Demonstration of specific cell-mediated anti-tumor immunity in lung cancer to autologous tissue extracts. Cell-mediated immunity (CMI) of lung cancer patients to autologous tumor antigens was assessed by mixed lymphocyte tumor interactions (MLTI) as measured in a microculture (200 microliter) lymphocyte proliferation (LP) assay. Positive lymphoproleferative responses were observed with cryopreserved intact mitomycin-C-treated autologous tumor cells (8/12 or 67% patients reactive) and with hypotonic membrane extracts (HMP) of tumor cells (28/40 or 70%). Good correlation was found between reactivity to tumor cells and extracts in parallel testing. In contrast, HMP of autologous normal lung tissue elicited very little LP reactivity, with only one patient giving a weak response by the SI criterion and low level of n cpm. Upon repeat testing, many patients gave reproducibly positive LP responses to tumor HMP. Patients at all clinical stages of disease and with different histologic tumor types had a similar proportion of HMP reactivity. Most reactive patients responded to a broad range of protein concentrations of tumor HMP, and LP responses were frequently elicited with 1 microgram or less of HMP. Thus, HMP appear to afford a convenient source of reactive tumor antigen for assessing anti-tumor immunity.


Recovery of transforming EBV from non-producer cells after superinfection with non-transforming P3HR-1 EBV. Cells of the Raji and NC37 lines can be induced by chemical inducers, such as BrdUrd and IdUrd, or the tumor-promoter TPA to EA-expression only, but do not reveal any VCA synthesis. After superinfection by nontransforming P3HR-1 EBV, however, a varying percentage of the cell population shows VCA synthesis and releases infectious viral particles. The recovered virus differs biologically from P3HR-1 EBV since it transforms human umbilical cord blood lymphocytes into EBNA-positive lymphoblastoid cell lines. Cells of these established lines are susceptible to renewed infection by P3HR-1 EBV which results in EA induction and VCA synthesis. Only cells of one line, NC37-R1, spontaneously produce VCA and EBV particles, which reveal transforming properties and do not induce EA upon superinfection of Raji cells. Infection of P3HR-1 EBV-converted BJA-B cells also leads to EA and VCA induction and the release of viral particles. In contrast to particles recovered from Raji and NC37 cells, no transforming activity was detectable in these virus preparations. According to these data, we propose that viral genomes persisting within Raji and NC37 cells are defective and become complemented by the superinfecting P3HR-1 virus.


Aryl hydrocarbon hydroxylase in persons with lung or laryngeal cancer. To test whether the distribution of AHH inducibility is shifted toward the high end of the range in patients who had lung and laryngeal cancer, we measured this trait in 59 patients (32 lung and 27 laryngeal) who had resectable tumors and had been disease-free for a period of time. The advantage of selecting patients who were free of clinical disease was that measurement of their AHH inducibility should not have been affected by the disease state. Patient and control populations showed no difference in basal and induced AHH activity of AHH inducibility. The mean AHH inducibility in patients who had lung cancer was 3.20 +/- 0.20; in patients who had laryngeal cancer 2.96 +/- 0.18, and for all controls 3.29 +/- 0.04 (no significant difference at p = 0 05). Further analysis of the distribution of AHH inducibility in the patient group compared to controls showed no suggestion of a shift toward the higher end of the range in patients who had lung and laryngeal cancer.


Influences on the density of beta-adrenergic receptors in the cornea and iris--ciliary body of the rabbit. By measurement of the specific binding of 3H-dihydroalprenolol, the densities of beta-adrenergic receptors on membranes prepared from homogenized corneas and iris--ciliary bodies of rabbits were studied. Sympathetic denervation, as a result of subconjunctival treatment with 6-hydroxydopamine, causes an increase in the density of beta-adrenergic receptors in membranes prepared from the ipsilateral iris--ciliary body but not the cornea. Topical treatment with epinephrine for 5 days causes a decrease in the density of beta-adrenergic receptors in membranes prepared from cornea and iris-ciliary body, whereas similar treatment with timolol causes an increase in the density of beta-adrenergic receptors. In the cornea, the decrease in receptor density that occurs following in vivo treatment with epinephrine is associated with a decreased ability to synthesize cyclic AMP, whereas the increase in receptor density that occurs following in vivo treatment with timolol is not associated with an altered ability to synthesize cyclic AMP. Our results indicate that the density of beta-adrenergic receptors in the anterior segment of the eye is inversely related to the level of adrenergic stimulation to the tissue but that the ability of a tissue to synthesize cyclic AMP does not necessarily parallel the change in receptor density.


Effects of epinephrine, indomethacin, acetylsalicyclic acid, dexamethasone, and cyclic AMP on the in vitro activity of lysosomal hyaluronidase from the rabbit iris. The effects of epinephrine, indomethacin, acetylsalicylic acid, dexamethasone, and cyclic AMP on lysosomal hyaluronidase activity in in the rabbit iris were studied in vitro. Indomethacin and acetylsalicylic acid inhibited the lysosomal hyaluronidase activity. Dexamethasone and cyclic AMP (adenosine-3',5'-cyclic monophosphate) had little effect on the enzyme activity. Epinephrine activated the enzyme activity. These drugs had no effect on the activities of either bovine testicular hyaluronidase or rabbit iridial acid phosphatase. The possible role of lysosomal hyaluronidase in the rabbit eye and the drug effects were considered.


Leukocyte migration inhibitory factor in HSV infections. The cell-mediated immune (CMI) response as measured by a direct assay of leukocyte migration inhibition factor (LMIF) was determined in a population of patients with recurrent herpes simplex virus (HSV) infections in the quiescent stage as well as in healthy volunteers. The migration of leukocytes incubated in the presence of HSV antigens was compared to that without viral antigens for the calculation of the migration index (MI). Eleven of 41 control subjects (16.8%) had a MI below 0.8, indicating a positive CMI response. In contrast, all the herpes patients tested had a MI above 0.8, suggesting an impairment in the production of LMIF at this stage of their disease. This difference was statistically significant (t = 4.296; p less than 0.001) and was not dependent on the age of the population. This study indicates that individuals with recurrent HSV infections have impaired CMI response betweeen attacks which may be associated with the stage of the disease.


Meningo-encephalitis presenting as an acute paranoid psychosis. A twenty-seven year old women with meningo-encephalitis and paranoid symptoms is presented. The patient was admitted to a psychiatric unit where, two months previously, her brother had been under treatment for a paranoid schizophrenic episode. The diagnostic lumbar puncture was performed after several days of increasing periods of disorientation, stupor, incontinence, posturing, and perseveration. There was no fever, no signs of meningeal irritation, and no localizing neurologic deficit. The problem of recognizing organic brain disease presenting as an acute psychotic episode is discussed. In this case, the positive family history was given inappropriate emphasis, while the good pre-morbid adjustment of the patient and the symptoms of confusion and headache were inadequately considered. Viral meningo-encephalitis, particularly that due to herpes simplex, often presents with a picture of behavioral abnormalities and minimal physical signs, and is easily confused with a functional process.


Free radicals produced in human lenses by a biphotonic process. Cataractous and noncataractous human lenses were irradiated with ultraviolet (UV) light, and the resulting triplet and radical species were studied by electron paramagnetic resonance spectroscopy. Evidence is presented that free radicals are produced by a biphotonic process involving tryptophan as an intermediate triplet. This makes free radical production energetically feasible. A possible link between incident UV light and cataract formation is suggested.


Wescodyne: lack of activity against poliovirus in the presence of organic matter. The National Institute of Health Guidelines for Recombinant DNA Research recommends 2% aqueous Wescodyne, an iodophore that is used in many hospitals and laboratories as a disinfectant, as a decontaminant for biological safety cabinets and 5% for a spill outside a cabinet. A contact time of 10 to 15 minutes was given for the 2% solution and 20 minutes was considered adequate for the 5% concentration. The results indicate: 1. Aqueous Wescodyne (5%) is ineffective when used for 80 minutes against poliovirus in a test mixture containing 8.5% bovine serum albumin (a mixture equivalent in protein concentration to the higher range in serum). 2. Wescodyne (10%) employed under the same conditions for 40 minutes is also ineffective. 3. Wescodyne (10% v/v) in 50% ethanol (w/w) was effective and this mixture, originally recommended for hand washing, should be considered for use in biohazard situations, particularly for decontamination of work surfaces and biological safety cabinets. These results are of significance for if a virucide cannot inactivate poliovirus one would be concerned about using the virucide against hepatitis B or SV40 viruses.


An organ culture model for study of biochemical development of fetal rat lung. We have developed a short-term organ culture model for the study of the biochemical and morphological development of late gestation fetal rat lung. Explants (1 mm3) of 19-day lung were cultured in an oxygen enriched environment in the presence of synthetic serum-free medium for 3 days. Morphological maturation continued in culture. The rate of incorporation of choline into disaturated phosphatidylcholine and the content of this phospholipid in the explants increased in vitro in a pattern very similar to that which occurs in vivo. The activities of choline kinase and cholinephosphotransferase were also similar in cultured lung and in vivo. Studies of glucose oxidation to CO2 provided additional evidence that the explants remained viable in culture. The explants retained the sensitivity of fetal lung to hormonal action. This was demonstrated by the stimulation of choline incorporation into phospholipid by cyclic AMP and an increase in the glycogen content after exposure to insulin.


Rotaviral infections in human neonates.

Rotaviral infections in human neonates. Between May 1, 1976 through May 14, 1977, feces from 1,056 five-day-old babies in newborn nurseries were examined by electron microscopy, of which 343 (32.5%) contained rotavirus. Rotaviruses were prevalent throughout the study period, but infection reached its peak during the winter months of December 1976 and January 1977, at which time 46% and 49% of babies, respectively, were excreting rotaviruses. Infection occurred substantially less frequently among breast-fed babies. Further, infected breast-fed babies excreted less virus than those who were bottle fed. Viral particles in feces of breast-fed babies frequently appeared in clumps, which usually contained complete smooth viral particles. Analysis of feeding charts in one of the wards revealed that infection was usually asymptomatic because only 15 of 189 (8%) babies had enteritis. None of these babies had symptoms of sufficient severity to necessitate treatment.


