Lipid composition of bovine serum lipoproteins.

The four classes of lipoproteins (one of very low density, two of low density, and one of high density) were isolated from the serum of a lactating Friesian cow. The proportions of protein and of the different lipid classes were determined in each lipoprotein. Triglycerides predominated in the very low density lipoprotein, and cholesteryl esters and phospholipids in the others. The triglycerides of the very low density lipoprotein were richer in oleic acid than were those of the low density lipoproteins, but its cholesteryl esters were relatively poorer in linoleic and linolenic acid than were those of any of the others. Phytanic acid (3, 7, 11, 15-tetramethylhexadecan-1-oic acid) was in all lipoproteins except those of very low density; it was not in cholesteryl esters but was abundant in triglycerides, particularly in those of the low density lipoproteins. Hydrolysis of the triglycerides of very low density lipoprotein with pancreatic lipase showed that 82% of their stearic acid was esterified to the 1- and 3-positions of glycerol and that 64% of their palmitic acid was esterified to the 2-position.


The use of the haemagglutination-inhibition test for detecting antibodies to type SAT 2 foot-and-mouth disease viruses in cattle sera.

Two strains of type SAT 2-foot-and-mouth disease virus which gave high titres of haemagglutinin activity reacted type-specifically in direct haemagglutination-inhibition tests with reference, bovine convalescent antisera. Comparisons of the haemagglutination-inhibition and the serum neutralization tests using cattle sera showed that both were equally specific and sensitive for detecting virus antibody.


The effects of humoral, cellular and non-specific immunity on intracerebral Bordetella pertussis infections in mice.

When mice were injected intracerebrally with doses of Bordetella pertussis vaccine greater than 5 ImD 50 and challenged intracerebrally 14 days later with virulent B. pertussis there was an immediate reduction in the numbers of organisms. An analysis of this in vivo bactericidal effect has shown that large doses of an unrelated vaccine, Salmonella typhosa, equivalent in cell mass to about 50 ImD 50 of B. pertussis vaccine can achieve this effect, so for such doses the effect must be partly non-specific. This action is not maintained and so is not ultimately protective. Local immunoglobulin was also demonstrable 14 days after 300 ImD 50 of B. pertussis vaccine but following smaller doses of 10-20 ImD 50 it could not be found until after the mice had been infected and the blood-brain barrier impaired. A similar immediate reduction in the numbers of infecting organisms inoculated 1 day after vaccination has been shown to follow very small, non-protective doses of vaccines unrelated to B. pertussis and to be achieved with lipopolysaccharide and endotoxin isolated from B. pertussis. Brains were not sterilized and only in mice receiving protective B. pertussis vaccine was the lowering of infection maintained beyond 2 days and the brains eventually sterilized. The antibody passively protecting mice against intracerebral infection was found in the 19S and 11 S globulin fractions of the serum of once-vaccinated mice and in the 11 S and 7 S fractions of the serum of rabbits and ascitic fluid of mice receiving repeated doses of vaccine. The IgM probably eliminated infections by immediate sterilization but had to be present locally to do so since it was unable to pass from the circulation into the brain, and was therefore inactive when injected intraperitoneally. The IgA and IgG were not so restricted and both the 11 S and 7 S globulins were capable of exerting an immediate suppressive effect on infecting organisms. The 7 S globulin was also capable of a maintained or delayed suppressive effect. Lymphocytes from fully protected once-vaccinated mice, transferred 2-3 weeks after intraperitoneal vaccination, were able to confer some protection when injected intraperitoneally or intracerebrally into recipient mice infected 2 weeks after transfer. Homologous, non-concentrated antiserum from once-vaccinated mice, injected intraperitoneally 1 hr. before infection sometimes augmented the transferred immunity, whereas alone it was inactive.


In vitro detection of immune responses to MTV-induced mammary tumors: qualitative differences in response detected by time studies.

Both BALB/cfC3H females neonatally infected with mammary tumor virus (MTV) and MTV-free BALB/c females possess spleen cells capable of significant activity against target-MTV-induced mammary tumor cells in in vitro microcytotoxicity tests. The responses of the BALB/cfC3H and BALB/c females are different, however, on the basis of time studies. Further, the reactivity characteristic of virgin BALB/cfC3H females is increased in parous females, and part of this reactivity is subsequently lost in multiparous females bearing large tumors. BALB/cfC3H females possess reactive spleen cells as early as 3 weeks of age, indicating that response to MTV antigens develops soon after birth in these neonatally infected mice.


Immunopathology of mouse hepatitis virus type 3 infection. Role of humoral and cell-mediated immunity in resistance mechanisms.

Humoral and cell-mediated immune responses were studied in resistant and susceptible strains of mice infected with mouse hepatitis virus type III (MHV 3). Virus was maintained by regular passages in susceptible DBA/2 mice and assayed in DBA/2 mice by LD-50 determination. Normal resistant A strain mice were able to clear the virus from liver, brain, and serum within 7 days after infection. No neutralizing antibody was found. Transfer of serum from immunized A strain mice was not effective in protecting susceptible DBA/2 mice against challenge with virus. In A strain animals resistance to MHV-3 developed rapidly during the 3rd week of life. During the period of susceptibility, newborns were protected neither by transplacental passages of anti-MHV-3 antibodies nor by injection of "educated" thymus cells.


In vitro studies on the cellular immune response of tumor-bearing mice to SV40-transformed cells.

