitor, increases both cyclic GMP levels and the dark permeability of the plasma membrane; and beta,gamma-methylene ATP increases the effectiveness of light in suppressing both permeability and cyclic GMP levels.


Single case study. A head movement disorder occurring in dreaming sleep. A movement disorder specifically associated with dreaming sleep was observed in an otherwise healthy young woman. It was characterized by gross head movements, nightly occurrence, and no awakening. The patient had a lifelong history of the disorder, as did her brother.


Fatal cases of lipid storage myopathy with carnitine deficiency. Three patients affected by a progressive myopathy with rapid lethal evolution are presented. Excessive lipid storage was found in type 1 fibres of muscle and in liver, kidney, and myocardium. Carnitine concentrations were markedly reduced in muscle, plasma, and heart, significantly lower in the liver, and normal in kidney. D-L carnitine replacement therapy was ineffective in the only case treated. The relationship of the present cases with the syndrome of lipid storage myopathy and carnitine deficiency is discussed.


Antagonism of the paralysis produced by botulinum toxin in the rat. The effects of tetraethylammonium, guanidine and 4-aminopyridine. The injection of botulinum toxin type A into the hind-leg of adult rats causes complete paralysis of the leg lasting for several weeks. In the extensor digitorum longus (EDL) muscle transmitter release is reduced to a level of less than 1% of normal. Tetraethylammonium (TEA) and guanidine in concentrations of about 3 mM restore, in EDL muslces in vitro, neuromuscular transmission to about the normal level, provided that the external calcium concentration is 4 mM or higher. 4-Aminopyridine (4-AP) has similar restorative effect but is about 20-30 times more potent. Unlike TEA and guanidine, 4-AP is effective when the ambient calcium concentration is 2 mM; this drug is therefore also active in vivo. The intravenous injection of 4-AP (5 mg/kg body weight) restores neuromuscular transmission from complete paralysis by botulinum toxin to a normal level as shown by the recording of almost normal twitch and tetanic tensions in the EDL muscle. In rats paralysed by a lethal dose of botulinum toxin, the intraperitoneal administration of 4-AP restores general motor activity, the effect lasting 1-2 hours. A study of the effects of these drugs on spontaneous and evoked transmitter release suggests that all three compounds increase the level of free calcium inside the nerve terminals. In botulinum poisoning the transmitter release mechanism appears to be intact, but a reduced sensitivity to calcium has been shown (Cull-Candy et al. 1976), and this could explain why the drugs restore evoked transmitter release in botulinum poisoning.


Immunobiology of primary intracranial tumors. The avian sarcoma virus-induced glioma model in rats was used to study the effectiveness of immunotherapy, namely, Bacillus Calmette-Guérin (BCG) with or without sarcoma cells and/or chemotherapy with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), upon survival times. The most consistent prolongation of survival time was produced by triple therapy: BCG + intraperitoneally administered sarcoma cells + intravenously administered BCNU.


Relative toxicity and metabolic effects of cholecalciferol and 25-hydroxycholecalciferol in chicks. The relative toxicity and metabolic effectiveness of cholecalciferol (CC) and 25-hydroxycholecalciferol (25-HCC) in chicks were evaluated by feeding six graded levels of each and observing gross and microscopic pathology as well as several metabolic parameters of calcium metabolism. Renal tubular calcification was observed when CC was fed at the rate of 10.0 mg/kg of diet and when 25-HCC was fed at the rate of 0.1 mg/kg diet. Thus, 100-fold increase in toxicity results when the hydroxylated form of CC is fed. Both microscopic renal lesions and increased renal calcium and inorganic phosphate concentrations occurred in chicks with normal serum calcium concentrations.


