A new system for studying molecular mechanisms of mutation by carcinogens. A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.


Total resection of distal femur or proximal tibia for bone tumours. Autogenous bone grafts and arthrodesis in twenty-six cases. Resection of the distal femur or proximal tibia en bloc has been performed on twenty-six patients with primary bone tumours. The gap was filled with autogenous bone grafts stabilised with a long intramedullary nail, thus arthrodesing the knee. In two cases temporary stabilisation with a Küntscher rod and acrylic cement was adopted because of adjuvant chemotherapy. Union was achieved in twenty-four cases (92 per cent). Infection was the main and practically the only major complication, occurring in five (19 per cent) of the cases: it healed with union in three, healed with non-union in one, and led to an above-knee amputation in the fifth case. Follow-up has been from one to eight years with an average of four years.


Development of second malignancies in rats after cure of acute leukemia L 5222 by single doses of 2-chloroethylnitrosoureas. After cure of rat leukemia L 5222 in 79 BD IX rats by single doses of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea (Hydroxyethyl-CNU) a total of 9 rats (11%) developed secondary malignomas.


Subcellular distribution in cerebral cortex of two proteins phosphorylated by a cAMP-dependent protein kinase. The subcellular distribution of Proteins Ia and Ib, two proteins which serve as specific substrates for protein kinases present in mammalian brain, was studied in the dog cerebral cortex. Proteins Ia and Ib were found to be most highly enriched in synaptic vesicle fractions; they were also present in postsynaptic density and synaptic membrane fractions in significant amounts. Proteins Ia and Ib present in the synaptic vesicle fraction appear to be similar, if not identical, to those present in the postsynaptic density fraction as judged by several criteria: (a) the ability to serve as substrate for cAMP-dependent protein kinase, (b) electrophoretic mobility in the presence of sodium dodecyl sulfate, (c) extractability with NH4Cl or EGTA, and (d) fragmentation to electrophoretically similar peptides by a purified Staphylococcus aureus protease. In addition, the postsynaptic density fraction has been found to contain cAMP-dependent Protein Ia and Protein Ib kinase activity. The subcellular localization of Proteins Ia and Ib suggests a role for these proteins in the physiology of the synapse.


Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia. Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501-520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.


Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus. A method was developed for directly observing the inner surfaces of plasma membranes by light and electron microscopy. Human erythrocytes were attached to cover slips (glass or mica) treated with aminopropylsilane and glutaraldehyde, and then disrupted by direct application of a jet of buffer, which removed the distal portion of the cells, thus exposing the cytoplasmic surface (PS) of the flattened membranes. Antispectrin antibodies and Sendai virus particles were employed as sensitive markers for, respectively, the PS and the external surface (ES) of the membrane; their localization by immunofluorescence or electron microscopy demonstrated that the major asymmetrical features of the plasma membrane were preserved. The fusion of Sendai virus particles with cells was investigated using double-labeling immunofluorescence techniques. Virus adsorbed to the ES of cells at 4 degrees C was not accessible to fluorescein-labeled antibodies applied from the PS side. After incubation at 37 degrees C, viral antigens could be detected at the PS. These antigens, however, remained localized and did not diffuse from the site of attachment, as is usually seen in viral antigens accessible on the ES. They may therefore represent internal viral antigens not incorporated into the plasma membrane as a result of virus-cell fusion.


Association of glycogen synthase phosphatase and phosphorylase phosphatase activities with membranes of hepatic smooth endoplasmic reticulum. A detailed investigation was conducted to determine the precise subcellular localization of the rate-limiting enzymes of hepatic glycogen metabolism (glycogen synthase and phosphorylase) and their regulatory enzymes (synthase phosphatase and phosphorylase phosphatase). Rat liver was homogenized and fractionated to produce soluble, rough and smooth microsomal fractions. Enzyme assays of the fractions were performed, and the results showed that glycogen synthase and phosphorylase were located in the soluble fraction of the livers. Synthase phosphatase and phosphorylase phosphatase activities were also present in soluble fractions, but were clearly identified in both rough and smooth microsomal fractions. It is suggested that the location of smooth endoplasmic reticulum (SER) within the cytosome forms a microenvironment within hepatocytes that establishes conditions necessary for glycogen synthesis (and degradation). Thus the location of SER in the cell determines regions of the hepatocyte that are rich in glycogen particles. Furthermore, the demonstration of the association of synthase phosphatase and phosphorylase phosphatase with membranes of SER may account for the close morphological association of SER with glycogen particles (i.e., disposition of SER membranes brings the membrane-bound regulatory enzymes in close contact with their substrates).


Subcellular localization of DNA-binding protein BA by immunofluorescence and immunoelectron microscopy. Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined.


Effect of enzymes on rotavirus infectivity. The infectivity of a bovine rotavirus was enhanced 140-, 8-, and 3-fold, respectively, by trypsin, protease, and lactase. Ficin, carboxypeptidases A and B, lysozyme, and beta-galactosidase had little effect on the infectivity. Chymotrypsin caused a threefold decrease in the infectivity. Trypsin acts directly on the rotavirus and not on the host cell.


Neutralization of human serum beta-lysin by sodium polyanetholsulfonate and sodium amylosulfate. Normal fresh and heat-inactivated (56 degrees C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 x 10(4) colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 mug of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed beta-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished beta-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded beta-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl(2) completely abrogated beta-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37 degrees C) completely removed beta-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both beta-lysin and lysozyme activity. Addition of either 63 to 500 mug of sodium polyanetholsulfonate per ml or 63 to 500 mug of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized beta-lysin activity for the entire observation period of 22 h.


Variable neutralization of several nonspecific antibacterial systems in fresh, defibrinated human blood by sodium polyanetholsulfonate and sodium amylosulfate. Fresh, defibrinated human blood (80 vol%, i.e., 80% [vol/vol] of a 2-ml final assay volume) from two healthy adult donors killed "delayed serum-sensitive" (DSS) and "promptly serum-sensitive" (PSS) strains of Serratia marcescens, PSS control strain Escherichia coli C, Bacillus subtilis strain ATCC 6633, and Micrococcus lysodeikticus ATCC 4698 in a kinetic manner comparable to that of fresh human serum (80 vol%). However, heat-inactivated (56 degrees C, 30 min), defibrinated human blood revealed markedly reduced or a total lack of beta-lysin activity against the B. subtilis assay strain. Similarly, lysozyme activity of defibrinated blood was diminished somewhat by heat treatment, as determined with the M. lysodeikticus assay strain. Addition of 500 mug of sodium polyanetholsulfonate (SPS) per ml to 80 vol% of fresh, defibrinated human blood completely neutralized blood bactericidal activity against all assay strains of S. marcescens, E. coli C, and B. subtilis; however, SPS at this concentration failed to abolish lysozyme activity for prolonged periods of incubation. Addition of 500 mug of sodium amylosulfate (SAS) per ml to 80 vol% of fresh defibtinated human blood resulted in protection of cell inocula of DSS strains of S. marcescens only; SAS failed to protect cell inocula of the PSS strains of S. marcescens, E. coli C, B. subtilis, and M. lysodeikticus for extended periods of observation. Based on these data, it is recommended that blood culture specimens that are first drawn into specimen containers (such as Vacutainer tubes or the like) at the patient's bedside, and which contain >/=250 mug of SPS per ml, be diluted into suitable broth media with at least >/=250 mug of SPS per ml by the receiving laboratory within 2 to 4 h after procurement of the specimen. This procedure would ensure continued, adequate neutralization of the specimen's inherent beta-lysin, lysozyme, and complement- and antibody-mediated bactericidal activities.


Comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses. Rhesus and cynomolgus monkey kidney tissue cultures and two continuous lines, Madin-Darby canine kidney (MDCK) and LLC-MK2, were compared in titrations and isolations of influenza and parainfluenza viruses. Tube cultures were inoculated with laboratory virus strains or stored patient specimens and observed for hemadsorption. Trypsin was added to the medium of the continuous lines to increase sensitivity. All four tissue cultures gave similar titers of influenza A/USSR (H1N1), A/Texas (H3N2), and B/HK, but lower titers of parainfluenza 1, 2, and 3 were observed with MDCK. Cynomolgus kidney was the best single tissue culture for reisolation of the six viruses, but foamy-virus contamination of many lots was a serious problem. Reisolation of influenza viruses was as successful with MDCK as with primary monkey kidney. LLC-MK2 was similar to rhesus kidney but less successful than cynomolgus kidney. For reisolation of parainfluenza viruses, LLC-MK2 was superior to rhesus monkey kidney and similar to cynomolgus kidney. MDCK was less useful for parainfluenza viruses. Thus, LLC-MK2 would be an acceptable single tissue alternative to primary monkey kidney. The combination of MDCK and LLC-MK2 would provide optimal sensitivity for isolation of all six viruses.


Improved indirect hemagglutination test for cytomegalovirus using human O erythrocytes in lysine. A modified indirect hemagglutination test for cytomegalovirus antibodies is described in which glutaraldehyde-fixed human O cells, rather than sheep cells, are used. Nonspecific hemagglutination was reduced by use of the optimal tannic acid concentration for each cell batch and the addition of 0.1 M lysine to the phosphate-buffered saline in which the fixed, tanned, sensitized cells were resuspended for use in the test. Three of 349 sera showed nonspecific hemagglutination by this technique. Antigen made by freezing phosphate-buffered saline (pH 7.2) over an infected monolayer can be used at dilutions of 1:8 to 1:15. Fixed, tanned, sensitized cells ready for use in the test can be stored in liquid nitrogen for up to 8 months. Use of cryoprotectants and washing after thawing is unnecessary. Simplification of the assay permits one person to screen 300 sera in 1 day or to determine the immune status of a potential donor or recipient in 45 min after the test is set up. The modified assay compares favorably in sensitivity with the complement fixation test and with previously described methods for performing the indirect hemagglutination test.