Specific cell-mediated immunity to SV 40 tumor-specific transplantation antigen (TSTA) in mice undergoing tumorigenesis by syngeneic SV 40-transformed BALB/C cells was investigated by the macrophage migration inhibition (MMI) and transplantation rejection tests. Specific cellular reactivity to SV40 TSTA could be demonstrated in BALB/c mice early after tumor cell inoculation. This activity was no longer detectable during the later stages of tumor growth but was again demonstrable 2 weeks after tumor excision. Addition of an equal number of non-reactive peritoneal exudate (PE) cells from tumor-bearing mice to PE cells from mice immune to SV40 TSTA specifically abrogated the reactivity of the latter cells to soluble SV40 TSTA. When lymphoid cells with blocking activity were cultured in vitro they not only lost their blocking capacity but also regained their reactivity to SV40 TSTA in the MMI test. These findings indicate that tumor-bearing hosts possess lymphocytes specifically sensitized to the TSTA of the tumor and that the specific reactivity of these cells can be regained after culture in vitro.


Modulation of in vivo antibody responses by cholera toxin.

Treatment of mice with an exotoxin (0.01 mug to 1.0 mug) purified from Vibrio cholerae culture filtrates markedly influenced the immune response to sheep erythrocytes (SRBC) and the Escherichia coli lipopolysaccharide (LPS). Simultaneous administration of the toxin (CT) with antigen resulted in a delayed appearance of antibody plaque-forming cells (PFC) during the first few days after immunization, followed by a marked enhancement of both IgM and IgG PFC. The secondary immune response to SRBC was also similarly affected when CT was given together with a second inoculation of SRBC; i.e., a delay in appearance of hemolytic PFC followed by a markedly enhanced IgM and IgG PFC response. Treatment of mice with cholera toxin 1 to 3 days before SRBC or LPS was immunosuppressive. The effect of CT on the level of splenic cyclic AMP appeared related to the effects on antibody formation.


The modulation of spontaneous and anti-Ig-stimulated motility of lymphocytes by cyclic nucleotides and adrenergic and cholinergic agents.

Translational movement in B lymphocytes was stimulated by anti-Ig antibody. Drugs that are presumed to elevate cyclic AMP stopped this stimulated motility. Such was the case with dibutyryl cAMP and theophylline, cholera enterotoxin, and isoproterenol, a beta adrenergic agonist. Conversely, in the absence of anti-immunoglobulin antibody, cyclic GMP and the cholinergic drugs acetylcholine and carbamylcholine increased spontaneous motility of lymphocytes, with the B class of lymphocytes demonstrating greater responsiveness. The increase in motility brought about by cholinergic drugs was totally stopped by atropine, suggesting that the B lymphocyte surface contains a cholinergic receptor. The inhibition of anti-immunoglobulin-stimulated movement produced by cyclic AMP was not observed if the cells were first incubated with colchicine, the microtubular-disrupting drug. This suggests that the cyclic AMP-decreased motility was brought about by the increased stabilization of microtubules. Lymphocyte motility was dissociable from other early events subsequent to binding of anti-immunoglobulin antibodies: patching, capping, and endocytosis of complexes were unaffected by cyclic AMP, cyclic GMP, or drugs of the adrenergic or cholinergic systems.


Induction of lymphocyte cytotoxicity by modification of the effector or target cells with periodate or with neuraminidase and galactose oxidase.

Treatment of mouse spleen cells with periodate (NaIO4) or with neuraminidase and galactose oxidase (NAGO) induces blastogenesis and renders the cells cytotoxic to mastocytoma (P815) target cells. Treatment of target cells (P815 cells and turkey erythrocytes) with NaIO4 or with NAGO renders them susceptible to cytolysis by untreated mouse spleen cells. The cytotoxicity induced by NaIO4 is reduced upon reacting the NaIO4-treated, effector or target cells with borohydride or hydroxylamine. Thus the formation of free surface aldehydes on either the effector or target cell induced a cytotoxic effect. It is postulated that cross-linkage via a Schiff base between effector and target cell initiates the cytotoxic effect. Cytotoxicity induced by NaIO4 or NAGO is immunologically nonspecific and is independent of major antigenic differences between effector and target cells. Phagocytic cells are not involved in NaIO4-or NAGO-induced cytotoxicity toward P815 target cells.


The initiation of mast cell degranulation: activation at the cell membrane.

The low molecular weight mast cell activator, polymyxin B, has been covalently bound to an insoluble matrix of Sepharose 4B. It has been demonstrated that mast cells in preparations of rat peritoneal cells bind to Sepharose 4B-polymyxin B beads but not to control beads. The bound cells are stimulated to degranulate by this interaction at the cell membrane with the resultant release of biogenic amines.


Quantitative aspects of the production of superoxide radicals by phagocytizing human granulocytes.

The production of superoxide radicals (O2-) by human granulocytes after phagocytosis of latex particles has been studied as a function of cell, latex, and cytochrome c concentration. Under the chosen conditions, latex phagocytosis stimulated the reduction of cytochrome c by O2- sevenfold to 420 mumoles per 10-10 granulocytes per hour. At high cytochrome c concentrations, this value increased to 800 to 1,000 mumoles per 10-10 granulocytes per hour. These amounts suggest that O2- plays an important role in the formation of H2O2 during the phagocytic process. We propose that the extra oxygen consumed in the respiratory burst is first reduced to O2-, possibly by ascorbate, before being converted to H2O2. No stimulation of the superoxide radical formation was found in the phagocytizing granulocytes of two patients with chronic granulomatous disease, and intermediate values were found for the heterozygotes of this deficiency.


The transport of triglyceride in the high-density lipoproteins of human plasma.