Beta adrenergic receptor labeling in intact animals with 125I-hydroxybenzylpindolol. After the intravenous administration to mice of 125I-hydroxybenzylpindolol (125I-HYP), a potent beta adrenergic antagonist, particulate bound radioactivity in brain, heart and lung is selectively associated with beta adrenergic receptor binding sites. The amount of total and bound radioactivity in these tissues is time dependent, reaching peak values at about 5 minutes after injection, and increases approximately linearly with increasing 125I-HYP doses. The lung has the highest levels of radioactivity as well as the highest proportion of bound to total radioactivity. The amount of specifically bound 125I-HYP is markedly reduced by simultaneously injecting a beta adrenergic bound 125I-HYP is markedly reduced by simultaneously injecting a beta adrenergic agonist or antagonist, although the total amount of radioactivity in the tissues is not affected. The beta antagonist (-)-propranolol reduces specific 125I-HYP binding 50% at doses of 0.01, 0.03 and 0.004 mg/kg in brain, heart and lung, respectively. Specific 125I-HYP binding is stereospecific in these tissues as (+)-propranolol is only 1 to 2% as effective as (-)-propranolol in reducing binding. The beta agonist (-)-isoproterenol has ID50 values in the range of 2 to 20 mg/kg, whereas the alpha adrenergic antagonist, phentolamine does not reduce 125I-HYP binding. Although some radioactivity is associated with particulate fractions from the liver, little, if any, specific binding of 125I-HYP to a beta adrenergic receptor is demonstrable. The characteristics of 125I-HYP binding in mouse heart, lung and brain are those expected for the recognition site of the beta adrenergic receptor and thus provide a method for labeling the beta adrenergic receptor in vivo.


Side effects of immersion-type cleansers on the metal components of dentures. Three metals were immersed in eight denture cleansing solutions for a period of 240 hours to determine the relative side effects of these cleansers upon various metal components of complete and removable partial dentures. Conclusions drawn from this study are: 1. Commercial bleaches and unbuffered hypochlorite solutions should not be used on dentures with metal components. 2. Routine use of buffered hypochlorite solutions may cause surface damage to aluminum-base material. 3. Stainless steel (orthodontic wire) will tarnish after continued exposure to buffered and unbuffered hypochlorite solutions. 4. Further research is necessary to delineate the effects of existing denture cleansers and to improve on their effectiveness and safety.


Microiontophoretic studies of the effects of cylic nucleotides on excitability of neurones in the rat cerebral cortex. 1. Responses of cerebral cortical neurones to the microiontophoretic application of acetylcholine, noradrenaline, cyclic adenosine 3',5'-monophosphate (cyclic AMP) and cyclic guanosine 3',5'-monophosphate (cyclic GMP) were examined.2. The application of acetylcholine and cyclic GMP to identified pyramidal tract neurones resulted in an increased frequency of firing in a large number of cells. Upon application of both substances to cells which could not be identified as pyramidal tract cells, a reduction in the frequency of spontaneous firing was sometimes observed.3. Careful current controls had no effect on the cells discussed here, indicating that the observed responses were not due to the iontophoretic currents. Also, the electro-osmotic ejection of cyclic GMP (outward current) produced similar changes of cell firing to those which followed iontophoretic application (inward current).4. The microiontophoretic application of atropine resulted in a blockade of acetylcholine responses while leaving responses to cyclic GMP unaffected. This suggests that cyclic GMP was not acting indirectly by releasing acetylcholine from presynaptic endings.5. Ejection of cyclic GMP from solutions containing calcium ions produced responses comparable to those produced by cyclic GMP alone. It is unlikely therefore that cyclic GMP was causing excitation by chelating calcium.6. Applications of noradrenaline and cyclic AMP produced a reduction in the spontaneous discharge rate of most neurones tested.7. Phosphodiesterase inhibitors such as ICI 63,197 caused a potentiation of the noradrenaline responses of pyramidal tract neurones.8. 5'-adenosine monophosphate produced a powerful depression of all cells to which it was applied. This action was blocked by aminophylline, suggesting the effect was mediated through an adenosine receptor. Responses to cyclic AMP were usually not abolished, but were reduced by about 50% in amplitude.9. These results are consistent with the hypothesis that cyclic AMP may mediate some neuronal effects of noradrenaline and cyclic GMP may mediate some effects of acetylcholine. The results are also consistent with the suggestion that the two nucleotides may sometimes mediate opposite cellular responses to humoral stimuli.