Economic impact of rotavirus and other neonatal disease agents of animals. Methods for estimating the economic impact of disease agents were developed and utilized to assess the relative economic importance of rotavirus and other disease agents in calves. Based on incidence data from 2 sources, Escherichia coli was responsible for the most devastating economic losses (50.9% and 74.6%). Coronaviral (17.5% and 29.7% loss) and rotaviral (3.2% and 9.1% loss) infections ranked 2nd and 3rd, respectively. In one study, cryptosporidial infections (6.5% loss) were estimated to be similar in economic impact to rotaviral infection. Salmonellosis, mycotic gastroenteritis, infectious bovine rhinotracheitis, and bovine viral diarrhea infections accounted for minor losses. The estimated average annual loss of calves for the 7-year period, 1970 through 1976, was $95,500,000/year. Based on data from 2 studies, the estimated average annual loss from E coli was $48.6 and 71.2 million; from coronaviral infection, $16.7 and 28.4 million; from rotaviral infection, $3.1 and $8.7 million; and from cryptosporidial infection, from 1 study, $6.2 million. Estimates of economic impact of disease agents on calves, and likely in other species, indicate that rotaviral infections have a relatively minor role with respect to E coli and coronaviral infections.


Pathogenic relationships of rotavirus, Escherichia coli, and other agents in mixed infections in calves. Infection with agents interpreted as causing or contributing to diarrhea (rotavirus, coronavirus, enterotoxigenic Escherichia coli, and cryptosporidia) were demonstrated in 24 of 32 newborn calves that had naturally occurring diarrheal disease. The calves were from 12 herds in Iowa. Infections as well as enteric lesions and hypoglobulinemia occurred more frequently among diarrheal calves than among nondiarrheal calves from these same herds. In most calves, infections were mixed; ie, both viruses, one or both viruses plus cryptosporidia, or rotavirus plus enterotoxigenic E coli.


Pathophysiologic changes due to coronavirus-induced diarrhea in the calf. Extensive water, sodium, chloride, bicarbonate, and potassium losses occur in the diarrheal calf. The water loss is entirely from the extracellular space. In severe cases, hypovolemic shock occurs with the blood volume decreased by as much as one-half. Acidosis, which results from fecal bicarbonate loss, lactic acidosis, and renal dysfunction, results in tissue buffering, which in turn causes the efflux of cellular potassium ions. Although there is a total body potassium deficit, plasma potassium concentration is increased. This, in conjunction with an intracellular deficit, causes weakness, lethargy, and potassium cardiotoxicosis resulting in death. Hypoglycemia also contributes to the weakness and lethargy seem as calves become moribund. These losses from the body and shifts in fluids and electrolytes must be understood to develop the most effective rationale for supportive therapy.


Small plaque variant transmissible gastroenteritis virus. A small plaque (SP) variant transmissible gastroenteritis (TGE) virus strain that may be useful in the control of TGE in swine has been developed and tested. This strain was derived from a persistently infected swine leukocyte cell line originally infected with a virulent TGE virus. The SP viral strain was avirulent for 3-day-old susceptible pigs and pregnant gilts. The SP virus elicited protective antibody when inoculated into pregnant gilts oral/intranasally, or intramammarily, or by both of these routes. The morbidity and mortality of their passively immune suckling pigs were 62% and 14%, respectively.


Fructose-1,6-biphosphatase of the small intestine. Purification and comparison with liver and muscle fructose-1,6-bisphosphatases. The occurrence of specific fructose-1,6-bisphosphatase [D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11] (Fru-1,6-P2ase) in the small intestine was confirmed. 1. Fru-1,6-P2ase was isolated from mouse small intestine by a simple method. The isolated enzyme preparation was an electrophoretically homogeneous protein. 2. The molecular weight and subunit molecular weight were 140,000 and 38,000, respectively. 3. The intestinal enzyme was electrophoretically distinct from the liver enzyme. 4. The kinetic properties of the purified intestinal enzyme were compared with those of the mouse liver and muscle enzymes. 5. Mouse intestinal and muscle Fru-1,6-P2ases hydrolyzed ribulose-1,5-bisphosphate in addition to fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate.


Decrease of rat liver cysteine dioxygenase (cysteine oxidase) activity mediated by glucagon. The hepatic cysteine dioxygenase activity of rats was markedly decreased by the intraperitoneal administration of glucagon. The enzyme activity was also decreased by either dibutyryl cyclic AMP or theophylline. The prior administration of actinomycin D completely blocked the glucagon-mediated decrease of enzyme activity, while administrations of this inhibitor of protein synthesis after glucagon injection did not block the decrease of enzyme activity. A single administration of actinomycin D resulted in a slight increase of cysteine dioxygenase activity in the rat liver. On the other hand, the injection of cycloheximide resulted in a rapid decrease of the hepatic cysteine dioxygenase with a half-life of 2.5 h. The half-life of the enzyme in rat liver after glucagon administration was one hour. The administration of hydrocortisone or insulin had no effect on the glucagon-mediated decrease of cysteine dioxygenase of rat liver. The enzyme activity of alloxan diabetic rat liver was almost the same as that of the intact rat liver. The evidence obtained here suggests that enhancement of degradation or inactivation of cysteine dioxygenase is responsible for the glucagon-mediated decrease of the enzyme activity in rat liver.


Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase. Cholera toxin, using [32P]NAD+ as substrate, specifically radiolabels at least two proteins in plasma membranes of wild type S49 mouse lymphoma cells. The toxin-specific substrates are detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as bands corresponding to molecular weights of 45,000 and a doublet of 52,000 to 53,000. Membranes of two other cell types exhibit similar patterns of radiolabeled bands specifically produced by incubation with cholera toxin: the "uncoupled" variant S49 cell, which possesses adenylate cyclase activity unresponsive to hormones, and the HTC4 rat hepatoma cell, which lacks detectable catalytic adenylate cyclase activity but contains components of the cyclase system necessary for regulation by guanyl nucleotides and NaF. Little or no toxin-specific radiolabeling is observed in membranes of a fourth cell type, the adenylate cyclase activity-deficient S49 variant, which functionally lacks components of the cyclase system involved in cholera toxin action and regulation by guanyl nucleotides and NaF. The toxin-specific labeling pattern is not observed in membranes prepared from wild type S49 cells previously treated with cholera toxin in culture. One or both of the toxin substrates thus appears to be involved in regulation of adenylate cyclase by guanyl nucleotides and fluoride ion.


Autophosphorylation of rabbit skeletal muscle cyclic AMP-dependent protein kinase I catalytic subunit. The catalytic subunit of rabbit skeletal muscle cyclic AMP-dependent protein kinase I can catalyze self-phosphorylation. The autophosphorylation reaction uses ATP as the phosphoryl donor, requires Mg2+, and is inhibited by polyarginine. Prior treatment of the catalytic subunit with Escherichia coli alkaline phosphatase in the presence of bovine serum albumin greatly enhances the autophosphorylation of the subunit. The protein-bound phosphate is stable in acid but labile in base. Incubation of the 32P-labeled phosphoenzyme with histones led neither to the phosphorylation of histones nor to a loss of radioactivity from the phosphoenzyme. The results suggest that the phosphoenzyme does not represent an intermediate of the phosphotransferase reaction.


Characteristics of lead ion-stimulated phosphorylation of Electrophorus electricus electroplax (Na+ + K+)-adenosine triphosphatase and inhibition of ATP-ADP exchange. Pb2+-stimulated phosphorylation of Electrophorus electricus electroplax (Na+ + K+)-adenosine triphosphatase is prevented by stoichiometric quantities of 2,3-dimercaptopropanol. The chelator in the same low concentrations does not block Na+-dependent phosphorylation. Both Pb2+-and Na+-dependent phosphorylation reactions show the same dependence on MgCl2. Phosphorylation in the presence of both Na+ and Pb2+ is cumulative suggesting that Pb2+ and Na+ bind at separate, independent sites. The enthalpy change due to binding of Pb2+ is about -1.76 kcal/mol. 32P-phosphopeptides obtained from pronase or pepsin digests of Pb2+-and Na+-dependent phosphoproteins are electrophoretically identical. Pb2+ does not stimulate but does inhibit ATP-ADP exchange activity under the conditions in which this activity is stimulated by Na+. Since the phosphorylation sites are identical, it is concluded that the differences in reactivity of the Na+- and Pb2+-phosphoenzymes are due to different conformational changes produced by binding of Na+ and Pb2+. The Pb2+-sensitive conformation is critical for Na+ specificity of phosphorylation, reversibility of phosphorylation, and for phosphatase activity but not for acceptor site phosphorylation by ATP. These findings have implications for enzyme reaction models.


Stimulation and inhibition of the protein synthetic process by NAD+ in lysed rabbit reticulocytes. NAD+ at 0.16 mM stimulates the initiation step of the protein synthetic process in lysed rabbit reticulocytes. This conclusion is based on the stimulation of (i) the transfer of formylmethionine from f[35S]Met-tRNAfMet into polypeptide, (ii) the accumulation of the initial dipeptide, methionylvaline, in the presence of pactamycin, and (iii) the formation of the 40 S initiation complex. The effect of NAD+ changes from a stimulatory role on protein synthesis to an inhibitory role at concentrations greater than 0.16 mM. At 4.0 mM NAD+, protein synthesis is inhibited. This has been demonstrated experimentally by using the same three assays described above. In addition, 4.0 mM NAD+ inhibits MettRNAfMet.initiation factor.GTP ternary complex formation. The elongation and termination steps of polypeptide synthesis are not affected by 0.16 to 4.0 mM NAD+. The data presented clearly show that the stimulatory activity of 0.16 mM NAD+ and the inhibitory activity of 4.0 mM NAD+ affects the initiation step of the protein synthetic process in lysed rabbit reticulocytes.


Inactivation of phosphorylase phosphatase by a factor from rabbit liver and its chemical characterization as glutathione disulfide. A factor inactivating phosphorylase phosphatase was isolated from rabbit liver. The isolation procedure consisted of heat treatment at 85 degrees C, extraction with n-butyl alcohol, and chromatography on Dowex 1 and DEAE-cellulose columns. The purified factor was different from the known protein inhibitors and was shown to be tripeptide composed of equimolar amounts of glutamic acid, cysteine, and glycine. The NH2-terminal and COOH-terminal amino acids were determined as glutamic acid and glycine, respectively. The factor was finally identified as glutathione disulfide by high voltage paper electrophoresis, paper chromatography, and liquid column chromatography using an amino acid analyzer. Addition of the purified factor or glutathione disulfide converted phosphorylase phosphatase to a stable, less active enzyme species, the extent of conversion depending on the amount added. The inactivated phosphatase was completely reactivated by addition of both glutathione (or 2-mercaptoethanol) and Mn2+ and partially reactivated by adding glutathione alone. Injection of glutathione disulfide into the portal vein of rabbits caused a rapid increase in phosphorylase alpha activity in the liver. These results suggest that glutathione disulfide is involved in regulation of phosphorylase activity in vivo, by causing inactivation of phosphorylase phosphatase in the liver.