The metabolism of the small pool of triglyceride in human high-density lipoproteins (HDL) has been studied. In 28 postabsorptive and unmedicated subjects the previously reported reciprocal relationships between the concentration of very low-density lipoprotein (VLDL) lipids and HDL cholesterol was confirmed. It was also found that the HDL triglyceride correlated positively and significantly with the concentration of VLDL triglyceride, and there was a significant negative correlation between the concentrations of triglyceride and cholesterol in HDL. Five normal and 3 hypertriglyceridemic subjects were given 5,000 units of heparin intravenously. Not only was there a pronounced reduction in VLDL triglyceride, but there was a 60 to 80 per cent reduction in HDL triglyceride; the low-density lipoprotein (LDL) triglyceride was unchanged. To test a possible precursor-product relationship between the triglyceride pools in VLDL and HDL, 5 subjects were given an intravenous pulse injection of (14-C)-palmitic acid. The specific activity-time curves of triglyceride in VLDL and HDL did not conform to a typical precursor-product relationship; rather the HDL curve reached its peak well before it crossed the VLDL curve, quite compatible with an origin of HDL triglyceride which was independent of VLDL. However, when the lipoproteins of density smaller than 1.006 (VLDL and chylomicrons) were labeled in the intestine after an oral tracer dose of (3-H)-palmitic acid in 2 subjects, the relationship of the triglyceride labeling in HDL to that in d smaller than 1.006 lipoproteins was very similar to the relationship observed after the intravenous dose. Although an independent intestinal origin of HDL triglyceride could not be excluded, this result did suggest that the labeling of triglyceride in d smaller than 1.006 lipoproteins and in HDL was related and was consistent with a transfer from the larger lipoproteins to the HDL. In three subjects given a constant intravenous infusion of (3-H)-palmitic acid, the specific activity of triglyceride in VLDL became constant after 3 to 4 hours; it also became constant in the HDL after about 4 hours but at a level of only 40 to 70 per cent of the VLDL level. It appeared that 30 to 60 per cent of the HDL triglyceride was either derived from an unlabeled source or else turned over too slowly to become labeled to any extent during the infusions. Whatever the explanation, the same phenomenon would be likely to influence the level of the peak specific activity of HDL triglyceride after the pulse intravenous injections of label. It is possible that the specific activity of the "labeled" fraction of HDL triglyceride may have reached its peak at the point at which it crossed the VLDL curve, which would be consistent with a precursor-product relationship between VLDL and one fraction of the HDL triglyceride. It has been concluded that HDL triglyceride is heterogeneous and includes at least one pool, possibly transferred from VLDL, which is relatively rapidly turning over.


A radioisotopic assay for proline oxidase activity.

We developed a radioisotopic assay for proline oxidase in which product deltal-pyrroline-5-carboxylate-14-C is reacted with o-aminobenzaldehyde and the radioactivity trapped as the dihydroquinazolinium compound is recovered by ion-exchange chromatography. The sensitivity of this method allows the measurement of proline oxidase activity in small specimens (10 to 20 mg.) of liver.


A case of oropharyngeal chordoma.

A case of cervical chordoma without vertebral involvement is presented. The diffculty in histological diagnosis is described. Treatment by radiotherapy and surgery is described and discussed.


Deoxypyrimidine kinases of herpes simplex viruses types 1 and 2: comparison of serological and structural properties.

The kinetics of formation, the stability at 40 degrees C and the serological properties of thymidine kinase and deoxycytidine kinase activities induced by herpes simplex virus have been examined. The results are consistent with the hypothesis that both activities are carried on the same molecule-a deoxypyrimidine kinase. Mutants deficient in deoxypyrimidine kinase have been used to produce, by absorption of general antisera, deoxypyrimidine kinase-specific antisera. Using immunoprecipitation and SDS-polyacrylamide gel electrophoresis, only one size of polypeptide (mol. wt. 42400 plus or minus 200) has been found, constituting the type 2 enzyme. This is close to published values for the type i enzyme but co-electrophoresis demonstrated that the polypeptide of the type i enzyme was slightly bigger.


Electron microscopic studies on assembly of herpes simplex virus upon removal of hydroxyurea block.

The release of hydroxyurea-treated, herpes simplex virus-infected cells from the drug-induced block resulted in the prompt assembly of infectious virus. Electron microscope observations at sequential intervals following removal of the drug revealed considerable synchrony of replication. This synchrony permitted stages in the complex process of core assembly to be examined in detail. The data suggest that after partial or complete assembly the nucleoprotein enters the differentiated capsid to become enfolded.


Virus development in enucleate cells: echovirus, poliovirus, pseudorabies virus, reovirus, respiratory syncytial virus and Semliki Forest virus.

A group of RNA viruses, echovirus, poliovirus, reovirus, respiratory syncytial virus and Semliki Forest virus have been examined for ability to grow in enucleate African green monkey kidney (BSCi) cells. Semliki Forest virus produced an almost normal yield of virus but poliovirus, echovirus, reovirus and respiratory syncytial virus, although showing clear evidence of virus replication when compared with a nuclear DNA virus (pseudorabies virus) gave much lower yields than those from nucleate cells. Analysis of enucleate cells infected with echovirus and reovirus showed no evidence of a specific block in the synthesis of any virus-specified polypeptide. Infection with vesicular stomatitis virus at intervals after enucleation demonstrated a diminishing ability to support virus growth with increasing time. It is suggested that the yield of virus obtained from an enucleate cell is related to the length of the growth cycle of the virus, the reduced yield obtained with some viruses reflecting the declining ability of the enucleate cell to support virus growth.


Effect of interferon on induction of S antigen by polyoma virus in BHK 21 cells.

A new surface antigen (S antigen) can be detected by immunofluorescence during the abortive transformation of hamster cells by polyoma virus. It was found that addition of interferon greatly reduces the percentage of cells positive for S antigen.


Nerve supply and experimental myotonia in rats.