Epstein-Barr virus-specific serum immunoglobulin A as an acute-phase antibody in infectious mononucleosis. Immunoglobulin A (IgA) antibodies to Epstein-Barr virus viral capsid antigen were assayed serially in 19 patients with infectious mononucleosis and in 38 controls. Seventy-four percent of infectious mononucleosis patients demonstrated IgA antibody, whereas this was found in 13% of controls. This antibody appeared early in infectious mononucleosis and was virtually gone 10 weeks after onset. Comparison of IgA antibody kinetics was made with IgG and IgM antibodies to viral capsid antigen, heterophile antibody, and antibody to Epstein-Barr virus early antigen and nuclear antigen. Failure to demonstrate IgA antibody was associated with severe illness, prolonged illness, delay in IgG and anti-Epstein-Barr virus nuclear antigen antibody, and low or absent heterophile and anti-early antigen antibody. Assay of IgA antibody to viral capsid antigen is a potentially useful adjunct in the serodiagnosis of infectious mononucleosis or recent Epstein-Barr virus infection, as are the other antibodies tested, but in this study IgM viral capsid antigen antibody was the only acute-phase antibody present in all patients.


Effects of fatty acids on growth of Bordetella pertussis in defined medium. The effects of saturated and unsaturated fatty acids on the growth of Bordetella pertussis strain 114 in defined medium were tested. Individual fatty acids were found to be either inhibitory or stimulatory to growth, depending on concentration. Myristic (C14), pentadecanoic (C15), and palmitic (C16) acids were the most inhibitory saturated fatty acids tested. B. pertussis 114 was extremely sensitive to the unsaturated fatty acids oleic (C18; cis-9), elaidic (C18; trans-9) and petroselinic (C18; cis-6).


Collagenase in equine cell culture preparation. Equine kidney cells disaggregated by treatment with 0.01% collagenase were used in the preparation of primary monolayer cell cultures. The primary cells could be stored for long periods in liquid nitrogen and subsequently subcultivated. These techniques provided a long-term supply of equine kidney cells, free of apparent contamination, from the kidneys of a single fetus.


Skeletal muscle calcium metabolism and contractile force in vitamin D-deficient chicks. The myopathy associated with vitamin D deficiency has not been well characterized, and it is not known if weakness is a result of a specific effect of vitamin D deficiency on skeletal muscle. Chicks were raised from hatching on a vitamin D-deficient diet, and by 3 wk of age were hypocalcemic and appeared weak. Tension generated by triceps surae during repetitive stimulation of posterior tibial nerve was significantly less than that developed by chicks given vitamin D(3) supplements (309 g tension/g wet weight of triceps surae, SD 60, for vitamin D-deficient chicks; 470, SD 77, for vitamin D(3)-treated chicks, P < 0.01). Histochemical and electron microscopic examination of skeletal muscles of these chicks showed no abnormalities, and there were no electrophysiologic evidences of motor nerve or neuromuscular junction dysfunction. The concentration of ATP in skeletal muscle of the vitamin D-deficient chicks (5.75 mumol/g wet weight, SD 0.17) was not significantly different from that in vitamin D-treated chicks (5.60, SD 0.50). There was no correlation between strength and serum calcium, serum inorganic phosphate, or skeletal muscle inorganic phosphate. Relaxation of tension after tetanic stimulation was slowed in the vitamin D-deficient chicks (20.6 ms, SD 1.7, vs. 15.4, SD 1.3, in vitamin D-treated chicks and 15.3, SD 1.0, in normal control chicks), and in vitro (45)Ca(++) transport by sarcoplasmic reticulum from the vitamin D-deficient chicks was reduced. Calcium content of mitochondria prepared from leg muscles of vitamin D-deficient chicks (24 nmol/mg mitochondrial protein, SD 6) was considerably lower than that of mitochondria from normal control chicks (45, SD 8) or from chicks treated with vitamin D for 2 wk or more (66-100, depending upon level and duration of therapy). Treatment of the vitamin D-deficient chicks from hatching with sufficient dietary calcium to produce hypercalcemia did not significantly raise skeletal muscle mitochondrial calcium content (31 nmol/mg mitochondrial protein, SD 7) and did not prevent weakness. These studies demonstrate objective weakness as a result of myopathy in vitamin D-deficient chicks, and provide evidence that vitamin D deficiency has effects on skeletal muscle calcium metabolism not secondary to altered plasma concentrations of calcium and phosphate.


Prostaglandin E modulation of the mitogenic response of human T cells. Differential response of T-cell subpopulations. Prostaglandins (PG) of the E series, PGE(1) and PGE(2) (PGEs), can induce elevations of intracellular cyclic AMP (cAMP) among thymus-derived (T) lymphocytes (T cells) and inhibit their reactivity. For example, 0.1 muM of PGEs induces a two- to threefold increase of intracellular cAMP among human peripheral blood T cells and a 20-30% suppression of their blastogenic response to phytohemagglutinin. However, this suppression actually represents the net reactivity of T-cell populations demonstrating quite different responses to PGEs. Fractionation of T-enriched populations on a discontinuous density gradient yields a population of high density cells whose phytohemagglutinin-induced blastogenic response is suppressed 60%; a population of intermediate density cells whose response is suppressed 20%; and a population of low density T cells whose response is not suppressed, but is enhanced 20% by both of the PGEs. The diametrically opposite responses of low and high density T cells to the PGEs is not related to any difference in their intrinsic mitogen reactivity nor is it influenced by interactions with other T cells, bone marrow-derived (B) cells, or monocytes. Moreover, the distinct blastogenic response of low and high density T cells to PGEs does not simply correlate with PGE-mediated activation of adenylate cyclase. PGE(2) induced comparable absolute and identical relative increases of intracellular cAMP among the low and high density T cells. Cholera toxin, a potent activator of adenylate cyclase, and exogenous 8-bromo cAMP mimicked the effects of the PGEs on these two T-cell populations. These data demonstrate that T cells are heterogeneous with regard to their response to the PGEs. Thus, PGEs should be considered as potential regulators rather than as universal suppressors for T-cell reactivity. Moreover, the effect of PGEs on the blastogenic response of a given T-cell population depends upon intracellular events which occur subsequent to elevations of cAMP.


Vascular and metabolic effects of circulating epinephrine and norepinephrine. Concentration-effect study in dogs. Vascular and metabolic effects of circulating epinephrine and norepinephrine have been studied in relation to the plasma concentration of these amines in dogs. Intravenous infusion of epinephrine or norepinephrine (0.1, 0.5, and 2.5 nmol x kg-1 x min-1) raised the plasma concentration of the infused amine by 2.5 , 13, and 63 nM from resting levels of 2.4 and 3.6 nM, respectively. Blood flow to isolated adipose tissue; skeletal muscle preparations; and plasma levels of glycerol, glucose, and cyclic AMP were measured. Epinephrine and norepinephrine displayed a distinct selectivity with regard to both vascular and metabolic effects. Epinephrine caused significant vasoconstriction in adipose tissue already at a plasma concentration of 5 nM, whereas no significant effect was seen on skeletal muscle vascular resistance. Norepinephrine, on the other hand, caused significant vasoconstriction in skeletal muscle at 5 nM but had no vasoconstrictor effect in adipose tissue. Epinephrine was more potent than norepinephrine in increasing plasma cyclic AMP and glucose, whereas the converse was true for plasma glycerol. Epinephrine had significant effects on plasma cyclic AMP at 5 nM and on plasma glucose and glycerol at 15 nM. Norepinephrine, on the other hand, had significant effects on plasma glycerol at 5 nM, plasma cyclic AMP at 15 nM and plasma glucose only at 65 nM. It is suggested that these response patterns are related to a preferential action of epinephrine on beta 2-adrenoceptors and a preferential action of norepinephrine on beta 1-adrenoceptors. Our results support the view that both epinephrine and norepinephrine may act as circulating hormones, because vascular and metabolic effects of both amines were seen at plasma concentrations encountered during various kinds of stress in animals and man.


Hepatic secretion of lipoproteins in the rat and the effect of experimental nephrosis. Livers from normal and nephrotic rats were perfused by the nonrecirculating technique. Nephrosis was studied on the 7th d after the injection of puromycin animonucleoside. Amino acid-labeled lipoproteins (d < 1.21) were isolated from the perfusion medium by agarose column chromatography or by sequential density ultracentrifugation. In both groups of animals, in addition to very low density lipoproteins and nascent high density lipoproteins, column chromatography revealed the presence of a peak of 2-3 x 10(6) daltons. This peak contained lipoproteins of densities corresponding to <1.006, 1.006 < d < 1.02, and 1.02 < d < 1.06, which indicated that rat liver secretes a heterogeneous mixture of triglyceride-rich lipoproteins. The amount of these lipoprotein density classes was measured and their lipid and apoprotein composition and their apoprotein specific activity were determined. In both groups of rats there was a progressive rise in phospholipid and decrease in triglyceride content as the isolation density increased from 1.006 and 1.06. The lipoproteins from the nephrotics had higher amounts of cholesterol. The livers from the nephrotic rats secreted two to three times as much lipoprotein as controls in all density classes in the first 20 min, but during the next 40 min only the 1.02 < d < 1.06 and nascent high density lipoproteins remained at this high level compared to controls. A larger total liver pool of apolipoproteins in nephrotic livers was inferred from their lower specific activities during the first 20 min. The apoprotein composition of liver perfusate lipoproteins from nephrotics differed from controls. There was a 40% decrease in the amount of low molecular weight apoproteins in all density classes, with corresponding increases in apo B and apo E in the triglyceride-rich fractions. The apo A-1 content of nascent HDL was increased from 16% in controls to 52% in nephrotics, with corresponding decreases in apo C and apo E. When these results were combined with specific activity measurements of the individual apoproteins and the net secretion rate of total protein in each lipoprotein class, it was possible to estimate the total amount of each apoprotein secreted and the total incorporation of labeled amino acids into each. The incorporation of label gave results similar to those obtained by direct measurement of the amounts of apoproteins. Apo E secretion was increased by a factor of 1.8, apo B by 2.8, and apo A-1 by 8.4, whereas the secretion of apo C was not significantly altered. We explain these results by postulating that the primary stimulus to hepatic plasma protein synthesis in response to proteinuria is general and that subsequent negative feedback regulation affects individual apolipoprotein synthesis rates. A corollary of this hypothesis is that the biosynthesis and secretion of an apoprotein may be regulated independently of the lipoprotein density class in which it is found.