A novel phosphodiesterase from Aspergillus niger and its application to the study of membrane-derived oligosaccharides and other glycerol-containing biopolymers. A novel phosphodiesterase has been found in commercially available extracts of Aspergillus niger and has been partially purified by fractionation with acetone and chromatography on carboxymethylcellulose. The enzyme attacks glycerophosphodiester bonds with the liberation of free glycerol only. The synthetic substrate glucose 6-phospho-sn-1'(3')-glycerol is hydrolyzed with production of equivalent amounts of free glycerol and glucose 6-phosphate. Similarly, the enzymic hydrolysis of sn-glycero-3-phosphocholine liberates glycerol and phosphocholine. The hydrophilic head groups of membrane phospholipids of Escherichia coli are continuously transferred to a closely related family of oligosaccharides ("membrane-derived oligosaccharides") containing glucose as the sole sugar (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368--1372). Oligosaccharide A-2 contains sn-1-glycerophosphate residues (derived from phosphatidylglycerol) in phosphodiester linkage. Treatment of this oligosaccharide with the phosphodiesterase led to the liberation of nearly all of the glycerol as free glycerol. Subsequent partial acid hydrolysis of the enzyme-treated oligosaccharide led to the recovery of glucose 6-phosphate in almost quantitative yield. The sn-1-glycerophosphate residues are therefore linked to position 6 of glucose units of the oligosaccharide. The activity of the enzyme is not restricted to glycerophosphodiesterases since it will hydrolyze phosphodiesters containing other polyols such as the synthetically prepared glucose 6-phospho-DL-1'(2'-hydroxy-3'-ethoxy)propane.


Photoreduction of copper chromophores in blue oxidases. The low temperature (77 K) irradiation of oxidized ceruloplasmin and Rhus vernicifera laccase at the 330 nm absorption which arises from type 3 copper leads to the reduction of type 1 copper as demonstrated by bleaching of the 610 nm chromophore and the decrease of the EPR signal associated with this species. Type 2 copper remains unaffected. Concomitant with the type 1 copper reduction, a new EPR signal which is possibly that of a biradical appears. Upon thawing, type 1 copper is reversibly oxidized and the radical signal disappears. Irradiation of oxidized protein at the absorption band of type 1 copper produces no spectral change. An EPR study at room temperature confirms the wave-length specificity and reversibility of the photoreduction of type 1 copper and radical formation. Radical appearance and disappearance at room temperature are extremely slow (tau1/2 approximately 30 min). Optical studies at room temperature show that upon anaerobic irradiation of laccase in the 330 nm absorption band, both type 3 and type 1 chromophores are slowly reduced. Upon return to the dark and in the presence of O2, both type 3 and type 1 centers are reoxidized. Oxidizing equivalents either from O2 or K3Fe(CN)6 are required for the reoxidation reaction. These studies demonstrate that there is a direct energy transfer between type 3 and type 1 copper sites in blue copper oxidases.


The synthesis of complex-type oligosaccharides. I. Structure of the lipid-linked oligosaccharide precursor of the complex-type oligosaccharides of the vesicular stomatitis virus G protein. The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide. Following transfer the oligosaccharide is "processed" by removal of glucose and mannose residues and the sugars that constitute the outer branches of the complex-type oligosaccharide are added. The structure of the oligosaccharide moiety of the lipid-linked precursor has been elucidated in order to further define the steps involved in processing. Since it was not feasible to obtain adequate amounts of material for standard structural studies, most of the structural studies were performed on radiolabeled material, with radioactivity incorporated differentially into glucose, mannose, and N-acetylglucosamine. Based on endo-beta-N-acetylglucosaminidase CII digestion, alpha-mannosidase digestion, acetolysis, Smith periodate degradation, methylation analysis, and periodate oxidation, we propose the following structure for the oligosaccharide moiety of the lipid-linked oligosaccharide.


Inhibition of the degradation of receptor-bound human choriogonadotropin by lysosomotropic agents, protease inhibitors, and metabolic inhibitors. A previous report from this laboratory showed that binding of iodine-labeled human choriogonadotropin to Leydig tumor cells is not a reversible process (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 4892--4899). Most of the cell-bound hormone was found to be degraded to 3'-monoiodotyrosine before being released from the cells, and the degradation process could be inhibited by the lysosomotropic agents NH4Cl, chloroquine, and Triton WR-1339. It is reported herein that the degradation of receptor-bound human choriogonadotropin is an energy-dependent process, which can be inhibited by compounds that interfere with glycolysis or oxidative phosphorylation (e.g. NaF, NaN3, NaCN, and 2-deoxyglucose). Hormone degradation is also inhibited by some protease inhibitors such as the chloromethyl ketones of lysine and phenylalanine, but not by specific trypsin inhibitors (e.g. p-aminobenzamidine and p-tosyl-L-arginine methyl ester). With the exception of NH4Cl, it was found that the compounds which inhibit hormone degradation also inhibit hormone-stimulated steroidogenesis. However, the present results involving dose dependency, and those given in the following paper (Ascoli, M. (1978) J. Biol. Chem. 253, 7839--7843), indicate that these two phenomena are not related.


Demonstration of a direct effect of inhibitors of the degradation of receptor-bound human choriogonadotropin on the steroidogenic pathway. Results presented in the previous paper (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 7832--7838) show that the degradation of receptor-bound 125I-labeled human choriogonadotropin can be inhibited by chloroquine, protease inhibitors, and metabolic inhibitors. These compounds were also shown to inhibit gonadotropin-stimulated steroidogenesis. It is reported herein that these inhibitors also block the stimulation of steroidogenesis by both cholera toxin and 8-Br-adenosine 3':5'-monophosphate, thus showing that they are not specific for the hormonal stimuli. These results, taken together with previous observations that show that NH2Cl can block hormone degradation without inhibiting hormone-stimulated steroidogenesis, strongly suggest that the degradation of choriogonadotropin is not required for its stimulatory action on progesterone production.


Reconstitution of escherichia coli succinoxidase from soluble components. 1. The membrane-bound succinoxidase of Escherichia coli was fractionated with deoxycholate into three soluble components, viz. succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase complex, and a factor identified as a phospholipid-containing component. 2. The dehydrogenase and cytochrome oxidase complexes were partially purified by filtration on Amicon membranes, Sepharose 4B chromatography, and sucrose gradient centrifugation. 3. Reconstitution of membranous succinoxidase, which catalyzes the oxidation of succinate by molecular oxygen by an integrated CN(-)-sensitive pathway, was achieved by mixing the soluble succinate dehydrogenase.cytochrome b1 complex with the soluble cytochrome oxidase complex in the presence of deoxycholate and then removing the detergent by gel filtration on Sephadex G-75. The phospholipid-containing factor stimulated the formation of succinoxidase by about 100% over that observed with the two complexes. 4. Isopycnic sucrose gradient centrifugation of succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase, and the reconstituted succinoxidase gave buoyant densities (p value) as 1.167, 1.229, and 1.194, respectively. 5. Electron microscopic evidence is presented for the vesicular nature of the reconstituted succinoxidase.


Bone changes induced by diphenylhydantoin in chicks on a controlled vitamin D intake. Florid rickets developed in chicks receiving doses of diphenylhydantoin analogous to doses used in humans as anticonvulsants, vitamin D3 being given in amounts sufficient for normal bone mineralization in controls. The changes in the bones were directly related to the dose of diphenylhydantoin and inversely related to the dose of vitamin D3. Bone mineralization was assessed by roentgenography, histological examination, microradiography, and measurement of bone ash. Of these methods, roentgenography was the least sensitive. Rachitic changes were detectable by light microscopy and microradiography in chicks whose skeletons appeared normal roentgenographically. Roentgenographic evidence of rickets became detectable only when the rickets was far advanced. Rickets developed at serum levels of diphenylhydantoin similar to those found in patients taking anticonvulsant medication.


Quantitative high-performance thin-layer chromatography of lipids in plasma and liver homogenates after direct application of 0.5-microliter samples to the silica-gel layer. Lipid profiles were determined by high-performance thin-layer chromatography (HPTLC) after direct application of 0.5 microliter plasma from capillary blood to the silica-gel layer. Coefficients of variation for the fluorescence measurements were 2.1% for phosphatidylcholine. The recovery of known amounts of lipid was 96--100%. A linear relationship between peak area and amount of lipid was found in the nmole range, corresponding to the amount of lipid in 0.125--0.75 microliter Lipid-Trol, which served as the standard reference sample. The plasma lipids of healthy subjects and of patients suffering from various illnesses were analyzed using reference methods and HPTLC. Identical values were obtained for cholesterol esters, triacylglycerols and phosphatidylcholine. Free cholesterol values determined by HPTLC were slightly lower (7%). The correlation between data obtained by reference methods and HPTLC was as follows: cholesterol, r = 0.938; cholesterol esters, r = 0.964; triacylglycerols, r = 0.985; phosphatidylcholine, r = 0.938. The separaiton and quantitation of liver lipids using HPTLC after direct application of the tissue homogenate to the silical-gel layer was carried out. Comparison with reference methods revealed that HPTLC gave higher cholesterol values (24%). The triacylglycerol concentrations, however, were identical under both methods and correalted satisfactorily (r = 0.959).


Analysis of antibody assay methods and classes of viral antibodies in serodiagnosis of cytomegalovirus infection. Forty-nine serum pairs with antibody to cytomegalovirus (CMV) were evaluated for rises in antibody titer (greater than or equal to fourfold) by indirect hemagglutination (IHA) and complement fixation (CF), using a freeze-thaw antigen (FT) and a glycine extract antigen (GE). In this sample CF-FT detected more rises in antibody titer than did CF-GE. IHA detected the least number. The apparent reason for stationary antibody titers with CF-GE and IHA was the presence of high antibody titers in the first serum specimen. Separation of immunoglobulin classes of 20 serum pairs by sucrose gradient centrifugation indicated that these antibodies with IHA were of the immunoglobulin M (IgM) class and those with CF-GE were of the IgG class. By separation of immunoglobulin classes, rises in IgG CMV antibody titers were seen with IHA, rises not observed in the whole serum because of high IgM antibody titers in the first serum specimen. Absence of rises in antibody titers with CF-FT was due in part to too early sampling of the second serum specimen (less than 21 days) and in part to an apparent inability of some individuals to respond with antibody reactive with FT antigen. CF-GE and CF-FT antibodies of the IgM class were detected in some sera, usually in specimens collected more than 10 days after the onset of symptoms. Although reactive with CMV antigen, the specificity of these IgM antibodies in relation to rheumatoid factor requires clarification.