20, 25-Diazacholesterol does not induce myotonia in denervated mammalian skeletal muscle, and cordotomy also renders the muscles resistant to the induction of myotonia. Denervation of muscle already rendered myotonic, has no effect on the intensity of the phenomenon. Finally, tenotomy and posterior rhizotomy leave the myotonic activity in the "deafferentated" muscles unchanged. These findings are discussed in relation to "trophic" influences of the motor pathways on the postsynaptic structures of skeletal muscle.


Stages in fibre breakdown in Duchenne muscular dystrophy. An electron-microscopic study.

A scheme is presented which divides the process of fibre breakdown in Duchenne muscular dystrophy into 5 sequential stages. In Stage 1 the fibre appears superficially normal although there are subtle changes in the relative volumes of the fibre components. In Stage 2 localised overcontraction of the myofibrils results in excessive stretching of the sarcomeres in other regions of the fibre. This continuing process results in the formation of contraction clumps in which the contractile filaments form an increasingly homogeneous mass. By the final stage the normal structural features of the fibre have completely disappeared and it is invaded by macrophages. It is suggested that the central process of myofibril clumping results from a localised inability of the sarcomeres to relax which implies a defect in the mitochondria, Z-line or sarcoplasmic reticulum. This is discussed in relation to other workers' findings of a decrease in the calcium-accumulating capacity of the sarcoplasmic reticulum in Duchenne muscular dystrophy.


Rebound phenomenon complicating cerebral dehydration with glycerol. Case report.

A patient with glioblastoma multiforme of the brain was treated with both intravenous and oral glycerol as well as intravenous mannitol in an attempt to reduce increased intracranial pressure. After an initial lowering of the cerebrospinal fluid (CSF) pressure to near normal values during continuous glycerol administration, a secondary rise in CSF pressure above the initial level occurred despite a persistent elevation of plasma osmolality (315 mOsm/kg) and glycerol level (30 mmole/l). Similarly, 4 hours after the administration of a single oral dose of glycerol, CSF pressure increased to levels higher (700 mm H2O) than the original baseline (400 mm H2O).


Uptake of 99mTc bone-scanning agent by lungs with metastatic calcification.

Uptake of 99mTc bone-scanning agent into lungs containing metastatic calcification was demonstrated in a patient with a carcinosarcoma of the bladder. This gives further support to the hypothesis that 99mTc bone agents are associated with hydroxyapatite.


Effects of semistarvation on rat liver, kidney, and heart mitochondrial function.

Two groups of rats were provided simultaneously with a commercial stock diet for a period of 7 days. One group was fed ad libitum (control), and the other was restricted to one-fourth of the daily intake of control animals (semistarved). Body weight declined significantly in semistarved rats whereas body weight of controls increased over the 7-day period. The following were determined in vitro on mitochondria isolated from liver, kidney, and heart tissues of both groups: substrate-stimulated and DNP-uncoupled respiratory rates; specific acivities of the Krebs cycle dehydrogenases, and cytochrome c oxidase. Degradative effects of reduced food intake on mitochondrial function were observed. Uncoupled respiratory rates of liver and kidney mitochondria (using succinate as substrate) and heart mitochondria (using alpha-ketoglutarate and pyruvate) were lower. Also lower were activities of isocitrate dehydrogenase, NADP: isocitrate dehydrogenases, transhydrogenase, succinate dehydrogenase, and cytochrome c oxidase of heart mitochondria, transhdrogenase of liver mitochondria, and isocitrate dehydrogenase and transhydrogenase of kidney mitochondria. Such decreases in enzyme activities under conditions of dietary protein deficiency might have their basis in breakdown rates exceeding synthesis rates or result from partial inactivation of existing enzyme protein. Thus, there is evidence that responses to semistarvation of such parameters of mitochondrial function may differ among various tissues. In addition, liver and kidney citrate levels were lower and heart citrate level higher with semistarvation.


Case 11, Part 2.  Angiokeratoma corporis diffusum (Fabry's disease).

A case of Fabry's disease is reported.  The patient had multiple supernumerary, impacted, and unerupted teeth and other developmental abnormalities of the facial skeleton.  The patient tolerated surgical removal of impacted and unerupted teeth without complications.  However, this did not relieve the facial pain.  The expected prognosis of this patient will be a continued vascular and renal degeneration with death probable before the age of 50 years if the disease is untreated.  Currently, the patient is being considered as a possible candidate for renal transplantation.


The role of adenoviruses in the pertussis syndrome.

To define the role of adenoviruses in the pertussis syndrome, a study was done of a group of 134 children with clinical pertussis and a healthy control population of similar age, race, sex, and socioeconomic status. Adenovirus infections occurred in 30 (22.4%) of 134 patients with the pertussis syndrome and 5 (4.9%) of 101 control subjects (p smaller than 0.001). B. pertussis was recovered from 46 (34.3%) patients, and from 18 (39.1%) of these patients adenoviruses were also isolated. Although adenovirus infections also occurred in patients with the pertussis syndrome with negative cultures for B. pertussis, the rate, 12 of 88 patients (13.6%), was significantly lower (p smaller than 0.001). The clinical course was similar irrespective of the results of bacterial or viral cultures. These data substantiate the frequent association of adenoviruses with the pertussis syndrome, It would appear that adenoviruses do not usually have an independent role in the pathogenesis of the pertussis syndrome since we found them so commonly to be one agent in a mixed infection.


Plasma lipid values and lipoprotein patterns during adolescence in boys.