Evidence for skeletal resistance to parathyroid hormone in magnesium deficiency. Studies in isolated perfused bone. Hypocalcemia during magnesium (Mg) depletion has been well described, but the precise mechanism(s) responsible for its occurrence is not yet fully understood. The hypocalcemia has been ascribed to decreased parathyroid hormone (PTH) secretion as well as skeletal resistance to PTH. Whereas the former is well established, controversy exists as to whether or not Mg depletion results in skeletal resistance to PTH. These studies examine the skeletal response to PTH in normal dogs and dogs fed a Mg-free diet for 4-6 mo. Isolated tibia from normal (serum Mg 1.83+/-0.1 mg/100 ml) and experimental dogs (serum Mg 1.34+/-0.15 mg/100 ml) were perfused with Krebs-Henseleit buffer during a constant infusion of 3 ng/ml of synthetic bovine PTH 1-34 (syn b-PTH 1-34). The arteriovenous (A-V) difference for immunoreactive PTH (iPTH) across seven normal bones was 37.5+/-3%. In contrast, the A-V difference for iPTH was markedly depressed to 10.1+/-1% across seven bones from Mg-depleted dogs. These findings correlated well with a biological effect (cyclic AMP [cAMP] production) of syn b-PTH 1-34 on bone. In control bones, cAMP production rose from a basal level of 5.8+/-0.2 to 17.5+/-0.7 pmol/min after syn b-PTH 1-34 infusion. In experimental bones, basal cAMP production was significantly lower than in controls, 4.5+/-0.1 pmol/min, and increased to only 7.1+/-0.4 pmol/min after syn b-PTH 1-34 infusion. Even when PTH concentrations were increased to 20 ng/ml, cAMP production by experimental bones was lower than in control bones perfused with 3 ng/ml. Histological examination of bones from Mg-deficient dogs showed a picture compatible with skeletal inactivity. These studies demonstrate decreased uptake of iPTH and diminished cAMP production by bone, which indicates skeletal resistance to PTH in chronic Mg deficiency.


Lipolysis produces changes in the immunoreactivity and cell reactivity of very low density lipoproteins. Smaller very low density lipoprotein (VLDL) remnants interact more readily with tissues than do larger "intact" VLDL. This may be related to changes in the availability of VLDL apoproteins on the surface of the lipoproteins. To test this hypothesis VLDL were incubated at 37 degrees C with bovine milk lipase (LPL), and the abilities of LPL-treated VLDL preparations to compete with (125)I-low density lipoproteins (LDL) for interaction with cultured normal human fibroblasts were measured. At the same time, the immunologic activities of these preparations were also tested by double antibody radioimmunoassay. Triglyceride (TG) contents of VLDL fell by 30-90% during incubation with LPL and, on zonal ultracentrifugation, VLDL of faster Svedberg unit of flotation (S(f1.063)) rates (>150) were gradually converted to smaller VLDL with lower S(f) rates (21-60). LPL-treated VLDL competed two to five times more effectively with (125)I-LDL for binding to cellular receptors than did control VLDL. Control VLDL incubated with heat-inactivated LPL at 37 degrees C, or with active LPL at 4 degrees C had unaltered cell reactivities and TG contents compared with VLDL incubated without any enzyme. The direct uptake and degradation of LPL-treated VLDL was also assessed by using VLDL (125)I-labeled in apoprotein (Apo)B. LPL-treated VLDL-(125)I-ApoB were taken up and degraded by fibroblast at greater rates than were control VLDL-(125)I-ApoB. Thus, hydrolysis of VLDL lipids was accompanied by an increased ability of VLDL to interact with fibroblasts. The immunoreactivity of ApoB in the same VLDL preparations, expressed as the "apparent ApoB contents" of LPL-treated VLDL, increased by 10-50% (P < 0.02) in those assays that contained anti-LDL antisera, but the ApoB of control VLDL remained constant. However, assays that contained antisera directed against ApoB isolated from VLDL did not distinguish between LPL-treated and control VLDL. Thus, VLDL lipid hydrolysis was accompanied by changes in the immunoreactivity of VLDL-ApoB, which probably reflect changes in the disposition of ApoB on the surface of VLDL. The altered disposition of ApoB on VLDL "remnants" may be related to their enhanced interaction with cells.


Low-density lipoprotein receptor activity in cultured human skin fibroblasts. Mechanism of insulin-induced stimulation. Low-density lipoproteins (LDL) receptor activity, as reflected by LDL degradation, was stimulated by the addition of insulin to cultures of human skin fibroblasts. These changes occurred independently of the glucose concentration of the incubation medium and occurred whether or not LDL receptor activity was suppressed. A comparison of the saturation kinetics of LDL receptor activity in the presence and absence of insulin indicated that insulin produced a 35% increase in Vmax with no difference in "apparent Km". These results suggest that insulin enhances LDL receptor activity by increasing the number of LDL receptors rather than by influencing binding affinity. In confirmation, LDL degradation by receptor negative cells was not enhanced by insulin. Sterol synthesis from [14C]acetate was also stimulated by insulin, but egress of cholesterol and cellular cholesterol content were unaffected by the hormone. The effect of insulin on LDL receptors was not dependent on its known ability to enhance cellular DNA synthesis and proliferation, because insulin stimulated LDL receptor activity in cells kept quiescent by maintenance in plasma-derived serum that was devoid of platelet derived growth factor. Nevertheless, the effect of insulin in enhancing LDL receptor number, coupled with stimulation of endogenous cholesterol synthesis, provides a mechanism whereby the cell could theoretically increase its supply of cholesterol during times of additional need.


Tubuloglomerular feedback and single nephron function after converting enzyme inhibition in the rat. Experiments were done in normal rats to assess kidney, single nephron, and tubuloglomerular feedback responses during renin-angiotensin blockade with the converting enzyme inhibitor (CEI) SQ 20881 (E. R. Squibb & Sons, Princeton, N. Y.) (3 mg/kg, per h). Converting enzyme inhibition was documented by complete blockade of vascular responses to infusions of angiotensin I (600 ng/kg). Control plasma renin activity was 12.5+/-2.7 ng angiotensin I/ml per h (mean+/-SEM) and increased sevenfold with CEI (n = 7). There were parallel increases in glomerular filtration rate from 1.08+/-0.05 to 1.26+/-0.05 ml/min and renal blood flow from 6.7+/-0.4 to 7.5+/-0.5 ml/min. During CEI infusion absolute and fractional sodium excretion were increased 10-fold. Proximal tubule and peritubular capillary pressures were unchanged. Single nephron glomerular filtration rate (SNGFR) was measured from both proximal and distal tubule collections; SNGFR based only on distal collections was significantly increased by CEI. A significant difference was observed between SNGFR values measured from proximal and distal tubule sites (6.0+/-1.6 nl/min) and this difference remained unchanged after CEI administration. Slight decreases in fractional absorption were suggested at micropuncture sites beyond the late proximal tubule, whereas early distal tubule flow rate was augmented by CEI. Tubuloglomerular feedback activity was assessed by measuring changes in proximal tubule stop-flow pressure (SFP) or SNGFR in response to alterations in orthograde microperfusion rate from late proximal tubule sites. During control periods, SFP was decreased 11.2+/-0.4 mm Hg when the perfusion rate was increased to 40 nl/min; during infusion of CEI, the same increase in perfusion rate resulted in a SFP decrement of 6.7+/-0.5 mm Hg (P<.001). When late proximal tubule perfusion rate was increased from 0 to 30 nl/min, SNGFR was decreased by 15.0+/-1.2 nl/min during control conditions, and by 11.3+/-1.3 nl/min during CEI infusion. Attenuation of feedback responsiveness during CEI was also observed at lower perfusion rates with both techniques. These results indicate that blockade of the renin-angiotensin system with CEI reduces the activity of the tubuloglomerular feedback mechanism which may mediate the observed renal vasodilation.


Participation of monocyte-macrophages and lymphocytes in the production of a factor that stimulates collagenase and prostaglandin release by rheumatoid synovial cells. Cultured mononuclear cells from human peripheral blood produce a soluble factor (MCF) that stimulates collagenase and prostaglandin E2 (PGE2) release by cultured rheumatoid synovial cells up to several hundred fold. These target rheumatoid synovial cells lack conventional macrophage markers. To determine which mononuclear cells are the source of MCF, purified populations of monocyte-macrophages, thymus-derived (T) lymphocytes, and bone marrow-derived (B) lymphocytes were prepared. The monocyte-macrophages alone produced levels of MCF that were proportional to cell density but unaffected by phytohemagglutinin or pokeweed mitogen. No detectable collagenase activity was produced by the cultured monocyte-macrophages or lymphocytes. Purified T lymphocytes produced levels of MCF approximately or equal to 1--3% those of purified monocyte-macrophages in the presence or absence of the above lectins. Purified T lymphocytes modulated the production of MCF by the monocyte-macrophages, however, in a manner dependent upon relative cell densities and the presence of lectins. For example, at optimal ratios of T lymphocytes: monocyte-macrophages, MCF production was markedly stimulated by pokeweed mitogen. Thus, interactions of T lymphocytes and monocyte-macrophages could be important in determining levels of MCF, which regulate collagenase and PGE2 production by target synovial cells in inflammatory arthritis.