Role of rheumatoid factor in complement fixation and indirect hemagglutination tests for immunoglobulin M antibody to cytomegalovirus. Absorption of immunoglobulin M (IgM)-rheumatoid factor (RF) from serum samples by reaction with insolubilized gamma globulin reduced the complement-fixing (CF) antibody titer to cytomegalovirus (CMV) antigen to less than 1:2 in the IgM fraction of some, but not all, sera. Thus, IgM-CF activity in some sera appeared to be due to specific IgM anti-CMV antibody and in other sera to complexes of IgM-RF with antiviral IgG antibody. Prozones were present in the CF tests on IgM fractions. Increasing the concentration of antigen from 2 to 4 U reduced the prozone titer by one or two double dilutions. This observation suggested that a competition for antigen may be operating at low dilutions of IgM antibody fractions. Removal of RF had little or no effect on the reaction of the IgM fraction of sera with CMV by the indirect hemagglutination test.


Rickettsial indirect hemagglutination test: isolation of erythrocyte-sensitizing substance. The erythrocyte-sensitizing substances (ESS) of Rickettsia prowazekii and R. conorii were characterized by biological and chemical criteria. ESS could be derived from either soluble or particulate complement-fixing antigens obtained by ether extraction of rickettsiae. The soluble complement-fixing antigen exhibited two peaks of serological activity in potassium tartrate density gradients. The particulate complement-fixing antigen coincided with the more dense peak but was distinguishable by its sedimentation in rate-zonal sucrose gradients. ESS was obtained from each of the complement-fixing-reactive gradient peaks by extraction with hot alkali and was quantified by a modified indirect hemagglutination test. These ESS preparations sedimented similarly in potassium tartrate gradients and were shown to contain protein and carbohydrate, both by colorimetric tests and by incorporation of radioactive precursors. The serological activity of ESS was unaffected by trypsin, but both antigenicity and erythrocyte-binding capacity were reduced after exposure to sodium metaperiodate. Highly purified ESS was rapidly inactivated by potassium tartrate and required stabilization with bovine plasma albumin.


Conjunctivitis due to adenovirus type 19. An outbreak of unusual conjunctivitis occurred in Seattle in the summer of 1974. We found evidence of adenovirus type 19 (Ad19) infection in 28 of 42 (67%) referred cases from whom specimens for virus isolation and/or serology were obtained. Virus was isolated from conjunctiva, throats, and/or stools, often in week 2 of illness. Ad19-related cases more frequently had severe ocular pathology, transient visual impairment, and active disease for longer than a week. Secondary illnesses were more frequent in relation to Ad19 infection (26%) than to conjunctivitis of other etiology (5%, P less than 0.05). Persons with Ad19 infection commonly experienced sore throats without coughs or fevers. No common source of infection was identified in the outbreak, and illness was unrelated to occupation, residence, or family characteristics. A 3- to 8-day incubation period was suggested by two related cases.


Synergistic lysis of erythrocytes by Propionibacterium acnes. Sheep and human erythrocytes, partially processed by Staphylococcus aureus or Clostridium perfringens, were susceptible to lysis in the presence of Propionibacterium acnes. P. acnes liberated a lipase that was detected on Tween 80 agar and also on phospholipase C-precipitated egg yolk agar. Such a lipase might have contributed in the process of an intensified cellular lysis. Similar reactions were attempted with Lactobacillus acidophilus, known to possess a nondiffusible lipase, and failed to produce any such reactions. The synergistic reactions, between P. acnes and C. perfringens, were compared with The classical CAMP reaction in an attempt to find a correlation with the established membrane composition of the erythrocytes involved. Synergistic reactions observed do seem to reflect the membrane composition. Such findings, besides being contributory to an understanding of the role of these organisms in the process of pathogenesis, are of importance in the elucidation of molecular organization of biomembranes. Detailed studies, involving a large number of representative anaerobic bacteria, may also help provide an avenue in anaerobic species identification.


Comparison of an enzyme-linked immunosorbent assay for quantitation of rotavirus antibodies with complement fixation in an epidemiological survey. The development of a micro-scale enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase as the marker enzyme for the detection and measurement of human rotavirus antibodies is described. A semipurified preparation of the serologically related simian agent, SA-11 virus, was used as the antigen. Test sera were reacted with antigen-sensitized wells in disposable poly-vinyl microplates. Any attached antibody was detected by the addition of peroxidase-labeled anti-species immunoglobulin (conjugate) followed by assay of the enzyme reaction with its substrate, hydrogen peroxide plus 5-aminosalicylic acid. This micro-ELISA was compared with complement fixation in a seroepidemiological study of the age prevalence of rotavirus antibody in Aboriginal and European populations living in the same outback area in Australia. The ELISA (results read with the naked eye) proved to be approximately 16 times more sensitive than complement fixation. Of Aborigines, 71% had rotavirus complement-fixing antibody, as compared to 45% of Europeans. By ELISA 100% of both populations had rotavirus antibodies. Mean antibody titers in the different age groups were higher in Aborigines than in Europeans. Antibody levels rose steeply throughout the first 20 years of life, remained high during the next 20 years, then increased again at least up to the age of 60 years. The micro-ELISA was practical, simple to perform, and more suitable than complement fixation for large seroepidemiological rotavirus studies. It also has potential for serodiagnosis of the disease, both in the laboratory and in the field.


Solid-phase enzyme-linked immunosorbent assay for detection of hepatitis A virus. An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of hepatitis A virus in human fecal specimens. Investigations with 88 fecal specimens from 77 patients with suspected viral hepatitis and 8 of their household contacts showed that ELISA was as specific and sensitive as radioimmunoassay and almost as sensitive as immune electron microscopy. The ELISA is quick and simple to perform, does not require sophisticated technical equipment, and can be read with the naked eye, making it suitable for field work and rapid diagnosis.


Measurement of rotavirus antibody by an enzyme-linked immunosorbent assay blocking assay. A new method for the measurement of rotavirus antibody is described, utilizing the system of enzyme-linked immunosorbent assay (ELISA). In this method, serum is incubated with a fixed amount of rotavirus antigen, and the amount of antibody is determined by measuring the amount of unneutralized antigen. Such an assay system proved to be as efficient as the other available rotaviral antibody systems. The ELISA blocking assay also has the advantages of not requiring purified or gnotobiotic antigen and of being able to measure rotaviral antibody in all animal species.


Neutralization of human serum lysozyme by sodium polyanethol sulfonate but not by sodium amylosulfate. Sodium polyanethol sulfonate (SPS) at 500 microgram/ml, but not sodium amylosulfate (SAS) at 500 microgram/ml, precipitated egg white lysozyme (1 mg and 50 microgram of lysozyme per ml) as determined with the assay strain Micrococcus lysodeikticus ATCC 4698. Fresh and heat-inactivated (56 degrees C, 30 min) human serum (80%, vol/vol) killed M. lysodeikticus (10(4) bacteria per ml at zero time) within 1 to 2 h after exposure. Addition of 250 to 500 microgram of SPS per ml to fresh human serum protected M. lysodeikticus for 22 h as effectively as absorption of either fresh or heat-inactivated human serum with bentonite (10 mg/ml of serum, 10 min, 37 degrees C); the latter procedure is known to remove serum lysozyme. In contrast, SAS at 250 and 500 microgram/ml of serum retarded killing of the assay bacteria for periods of 4 h; after overnight (22 h) incubation, however, the number of M. lysodeikticus survivors had decreased significantly. The finding that SPS, but not SAS, at 250 to 500 microgram/ml effectively neutralized serum lysozyme-mediated killing of a lysozyme-sensitive assay strain may be of relevance with respect to laboratory processing of human blood culture specimens.


GABA-ergic pathways in the goldfish retina. A high-affinity uptake mechanism for [3H]-gamma-aminobutyric acid (GABA) has been localized to type H1 cone horizontal cells and type Ab pyriform amacrine cells in the retina of the goldfish by light and electron microscopy autoradiography. By stimulating isolated retinas with colored lights during incubation we have been able to use [3H]-GABA uptake as a probe of light-evoked changes in membrane potential. All colors of lights increase and darkness decreases [3H]-GABA uptake by H1 cone horizontal cells. Our model of voltage dependence of GABA uptake predicts that all colors of light should hyperpolarize H1 cone horizontal cells and other investigators have shown by intracellular recording and dye-marking that type H1 cone horizontal cells hyperpolarize to all wavelengths of light. We have also obtained evidence that dark-induced depolarization of cone horizontal cells leads to release of GABA. Type Ab pyriform amacrine cells show maximal [3H]-GABA uptake in darkness and when exposed to green or blue lights, but red lights dramatically suppress uptake. We predict these neurons to be red-depolarizing, and recent intracellular recordings and dye-marking by Famiglietti et al. ('77) support our conclusions. Synaptic relations of apparently GABA-ergic neurons were investigated in the electron microscope. We propose type H1 cone horizontal cells to be both pre- and post-synaptic to red-sensitive cones and type Ab pyriform amacrine cells to be both pre- and post-synaptic to red-sensitive center-depolarizing bipolar cells.


Electron microscopy of neoplasms of pancreatic islet cells. Neoplasms of pancreatic islet cells that produce widely divergent clinical syndromes cannot be distinguished from one another by conventional microscopy. Differentiation among these tumors by electron microscopy is possible in some cases only. Coordinated clinical evaluation, histochemical and ultrastructural studies, and assay of serum and neoplastic tissue for various polypeptide hormones afford the best characterization of these neoplasms.


Ulcers associated with the swollen leg.

Ulcers associated with the swollen leg. A number of disparate conditions may produce ulcers in swollen legs. The most frequent causative condition is venous stasis; other causal disorders are lymphatic obstruction, mixed vascular anomalies, benign and malignant neoplasms and a number of miscellaneous conditions. Careful attention to cutaneous signs of these conditions may be helpful in arriving at an accurate diagnosis and appropriate therapy.


Phospholipids of a bone matrix calcification nucleator. Phospholipids of a bone matrix calcification nucleator are identified as mono and diphosphoinositides and phosphatidyl serine. The nucleator, a protein-phospholipid complex, was dissociated by acidified-solvent porous-glass column chromatography. Analysis was by gas-liquid chromatography.


Significance of high-density lipoprotein cholesterol. The central importance of elevated blood cholesterol levels in the process of atherosclerotic heart disease (ASHD) is firmly established. Cholesterol is transported in the blood in conjunction with protein as a lipoprotein complex. The low-density lipoproteins (LDL) carry the bulk of the blood cholesterol. The high-density lipoproteins (HDL) occur in much smaller amounts. Recently it has been demonstrated that not only is the cholesterol carried in the HDL fraction not harmful, but appears to be protective against the development of ASHD. Data from Framingham and elsewhere seem consistent with this observation. The HDS apparently represent the fraction concerned with the efflux of cholesterol from the tissues, so that higher levels may represent a heightened cholesterol-riddance mechanism. Thus far, two circumstances seem to be associated with elevated levels of HDL--physical exercise and ethanol ingestion. The importance of these observations in the clinical management of the patient at risk is discussed.