Lipid studies were performed on fasting plasma samples in 214 healthy adolescent boys, 11-19 years of age. Ranges of normal values for plasma cholesterol and triglyceride concentrations, and normal lipoprotein patterns were established for this age group. The boys were grouped according to puberty rating. A significant increase in triglyceride values was obtained with adolescent maturity. Plasma cholesterol values did not change with age or puberty rating. One boy had hypercholesterolemia and a lipoprotein pattern consistent with Type II hyperlipoproteinemia (Fredrickson). Seventeen boys, all of whom had reached adolescent maturity, had elevated triglyceride values and a marked increase in pre-beta-lipoprotein. It appears from this study that increased concentrations of triglyceride and Type IV hyperlipoproteinemia of Fredrickson may be detected as boys reach maturity at adolescence.


A laboratory procedure for copperplating rubber base impressions.

A method for producing electroformed copper dies from rubber base impressions has been described. Castings made by the use of such dies have met the standards set by several critical dentists.


Luteoma of pregnancy: report of a case and review of the literature.

A case of a unilateral, non-virilizing pregnancy luteoma in a 28 year old Caucasian female resulted in good health more than six years after removal of the lesion. Theories on the histogenesis of the lesion are briefly recounted and the literature is reviewed. This case appears to be number 62 in the world literature.


Modification of nuclear restriction in vitro by plasma from tumor-bearing animals.

Putative regulatory macromolecules, which may be useful in diagnosis or tumor detection, were identified in the peripheral blood plasma of tumor-bearing animals. We monitored the components by measuring their ability to stimulate messenger RNA (mRNA) release from isolated nuclei in a cell-free system of rat liver nuclei in fortified homologous cytosol. This in vitro test system exhibited near-normal in vivo nuclear RNA restriction. When added to the assay at a protein concentration of 3.0 mg/ml, dialyzed plasma from rats or mice with chemically induced transplantable or primary tumors stimulated mRNA release from 87% to more than 300% over control plasma from normal rats. Plasma from partially hepatectomized rats stimulated only 26% over control plasma. The test system derived from rat liver seemed to permit the monitoring of plasma from other species. Available evidence, particularly relating to tumor-host-interaction, suggests, but does not prove, that regulatory components are released from the tumor cells to the circulation.


Effect of beta-oxidized nitrosamines on syrian hamsters. III. 2,2'-Dihydroxydi-n-propylnitrosamine.

2, 2-Dihydroxy-di-n-propylnitrosamine (DHPN), an assumed metabolite of di-n-propylnitrosamine (DPN), injected subcutaneously once weekly for life, was carcinogenic in Syrian hamsters. The main target organs were the respiratory tract, pancreas, liver, and kidneys. In the respiratory system the most affected segments were the nasal cavities and the lungs. Adenomas and adenocarcinomas, mostly of ductal origin, were induced in the pancreas. Liver neoplasms were hemangloendotheliomas, angiosarcomas, hepatocellular adenomas, cholangiomas, and cholangiocarcinomas. Kidney neoplasms were adenomas and adenocarcinomas. The morphology of the induced neoplasms was described, as well as the effects of DHPN, compared to those another possible metabolite of DPN, 2-hydroxypropyl-n-propylnitrosamine (2-HPPN), which is formed in vivo with only 1 aliphatic chain degraded via theta-oxidation.


Immunization of Rous sarcoma virus-inoculated marmosets with BCG and transformed allogeneic cells.

The effects of specific immunotherapy with allogeneic cells transformed by Schmidt-Ruppin Rous sarcoma virus (SR-RSV), of treatment with BCG, and of surgery on the growth of SR-RSV-induced sarcomas in white-lipped marmosets were studied. Tumor incidence, tumor progression, and survival did not differ between control and treated animals. Animals immunized with BCG developed lymphocyte reactivity to tuberculin, which remained until the animals died. BCG was isolated from the spleen of one tumor-bearing animal.


Effect of a normal serum protein absent from hepatoma-bearing animals on cell cultures.

The effect of a serum factor purified from normal rat serum on growth and incorporation of 3-H-thymidine and 3-H-leucine was investigated with the use of 3T3 and L cells. The factor was not present in serum of hepatoma-bearing animals at a time when the weight of the hepatomas was equal to or greater than liver weight. The factor disappeared gradually from the serum of hepatoma-bearing animals, and the disappearance was proportional to the increase in the size of the hepatomas. Diminished amounts of the factor could be detected as early as 10 days after hepatoma transplant, and the factor was absent at 30 days after hepatoma transplant from the serum of rats bearing hepatoma 7777. Medium supplemented with 5% normal rat serum supported incorporation of lavel into 3T3 and L cells and growth of these cells, as did medium supplemented with 10% calf serum. Serum from hepatoma-bearing animals was only approximately equal to 50% as efficient as normal rat serum in supporting growth and label incorporation. The addition of purified factor to the serum  from hepatoma-bearing animals restored the ability of the serum to support growth and label incorporation to between 70 and 90% of that found with normal rat serum as a medium supplement. The factor also enhanced incorporation of 3-H-thymidine following release of 3T3 cells from contact inhibition.


Effect of pyridoxine availability on the activity of serine dehydratase of normal liver, host liver, and three Morris hepatomas.

The effect(s) of dietary pyridoxine availability on serine dehydratase (SD) specific activity levels of normal liver. Morris hepatomas "5123A, 7316B, 7800, and of respective host livers was studied. Buffalo female weanling rats were fed ad libitum a pyridoxine-free diet or the same diet supplemented with the vitamin. They were inoculated intramuscularly in the hind leg muscles with hepatoma cells after 3 weeks on the respective diets, and those bearing hepatomas "5123A, 7316B, 7800 were killed at 28, 30, and 48 days, respectively, after inoculation. SD activity was highly affected by pyridoxine. Absence of the vitamin from the diet resulted in greatly reduced activity levels in normal liver and the three hepatomas. Tumors grown in animals fed the pyridoxine-supplemented diet had 39j ("5123A), 3.5 ("7316B), and 2.1 ("7800) times more SD specific activity tan respective tumors grown in animals fed the deficient diet. A 1.7-fold increase was observed in normal liver. In contrast to these findings, the specific activity of the enzyme was reduced by 6.3, 1.5, and 3.0 times, respectively, in the host livers of animals fed the vitamin-supplemented diet and bearing hepatomas "5123A, 7316B, and 7800. Serine dehydratase activity depends greatly on dietary vitamin B6 and hence I propose that activity levels in vivo are regulated by its presence or absence.