Physiological and pharmacological influences on thyroxine to 3,5,3'-triiodothyronine conversion and nuclear 3,5,3'-triiodothyronine binding in rat anterior pituitary. Our recent in vivo studies have suggested that intrapituitary l-thyroxine (T(4)) to 3,5,3'-triiodo-l-thyronine (T(3)) conversion with subsequent nuclear binding of T(3) is an important pathway by which circulating T(4) can inhibit thyrotropin release. The present studies were performed to evaluate various physiological and pharmacological influences on these two processes in rat anterior pituitary tissue. Intact pituitary fragments were incubated in buffer-1% bovine serum albumin containing 0.14 ng/ml [(131)I]T(3) and 3.8 ng/ml [(125)I]T(4). Nuclei were isolated after 3 h of incubation and the bound iodothyronines identified by paper chromatography. There was 0.3-1% [(125)I]T(3) contaminating the medium [(125)I]T(4), and this did not change during incubation. Nuclear [(125)I]T(4) was not decreased by 650-fold excesses of medium T(3) or T(4), suggesting that it was nonspecifically bound. The ratio of nuclear to medium [(131)I]- and [(125)I]T(3) were expressed as nuclear counts per minute per milligram wet weight of tissue:counts per minute per microliter medium. Intrapituitary T(4) to T(3) conversion was evidenced by the fact that the nuclear:medium (N:M) ratio for [(131)I]T(3) was 0.45+/-0.21, whereas that for [(125)I]T(3) was 2.23+/-1.28 (mean+/-SD, n = 51). A ratio (R), the N:M [(125)I]T(3) divided by the N:M [(131)I]T(3), was used as an index of intrapituitary T(4) to T(3) conversion. Increasing medium T(3) concentrations up to 50 ng/ml caused a progressive decrease in the N:M ratio for both T(3) isotopes, but no change in the value for R, indicating that both competed for the same limited-capacity nuclear receptors. Increasing concentrations of medium T(4) caused no change in the N:M [(131)I]T(3) but did cause a significant decrease in R in three of four experiments. These results suggest saturation of T(4)-5'-monodeiodination occurred at lower T(4) concentrations than saturation of nuclear T(3) binding sites. In hypothyroid rats, the N:M ratios for both [(131)I]T(3) and [(125)I]T(3) were increased (P < 0.005), but R was three-fold higher than in controls (P < 0.005). Animals given 10 mug T(4)/100 g body wt per d for 5 d had significantly decreased N:M ratios for both [(131)I]T(3) and [(125)I]T(3), as well as a decreased value for R. In fasted rats, neither N:M ratio was depressed, although hepatic T(4) to T(3) conversion in the same animals was 50% of control (P < 0.005). Iopanoic acid (13 muM), but not 6-n-propylthiouracil (29 muM), decreased the N:M [(125)I]T(3) with a significant decrease in the value for R (P < 0.025 or less). Neither sodium iodide (6 muM) nor thyrotropin-releasing hormone (7-700 nM) affected the T(3) N:M ratios. These results indicate that intrapituitary T(4) to T(3) conversion is stimulated in hypothyroidism and depressed in T(4)-treated animals, whereas opposite changes occur in hepatic T(4)-5'-monodeiodination. Unlike liver, anterior pituitary T(4)-5'-monodeiodination is not affected by fasting or incubation with 6-n-propyl-2-thiouracil, but T(4) to T(3) conversion is inhibited in both by iopanoic acid. These results indicate that there are important differences between anterior pituitary and other tissues in the regulation of T(4)-5'-monodeiodination.


Beta adrenergic and muscarinic cholinergic receptors in canine myocardium. Effects of ischemia. Experimental myocardial ischemia produced in dogs by proximal left anterior descending coronary artery ligation is accompanied by relatively rapid (1 h) increases in the number of (-) [3H]dihydroalprenolol binding sites without changing their dissociation constants in ischemic left ventricular tissue. The changes, persist for at least 8 h and are accompanied by marked decreases in myocardial tissue ischemic region norepinephrine content. In contrast, in the same canine model 1 h of proximal left anterior descending coronary artery ligation did not result in a significant change in the number of [3H]quinuclidynl benzilate binding sites of their dissociation constants. However, the number of [3H]quinuclidynl benzilate binding sites (muscarinic cholinergic receptors) are 50--70% greater than (-) [3H]dihydroalprenolol binding sites (beta adrenergic receptors) in canine left ventricular tissue. Thus, the data suggest that proximal left anterior descending coronary artery occlusion for 1 h significantly increases the number of beta adrenergic receptors in ischemic left ventricular tissue without changing the number of muscarinic cholinergic receptors. Whether the ischemia-produced increase in cardiac beta-receptor content is causally related to increased cyclic AMP levels that develop in ischemic tissue and/or an etiologic factor in arrhythmias originating from ischemic myocardial tissue will have to be determined in additional studies.


New function for high density lipoproteins. Their participation in intravascular reactions of bacterial lipopolysaccharides. The addition of bacterial lipopolysaccharide (LPS) from Escherichia coli 0111:B4 or Salmonella minnesota R595 to plasma (or serum) resulted in a marked reduction of the hydrated buoyant density of the parent LPS (0111:B4 [d = 1.44 g/cm3] and R595 [d = 1.38 g/cm3]), to d less than 1.2 g/cm3. This reduction in buoyant density to less than 1.2 g/cm3 of the LPS required plasma (or serum) lipid. Delipidation of plasma (or serum) by extraction with n-butanol/diisopropyl ether (40/60, vol:vol) prevented the conversion of the parent LPS to a form with d less than 1.2 g/cm3. Reversal of the effect of delipidation was accomplished by the addition of physiologic concentrations of high density lipoprotein (HDL). In contrast, as much as two times normal serum concentration of low density or very low density lipoprotein were ineffective. The ability of normal plasma (or serum) to inhibit the pyrogenic activity of LPS, lost after delipidation, was also restored after the addition of HDL. Preliminary results suggested that prior modifications of the LPS, probably disaggregation, may be required before interaction with HDL.


Studies of relationship among bile flow, liver plasma membrane NaK-ATPase, and membrane microviscosity in the rat. Liver plasma membrane (LPM) NaK-ATPase activity, LPM fluidity, and bile acid-independent flow (BAIF) were studied in rats pretreated with one of five experimental agents. Compared with controls, BAIF was increased 24.6% by thyroid hormone and 34.4% by phenobarbital, decreased by ethinyl estradiol, but unchanged by propylene glycol and cortisone acetate. Parallel to the observed changes in BAIF, NaK-ATPase activity also was increased by thyroid hormone (40.8%) and decreased by ethinyl estradiol (26.2%). In contrast, NaK-ATPase activity failed to increase after phenobarbital but did increase 36% after propylene glycol and 34.8% after cortisone acetate. Thus BAIF and NaK-ATPase activity did not always change in parallel. The NaK-ATPase K(m) for ATP was not affected by any of these agents.LPM fluidity, measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene, was found to be increased by propylene glycol, thyroid hormone, and cortisone acetate, decreased by ethinyl estradiol, and unaffected by phenobarbital. Thus in these cases, induced changes in LPM fluidity paralleled those in NaK-ATPase activity. In no case did Mg-ATPase or 5'-nucleotidase activities change in the same direction as NaK-ATPase, and the activity of neither of these enzymes correlated with LPM fluidity, thus indicating the selective nature of the changes in LPM enzyme activity caused by the agents. These findings indicate that LPM fluidity correlates with NaK-ATPase activity and may influence the activity of this enzyme. However, the nature of the role of LPM NaK-ATPase in bile secretion is uncertain and needs further study.


Appearance and characterization of lipoprotein X during continuous intralipid infusions in the neonate. The development of hyperphospholipidemia and hypercholesterolemia was studied in infants that required total parenteral nutrition and given a continuous infusion of Intralipid, (1-4 g/kg body wt per 24 h. Detailed studies were carried out on infusion periods lasting 1-10 d. After 24 h there was a marked increase in plasma free cholesterol (68%) and phospholipid (77%) concentrations. Based on the amount of cholesterol in Intralipid, and the rate of infusion, it was estimated that at least 50% of the plasma cholesterol increment during 64-h infusions was derived from endogenous sources. By contrast, the hyperphospholipidemia could be attributed to the Intralipid as the rise in plasma was calculated to be equivalent to only 16% of the exogenous phospholipid infused. Approximately 10% of the phospholipid in Intralipid was in a triglyceride-free mesophase form with a free cholesterol:phospholipid molar ratio of 0.063. There were no systematic changes in plasma concentrations of cholesterol ester or triglyceride during Intralipid infusions. The increase in free cholesterol and phospholipid was localized in the low density lipoproteins (d = 1.006-1.063 g/ml). The presence of lipoprotein X (Lp-X) in the low density lipoprotein fraction was demonstrated by electrophoresis in agar and by isolation and chemical characterization with hydroxylapatite chromatography. Isoelectric focusing of urea-soluble protein of Lp-X revealed that albumin and apolipoproteins CII and CIII were major components, whereas apolipoprotein E and AI were minor constituents. The abnormal lipoprotein was apparent by 16 h during 64 h of infusion. After 6 d of continuous infusions the free cholesterol in Lp-X was 30+/-10 mg/dl (mean+/-SD), which represents a total Lp-X mass of 90 mg/dl. After cessation of the infusion, Lp-X, as monitored by electrophoresis in agar, disappeared within 72-96 h. Thus, during infusion of Intralipid in infants at rates commonly employed, the capacity of the clearance mechanisms for phospholipid are exceeded, which causes the accumulation of phospholipid and free cholesterol in the form of Lp-X particles. It is suggested that mesophase phospholipids in Intralipid may play a significant role in this process.


Hydroxyl radical generation by polymorphonuclear leukocytes measured by electron spin resonance spectroscopy. Electron spin resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed to detect the formation of hydroxyl radicals (OH.) by phagocytosing polymorphonuclear leukocytes (PMN). An electron spin resonance signal with the identical g value and splitting characteristics of the DMPO/OH). adduct was detected on incubation of normal PMN with opsonized zymosan. Adduct formation was strongly inhibited by superoxide dismutase and by the OH. scavenger mannitol, but catalase had little or no effect. (DMPO/OH). was not formed by PMN from a patient with chronic granulomatous disease; in contrast, adduct formation by PMN which lack myeloperoxidase was greater than normal. These findings are discussed in relation to the formation of OH. by PMN.


Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces. Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus, and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy.