Differential inhibitory effects of 5-bromodeoxyuridine on vaccinia and monkeypox viruses. Replication of vaccinia and monkeypox viruses was impeded in the presence of 5-bromodeoxyuridine (BUdR) in RL-33 cells derived from rabbit lung tissue. The different degree of inhibition was found among strains of those viruses, in which it was evident that five strains (CV-1, Lister, IHD, Dairen-I and Ikeda) of vaccinia viruses resulted in more reduced yields of infectious virus than four strains (Sen-19, Orang Utan, Copenhagen and Sierra Leone) of monkeypox viruses. There was also strain variation in BUdR sensitivity within vaccinia viruses but not in the case of monkeypox viruses.


Possible windborne spread of bluetongue to Portugal, June-July 1956. The possible sources for the epidemic of bluetongue in Portugal at the beginning of July 1956 were examined. Introduction through authorized importation of domestic or wild ruminants was not feasible, since no cattle, sheep or goats were imported and the wild ruminants were confined to Lisbon Zoo, which was too far from the initial outbreaks. Weather maps were examined to see if the wind could have carried infected Culicoides midges from North Africa. On 21 June 1956 infected midges in Morocco could have been taken offshore by southeast winds and then carried by south winds unusual at that time of year to the south coast of Portugal. The 200-300 km sea crossing would have taken some 10 h and been by day when air temperatures near the sea surface were about 18-20 C. Bluetongue had not been reported at that time in Moroccco, and the possibility of the presence of the virus in moroccan animals without clinical signs is discussed.


The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice. The pathogenicity for mice of two mutants of herpes simplex virus (type 1 and type 2), which fail to induce thymidine kinase, were compared with their respective parent strains. The mutants were much less virulent than the parents following either intracerebral or peripheral inoculation. The replication of the virus at the site of inoculation and its progression into the nervous system were studied. Following a very large inoculum in the ear, the type 1 mutant was found to establish a latent infection in the cervical dorsal root ganglia. Mice inoculated intracerebrally with small doses of the mutant viruses were solidly immune to challenge with lethal doses of the parent strain.


A search for faecal viruses in new-born and other infants. Faecal specimens were collected at weekly intervals over the winter months from 141 new-born infants without diarrhoea. Contrary to the findings in other studies, no viruses were detected by electron micriscopy or culture in any of these specimens. Over the same period faecal specimens were collected from 84 infants up to four years of age admitted to hospital. Rotaviruses or adenoviruses were found in 48% of infants with gastroenteritis. Enteroviruses and other small round virus-like particles were found in infants both with and without gastroenteritis. No viruses or pathogenic bacteria could be found in 34% of specimens from infants with gastroenteritis.


Stool viruses in babies in Glasgow 2. Investigation of normal newborns in hospital. The stools of 37 newborn babies born in hospital were examined for the presence of virus. An extract of every stool passed by each baby was examined in the electron microscopy and inoculated into cell cultures. The babies were delivered in four separate maternity units (A-D). All the babies from unit A and C(9 babies) were found to be excreting rotavirus though none showed any evidence of diarrhoea. Two of the babies also excreted astrovirus. Subsequently unit A was closed for cleaning and, on reopening with more restrictions on visitors, a further 19 babies were examined. No virus was found in any of their stools, nor was virus observed in the stools of babies from units B and D, where visiting was also more restricted. Visiting restrictions in these units excluded older siblings of the babies. No virus was cultured from any stools in this study.


Studies of louping-ill virus (Flavivirus group) in wild red grouse (Lagopus lagopus scoticus). Studies were made to find evidence of louping-ill virus infection in free-living red grouse and relate this to their breeding success. In areas where ticks were abundant 61 (84%) adult grouse had antibody to the virus compared with 1 (10%) in areas where ticks were relatively scarce. Of 162 chicks tested 25 were shown to be viraemic. Infected chicks were of significantly less weight than comparably aged uninfected birds and the probability that they died was much greater than that of uninfected birds. It is concluded that the relatively poor breeding success in areas of high tick numbers was principally due to infection with louping-ill virus. The susceptibility of the red grouse to infection is discussed.


Immunofluorescent analysis of murine leukemia virus-infected cells by flow microfluorometry. Flow microfluorometric assay with a Cytofluorograf model 4801 in combination with immunofluorescence was applied to the quantitative assay of cells exogenously infected with mouse leukemia viruses and to the chemical induction of virus in AKR cells. The Cytofluorograf provides sensitivity equal to the visual method and is capable of assaying up to 5000 cells/sec with specificity equivalent to that of the direct visual immunofluorescence assay, thereby providing a large, statistically valid sampling in a fraction of the time required by visual counting. Flow microfluorometry also provides a method of quantitatively resolving fluorescent cell populations on the basis of relative size and degree of fluorescence.


Sensitive new in vitro bioassay for melanocyte-stimulating activity using the skin of Anolis carolinensis. An accurate, highly reproducible and sensitive bioassay for melanocyte-stimulating hormone (MSH) using the skin of Anolis carolinensis in vitro is described. The time taken for green Anolis skin fragments to change to a specific, visually assessed, green-brown color is dose-related, and this forms the basis of the new assay. The method is simple to perform, and 1 person may assay 20 samples in a day using the dorsal skin from a single adult lizard. The mean dose-response ranges between 48 X 10(-12) and 375 X 12(-12) M (38 to 625 pg/ml). Using the assay, alpha-MSH, beta-MSH, ACTH (4-10), and ACTH (1-10) were equipotent on a molar basis. For repeated bioassay of rat pituitary extracts, the dose-response curves were highly significant, and only 1 of the 9 pituitary dose-response curves deviated significantly from the slope of the standard alpha-MSH curve. The index of precision, lambda, for the 9 pituitary bioassays ranged between 0.037 and 0.081, while the mean 95% fiducial limits were -6.6 and 7.1% on either side of the estimated potency. The new rate method is compared with an earlier quantal method which also uses the isolated skin of Anolis carolinensis. The quantal method does not have dose-response characteristics and is therefore less accurate and reproducible than the new method; the coefficient of variation for repeated bioassay of the same pituitary extracts ranged from between 12 to 20% for the quantal method and between 2.9 to 5.7% for the new rate method.


Theophylline incorporation into the nucleic acids of theophylline-stimulated melanoma cells. Theophyllin, an inhibitor of cAMP-degrading phosphodiesterase, stimulates melanin biosynthesis in cultures of RPMI 3460 hamster melanoma cells. Although theophylline does produce an initial transient elevation of intracellular cAMP levels, long-term treatment with theophylline produces a significant decrease in cAMP content. There is an inhibition of the theophylline stimulation by dibutyryl-cAMP; this is apparently caused by interference of dibutyryl-cAMP with the uptake and incorporation of theophylline, as shown by experiments with 3H-theophylline. An alternative theory is that theophylline, being a methylxanthine compound, is metabolized by the cell and possibly causes melanotic stimulation by becoming incorporated into cellular nucleic acids or by altering the normal nucleic acid metabolism. The following observations are consistent with this theory: (u) 3H-theophylline was incorporated into both trichloroacetic acid (TCA)-soluble and TCA-insoluble cell fractions; most of the insoluble label became soluble after digestion with ribonuclease and deoxyribonuclease. (2) These nuclease digests of the 3H-theophylline-labeled TCA-insoluble cell fractions contained 3H-labeled material that chromatographed differently from normal nucleotides on ion exchange thin layer sheets. (3) The acid-soluble pool of 3H label disappeared rapidly while both the insoluble label and the induction of melanogenesis remained stable for 50 hr after the removal of exogenous 3H-theophylline.


Viral immune complexes in systemic lupus erythematosus: C-type viral complex deposition in skin. Punch biopsies were examined by indirect immunofluorescence for immune complex deposits containing C-type viral antigen. Antisera specific for immunoglobulins and HEL-12 virus mediated fluorescence at the dermal-epidermal junction and in vessel walls of 16 of 16 biopsies involved skin from patients with systemic lupus erythematosus (SLE). Preimmune sera did not mediate fluorescence and gradient purified HEL-12 virus, simian sarcoma virus and baboon endogenous virus but not Rous sarcoma virus blocked the reaction of anti-HEL-12 virus serum with SLE tissue. Ten biopsies from uninvolved skin of the patients with SLE did not react with the antiviral serum, nor did tissue from 9 patients with discoid lupus erythematosus, psoriasis, bullous pemphigoid or normal skin. These data support the hypothesis that C-type viral immune complexes participate in the pathogenesis of SLE.


Localization of hepatitis A antigen in marmoset organs during acute infection with hepatitis A virus. Twelve marmosets (Saguinus mystax) were inoculated intravenously (iv) with hepatitis A virus (HAV). One died early (day 12); seven were sacrificed at the time of elevation in level of alanine aminotransferase (serum glutamic-pyruvic transaminase), and four without elevation were not sacrificed but seroconverted. In the seven marmosets sacrificed during the acute stage of illness, hepatitis A antigen (HA Ag) was detected in the liver by immunofluorescence in all cases, by immune electron microscopy in four, and by enzyme-linked immunosorbent assay (ELISA) in three. The HA Ag appeared by immunofluorescence as very fine granules in the cytoplasm of hepatocytes and Kupffer cells. The HA Ag could not be detected by immunofluorescence in biopsy specimens taken from the duodenum, jejunum, ileum, or transverse colon in any of eight marmosets in which necropsy was performed during the acute or preacute stage of illness. These findings suggest that the gut is not involved during the acute phase of HAV infection following iv inoculation into marmosets. The ELISA results showed that only three of 12 marmoset livers obtained during the acute phase of HAV infection could be used as an antigen source in serologic testing for antibody to HA Ag. Thus, marmoset livers were no better as a source of HA Ag than acute-phase stools from patients with type A hepatitis.


The adrenal response to exogenous adrenocorticotrophin in patients with infections due to Neisseria meningitidis. The adrenal response to a soluble form of beta1-24-corticotropin (tetracosactrin [ACTH]: 250 microgram administered intramuscularly) was studied in 28 patients with meningitis due to Neisseria meningitidis (21 with petechiae and seven without) and in six patients with Salmonella typhi bacteremia. Six normal subjects also were tested for adrenal responsiveness at four different times of the day (8 A.M., 12 noon, 4 P.M., and 10 P.M.) and served as controls. The results showed that, whatever the time of testing, patients with meningococcal infections and typhoid fever had unstimulated (basal) levels of plasma cortisol above the 99% confidence limits for the mean unstimulated cortisol levels for the normal subjects. Furthermore, although patients with meningitis without petechiae and subjects with S. typhi bacteremia responded to ACTH stimulation in a manner similar to that of the normal subjects, most subjects with meningitis with petechiae did not have increased levels of plasma cortisol after treatment with ACTH. This lack of response could not be ascribed entirely to the higher basal levels of plasma cortisol in these patients. Patients with meningitis associated with petechiae may have a relatively decreased adrenal response to stimulation with exogenous ACTH.