Anaerobiosis and symbiosis with bacteria in free-living ciliates. Marine, sediment-dwelling ciliates were examined for cytochrome oxidase activity by a cytochemical method and for fine structural details. Species of Plagiopylidae (Trichostomatida), i.e. Plagiopyla frontata, Sonderia vorax and Sonderia sp., and of Heterotrichda, i.e., Parablepharisma pellitum, Parablepharisma sp., Metopus contortus, Metopus vestitus and Caenomorpha capucina; previously considered to be obligate anaerobes because of their sulfide-containing habitat, do not have cytochrome oxidase activity or mitochondria with cristae or tubuli. The evolutionary origin and significance of anaerobic ciliates is discussed. Most of the anaerobic ciliates harbor a flora of ecto- and endosymbiotic bacteria as demonstrated by transmission and scanning electron micrographs. It is speculated that the bacteria may utilize the metabolic end products of the protozoa for growth and energy yielding processes. These associations are also compared with other, previously described cases of symbiosis involving prokaryotes and protozoa.


Some biochemical properties of mitochondria isolated from Euglena gracilis. Mitochondria were isolated from Euglena gracilis strain Z by pressure-breakage of the cells and sucrose-cushion centrifugation. Multiple peaks (2-4) were observed in the rate of phosphorylation with Mg-ADP-phosphate concentration curves. The phosphorylative and oxidative activities were highest with NADH as the substrate, moderate with succinate, and lowest with glutamate. Inhibition of phosphorylation with 2,4-dinitrophenol and carbonyl cyanide, m-chlorophenylhydrazone gave sigmoidal concentration curves, with the extent of inhibition by DNP depending on the substrate used. Inhibition of phosphorylation by valinomycin, atractyloside, or carboxyatractyloside was only approximately 60%. Oligomycin inhibited phosphorylation in 2 phases at low and high concentrations; it inhibited Mg-ATPase in a sigmoidal fashion. Both phosphorylation and oxidation had discontinuities in Arrhenius plots at 34 C and 18 C. The relative Mg2+-dependent nucleoside triphosphatase activity was: 1 for ATP and GTP, 0.6 for ITP, 0.15 for CTP and UTP; with Ca2+ in place pf Mg2+ this activity was 0.35. Both DNP and CCCP stimulated the Mg-ATPase 50-200%. The optimal pH for the stimulation was approximately 7 regardless of the uncoupler used, and approximately 8 without the uncouplers. The few differences observed between mitochodria from Euglena and those from other sources are probably due to the fragmentation of the reticular mitochondrial structure during isolation and not to unique characteristics of these mitochondria.


Cyclic AMP and the preparation of the mouse uterus for implantation. Experiments are described that demonstrate the ability of dibutyryl cyclic AMP (dcAMP) to induced oestrogen-like changes in the uteri of ovariectomized mice treated with progesterone. A single intraluminal injection of 6-14 micrograms dcAMP in 0-02 ml phosphate-buffered saline promoted cell division in the uterine stroma and sensitized the endometrium so that a decidual reaction could be induced by a subsequent injection of arachis oil into the uterine lumen. Since dcAMP has already been shown to induce implantation of diapausing mouse blastocysts in the absence of oestrogen, it is suggested that cyclic AMP is involved in egg implantation in rodents.


Spontaneous malignant histiocytoma with metastasis in a rhesus monkey [Macaca mulatta]. A malignant histiocytoma was observed on the left arm of a recently imported rhesus monkey. Rapid invasion of deep tissues and tumor growth on the face prompted euthanasia. Necropsy revealed additional neoplastic foci in the lungs, diaphragm, intercostal muscles, pericardium and liver. Microscopically the predominant cell type was histiocytic; some cells had a distinct epithelioid appearance and other were spindle-shaped.


Expression of Thy 1 antigen in normal and neoplastic mammary cells of mice. The expression of Thy 1.2 (theta C3H) antigen was measured on the membranes of normal and neoplastic RIII and C3H mammary cells. Competitive inhibition assays revealed that the average membrane content of Thy 1.2 in mammary tissues was about equal to that of lymph node cells. Higher percentages of Thy 1.2-positive cells than mammary tumor virus (MuMTV)-positive cells were observed by immunofluorescence, which suggested that not all the Thy 1.2-positive cells recovered from tumors were also MuMTV-positive. Established tissue culture cell lines C3H and RIII MT expressed lower levels of Thy 1.2 than did cells from mammary tumors. Treatment with the synthetic gluco-corticoid dexamethasone increased the average Thy 1.2 expression in cultured mammary tumor cells as well as the levels of RNA-directed DNA polymerase. Since the percentages of Thy 1.2-positive cells also were greater in steroid-treated cultures, while fewer cells were needed to absorb a standard amount of anti-Thy 1.2 activity, it was concluded that dexamethasone enhanced membrane Thy 1.2 expression as well as MuMTV production by the cultured cells.