Alpha-1-antitrypsin Pi S phenotype and liver cell inclusion bodies in alcoholic hepatitis. Typical liver cell inclusions of alpha-1-antitrypsin deficiency were found in a patient with the Pi S phenotype and acute alcoholic hepatitis. It is suggested that the inclusions resulted from the combined effects of the S phenotype and alcohol and, on the basis of the known chemistry of the variant antitrypsins, that the lowered serum concentration of antitrypsin associated with the S allele may be due to hepatic holdup.


A radioimmunoassay for hepatitis A antigen and antibody. The HAVAB radioimmunoassay for the detection of antibody to hepatitis A is assessed. Its modification to detect virus in faecal samples is described and in this it was found to be more sensitive and specific than electron microscopy.


Toxic effects of metals from the environment on hair growth and structure. Diffuse alopecia related to ingestion of toxic metals from the environment has been observed in 36 patients. Copper, arsenic, mercury and cadmium were involved and the intensity of disturbance of the hair cycle, heralded by presence of dystrophic hairs, was proportional to the amount of toxic material detected in blood and urine. Toxicity can be observed in vitro by measuring labeled cells on squash preparations after incorporation of tritiated uridine and thymidine in plucked hairs. The labeling indexes, as well as the ratio between cells entering the S phase and the viable cells were always lower in toxic alopecia than in normal hair. Toxic metals do not seem to affect the morphology of the hair shaft.


Mechanisms involved in alpha-adrenergic effects of catecholamines on liver metabolism. Recent results have indicated that alpha-adrenergic receptors are the major mediators of catecholamine actions on liver metabolism in several species. It is well-established that cAMP and cAMP-dependent protein kinase are not involved in hepatic alpha-adrenergic effects. This review presents evidence that alpha-adrenergic stimulation of glycogenolysis in rat liver involves the mobilization of Ca2+ ions from mitochondria and stimulation of phosphorylase kinase by the resulting increase in cytosolic Ca2+ concentration. Possible mechanisms by which activation of alpha-adrenergic receptors causes release of mitochondrial Ca2+ and affects other cell processes are discussed.


Inhibition of lipolysis in hamster adipocytes by the cation ionophor X537A. The present study reports the effects of the lipophylic ionophore X537A on lipolysis and accumulation of cAMP in isolated hamster epidiymal adipocytes. X537A inhibited lipolysis activated with norepinephrine, isoproterenol, dibutyryl cAMP or theophylline but failed to influence basal lipolysis. The minimum effective concentration of X537A required to inhibit lipolysis was between 1 and 3 micrograms/ml; at a concentration of 10 micrograms/ml, X537A inhibited lipolysis by approximately 50%. The antilipolytic effect of X537A does not result from decreased formation of cAMP because the accumulation of cAMP in response to isoproterenol or theophylline was significantly potentiated in the presence of the ionophore. Most of the additional cAMP that accumulated in the presence of X537A was found to be intracellelular, the distribution of cAMP between cells and incubation medium not being influenced by X537A. Neither the basal activity of cAMP dependent protein kinase nor the activity in the presence of isoproterenol or theophylline was influenced by X537A. The effects of X537A on lipolysis and on accumulation of cAMP were found to persist in the absence of extracellular calcium, but adipocytes that were preincubated in a calcium free media containing 4.0 mM EGTA failed to respond to X537A with an increase in cAMP levels. It is concluded that X537A inhibits lipolysis by uncoupling cAMP accumulation from activation of triglyceride lipase by a mechanism unrelated to activation of protein kinase.


Human gingival lymphocytes. I. Methodology for the isolation of human gingival lymphocytes. Various methodologies were examined for the isolation of inflammatory cells from diseased human gingiva. The best recovery of viable gingival lymphocytes (gMNC) was achieved by a method which combined initial collagenase digestion followed by gentle teasing with an 18-gauge needle.


Lack of effect of fluoride on urinary cAMP excretion in rats. The urinary excretion of cyclic 3'5'-adenosine monophosphate (cAMP) was measured in rats provided a low fluoride diet (less than 0.5 ppm) and drinking water containing either 0 or 25 ppm of fluoride. The pMole cAMP/microgram creatinine ratio decreased with age but was not influenced by fluoride intake.


Granular-cell tumors of the vulva: a report of three cases. Three black women were found to have granular-cell tumors on the vulva; in one, there were several additionally in other cutaneous sites. Although this benign neoplasm is uncommon on the vulva, it should be considered in the differential diagnosis of nodular lesions on the vulva. Granular-cell tumors in multiplicity occur frequently and are not a sign of metastatic disease. This lesion has predilection for blacks. Pseudocarcinomatous hyperplasia occurs typically in overlying squamous epithelium and may be misinterpreted as squamous-cell carcinoma.


Antibody-independent neutralization of vesicular stomatitis virus by human complement. II. Formation of VSV-lipoprotein complexes in human serum and complement-dependent viral lysis. Vesicular stomatitis virus (VSV) is efficiently neutralized by normal, nonimmune human serum without the participation of antibody. Neutralization is complement- (C) dependent and requires the early-acting components of the classical pathway, C1, C4, C2, and C3, but not later-acting C components. In further studies, normal human serum was found to markedly increase the density of a variable but significant proportion of virus-associated RNA and to markedly decrease the density of the remainder of virus-associated RNA. The RNA of increased density was found to be dense ribonucleocapsid cores released from VSV by C-dependent viral lysis mediated through the classical pathway. The released ribonucleocapsid cores found at the bottom of sucrose density gradient after incubation of VSV with human serum were resistant to degradation by proteolytic enzymes. The VSV-derived RNA found floating on the tops of sucrose density gradients performed on serum-treated VSV was infectious virus. The decreased density was due to binding of VSV to human serum lipoproteins (LP), primarily very low density lipoproteins (VLDL). Binding of VLDL to VSV required the presence of the viral envelope and the external glycoprotein, G. Despite the binding of LP to VSV, LP did not neutralize VSV, and LP-depleted sera were fully active in neutralizing VSV. Thus, LP do not represent an accessory factor for the C-dependent neutralization of VSV.


Target-effector interaction in the natural killer cell system. IV. Modulation by cyclic nucleotides. Dibutyryl cAMP (dB-cAMP) and the cAMP elevating agents, prostaglandin E1, theophylline, and histamine markedly suppressed NK cytolytic function in a dose- and rate-dependent manner. The inhibition was rapidly induced and persisted in the presence of the drugs. Separate pretreatment of targets and highly purified NK cells, isolated by a target binding and velocity sedimentation technique, revealed that PGE1 and dB-cAMP acted at the level of the effector cell in a short-term cytolytic assay. In contrast to the inhibitory effects of cAMP elevating agents, dB-cGMP and carbamylcholine caused a small but significant acceleration in the rate of lysis and could compete with inhibitory doses of dB-cAMP to reduce the level of suppression thereby suggesting that the cAMP-cGMP ratio might be important in NK-mediated lysis. Insulin had no effect on NK activity, whereas T cell-mediated cytolysis was augmented by insulin and cGMP if the effector cells were taken early after alloimmunization but not later. Neither cAMP- nor cGMP-elevating agents affected the frequency of NK-target cell conjugates. These results are compatible with the hypothesis that cyclic nucleotides may be involved in triggering the lytic event within NK cells.


Production of human lymphocyte mediators after activation with periodate or neuraminidase and galactose oxidase. Treatment of human mononuclear cells with sodium metaperiodate (NaIO4) or neuraminidase and galactose oxidase (NGO) results in lymphocyte activation and subsequent generation of supernatants rich in migration inhibitory factor (MIF) and leukocyte inhibitory factor (LIF). Preliminary characterization of these mediators by Sephadex G-100 gel filtration suggests that they are similar to antigen- and concanavalin A-induced MIF and LIF, eluting in the 25000 m.w. and 68000 m.w. regions, respectively. The possibility of galactose oxidase carryover into the supernatants has been studied and conditions are described that minimize this eventuality. A method is presented for producing control and lymphokine-rich supernatants both of which have been exposed to identical concentrations of NGO although in the control activation is blocked by the addition of 0.1 M galactose to the incubation. These findings establish NaIO4 and NGO as useful mitogens for generating human lymphokine-rich supernatants that can be used without further purification.


Natural killing in estrogen-treated mice responds poorly to poly I.C despite normal stimulation of circulating interferon. Natural killing by mouse spleen cells can be stimulated in vivo by interferon or by agents that stimulate interferon, such as poly I.C. Natural killing can be suppressed in vivo by the sustained administration of 17 beta-estradiol. In BALB/c mice that had been treated with 17 beta-estradiol for 10 weeks, natural killing did not respond to intravenous poly I.C, although stimulation of circulating interferon was equal to controls. Estradiol, then, does not block interferon production but does suppress the response of natural killer cells to interferon. It is suggested that estrogens either block the maturation of natural killer cells or reduce the number of natural killer cell precursors.


Immobilization of viral and mycoplasma antigens and of immunoglobulins on polystyrene surface for immunoassays. The immobilization of purified influenza virus, rubella virus, crude nuclear cytomegalovirus antigen and of mycoplasma on polystyrene tubes was studied using radio-iodinated preparations. The antigen activities on tube surfaces were determined using sequentially specific human antibodies and alkaline phosphatase-conjugated anti-human IgG in an enzyme-immunoassay (EIA) reaction. In addition, immobilization of radio-iodinated human IgG, IgM, and IgA, serving as model proteins, was studied using the respective anti-immunoglobulin conjugates in EIA directly. Pretreatment of the surface with albumin and glutaraldehyde inhibited the adsorption and antigenicity of IgG. Increase of temperature and thus of speed of adsorption did not affect the fraction of antigen eluted during the test procedure. Only with IgG and IgA was it necessary to saturate the polystyrene surface in order to achieve maximal reactivity in EIA. With other antigens, maximal reactivity in EIA was obtained with amounts of protein much lower than the maximal amount that could be adsorbed per tube. IgM was found to have an exceptionally high affinity to polystyrene.