Peroxidase-labeled antibody technique for rapid detection of mouse hepatitis virus in cases of natural outbreaks. A recent outbreak of hepatitis in mice at the Laboratory Animals Centre (Surrey, England) is described. The only strains of mice that showed clinical symptoms and typical liver lesions were the nude (nu/nu) and obese mice. Although other inbred strains showed no clinical symptoms, all strains seroconverted. Neutralization typing of the mouse hepatitis virus showed it to be type 1, a hepatotropic strain of low virulence, a finding that was consistent with the pathology of the disease. Because of recurring outbreaks of disease caused by this strain and other strains of MHV in the United Kingdom, a rapid method for diagnosis in tissue culture was devised, employing the peroxidase enzyme-labeled antibody technique for detection of mouse hepatitis virus in L-929 cells.


Depressant action of lithium at the crayfish neuromuscular junction: pre- and postsynaptic effects. 1. The effects of replacement of external sodium ions with lithium have been studied at the excitatory neuromuscular junction of the crayfish. 2. Intracellularly recorded excitatory junctional potentials fall 45% in amplitude in the first 10 min after lithium substitution, and fail irreversibly in 48--120 min. 3. The quantal content of extracellularly recorded excitatory junctional potentials declines 25--70% within the first 10 min of Li+ exposure. During the next 40--120 min the nerve terminal potentials and quantal release at individual synapses fail simultaneously and irreversibly. 4. The mean amplitude of the spontaneous miniature excitatory junctional potentials (m.e.j.p.s) is reduced 13% by Li+ substitution, but recovers upon restoration of sodium. The mean frequency of m.e.j.p.s rises steadily during Li+ exposure, and continues to increase after reintroduction of Na+. 5. The postsynaptic response to iontophoretically applied L-glutamate falls 35--40% in 10 min, but never falls below 45% of the control level. The effect on the glutamate response is completely reversible with sodium restoration. 6. The effective resistance of the postsynpatic cells is unaffected or only increased slightly by lithium substitution. 7. Thus, the primary mechanism of transmission block by lithium appears to be decreased transmitter release and inexcitability of presynaptic terminals, probably as a result of intracellular accumulation of lithium.


Assignment of the receptor for ecotropic murine leukemia virus to mouse chromosome 5. The gene for the receptor for ecotropic murine leukemia virus (Rev) has been assigned to mouse chromosome 5. This determination was made possible by an analysis of somatic cell hybrids between mouse and Chinese hamster cells. The parents of these hybrids were A/HeJ or Mus poschiavinus peritoneal exudate cells or BALB/c primary embryo fibroblasts and E36, a Chinese hamster lung fibroblast deficient in hypoxanthine guanine phosphoribosyltransferase. Segregation of mouse chromosomes in these hybrids was analyzed by chromosome banding and isozyme expression. Cells were tested for their ability to absorb and replicate vesicular stomatitis virus (murine leukemia virus [MuLV]) pseudotype particles and ecotropic MuLV as measured by the XC test. The presence of chromosome 5 was essential for receptor expression as determined by three statistical procedures. Segregation of the receptor for ecotropic murine leukemia virus was also followed in two series of subclones. In both, receptor expression was syntenic with phosphoglucomutase-1, an isozyme which has been mapped to mouse chromosome 5.


Ethylene formation by polymorphonuclear leukocytes. Role of myeloperoxidase. Ethylene formation from the thioethers, beta-methylthiopropionaldehyde (methional) and 2-keto-4-thiomethylbutyric acid by phagocytosing polymorphonuclear leukocytes (PMNs) was found to be largely dependent on myeloperoxidase (MPO). Conversion was less than 10% of normal when MPO-deficient PMNs were employed; formation by normal PMNs was inhibited by the peroxidase inhibitors, azide, and cyanide, and a model system consisting of MPO, H2O2, chloride (or bromide) and EDTA was found which shared many of the properties of the predominant PMN system. MPO-independent mechanisms of ethylene formation were also identified. Ethylene formation from methional by phagocytosing eosinophils and by H2O2 in the presence or absence of catalase was stimulated by azide. The presence of MPO-independent, azide-stimulable systems in the PMN preparations was suggested by the azide stimulation of ethylene formation from methional when MPO-deficient leukocytes were employed. Ethylene formation by dye-sensitized photooxidation was also demonstrated and evidence obtained for the involvement of singlet oxygen (1O2). These findings are discussed in relation to the participation of H2O2, hydroxyl radicals, the superoxide anion and 1O2 in the formation of ethylene by PMNs and by the MPO model system.


Ir-genes in H-2 regulate generation of anti-viral cytotoxic T cells. Mapping to K or D and dominance of unresponsiveness. H-2 dependent and virus-specific Ir genes regulate the generation of primary virus-specific K or D restricted cytotoxic T-cell responses in vivo. The following examples have been analyzed in some detail: first, Dk restricted responses to vaccinia in Sendai viruses are at least 30 times lower than the corresponding K-restricted responses irrespective of the H-2 haplotypes (k, b, d, dxs, dxq) of K and I regions; in contrast, LCMV infection generates high responses to Dk. These findings are consistent with but do not prove that this Ir gene maps to D. Second, Db restricted responses to vaccinia and Sendai viruses are high in strains possessing the Kq or KbIb, KbaIb haplotype, are very low in strains with Kk, and relatively low in mouse strains of the KdI-Ad haplotype; LCMV generates high Db restricted response in the presence of Kk. This Ir gene for the response to vaccinia and Sendai viruses maps to K since B10.BYR (KqIkdDb) is a responder and B10.A (2R) is a nonresponder (KkIkdDb). Third, virus and K or D allele specific nonresponsiveness is dominant with variable penetrance; in heterozygous mice the nonresponder Kk allele over-rides responsiveness normally found in KbDb or KqDb combinations. Fourth, when (responder X nonresponder)F1 lymphocytes are stimulated in an environment expressing vaccinia virus plus only a high responder Kb or Kq allelle and Db, response to vaccinia Db is high; in contrast when the same F1 cells are stimulated in an environment expressing the low responder allele Kk, response to vaccinia Db is low. Thus absence of Kk during immunization allows generation of high responsive Db restricted vaccinia specific cytotoxic T cells. The Dk dependent low response to vaccinia Dk can be explained by a preclusion rule or by failure of vaccinia to complex with Db; however the analysis of Kk dependent low response to vaccinia Db does not support these explanations or that self-tolerance is responsible for this Ir effect but is compatible with the interpretation that Kk vaccinia is immunodominant over Db vaccinia. These results are discussed with respect to (a) possible mechanisms of regulation by Ir genes and (b) H-2 polymorphism and HLA-disease association.


Helper-independent mink cell focus-inducing strains of Friend murine type-C virus: potential relationship to the origin of replication-defective spleen focus-forming virus. Recent studies have indicated that both the replication-defective spleen focus-forming virus (SFFV) in the Friend virus complex and the helper-independent mink cell focus-inducing (MCF) viruses derived from AKR-murine leukemia virus (MuLV) are env gene recombinants between ecotropic virus and xenotropic virus. In an attempt to isolate additional env gene recombinants between Friend murine leukemia virus (F-MuLV) and xenotropic virus, we have inoculated cloned ecotropic F-MuLV into newborn NIH Swiss mice and analyzed MuLV released from preleukemic and leukemic spleens of infected mice. Two helper-independent MCF strains of F-MuLV have been isolated. Like the previously described AKR-MCF viruses, the Friend MCF viruses are env gene recombinants between an ecotropic virus (F-MuLV) and a mouse xenotropic virus, as shown by host range, interference pattern, and tryptic peptide analysis of the gp70s of these MuLV. Furthermore, RNA from the Friend MCF viruses hybridizes completely to cDNAsffv, a nucleic acid probe which detects that portion of SFFV which was not derived from P-MuLV. The ability to isolate replicating MCF viruses derived from F-MuLV FURTHER strengthens the parallels between the Friend erythroleukemia system and the AKR thymic leukemia system. Finally, the potential relationship of helper-independent env gene recombinants between F-MuLV and xenotropic virus to be highly leukemogenic SFFV is discussed.


Source and hormone-dependence of Gix-gp70 in mouse serum. The gp70 family of glycoproteins is distinguished by the role of these molecules as constituents of C-type viral envelopes and also as Mendelian cellular constituents expressed independently of virus production. The source of G(IX)-gp70 in the serum of 129 strain mice, which are not overt producers of virus, could not be traced to any organ or tissue that is known to be G(IX)-positive by serological tests. Hematopoietic tissues were excluded as source of serum G(IX)-gp70 by tests with reciprocal radiation chimeras made from 129 and 129-G(IX)(-) donors and recipients. Thymus and spleen were excluded because excision of these organs did not affect levels of G(IX)-gp70 in the serum. The serum of young adult 129 males contains roughly four times as much G(IX)-gp70 as adult 129 females and the levels rise in both sexes with increasing age. Castration of 129 males reduced the level of serum G(IX)-gp70 to that of females, and the level was fully restored by testosterone. Thus the epididymis and seminal fluid, though rich in G(IX)-gp70, do not contribute significant amounts of G(IX)-gp70 to the serum. The level of G(IX)-gp70 in the serum of testosterone-treated females, though more than double that of untreated females, did not reach the level of normal males, under the conditions tested. This may signify that G(IX)-gp70 production by males is subject to imprinting by testosterone in early life. Evidently the main source of serum G(IX)-gp70 is a tissue or organ that is common to males and females, is directly or indirectly responsive to testosterone, and has not so far been identified serologically as G(IX)- positive.


Virus-induced diabetes mellitus. XV. Beta cell damage and insulin-dependent hyperglycemia in mice infected with coxsackie virus B4. Coxsackie virus B4 that had been passaged in cultures enriched for pancreatic beta cells produced a diabetes-like syndrome when inoculated into SJL/J mice. The infection resulted in insulitis and destruction of beta cells. Viral antigens were found in beta cells by staining with fluorescein-labeled antibody to Coxsackie virus B4. The destruction of beta cells led to a decrease in the immunoreactive insulin content of the pancreas and hypoinsulinemia. The reduction in immunoreactive insulin correlated inversely with the elevation of glucose in the blood and over 80% of the animals were found to be hyperglycemic within 14 days after infection. The percentage of animals with hyperglycemia decreased with time and at the end of 60 days, less than 5% of the animals were still hyperglycemic. However, many of the normoglycemic mice were found to be metabolically abnormal when evaluated by glucose tolerance tests. Studies on the susceptibility of the host showed that only certain inbred strains of mice became diabetic when infected with Coxsackie virus B4. It is concluded that both the passage history of the virus and the strain of the host influence the development of diabetes.