Vitamin A contents of rat intestinal epithelium and jejunal mucinous adenocarcinoma. Concentrations of retinol and tetinyl esters were assayed in rat intestinal mucosa and in chemically induced transplanted mucinous adenocarcinoma of the jejunum. Lipid extract from the tissues was chromatographed on deactivated alumina to isolate retinol and retinyl esters, which were determined by specific spectrofluorometry. Normal intestinal mucosa tissue contains 556 ng of retinol equivalents as retinyl esters and 303 ng of free retinol/g of wet tissue. The concentration of retinyl esters in the intestinal mucosa from rats carrying the transplanted tumor was 341 ng/g wet tissue; no free retinol was detected in the small intestinal epithelium of these rats. Liver tissue from the tumor-bearing rats contained 157 microng of retinol equivalents as retinyl esters and 136 microng of free retinol/g of wet tissue. The concentration of vitamin A per cell in the adenocarcinoma tissue was about 20 times less than that in intestinal epithelium.


Growth of murine sarcoma virus-transformed rat kidney cells in nude mice: absence of induction of host endogenous viruses. Clonal isolates of the normal rat kidney cell line (NRK) transformed by a defective murine sarcoma virus (Kirsten strain) were injected into nude mice of BALB/c background to determine whether the growth of these cells as tumors was accompanied by the induction of host endogenous type C viruses. All the virus-transformed clones produced rapidly growing tumors in nude mice, but neither the induction of mouse endogenous viruses nor the rescue and spread of the transforming sarcoma virus were observed during the growth of tumors. The degree of expression of the tumor virus structural proteins in the transformed cells did not determine the cellular phenotype with regard to tumorigenicity in nude mice, nor did it modify the cellular growth properties in vitro. Consistent with earlier observations with simian virus 40-transformed mouse and rat cells, the ability of sarcoma virus-transformed NRK cells to initiate tumor growth in nude mice appeared to be correlated with anchorage-independent growth in vitro.


Growth curve of an experimental solid tumor following radiotherapy. Because it has long been recognized that the growth of most untreated tumors is well described by Gompertzian or exponential growth curves but that the growth of treated tumors has never been well characterized mathematically, we developed and applied an equation that, while not dependent on restrictive assumptions or unmeasurable variables, was nevertheless capable of describing perturbed as well as unperturbed growth. For the 3964A rat hepatoma, a functional relationship between dose level of radiotherapy and growth-curve response was derived and used for predictive purposes. An implied relationship between tumor size and sensitivity to therapy and some clinical implications were examined.


Quantitative in vivo studies of soluble simian virus 40 tumor-specific transplantation antigens of the mouse. Quantitative studies have been performed on the immunogenicity of a membrane-bound antigen of a simian virus 40 (SV40) -induced sarcoma in syngeneic BALB/c mice and of subcellular fractions derived from this tumor. The objectives of the investigation were: a) to develop a quantitative in vivo assay of the tumor-specific transplantation antigen (TSTA) and b) to compare the distribution of histocompatibility antigens, H-2, with that of the SV40 TSTA during several fractionation steps. The immunogenicity of the TSTA-containing fractions was assessed from dose-response curves relating tumor size and the amount of protein used for immunization. After digestion of the tumor cell membranes with a limited amount of papain, H-2 as well as TSTA were present in a soluble form. A single immunization with only 2 microng of the solubilized TSTA reduced the tumor size by 70% compared to that in nonimmunized control animals. The results of several fractionation steps suggest that H-2 and the TSTA are not tightly associated in the solubilized immunogenic material.


Bioactivity of C3H and RIII mammary tumor viruses in virgin female BALB/c mice. The bioactivities of C3H and RIII mammary tumor virus (MuMTV) in virgin female BALB/c mice differed. The average number of mammary hyperplastic alveolar nodules per mouse after noduligenic tests was 20.1 in BALB/cfC3H and 10.7 in BALB/cfRIII females. Spontaneous mammary tumor incidence after 20 months of observation was 47.5% in BALB/cfC3H and 14.6% in BALB/cfRIII females (P less than 0.01).  The frequency of lung metastases in mammary tumor-bearing mice was 63.1% in BALB/cfC3H and 16.6% in BALB/cfRIII females (P less than 0.05), although the clinical duration of mammary tumors was the same. These data demonstrated a lower bioactivity of RIII MuMTV when compared to C3H MuMTV in BALB/c mice and suggested that the causative virus may control all the steps of mouse mammary tumor development, including metastasis.