Adenosine-3',5'-cyclic monophosphate levels and adenosine-3',5'-cyclic monophosphate phosphodiesterase activity in metastasizing and nonmetastasizing rat mammary carcinomas.

The adenosine-3, 5-cyclic monophosphate phosphodiesterase (cPDE) activity in the homogenates of 6 spontaneously metastasizing, nonimmunogenic, glycocalyx-shedding rat mammary carcinomas (MT) was assayed and compared with four histologically and growth rate-matched nonmetastasizing, immunogenic MT. The levels of this enzyme were 2.5 times higher in the nonmetastasizing tumors. To rule out the possibility of the presence of inhibitor(s) or stimulator(s) of cPDE, homogenates from a nonmetastasizing and from a widely metastasizing tumor were mixed. cPDE from both nonmetastasizing and metastasizing MT showed two apparent Km and two corresponding Vmax. The activity of the enzyme at concentrations of 1 muM (low Km) and 100 muM (high Km) adenosine-3, 5-cyclic monophosphate (cAMP) decreased in parallel with increasing metastasizing capacity. About 50% of the low and the high Km cPDE was in the cytosol in both groups, whereas the rest was particulate. The proportion of low and high Km activity was similar in all the fractions except in the plasma membrane of the metastasizing tumors where the percent of low Km enzyme was three times higher than that of the high Km. The steady-state levels of cAMP were 1.3-2.0 times higher in the metastasizing tumors, inversely proportional to their cPDE activities.


A new common marker for premalignant and malignant hepatocytes induced in the rat by chemical carcinogens.

A new common antigen, preneoplastic (PN), was found in every early and late hyperplastic nodule and in every primary hepatoma induced by N-2-fluorenylacetamide, ethionine, 3-methyl-4-dimethylaminoazobenzene, dimethylnitrosamine, or diethylnitrosamine in three strains of rats (CFN, F344, and BUF). It so far has not been found in normal adult rat liver, liver surrounding nodules or cancer, fetal liver, amniotic fluid, adult rat serum, sera from rats with hyperplastic nodules or primary hepatomas, or various normal rat tissues. Immunofluorescent staining indicated the PN antigen was present in the cytoplasm of hepatocytes in hyperplastic nodules and in primary hepatomas.


Anemia associated with feline leukemia virus infection in cats.

The types of anemia associated with natural and experimental feline leukemia virus (FeLV) infection in cats were investigated. In one experiment, 10 kittens were inoculated neonatally with Jarrett FeLV-1, an isolate of subgroup A; 6 developed anemia a few weeks later. This anemia was characterized by macrocytosis, normoblastosis, increased erythropoiesis in the bone marrow, and extramedullary hematopoiesis in the spleen. Anemia was transient and nonfatal and occurred before the onset of lympoid malignancy. The same type of anemia was also seen in 9 of 24 kittens inoculated with Jarrett FeLV-9 of subgroups A and B. A different form of anemai occurred in another experiment in which 10 kittens were inoculated with FeLV-C of subgroup C only. All 10 kittens developed a profound aplastic or erythroblastopenic anemia in which the bone marrow became depleted of erythroid tissue; all kittens died within 16 weeks, most as a direct result of anemia. In an experiment in which kittens were inoculated with FeLV-B of subgroup B only, no kitten showed anemia. Cats with naturally acquired, nonleukemic lymphosarcoma were also studied. Of 33 lymphosarcomas in which myelophthisis was excluded as a cause, 54% of the affected cats had anemia, the features of which were consistent with hemolytic origin. When virus could be grown from these lymphosarcomas, it was of subgroup A alone or a combination of A and B. With one exception, anemic cats had low or negative titers to feline oncornavirus-associated cell membrane antigens. Until more isolates have been tested, it is not known if the various hematologic changes reflected differences in the pathogenic effects of the subgroups of the virus or of types of strains within them.


Evaluation of dye exclusion and colony inhibition techniques for detection of polyoma-specific, cell-mediated immunity.

Cellular immunity directed against polyoma virus-induced antigen was observed with C3H/HeJ splenic lymphoid cells from mice sensitized by a short-term immunization schedule with syngeneic polyoma 4198 and 4198V tumor cells. Polyoma specificity of the response was shown by demonstration that splenic cells from DBA/2J animals with polyoma virus-induced tumors were cytotoxic for the C3H 4198 and 4198V cells, but not for the L-M cell, another cell line of C3H origin. The polyoma-specific response in the syngeneic system was detectable with the dye exclusion assay but not with the colony inhibition procedure. Colony inhibition with the L-M cell was observed with sensitized lymphoid cells in both allogeneic and syngeneic systems.


Potentiation of an adenovirus-associated virus by herpes simplex virus type-2-transformed cells.