Detection of virus and cellular-determined antigens in situ using [125I]protein A and autoradiography. The present paper describes the use of [125I]Protein A, isolated from Staphylococcus aureus, in detecting antigen-antibody complexes by autoradiography on single cells. the method is relatively quick, reproducible, potentially more sensitive than immunofluorescence, and should be useful in combination with conventional radioimmuno-assays. We have used it to detect the cellular expression of IgM, kappa, lambda, and beta 2-microglobulin, as well as the expression of Epstein-Barr Virus (EBV)-associated antigens expressed in human lymphoblastoid cell lines.


Periodate or glutaraldehyde for preparing peroxidase conjugates? Conjugates of horseradish peroxidase with antibodies (anti-human IgG (H + L)) or their Fab' fragments were prepared according to the newest modification of the periodate (P-) method or the two-step glutaraldehyde (G-) method. The conjugates were analysed by gel chromatography and subsequently tested in three different applications. For tissue immunohistochemistry on sections of lupus erythematosus skin, G-conjugates were preferred to polymeric P-conjugates. In ELISA for detection of human antibodies against penicillin P-conjugates were superior to G-conjugates. For the detection of surface Ig on lymphoid cells both types of conjugate were more or less equally suitable. A scheme for the most suitable combinations of method of preparation and field of application is given.


Characterization of human papillomavirus 3 in warts of a renal allograft patient. Multiple flat wart-like lesions of a renal allograft recipient were shown to contain HPV 3 or a serologically crossreacting virus by indirect immunofluorescence with monospecific animal antisera against HPV [1--5]. The patient's serum revealed virus specific antibodies (immunofluorescence titer 1/80). Papillomaviruses were isolated and after in vitro iodination 3 major proteins (MW 70.000, 56.000 and 43.000) were detected by SDS polyacryalmide gel electrophoresis. DNA was extracted from the warts and cleaved with the restriction endonuclease Hae III. Distinct bands were discernible within the background of cellular DNA and these fragments were identified as papillomavirus DNA by blot hypbridization with 32P-labeled viral DNA.


The influence of defective-interfering particles of the PR-8 strain of influenza A virus on the pathogenesis of pulmonary infection in mice. Homologous autointerference mediated by defective-interfering (DI) particles was first described with the PR-8 strain of influenza virus. However, little is actually known about the influence of DI particles of influenza virus on the pathogenesis of pulmonary infection. The present studies were designed to determine (1) the requirements for successful autointerference in vivo with DI particle-enriched PR-8 influenza virus and (2) the effects of DI particle-enriched virus on the development and progression of otherwise lethal pulmonary infection in mice. PR-8 influenza virus passaged in chicken eggs, but not that passaged in Madin-Darby bovine kidney cells, and enriched in DI particles markedly attenuated pulmonary infection in seven-week-old Swiss and four-week-old C57B16/Cr mice but not in three- to four-week-old Swiss mice. In addition, replication of influenza virus, straining of viral antigen, and numbers of infiltrates in lungs of mice infected with DI particle-enriched influenza virus were reduced in comparison with values in mice infected with comparable amounts of wild-type influenza virus. The protection mediated by DI particle-enriched virus appeared to be related to augmented humoral immune responses in infected mice rather than to autointerference with replication of wild-type virus.


Effect of antiviral lipids, heat, and freezing on the activity of viruses in human milk. Semliki Forest virus (SFV), herpes simplex virus type 1 (HSV-1), coxsackievirus B4, and cytomegalovirus (CMV) were added to human milk, which was then subjected to treatments that approximated those required for the decontamination or storage of milk. Boiling was the only treatment that eliminated these viruses from the milk. Pasteurization (at 62.5 C for 30 min) did destroy CMV, but the other viruses could still be detected. All of the viruses except HSV-1 were detectable after the contaminated milk samples had been stored at -15 C for 10 days. SFV, HSV-1, and CMV were also decreased by lipid antiviral activity, which was present in 78% of samples that were obtained on day 5 postpartum. Naturally excreted CMV was detected at the highest levels in milk that lacked this antiviral factor; this CMV also survived freezing for 10 days. Thus, whereas the lipid antiviral activity decreased the level of enveloped viruses, whether they were added to or naturally excreted into human milk, further reduction in the levels of these viruses required heat treatment.


Common exposure outbreak of gastroenteritis due to type 2 rotavirus with high secondary attack rate within families. A sharp outbreak of gastroenteritis associated with human rotavirus type 2 involved not only all of nine infants and young children in a playgroup but also seven of 10 parents and grandparents studied. The source of the outbreak appeared to be two non-playgroup siblings. Six of 11 individuals studied shed human rotavirus type 2, and each of seven from whom paired sera were obtained developed a type 2 sero-response. Overall, evidence of infection with rotavirus type 2 was demonstrated in 10 of 11 individuals by detection of virus in stools and/or a serologic response in an enzyme-linked immunosorbent assay.


Occurrence of infection with group B coxsackievirus in rheumatic and nonrheumatic Filipino children. The pathogenesis of rheumatic fever has been linked to various immune mechanisms involving streptococcal antibodies and heart tissue antigens. Latent myocarditis due to group B coxsackievirus has also been considered as a possible conditioning factor. The validity of the role of infection with group B coxsackievirus in rheumatic fever was tested by determining the incidence of type-specific neutralizing antibodies in sera of Filipino children. Analysis of the results by means of a normal Z-test showed that the incidence in rheumatic children was not statistically significant in comparison to the incidence in asymptomatic children.


Comparative prevalence of subclinical cytomegalovirus and herpes simplex virus infections in the genital and urinary tracts of low-income, urban women. The prevalence of subclinical infections caused by cytomegaloviruses (CMV) and herpes simplex viruses (HSV) was assessed simultaneously in the genital and urinary tracts of 1,101 indigent women. Shedding of CMV from the genital and urinary tracts exceeded that of HSV by ninefold (8.89% vs. 0.95) and sevenfold (3.8% vs. 0.51%), respectively. Pregnancy, regardless of gestational age, had no discernible influence on productive infection with either virus in either the genital or the urinary tract. In contrast, age was a significant variable, especially with CMV. The prevalence of CMV shedding decreased steadily with age, from peak values of 15% and 8% in the genital and urinary tracts, respectively, of females 11-14 years of age to undetectable levels in females 31 years of age and older. Productive HSV infection was more prevalent in postmenopausal than in premenopausal women: 2.4% vs. 0.8% and 3.1% vs. 0.2% in the genital and urinary tracts, respectively. Likewise, HSV infection of both the genital and the urinary tract combined was more common in older than in younger females.


[Scanning electron microscopic observations of calcium pyrophosphate crystals of joint tissues and synovial fluid (author's transl)]. The deposition and ultrastructure of calcium pyrophosphate (CPPD) crystals in joint tissues of pseudogout patients and cadavers were studied. Nine calcified menisci, 2 articular cartilages and 6 samples of synovial fluid were examined by scanning electron microscopy (SEM). Some of them were examined by analytical electron microscopy (EMMA). In the samples of menisci and cartilages, the findings were compared with those in the soft X-ray examinations and polarized light microscopy. The results are summarized as follows: 1) SEM observation of the cut surfaces of calcified menisci and cartilages showed a three-dimensional ultrastructure for the CPPD crystals. The crystals in the synovial fluid taken from pseudogout knees were also clearly demonstrated by this method. The EMMA analysis provided the possibility to examine the structure and content of the crystals simultaneously. 2) Crystal deposition in the meniscus varied with the depth of the tissues; it was diffuse in the collagen framework of the superficial layer, but showed accumulation in the deep layer where a clear line of demarcation between the collagen framework and crystals was seen. 3) The crystals in the meniscus were rod, granular or rectangular in shape, and 0.2-6.5 micro by 0.2-3.5 micro in size. Crystals from the articular cartilage were granular or rod-like in shape, and 0.2-3.5 micro by 0.2-1.0 micro in size. Most of the crystals found in the synovial fluid were rod-shaped. 4) X-ray microanalysis of the meniscus crystals by EMMA showed the same pattern of PK alpha, CaK alpha, and CaK beta content as that of CPPD crystals commercially available. The P/Ca ratio was about 0.7. 5) SEM and EMMA examination can be very useful for accurate identification of the form and content of the tiny crystals in joint tissues and synovial fluid. This can also be useful in proving a diagnosis of crystal-induced synovitis.


Effect of chlorpromazine on hepatic perfusion and bile secretory function in the isolated perfused rat liver. The hepatotoxicity of CPZ was studied in the isolated perfused rat liver in order to more closely define possible mechanisms of phenothiazine-induced cholestasis. Perfusate concentrations of CPZ were increased from 5 x 10(-6) M to 5 x 10(-4) M until bile secretion was significantly inhibited. Measurements were then made of determinants of bile secretory function, including the magnitude of lobar distribution of perfusate flow, BAIF, and liver plasma membrane enzyme activity, Na+,K+-ATPase, Mg++-ATPase and 5'-nucleotidase. BAIF diminished significantly from control values of 1.76 +/- 0.07 microliter min-1gm-1 of liver to 1.34 +/- 0.15 and 0.80 +/- 0.09 following 2.5 and 5 x 10(-4) M CPZ, respectively. Perfusate flow also diminished from 5.64 +/- 0.44 to 1.24 +/- 0.12 ml min-1 gm-1 of liver 20 min following 5 x 10(-4) M CPZ and was associated with reduced flow to peripheral areas of the hepatic lobes as demonstrated by Tc-HAM. By 30 min, perfusate flow had returned to baseline values. CPZ also transiently diminished the excretion of bile acids in livers receiving a constant infusion of 40 mumol hr-1 sodium taurocholate. Defects in hepatic perfusion could not account entirely for the impairment in BAIF, since comparable mechanical restriction of perfusate flow in controls only diminished BAIF to 1.49 +/- 0.08 microliter min-1gm-1 of liver. CPZ signofocamt;u rediced tje secofoc actovotu pf Mg++-ATPase and 5'-nucleotidase but did not affect Na+,K+-ATPase in liver plasma membrane isolated 20 min after 5 x 10(-4) M CPZ. CPZ also resulted in a profound shift in the recovery of protein in isolated liver plasma membrane fractions from the light (density = 1.16) to heavier (density = 1.18) fractions. These findings, together with previous observations demonstrating alterations in hepatic ultrastructure, indicate that CPZ interacts in a complex manner with hepatocyte plasma and cytoplasmic membrane components and suggest that these drug-membrane interactions independently result in diminished hepatic perfusion, impairment of bile acid excretion, and inhibition of bile acid-independent bile secretion.