Suppression of infectious murine leukemia virus in wild mice (Mus musculus) by passive immunization. Passive immunization with heterologous antivirus antiserum beginning at birth successfully suppressed infectious murine leukemia virus expression in Lake Casitas wild mice (Musmusculus) at 5-7 weeks of age.


Antibody production and complement system in protein energy malnutrition. Antibody production in kwashiorkor and marasmic infants was studied by dividing them into three groups and giving the first group a single dose of trivalent live attenuated polio virus, the second group live attenuated measles virus and the third group diphtheriatoxoid. The production of antibodies was found to be deficient in PEM as compared to normals and diminished more in kwashiorkor than in marasmus cases. The individuals of the complement system were significantly lower in kwashiorkor than in normal controls. However, C3 was the only fraction which is significantly diminished in marasmic infants. The results are discussed and as a conclusion it is suggested that deficient production of antibodies and diminution in the complement system in PEM may explain the susceptibility of such infants to repeated attacks of infection.


Properties of permissive monkey cells transformed by UV-irradiated simian virus 40. African green monkey cells (CV1 line) were infected with UV-irradiated simian virus 40 (SV40), and permissive lines of stably transformed cells were established. These cell lines display the SV40 T-antigen and the growth characteristics typical of nonpermissive transformed cells (e.g., reduced cell density inhibition, reduced serum dependence, ability to overgrow normal cells, and colony formation in soft agar). The level of permissiveness to superinfecting SV40 is fully comparable with that of nontransformed CV1 and BSC-1 lines. The transformed monkey lines also support SV40 plaque production under agar. By Cot analysis, the transformed permissive cells contain, on an average, 1 to 2 SV40 genome equivalents, and the majority of the viral sequences are associated with the high-molecular-weight cellular DNA. No spontaneous production of infectious SV40 has been observed. The transformed permissive monkey cells failed to support the replication of SV40 tsA mutants at the restrictive temperature. To account for this, it is suggested that the gene A product has separate functions for transformation and initiation of viral DNA synthesis, and only the former function is expressed in the transformed permissive monkey cells.


Cell cycle dependency of murine cytomegalovirus replication in synchronized 3T3 cells. Synchronized murine 3T3 cells have been used to investigate the possible dependency of murine cytomegalovirus replication upon the cell cycle. The normal latent period of 12 h characteristic of asynchronous 3T3 cells was protracted to more than 24 h after an early G1 infection in synchronous cells. In this case viral progeny were not detected until after the initiation of the host S-phase. Cells maintained in the G1 phase did not replicate virus. This failure could not be explained by a decrease in virus penetration but was apparently due to a requirement for an event associated with the host S-phase. Thymidine-induced inhibition of cell cycle traverse also blocked virus replication. Viral DNA synthesis did not initiate until after the initiation of host DNA. In contrast, herpes simplex virus type 1 replicated in 3T3 cells independently of the cell cycle.


Expression of tumor-specific transplantation antigen in cell lines transformed by wild-type of tsA mutant simian virus 40. The simian virus 40-induced tumor-specific surface antigen(s) (TSSA) and tumor-specific transplantation antigen(s) (TSTA)were detected in cells transformed by wild-type or temperature-sensitive mutant simian virus 40 by an antibody-mediated cytolytic assay for TSSA and an immunization test for TSTA. Cells transformed by tsA mutants, which lose their transformed phenotype when grown at nonpermissive temperatures, nonetheless do express TSSA and TSTA as well as T-antigen at both temperatures.