The herpes simplex virus type-2 (HSV-2)-transformed cell line 333-8-9 was inoculated with adeno-virus-associated virus type 3 (AAV-3) and examined by immunofluorescent (FA) techniques for evidence of AAV-3 antigen synthesis. Antisera prepared against the three sodium dodecyl sulfate-dissociated structural polypeptides (VP-1, VP-2, and VP-3) of AAV-3 and against the whole AAV-3 virion were used as probes to search for AAV-3 FA-stainable antigens synthesized by these cells. The 333-8-9 cells inoculated with AAV-3 ALONE PRODUCED FA-stainable antigens detected by VP-1, VP-3, and whole virion antisera, but not by the VP-2 antiserum. Coinfection of AAV-3-INOCULATED 333-8-9 CELLS WITH EITHER ADENOVIRUS TYPE-2 (Ad.2) or herpes simplex virus type-1 (HSV-1) helper permitted the formation of FA-stainable antigens detected by all antisera, including the VP-2 antiserum. In contrast to 333-8-9 cells, BHK-21 cells failed to produce AAV-3 FA-stainable antigens when inoculated with AAV-3 alone; addition of either Ad.2 or HSV-1 helper led to formation of antigens detected by all VP and whole virion antisera. These results suggest that 333-8-9 cells enhance the formation of two of the three AAV-3 structural polypeptides and that this incomplete potentiation may be related to HSV-2 genetic material possessed by these cells.


Biochemical properties of simian virus 40-transformed 3T3 cell mitochondria.

Mitrochondria isolated from simian virus 40-transformed 3T3 and nontransformed 3T3 cells were compared by various biochemical criteria. Transformed and nontransformed cell mitochondria had identical densities in linear sucrose and discontinuous bovine serum albumin gradients. The activities of several mitochondria-specific enzymes including cytochrome oxidase, adenylate kinase, nicotinamide adenine dinucleotide (NADH)-cytochrome c reductase, and NADH oxidase were similar in both cell types. However, the activity of the mitochondrial outer membrane enzyme, monoamine oxidase. In the virus-transformed cell mitochondria was reduced to 50% of that in nontransformed cell mitochondria.


Type-C virus particles in placenta of the cottontop marmoset (Saguinus oedipus).

Electron microscopy of near-term placentas of two cottontop marmosets (Saguinus oedipus) revealed, in one placenta, the presence of budding and mature C-type virus particles associated with the basal trophoblast. The particles were morphologically similar to those observed by other investigators in placentas of other primate species.


B-cell origin of hamster lymphoid tumors induced by simian virus 40.

Two lymphoid neoplasms induced by simian virus 40 (SV40) in Syrian hamsters were analyzed for lymphocyte characteristics. The hymphocytes from both tumors contained membrane-bound Ig that was exclusively of 7Sgamma2 class. Furthermore, neither lymphoid tumor had a complement receptor. Thus both tumors oringinated from a particular B-cell population, which suggests that this B-cell type is associated with an SV40 receptor.


Elevated immunofluorescence antibody titers to several herpesviruses in Burkitt's lymphoma patients: are high titers unique?

Antibody titers for viral capsid antigens of all four human herpesviruses were measured by immunofluorescence in the sera of 16 Burkitt's lymphoma (BL) PATIENTS, 16 AGE-, SEX-, AND LOCALITY-MATCHED CONTROLS, AND 136 FAMILY MEMBERS FROM THE West Nile District of Uganda. Among family members, titers greater than 1:4 were found in 98% for herpes simplex virus (HSV), 86% for varicella-zoster virus (VZV), 100% FOR CYTOMEGALOVIRUS (CMV), AND 94% FOR Epstein-Barr (EBV). Titers in patients averaged approximately equal to 2 logs (fourfold) higher than those in matched controls for EBV, VZV, and CMV (P EQUALS 0.001); titers for HSV were only slightly higher in cancer patients. The mothers of patients had someuhat higher EBV titers (0.05 smaller than or equal to P SMALLER THAN OR EQUAL TO 0.01) than the mothers of controls, but no other differences between patient and control families were found. By immunofluorescence, a method which apparently has not been used for all four human herpesviruses in BL patients, the patients had elevated antibody titers not only to EBV but also to CMV and VZV. The elevated titers to three of the four human herpesviruses were not due to serologic cross reactions.


Unexpected high incidence of primary liver cancer in Geneva, Switzerland.

The age-standardized incidence rate of primary liver cancer (PLC) in Geneva was 9.7 per 100,000 in males, a figure four or five times higher than incidence rates reported elsewhere in Europe . These PLC's were often associated with cirrhosis and alcoholism. This indicated that the toll of PLC related to alcoholism and to alcoholic cirrhosis may be greater than anticipated.


Induction of Epstein-Barr virus-associated nuclear antigen during in vitro transformation of human lymphoid cells.

Human lymphoid cells isolated from the peripheral blood of adults, from cord blood, and from fetal liver, spleen, bone marrow, and thymus were cultivated with or without a cell-free preparation of Epstein-Barr virus (EBV) with demonstrated transforming activity. The cultures were examined for the EBV-associated nuclear antigen (EBNA) and for transfromation into permanent lymphoblastoid cell lines (LCL). EBNA, seen only in cultures that had received exogenous EBV, was detected between days 1 and 6 after addition of EBV, most frequently on day 3. EBNA-positive cells had a lymphoblastoid appearance. Transformation into established LCL became apparent between days 12 and 19. The addition of pokeweed mitogen to cultures containing EBV enhanced the development of EBNA, whereas phytohemagglutinin or concanavalin A had no such effect. Neither EBNA nor transfomration was observed in lymphoid cells from fetal thymus. In fetal spleen, bone marrow, and liver cells, EBV regularly induced EBNA and LCL transformation.


Isolation of a B-tropic type-C virus from reticulum cell neoplasms induced in BALB/c mice by SJL/J type-C virus.