Virus-replicating T cells in the immune response of mice. II. Characterization of T cells capable of replicating vesicular stomatitis virus. Immunocytological properties of the splenic T cell (Tv) which develop into virus plaque-forming cells in response to the antigenic challenge in vitro were investigated in relation to the properties of helper T cells and suppressor T cells in antibody response. Tv was observed in spleen around 1 wk after the intravenous injection of mice with 10(7) sheep erythrocytes. This contrasted with the finding that both helper T cells and suppressor T cells developed as early as 3 days after the immunization. Tv was proliferative in response to the antigenic stimulation, whereas helper T-cell activity could be expressed without cell division. Development of Tv to virus plaque-forming cells was much more dependent on macrophages than the generation of helper activity. Tv was found in nylon wool adherent fraction, whereas helper T cell was found in both nylon adherent and nonadherent fractions. Tv belongs to the short-lived and nonrecirculating T-cell population (T1), whereas the major part of helper T cells belongs to the long-lived and recirculating T-cell population (T2). These results strongly suggest that vesicular stomatitis virus infect and replicate in the different subset(s) of T cell(s) to which the major part of helper T cells belong.


Virus-replicating T cells in the immune response of mice. III. Role of vesicular stomatitis virus-replicating T cells in the antibody response. The functional role of the T cell (Tv) which can replicate vesicular stomatitis virus (VSV) on activation by the antigen was investigated in antibody response in vitro. By the inoculation of VSV into the culture, marked augmentation of antibody response to sheep erythrocytes (SRBC) was observed in the culture of spleen cells taken more than 3 days after the immunization with SRBC, suggesting that the VSV-susceptible suppressor cells were included in these spleen cells and the activity was eliminated by the effect of VSV. Development of two distinct types of suppressor T cells was revealed in the spleen of mice after the priming with SRBC. First, nylon wool nonadherent (NAd) suppressor T cells found in the spleen cells taken 3 days after immunization, and second, nylon wool adherent (Ad) suppressor T cells found in the spleen cells taken approximately 1 wk after immunization. The activity of nylon Ad suppressor T cells was completely abolished by VSV-preinfection, whereas that of nylon NAd suppressor T cells was unaffected. It was also shown that the helper T-cell activity was not influenced by VSV-preinfection. These results provided direct evidence that nylon Ad suppressor T cell but not nylon NAd suppressor T cell nor helper T cell can actually replicate VSV after antigenic stimulation. Thus it was strongly suggested that Tv represents the nylon Ad suppressor T cells.


Chronic pain: a review for the family physician. The challenge of effective management of chronic pain frequently confronts the family physician. Successful management relies on the physician's skill in integrating fundamental concepts in the pathophysiology, psychodynamics, and diagnostic and therapeutic modalities associated with chronic pain syndromes. The use of time-contingent rather than pain-contingent therapy in the prevention of the chronic pain state is advocated.


Suppression of interferon production in mouse spleen cells by cytochalasin D. Cytochalasin D is thought to impair microfilament function. The present study has investigated its effects on four different systems in which interferon is formed, namely (1) mouse fibroblasts induced with virus (2) mouse spleen cells induced with virus, or (3) with endotoxin or (4) by allogeneic stimulation. Cytochalasin D did not suppress formation of interferon by fibroblasts (L cells) or spleen cells stimulated with either HVJ or NDV. However it did suppress production of interferon by spleen cells in response to endotoxin or an allogeneic stimulation; here its action was apparently not on the secretion of interferon, but on some earlier event. It also suppressed the production of interferon by mouse spleen cells induced with HVJ if this had been u.v. irradiated for more than 15 min: this suggests that cytochalasin D sensitive structures do play some role in interferon production by mouse spleen cells when stimulated with HVJ, as well as when they are stimulated with endotoxin or an allogeneic stimulus.


The role of macrophages in mice infected with murine cytomegalovirus. Quantitative studies were made of the infection of mouse peritoneal macrophages in vitro by cytomegalovirus, using virus assays and immunofluorescence. The efficiency of infection was low. Broth-induced peritoneal macrophages were about four times more resistant to infection than unstimulated macrophages and it was even more difficult to infect activated macrophages taken from mice 6 days after intravenous infection. Peritoneal macrophages (unstimulated) were infected at least 15 times more readily by tissue culture-passed (attenuated) virus than by salivary gland (virulent) virus, but macrophages prevented the spread of tissue culture virus to underlying susceptible mouse embryo fibroblasts, whereas they did so much less effectively with virulent salivary gland virus. The pathogenesis of infection was studied in intact mice by immunofluorescence, and the observations paralleled the in vitro findings. When large doses of salivary gland virus were injected intravenously, infected Kupffer cells (liver macrophages) were occasionally seen and the inoculated virus directly infected large numbers of hepatic cells. In similar experiments with tissue culture-passed virus, there was initial infection of occasional Kupffer cells, which only rarely gave rise to infected hepatic cells. Differences in the extend of Kupffer cell infection by the two strains of virus were not detected in these experiments. Salivary gland virus also usually failed to infect splenic or lymph node macrophages. Occasional infected mononuclear cells were seen in the blood, lung and bone marrow, but were not identified. Infected cells were very rarely seen in the thymus, even in suckling mice.


Comparison of interferon action in interferon resistant and sensitive L1210 cells. Translation inhibition, leu-tRNA aminoacylation and double-stranded RNA and ATP dependent phosphorylation were examined in interferon-treated and control cell-free lysates of leukaemic mouse L 1210 R and L 1210 S cells. No differences were observed between the respective interferon-treated and control cell-free extracts, except for the presence of an enhanced 67K dalton phosphoprotein fraction in interferon-treated L 1210 S cell-free extracts. In non-responding cell-free lysates, the lack of stimulation of a 67K dalton phosphoprotein fraction cannot be explained by the presence of an increased level of some inhibitory activity, such as a phosphatase.


Optical diffraction analysis of electron microscope images of tubular structures found in cells infected with herpes simplex virus type 2. Optical diffraction analysis was done on the electron micrographs of tubular structures found in cells infected with herpes simplex virus type 2. By this method, the helical arrangement of subunits forming a tubular structure was demonstrated.


Tumour specific transplantation antigen in hamster tumour cells induced with BK virus. Immunization of hamsters with purified BK virus (BKV) followed by transplantation of BKV-induced hamster tumour cells revealed a tumour specific transplantation antigen (TSTA) in these cells. The antigen did not cross-react with the TSTA of SV40 since immunization with BKV did not protect against challenge of SV40 tumour cells.


Mink lung cells: a non-primate cell line highly susceptible for varicella-zoster virus. Mink lung cells (MvILu) are highly susceptible to varicella-zoster virus (VZV). The titres of cell-free VZV suspensions reached 1.0 x 10(7) p.f.u./ml at 3 days post-infection, with subsequent cell degeneration, if MvILu cells were infected with a multiplicity of infectious virus of 0.01 p.f.u./cell. In contrast, during the same period and under the same conditions the titres of cell-free VZV were 10(2) to 10(3) times lower when grown on human foreskin fibroblasts. A fast and reliable plaque assay and a neutralization test for VZV on MvILU cells, were developed.


Structural polypeptides of Machupo virus. The structural proteins of an arenavirus pathogen, Machupo virus, were compared to the structural proteins of two previously characterized non-pathogenic arenaviruses, Pichinde and Tacaribe, in SDS-polyacrylamide gels. Similarities in mol. wt. of the major structural proteins from both pathogenic and non-pathogenic viruses were apparent; however, some differences in the number of glycosylation properties of minor proteins were observed. Machupo virions contain two major protein species. The most prominent is a non-glycosylated protein with a mol. wt. of 68000, while the other was glycosylated protein with a mol. wt. of 41000. Minor amounts of other proteins (mol. wt. 84000, 74000, 50000 and 15000) and a glycolipid were also observed.


Induction of infectious virus DNA and virus particles by mitomycin C in SV40-transformed mouse cells. A line of SV40-transformed mouse cells (SV/3T3-4E) was isolated and four clones were derived from this line. Spontaneous production of small amounts of infectious SV40 DNA was detected in the parental line and in three out of the four clones tested, although no synthesis of virions could be demonstrated. The yields of SV40 DNA were significantly enhanced following treatment of these cells with mitomycin C and infectious virus particles became detectable occasionally.


Suppression of the synthesis of cellular macromolecules by herpes simplex virus. Synthesis of cellular protein was substantially inhibited within 1 h of infection with herpes simplex virus, type 2, strain G (HSV-2). The inhibition also occurred, although no virus-specific protein synthesis was detected, after infection with u.v. irradiated virus and in cytoplasts that had been enucleated before infection. The inhibitory activity could not be distinguished from infectivity by dilution, sedimentation or reaction with gamma-globulin. HSV-2 also suppresssed the synthesis of Sendai virus proteins, but not those specified by HSV-1. Host protein synthesis was no more sensitive than virus protein synthesis to an increased concentration of NaCl in the medium, nor could the suppression of host synthesis be prevented by adding excess MgCl2 to the medium or by omitting CaCl2 or NaCl. It was accompanied by the breakdown of polyribosomes, which also occurred in the presence of cycloheximide but not at 4 degrees C. The breakdown yielded ribosomes that were sensitive to a high salt concentration, unlike those produced by treatment of polyribosomes with RNase. The synthesis of cellular DNA and RNA was also inhibited following infection with u.v.-inactivated virus. It is concluded that the suppression of host protein synthesis (and probably also of host DNA and RNA synthesis) is caused by a constituent of the infecting virus particles. The mechanism is obscure but probably does not depend on the leakage out of the cell of Mg2+ or into the cell or Ca2+ or Na+ ions, nor on the specific inhibition of initiation of host polypeptide chains, nor on RNase-like attack on host polyribosomes.


Prostanglandins enhance spread of herpes simplex virus in cell cultures. Stimuli such as u.v. light or trauma which induce recurrence of herpes simplex may act by affecting virus replication in the skin. Such stimuli release pharmacologically active agents in the skin, including prostaglandins (PGs) such as PGE2. These agents, and other compounds which alter levels of adenosine cyclic monophosphate (cyclic AMP), were tested for their effect on the replication of herpes simplex virus (HSV) in Vero cells. Prostanglandin E2 (PGE2) and prostaglandin F2alpha both increase the size of HSV plaques; PGE2 also increases the yield of virus inoculated at low m.o.i. Moreover, inhibitors of prostanglandin synthesis decrease plaque size and inhibit the growth of virus inoculated at low m.o.i.; such inhibition can be partially overcome by adding PGE2. Analysis of the results suggest that prostaglandins can enhance cell-to-cell spread of HSV, but that cyclic AMP is probably not involved in this effect.