Epstein-barr virus-specific RNA. II. Analysis of polyadenylated viral RNA in restringent, abortive, and prooductive infections. The complexity and abundance of Epstein-Barr (EBV)-specific RNA in cell cultures restringently, abortively, and productively infected with EBV has been analyed by hybridization of the infected cell RNA with purified viral DNA. The data indicate the following. (i) Cultures containing productively infected cells contain viral RNA encoded by at least 45% of EBV DNA, and almost all of the species of viral RNA are present in the polyadenylated and polyribosomal RNA fractions. (ii) Restringently infected Namalwa and Raji cultures, which contain only intranuclear antigen, EBNA, and enhanced capacity for growth in vitro, contain EBV RNA encoded by at least 16 and 30% of the EBV DNA, respectively. The polyadenylated and polyribosomal RNA fractions of Raji and Namalwa cells are enriched for a class of EBV RNA encoded by approximately 5% of EBV DNA. The same EBV DNA sequences encode the polyadenylated and polyribosomal RNA of both Raji and Namalwa cells. (iii) After superinfection of Raji cultures with EBV (HR-1), the abortively infected cells contain RNA encoded by at least 41% of EBV DNA. The polyadenylated RNA of superinfected Raji cells is enriched for a class of EBV RNA encoded by approximately 20% of EBV HR-1 DNA. Summation hybridization experiments suggest that the polyadenylated RNA in superinfected Raji cells is encoded by the same DNA sequences as encode RNA present in Raji cells before superinfection, most of which is not polyadenylated. That the same EBV RNA sequences are present in the polyadenylated and polyribosomal fractions of two independently derived, restringently infected cell lines suggests that these RNAs may specify functions related to maintenance of the transformed state. The complexity of this class of RNA is adequate to specify a sequence of a least 5,000 amino acids. That only some RNA species are polyadenylated in restringent and abortive infection suggests that polyadenylation or whatever determines polyadenylation may play a role in  the restricted expression of the EVB genome.


Increase in lipid fluidity of cellular membranes induced by adsorption of RNA and DNA virions. Changes in the dynamic behavior of membrane lipids of mammalian cells induced by adsorption of animal viruses were quantitatively monitored by fluorescence polarization analysis with the aid of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene embedded in the surface membrane lipid core of intact cells. Adsorption of encephalomyocarditis, West Nile, and polyoma viruses to hamster (baby hamster kidney) and mouse (3T3) cells is accompanied by a rapid and significant increase in the degree of fluidity of membrane lipids of the infected cells. These changes in membrane fluidity, which are virus dose dependent, are inhibited by low temperature and by treatment of the cells before-hand with compounds known to block viral receptors on the cell surface. It is suggested that increase in membrane lipid fluidity, induced by the adsorption of virions, is an early event in the process of cell-virus interactions.


Friend strain of spleen focus-forming virus: a recombinant between mouse type C ecotropic viral sequences and sequences related to xenotropic virus. The genome of the Friend strain of the spleen focus-forming virus (SFFV) has been analyzed by molecular hybridization. SFFV is composed of genetic sequences homologous to Friend type C helper virus (F-MuLV) and SFFV-specific sequences not present in F-MuLV. These SFFV-specific sequences are present in both the Friend and Rauscher strains of murine erythroleukemia virus. The SFFV-specific sequences are partially homologous to three separate strains of mouse xenotropic virus but not to several cloned mouse ecotropic viruses. Thus, the Friend strain of SFFV appears to be a recombinant between a portion of the F-MuLV genome and RNA sequences that are highly related to murine xenotropic viruses. The implications of the acquisition of the xenotropic virus-related sequences are discussed in relation to the leukemogenicity of SFFV, and a model for the pathogenicity of other murine leukemia-inducing viruses is proposed.


Circular Epstein-Barr virus genomes of reduced size in a human lymphoid cell line of infectious mononucleosis origin. Circular Epstein-Barr virus (EBV) DNA molecules have been purified and characterized from a human lymphoid cell line derived from a case of heterophile antibody-positive, blood transfusion-induced infectious mononucleosis, 883L. The circular EBV DNA in three cell lines obtained by transformation of human umbilical cord blood leukocytes with a strain of EBV originally derived from 883L was also studied. As estimated from sedimentation velocity data and electron microscopy, the circular EBV DNA molecules are 10 to 15% smaller than either the circular EBV DNA previously found intracellularly in several other types of EBV-transformed cells or the linear EBV DNA present extracellularly in virus particles. In addition, the EBV-transformed cord blood cell lines studied here differed from other EBV-transformed cells in that integrated virus DNA sequences could not be detected.