Decreased nitroblue tetrazolium reduction of granulocytes in type IV hyperlipoproteinemia. NBT reduction of granulocytes was determined in 40 patients with primary hyperlipoproteinemia. The reaction was measured in granulocytes both in the resting state and after stimulation with E. coli, as well as with and without addition of plasma. In 17 patients with type IV hyperlipoproteinemia (endogenous hypertriglyceridemia), the NBT reduction was significantly decreased in all these types of experiments when compared with 12 healthy controls. Sera from hypertriglyceridemic patients decreased NBT reduction of normal granulocytes. Increasing concentration of VLDLs from normolipidemic donors had the same effect. It is suggested that plasma lipoproteins may influence granulocyte function.


Central generation of bursting in the feeding system of the snail, Lymnaea stagnalis. The central generation of burst activity was investigated in the buccal ganglia of Lymnaea. Eight different patterns of burst activity were generated by one or two consecutive phases of compound synaptic potentials resulting from activity of neurones. These inputs acted upon the different endogenous properties of buccal neurones such as post-inhibitory rebound and spike adaptation. Effects of synaptic inputs were reinforced by by electrotonic coupling of some buccal neurones of the same type.


Synthetic phospholipid vesicles containing a purified viral antigen and cell membrane proteins stimulate the development of cytotoxic T lymphocytes. Synthetic phospholipid vesicles (liposomes) containing the purified glycoprotein (G) of vesicular stomatitis virus (VSV) and solubilized membrane proteins from cells of the appropriate H-2 haplotype elicited H-2-restricted cytotoxic T lymphocytes (CTL) that lysed VSV-infected target cells. The CTL were elicited by intact liposomes, not by released components. Thus, when spleen cells from VSV-primed H-2d X H-2b hybrid mice were stimulated with liposomes having G protein + membrane proteins from cells with one of the parental H-2 haplotypes, the resulting CTL lysed only VSV-infected target cells with that parent's H-2 type. This result argues against the view that T cells in general recognize only processed antigenic fragments on macrophages. Moreover, liposomes were only effective when G protein and cell membrane proteins were included in the same vesicles. This result suggests that for effective interaction with CTL precursors the antigen (G protein) and products of the H-2 complex must be closer to each other than 600--1,000 angstrom, the diameter of the lipid vesicles used in this study.


A T helper cell for anti-viral cytotoxic T-cell responses. We demonstrate here: (a) the existence of T helper (Th) cells that augment the generation of virus-specific cytotoxic T cells in vitro, (b) that the helper cells carry the theta and Ly 1 membrane antigens, (c) that activation of the Th effect is specific for viral antigens, and (d) that the delivery of help is not H-2 restricted.


Cyclic adenosine monophosphate response to prostaglandin E2 on subpopulations of human lymphocytes. Receptors for prostaglandin E2 or histamine were measured on subpopulations of human lymphocytes, using the cyclic AMP increase after exposure to prostaglandin or histamine as an indicator for the presence of receptors. The cyclic AMP response to prostaglandin E2 was similar in unfractionated lymphocytes and the T-enriched and T-depleted fractions. Within the T-enriched population, T cells bearing a receptor for the Fc portion of IgG (T gamma-cells) had a 27.4-fold rise in cyclic AMP after exposure to prostaglandin E2, whereas the remaining T cells (non-T gamma cells) had a fourfold increase. It would appear that prostaglandin receptors are concentrated on a small subfraction of T gamma cells, comprising approximately 15% of the T-cell population. The cyclic AMP response to histamine was less than twofold in all lymphocyte fractions.


[The microanalysis of serum lipoprotein lipids (author's transl)]. A method is described which allows the determination of phospholipids, free and esterified cholesterol, triglycerides and free fatty acids in lipoprotein fractions starting from 50 microliter of serum. Lipoproteins were separated by successive precipitation: VLDL with Heparin/Mg++, LDL with Dextran sulfate/Mg++ and finally HDL with Dextran sulfate/Mn++. Lipids extracted from the precipitated lipoproteins were determined gravimetrically and by densitometry after thin layer chromatography and charring (van Gent, C.M. (1968), Z. Anal. Chem. 236, 344--350; Egge, H. et al. (1970) Z. Klin. Chem. Klin. Biochem. 8, 488--491). The results obtained from the serum of 12 adult healthy persons were compared with those from lipoprotein fractions separated by preparative ultracentrifugation (Havel, R. J. et al. (1955) J. Clin. Invest. 34, 1345--1353). The distribution of lipids in beta-lipoproteins (d less than 1.063 g/ml) and HDL (1.063 less than d less than 1.21 g/ml) prepared by both methods showed good agreement. Some differences were observed between VLDL (d less than 1.006 g/ml) and VHDL (d greater than 1.21 g/ml) prepared either by precipitation or ultracentrifugation. Compared to the total lipid of the sera, recovery rates were 95--105%. Variation coefficients were in the range of 15--20% for VLDL lipids, 5--10% for LDL and HDL lipids and 10--15% for VHDL lipids. Gravimetrically determined total lipids had a variation coefficient of 4 and 6% for LDL and HDL respectively.


Comparison of four commercially available cAMP kits. In this study four commercially available cAMP kits were compared and shown to differ with respect to reliability, sensitivity, simplicity and expiration time. It is not possible to recommend a particular kit, because the choice depends on the demands of the investigator, which, of course, may vary. However, in our opinion, one of the kits was inferior to the others.


Feline syncytium-forming virus: DNA provirus size and structure. An infectious DNA assay has been used to investigate the size and structure of the genome of feline syncytium-forming virus (FSFV). The dose response between DNA extracted from FSFV-infected cells and plaque number on feline embryo cells followed two-hit kinetics and the mol. wt. of the proviral DNA was estimated as approx. 6 x 10(6).


Initial interaction of human fibroblast and leukocyte interferons with FS-4 fibroblasts. Human FS-4 cells were exposed to human fibroblast interferon for various times and further incubated in the absence of interferon until challenged with vesicular stomatitis virus. Addition of antibody to fibroblast interferon at the time of removal of interferon did not alter the development of the antiviral state. If cells were exposed to interferon for 45 min at either 0 or 37 degrees C, they developed resistance upon subsequent incubation at 37 degrees C. However, less resistance developed if the cells were initially incubated at 0 degrees C. Our results indicate that a single interaction of fibroblast interferon with susceptible cells, either at 0 or 37 degrees C, is sufficient for the subsequent development of an antiviral state, at least in the short term experiment. The kinetics of development of the antiviral state were compared with fibroblast and leukocyte interferon. The rise in the degree of antiviral resistance was steeper and maximal levels of resistance were reached sooner when FS-4 cells were incubated with increasing concentrations of fibroblast interferon than with leukocyte interferon. This suggests a greater affinity of fibroblast interferon for these cells.


A fluoroimmunoassay for measurement and visualization of antibody bound to surface antigens of HSV-infected cells. An assay based on quantitative spectrofluorometry of surface immunofluorescence was applied to the study of reactions between antibody and surface antigens of viable HSV-infected cells in suspension. Fluorescence was expressed in terms of photon counts per second. Both the direct and indirect fluorescent antibody techniques proved acceptable for assay of surface reactions, yielding values of specific fluorescence as high as eight times those of controls. Fluorescence microscopy of cell populations assayed by spectrofluorometry allowed for simultaneous visual examination of surface antigens. Comparison of the fluoroimmunoassay with the 51Cr-release test for cytolytic antibody to HSV-induced surface antigens revealed the latter to be the more sensitive, with antibody titres ranging up to four times those detected by fluoroimmunoassay. General correlation between the two assays was found using both rabbit and human sources of antisera.


Rotavirus polypeptides.

Rotavirus polypeptides. Rotavirus infected monkey kidney cells (LLC-MK2) have been labelled with 35S-methionine in the presence of actinomycin D. The cells have been lysed with SDS and the polypeptides separated by discontinuous polyacrylamide gel electrophoresis. Rotavirus polypeptides began to appear 4 to 5 h p.i.; incorporation was maximum at 8 h, but all the polypeptides were still being made 15 to 18 h p.i. Tissue culture adapted calf rotavirus particles were labelled with 35S-methionine and the polypeptides compared with cell associated rotavirus polypeptides. There were four inner coat, four outer coat and three non-structural polypeptides. Several of the outer coat polypeptides have altered mol. wt. on maturation. The polypeptides of rotavirus from seven species (human, pig, calf, lamb, mouse, foal and rabbit) have been compared and their mol. wt. calculated. The polypeptides fell into the same relative groupings for each virus, but there were variations in the mol. wt. of most comparable polypeptides. The polypeptides of tissue culture adapted and non-adapted calf rotavirus from the same original isolate varied only in one of the non-structural polypeptides.


Expression of retrovirus-associated 8S RNA in mammalian cells. A comparative study of the expression of retrovirus-associated 8S RNA was made in different mammalian cells. This RNA is found in cultured cells from all mammalian species analysed but its expression varies. An increase of 8S RNA is observed in sarcoma virus-transformed cells as compared to control uninfected cells or cells infected with leukaemia virus. No increased quantities of 8S RNA were detected in cells transformed by SV40 or by methylcholanthrene. These data show that the level of 8S RNA was augmented following transformation by sarcoma viruses.