Protection of mice against viral infection by Corynebacterium parvum and Bordetella pertussis. Mice could be significantly protected against infection with herpes simplex virus (HSV) by i.p. or i.v. injection of killed Corynebacterium parvum 7 days before infection. This protection was seen in inbred strains of mice with a different degree of sensitivity to HSV and after both i.p. and i.v. infection. Resistant mice immunosuppressed by X-irradiation and showing an increased susceptibility to HSV could also be protected by a previous injection of C. parvum. Elevated levels of interferon were demonstrated in the serum of mice injected with C. parvum 5 to 12 days previously. Four different strains of anaerobic coryneforms were compared and only those which were able to induce a systemic activation of the lymphoreticular system (as reflected by splenomegaly) protected against HSV infection. Protection against HSV-infection could also be demonstrated by using killed Bordetella pertussis. C. parvum also protected against Semliki Forest virus infection in two different strains of mice.


Detection of Epstein-Barr virus antigens and antibodies by peroxidase-labeled specific immunoglobulins. Detection of the Epstein-Barr (EBV) antigens, early antigen (EA), viral capsid antigen (VCA), and nuclear antigen (EBNA) by the indirect immunoperoxidase technique was highly sensitive. Antibody titers to EBNA, EA, and VCA were determined in more than 25 sera of patients with Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), or normal persons. A good correlation between the titers of these antigens was obtained by the immunoperoxidase and immunofluorescence methods. The indirect (anti-IgG) immunoperoxidase technique for the detection of EBNA is, in contrast to the indirect immunofluorescence method, highly sensitive. EBNA was associated with the chromosomes in cells arrested in the metaphase with colchicine.


Thermal treatment and infectivity of hepatitis A virus in human feces. The susceptibility of white-lipped marmoset monkeys (Saguinus sp) to human hepatitis A virus (HAV) provides a system for evaluation of thermal inactivation of HAV in feces and contaminated shellfish. Intramuscular or oral administration of HAV derived from feces of four patients with acute hepatitis A induced hepatitis in 28--100% of the inoculated marmosets. A 10% (w/v) fecal pool (GBG-BM) prepared from two patients (GBG and GBM) induced hepatitis in marmosets (2/4 with 1 ml; 2/2 with 3 ml) when given orally as a 1 : 3 dilution. A HAV-baby food raw oyster mixture fed to fasted marmosets induced hepatitis in 1/4 and seroconversion in 2/4 animals. Two groups of oysters were injected with HAV (concentrated 3 : 1 by centrifugation of the GBG-BM pool); one group was treated at 140 degrees F for 19 minutes and the other served as an untreated control. In animals fed the untreated inoculum, 4/6 developed hepatitis and 6/6 seroconverted, whereas of those fed the heat-treated inoculum 1/7 developed hepatitis and 2/7 seroconverted. These data suggest that pasteurization methods could be developed that would eliminate shellfish-associated hepatitis A and retain the palatability of the shellfish.


Hepatitis B virus DNA in primary hepatocellular carcinoma tissue. Tumour, cirrhotic, and metastatic tissues from four patients with primary hepatocellular carcinoma have been investigated for the presence of hepatitis B viral DNA by nucleic acid hybridization. Tumours from two of three patients with a current HBV infection contained 1--2 genomes per cell of unintegrated viral DNA, while tumours from the third HBs antigen-positive patient contained less than one genome equivalent per ten cells. A tumour from one patient with anti-HBs contained no detectable HBV DNA. A variety of models involving HBV as an etiologic agent may be advanced to explain the statistical correlation of HBV infection with primary hepatocellular carcinoma (PHC). The data presented here argue against the model that HBV DNA integrated into every cell is required to maintain the oncogenic transformation of hepatocytes, but they do not rule out other models.


Effects of phosphonoacetic acid on subacute myeloopticoneuropathy virus in vitro and in vivo. The effect of phosphonoacetic acid (PAA) in vitro and in vivo on subacute myeloopticoneuropathy (SMON) virus isolated from the spinal fluid of SMON patients was studied. PAA inhibited multiplication of SMON virus in cultures, but it did not show a direct effect on the virus. The drug did not influence the disease when the medication was started from 10 days after infection of suckling mice. However, the drug did elicit a delay in the incubation period.


Steroid hormone alteration of herpes simplex virus type 1 replication. The effect of steroid hormones on herpes simplex virus type 1 replication was examined. Virus replication studies revealed that various concentrations of prednisolone, hydrocortisone, dexamethasone, or progesterone could decrease virus yields up to a maximum of 99%. Using isopycnic centrifugation in CsCl to separate viral from cell DNA, it was found that virus-specific DNA synthesis was decreased by 30 to 100% depending on the hormone and concentration used. Cell-specific DNA synthesis was also adversely affected, but this did not alter cell viability or plating efficiency.


Human rotavirus infection in infants and young children with intussusception. Human rotavirus was detected by electron microscopy in 11 of 30 infants and young children with intussusception (37% of subjects under study). Serologic complement fixation tests revealed evidence of infection with the rotavirus in 70% of the patients examined who eliminated the rotavirus in their stools. These results indicate that human rotavirus, in addition to adenovirus, may be an infectious agent causing intussusception in infants and young children.


Homotransplantation of the liver in a patient with hepatoma and hereditary tyrosinemia. A girl with hereditary tyrosinemia, diagnosed at 6 months of age, was treated with a diet restricted in phenylalanine and tyrosine. At 9 1/2 years of age she developed an acutely enlarged liver and spleen, and the diagnosis of hepatocarcinoma was made. The patient received a liver transplant and tyrosine metabolites became normal while she was receiving a regular diet. Three months later, an infected thrombosis of the portal vein caused her death. Liver transplant appears to be an effective method of enzyme replacement in tyrosinemia and should be considered for prevention of hepatoma.


The basis of beta adrenergic bronchodilation. The effects of varying external K and Ca on isometric tension of the tracheal smooth muscle (TSM) of the dog were measured and the relaxant action of isoprenaline IPn) was noted in the same system. The results were similar to those reported for other smooth muscles. Oxygen consumption of the TSM was measured by a reproducible semimicro method simultaneously with mechanical tension under various conditions as above. Depolarized TSM required the energy equivalent of about 0.005 microliter of O2 min -1 . g -1 . t-1 (t = tension in g. cm-2) for sustained contraction. Relaxation of the depolarized TSM by IPn apparently was due to the antagonism of Ca and required extra energy equal to over half that required for contraction. Dibutyryl cyclic adenosine monophosphate failed to relax the depolarized TSM while tissue cyclic adenosine monophosphate level was raised by exposure to IPn. The subcellular energy consuming processes by which IPn antagonizes the contractile effect of Ca in depolarized TSM require further investigation.


Postsynaptic facilitatory effects of theophylline on amphibian neuromyal transmission. This study concerns the effects of theophylline on nerve-muscle transmission of the frog; it was of particular interest to evaluate the facilitatory actions of theophylline at the postsynaptic sites. At concentrations of up to 5 mM, theophylline exerted negligible effects on the end-plate resting potential or on the passive membrane characteristics. The major effects of theophylline (0.5--5.0 mM) were exerted on the end-plate potentials (EPPs), miniature EPPs, acetylcholine (ACh) potentials, and on the end-plate current. The amplitude of these parameters was markedly increased; furthermore, the half-decay time of the EPP and, particularly, of the end-plate current were markedly affected. On the other hand, the time course of the ACh potentials was not significantly affected by theophylline. Spontaneous and evoked release of ACh were not affected by theophylline (0.5--5 mM). Altogether, these results indicate that, in amphibia, the neuromyal facilitation induced by theophylline is mainly due to its postsynaptic actions. Furthermore, some of these data as well as results of others indicate that these effects of theophylline are not due to its anticholinesterase properties. It is suggested that theophylline may act directly on the cholinergic receptor or ionic conductance modulator and that it may stabilize the ACh-receptor complex.


2,3-Dimercaptosuccinic acid: a new agent for the treatment of lead poisoning. Using minimally lead-poisoned rats, we have measured urinary and fecal lead excretion in response to 2,3-dimercaptosuccinic acid (DMS) administered i.p. or p.o. and compared it to that induced by dimercaptopropanol (BAL) (i.p.), EDTA (i.p.), D-penicillamine (p.o. and i.p.) and the combination of BAL and EDTA (i.p.). At doses of 30 mg/kg, parenterally administered DMS was as effective as i.p. BAL and these two drugs were more effective than the other treatment groups. However, p.o. DMS was only 20% less effective and was as effective as i.p. EDTA and the combination of EDTA + BAL i.p. and significantly more effective than D-penicillamine p.o. or i.p. Unlike BAL, most lead excretion in response to DMS was via the urine, undoubtedly reflecting the greater water solubility of DMS. When mice were fed a diet containing both lead and DMS, the drug prevented the accumulation of porphyrins in erythrocytes. Studies with 210Pb indicate that this prophylactic effect is not due to an inhibition of lead absorption but rather to enhanced excretion of lead. The residual tissue distribution of 210 Pb administered simultaneously with DMS was not different form that of 210Pb alone. Since DMS is orally effective and its LD50 is 30 times greater than that of BAL, we expect this compound to be clinically useful in the treatment of lead poisoning.


Effects of adenosine triphosphate and prostaglandins on vascular adrenergic transmission. The effects of prostaglandins (PGs) on the adrenergic neuroeffector transmission and their relationship to the action of ATP were investigated. This was in view of the putative negative feedback mediated by ATP or a related purine compound, and the reported stimulation of PG synthesis by adenine nucleotides. The central ear artery and saphenous vein of the rabbit were isolated and their contractile responses to adrenergic nerve stimulation monitored. These responses were markedly reduced by PGE1 and PGE2 and significantly augmented by indomethacin and aspirin, suggesting the occurrence of the PGE-mediated negative feedback. PGF2a facilitated the response of the vein but was without affect on the artery, while arachidonic acid was facilitatory on the former and inhibitory on the latter. Possibly a PGF2a-like substance is formed in the presence of arachidonic acid and, in the vein, masks the effect of any PGE. ATP depressed the arterial and venous contractile response to adrenergic nerve stimulation. This inhibition was not significantly affected by indomethacin or aspirin. It was enhanced in the artery and diminished in the vein by arachidonic acid, but only to the extent of algebraic sum of the effects of ATP and the acid. It seems possible that the purine- and PGE-mediated feedback mechanisms are independent and parallel pathways.