Acute and latent infection of sensory ganglia with herpes simplex virus: immune control and virus reactivation. The role of antiviral antibody in controlling the acute and latent phases of herpes simplex virus (HSV) infection in sensory ganglia of mice was studied in vitro and in vivo. Organ cultures of ganglia inoculated in vitro with HSV produced infectious virus for at least 3 weeks. In the presence of antiviral antibody, the titre of virus was markedly reduced, but the infection was not eliminated. Similarly, passive administration of antibody to HSV-infected immunodeficient (nude) mice reduced the virus titre but did not eliminate the acute phase of the ganglionic infection. Suppression of the cell-mediated immune response in latently infected immunocompetent mice by treatment with cyclophosphamide and/or X-irradiation resulted in reactivation of HSV in up to 70% of the animals. Reactivation was demonstrated by recovering infectious virus in cell-free homogenates of ganglia and eye globes and by finding virus antigens in ganglia by immunofluorescent staining. Reactivation occurred both in vitro and in vivo in the presence of high concentrations of neutralizing antibody. It is concluded that antibody alone is not sufficient to eliminate the acute phase of the ganglionic infection and that cytotoxic agents known to suppress the host's cellular immune response can reactivate virus in the presence of neutralizing antibody.


Evidence for herpes simplex virus type-selective receptors on cellular plasma membranes. Herpes simplex virus type 1 (HSV-1) interfered with the adsorption of subsequently added homotypic but no heterotypic HSV, suggesting that the cellular receptors involved were type-selective. Both infective and u.v.-irradiated virus could block the attachment of virions to cellular surface receptors. The adsorption rate was studied by assaying non-adsorbed infective virus remaining in the fluid medium and cell-associated 3H-thymidine labelled HSV, and HSV mutants assayed in presence of phosphonoformic acid (PFA). The adsorption profiles indicated that GMK AH-1, Vero and SIRC cells all exhibited more HSV type 1-than type 2-selective receptors while HeLa S3 cells displayed more receptors with affinity for type 2 than for type 1. On HEp-2 and human embryonic lung cells HSV type 1- and type 2-selective receptors were about equally represented.


Characterization of interferon messenger RNA from human lymphoblastoid cells. After treatment with Sendai virus, Namalwa cells form large amounts of interferon. RNA extracted from treated whole cells or from their polysomes was injected into Xenopus laevis oocytes and the interferon formed was titrated. The results show that the amount of interferon mRNA was maximal by 9 h after treatment of the cells with Sendai virus and then declined. Sucrose gradient centrifugation of the mRNA gave substantial purification and showed that its size was 12 S.


Comparison of the antigens produced by foamy virus in a cytolytic and a persistent infection of HEp2 cells. The antigens from cytolytic infections of HEp2 cells by type I simian foamy virus produced two multicomponent precipitation lines when tested by immunodiffusion with the homologous hyperimmune rabbit antiserum. The antigens obtained from a non-productive infection of MK5 virus in HEp2 cells produced only those precipitation lines which corresponded with the inner lines obtained from the cytolytic infection. Similarly, hyperimmune rabbit antiserum against antigens extracted from the persistent infection lacked the antibody which was responsible for the outer lines of precipitation. Indirect immunofluorescence with acetone-fixed and unfixed cells using the homologous and heterologous sera confirmed the absence of antigens in the persistent infection and showed that an antigen is produced in the persistently infected cells which is either absent or present in very small amounts in cytolytically infected cells. Neutralization experiments and ether treatment suggested that the missing antigens in the persistent infection were the envelope components of foamy virus. It is proposed that the persistent infection has properties in common with some infections by RNA tumour viruses.


Loss of proviral DNA sequences in a revertant of Kirsten sarcoma virus-transformed murine fibroblasts. A previously described revertant cell line (K-BALB SR1212), derived as a single cell clone from a clonal line of murine fibroblasts (K-BALB) transformed by a nonproductive infection with the Kirsten strain of murine sarcoma virus, has normal morphology and growth kinetics and, unlike the transformed parent cell line, lacks a sarcoma virus that can be rescued. We report here that this reversion correlates with low to undetectable levels of expression of cellular Ki-MSV-specific RNA and a reduction of proviral sequences in the cell DNA to a level equivalent to that found in the uninfected BALB cells with a normal phenotype. The data indicate that phenotypic reversion has occurred as a consequence of the loss of part or all of the sarcoma provirus, either by chromosomal rearrangement or provirus excision.


Characterization of virus DNA synthesized in KB cells infected with two temperature-sensitive mutants of adenovirus type 5. KB cells were infected with H5ts36 or H5ts125, two adenovirus type 5 (Ad5) mutants with a temperature-sensitive synthesis of virus DNA. Infection was started at the nonpermissive temperature and at 16 h p.i. the temperature was shifted down to the permissive temperature. Shortly after the shift-down H5ts125-infected cells showed an accumulation of purely single-stranded DNA of virus origin, which was not observed in H5ts36-infected cells. This single-stranded DNA has been characterized by hybridization and sedimentation analysis. It was found that the single-stranded DNA was derived from both complementary strands and consisted of short fragments. The observation that the single-stranded DNA accumulates in H5ts125-infected cells under conditions in which the amount of DNA binding protein is reduced, suggests that the DNA-binding protein is not only involved in initiation, but also in elongation of nascent strands.


Protection of mice against infection with wild-type Mengo virus and an interferon sensitive mutant (IS-1) by polynucleotides and interferons. Single-stranded polynucleotide preparations [tRNA, poly(rI) plus poly(ho5C)-copolymer] which protect mice against picornavirus infections without inducing interferon, protected mice equally against infection with an interferon-sensitive mutant (IS-1) of Mengo virus and with wild-type virus (IS+). Poly(rI) . poly(rC), and mouse macrophage interferon [i.e. serum from mice treated with poly(rI) . poly(rC)] protected mice equally against infections with the two viruses, but fibroblast interferon protected better against infection with the interferon-sensitive mutant than with the wild-type virus. These and other results indicate that: Mengo virus had a genetic locus affecting sensitivity to fibroblast but not macrophage interferon; these two types of interferon have different mechanisms of action against Mengo virus infections in mice; Mengo virus genes controlling sensitivity to fibroblast interferon may modulate disease since infection in vivo induces only fibroblast interferon; the antiviral activity of the single-stranded polynucleotides is unlikely to be mediated by induction of either macrophage or fibroblast interferon.


Production of vesicular stomatitis virus with low infectivity by interferon-treated cells. In cultures of Ly cells treated with 10 or 30 reference units/ml of mouse interferon there was a 30 to 200 times reduction in the production of infectious vesicular stomatitis virus (VSV); virus particle production, as measured by VSV particle associated virus RNA, virus protein, and virus transcriptase, was inhibited by, at most, six times. These results suggested that interferon-treated cells produce VSV particles with low infectivity and resemble the findings in interferon-treated cells infected with murine leukaemia viruses.


Orthopoxvirus strains defective in surface antigen induction. Various strains of vaccinia, variola, whitepox, monkeypox and cowpox viruses were examined for their capacity to induce a specific early antigen detectable on the surface of infected cells. The Elstree strain of vaccinia, two strains of variola minor and white variants of cowpox and monkeypox viruses lacked the capacity to induce the antigen. Variation of the parent cowpox and monkeypox viruses to white variants was always accompanied by the loss of the antigen-inducing capacity.


Enhanced proliferation of endogenous virus in Chinese hamster cells associated with microtubules and the mitotic apparatus of the host cell. Chinese hamster ovary cells harbour intracytoplasmic virus-like particles of type A which are closely associated with sites of microtubule formation. We report here the enhanced proliferation of these particles and their release at the cell membrane by using either 5-bromodeoxyuridine or dibutyryl cyclic AMP. The extracellular mature particles are similar in morphology to retroviruses of type B. Close association of the type A virus precursors with microtubule organizing centres, i.e. kinetochores, centrioles and basal bodies, and with microtubules per se, is confirmed by studying the effects of the microtubule inhibitors Colcemid and vincristine sulphate. The role of microtubules in the activation and transport of the intracytoplasmic type A particles is discussed.


Production of tubular structures in Vero cells infected with herpes simplex virus type 2; effects of ultraviolet light irradiation and antimetabolites. In order to investigate the nature of tubular structures specifically found in herpes simplex virus type 2 (HSV-2)-infected cells, the multiplication of HSV-2 was studied in Vero cells cultured in the presence of varying concentrations of cytosine arabinoside (Ara-C) and cycloheximide (CH), inhibitors of DNA synthesis and protein synthesis respectively. Ara-C, at a concentration of 60 micrograms/ml, inhibited the multiplication of HSV-2 by more than 99% and also prevented the appearance of tubular structures and virus particles in the nuclei of infected cells. Nevertheless, the synthesis of virus specific surface antigens of HSV-2-infected Vero cells was not reduced, as revealed by the fluorescent antibody technique. On the other hand, 10 micrograms/ml of CH inhibited both the appearance of tubular structures and virus particles and the synthesis of virus specific surface antigens by more than 99%. These observations strongly suggest that the appearance of tubular structures is one of the late events in the process of virus multiplication. To measure the comparative genome size needed to produce membrane antigens, tubular structures and infectious centres, the effect of u.v.-inactivation of HSV-2 on these processes was studied. After u.v.-irradiation, the capacity to induce tubular structures was inactivated at a slower rate than the capacity to form infectious centres, but at a faster rate than the induction of surface antigens. Furthermore, more tubular structures could be induced by u.v.-inactivated virus than by the non-irradiated virus which was diluted to the same infectivity as the u.v.-irradiated virus. These results indicate that expression of the entire genome is not required for the production of tubular structures.


Mouse hybrid cell lines produce antibodies to herpes simplex virus type 1. A solid-phase radioimmunoassay procedure has been devised for the assay of antibodies produced in the mouse to herpes simplex virus type 1 (HSV-1). It is based on the adsorption of virus to flexible micro-well plates and uses radio-iodine-labelled rabbit antibody against mouse immunoglobulin to assess antibody binding. Using this assay for screening, cell hybrids have been obtained which yield monoclonal antibody to HSV-1. The hybrids are between spleen cells from hyperimmune mice and an immunoglobulin-non-secreting, azaquanine resistant myeloma cell line (NS-1). From 480 hybrid cell lines initially examined, five stable cell lines were obtained which released HSV-1-specific antibody in vitro and in vivo. Mice carrying transplants of these cell lines yield binding titres in serum of up to 1/25000. Both IgG and IgM antibodies were obtained in this way.