D1-murine leukemia virus (MuLV), an N-tropic type-C virus isolated from a spontaneous reticulum cell neoplasm, type B (RCN-B) of an SJL/J mouse was propagated in NIH Swiss mouse embryo cell cultures. When injected into BALB/c mice 1 day after neonatal thymectomy, 30% of the inoculated mice developed RCN-B in 5 months, whereas none of the uninoculated controls did. From the spleen and lymph node extracts of all RCN-B-bearing mice tested, B-tropic type-C viruses (designated E1-MuLV) were isolated in high titers (10-5 minus 10-6 XC plaque-forming units/ml). Only low titers (10-1 minus 10-2 XC plaque-forming units/ml) of N- or B-tropic viruses were isolated from those thymectomized mice, inoculated but nontumorous, whereas only N-tropic viruses were detected in the uninoculated thymectomized mice. No virus was isolated from the nonthymectomized, untreated controls. Antigenically, the viral envelope antigen (VEA) of E1-MuLV was distinct from those of DU-MuLV, xVEA, or Gross-VEA, but some cross reaction with AKR-MuLV-VEA was observed. The relationship of D1-MuLV to E1-MuVL with respect to oncogenesis and viral genome activation was discussed.


Chromosome aberrations in Syrian hamster embryo cells transformed after exposure to ultraviolet-irradiated herpes simplex virus type 1 or 2.

Six Syrian hamster embryo cell lines (14-012-8-1, KOS-6-1, 333-8-9, 333-2-29, MS-4-1, FR-6-1), developed after exposure of primary cultures to different strains of UV-irradiated herpes simplex virus (HSV) type 1 or 2, were analyzed for chromosome aberrations. All the cell lines showed chromosome stability (number of chromosomes were maintained within a narrow range of variation in the diploid region) and a low incidence of polyploids, endoreduplications, and metaphases with pulveration or extensively fragmented chromosomes. The cell lines, passaged over long periods of time in vitro, developed marker chromosomes that suggested a clonal-type evolution of the cell populations. Two cell lines, 333-8-9 and 14-012-8-1, showed two different marker chromosomes with large heterochromatic regions. Chromosomes with abnormal heterochromatic regions, which often appeared like prominent secondary constrictions, were found in all the cell lines we examined. The level of chromosome breakage was low in all the cell lines except the highly tumorigenic cell line 333-2-29, which had a high incidence of cells with single or double chromatinic bodies. The abnormal heterochromatic regions that occurred on marker chromosomes and prominent secondary constrictions were interpreted as a possible chromosomal effect of the HSV. The karyotypic stability and low incidence of open breaks might have been the result of UV irradiation of the HSV.


Blood group MN antigens and precursors in normal and malignant human breast glandular tissue.

Blood group M and N active substances were demonstrated in human mammary gland tissue in both benign and malignant lesions. The precursor T (Thomsen-Friedenreich) antigen occurred only in cancerous tissue, where it was found regularly in the 15 gross cancers tested. The precursor Tn antigen also was found regularly in cancerous breast tissue; Both antigens were reactive in breasts with in situ carcinoma. The T antigen was not demonstrable in the 6 benign mammary glands studied; similarly, the Tn antigen was unmasked by sialidase treatment of healthy breast tissues. Anti-T antibody, present in all human sera, was severely depressed in many breast cancer patients compared to controls.


A quantitative autoradiographic study of nucleolus-associated RNA and DNA synthesis during the eclipse phase in Rous sarcoma virus-infected chicken fibroblasts.

Functional and morphologic differences between the sensitivity of nucleoli of Rous sarcoma virus-transformed cells and that of newly infected cells to the action of actinomycin D (AD) have been demonstrated by quantitative light and electron microscope autoradiography and utilized to investigate the function of the nucleolus in the early stages of infection. After a pulse exposure to low doses of AD, increased RNA synthesis is induced within 80 minutes in the fibrillar portion of the nucleolus by infection. A concomitant increase in the retention of tritiated AD in the nucleolus and a quantitative redistribution of intranuclear and cytoplasmic DNA label are interpreted as evidence for a virus-induced amplification of the binding sites of AD in nucleolar chromatin.


Effect of simian virus 40 subcutaneous tumors on circulating lipids and lipoproteins in the Syrian hamster.

Circulating lipid levels and lipoprotein patterns in the Syrian hamster were determined at various times after subcutaneous inoculation with simian virus 40 (SV40) strain F, strain A-2895, or Fortner melanoma tumor cells. SV40 F tumors induced a rapid triphasic elevation of serum total lipids through inhibition of prebeta lipoprotein catabolism. Alpha lipoprotein levels declined in proportion to tumor mass. Liver wet weight and total lipid content increased significantly, but a normal rate of 3H-glycerol incorporation into polyanion precipitable (prebeta) serum lipoprotein was maintained. Determination of serum endogenous lipase, lecithin:cholesterol acyltransferase (LCAT), and cholinesterase activities indicated that these enzymes were not primarily responsible for the tumor-induced hyperlipidemia. Tumor-bearing animals also had selectively increased rates of protein and lipid excretion into the urine, with no evidence of gross hepatocellular or kidney damage. Growth of SV40 A-2895 tumors in hamsters resulted in a large increase in the rate of prebeta lipoprotein synthesis and degradation. Circulating prebeta lipoprotein levels were elevated much later in these animals, subsequent to a marked decrease in LCAT activity. Quite different results were obtained with Fortner melanoma, even large tumors having only a moderate effect on serum total lipid levels and lipoprotein patterns in the Syrian hamster.


Inhibition of the cellular immune response to simian virus 40 tumor cells in tumor-bearing and tumor-immune mice by concanavalin A.

The effects of in vivo-administered concanavalin A (Con A) on the kinetics of the primary and secondary cellular immune responses to simian virus 40-transformed tumor cells were investigated in BALB/c mice. Either a single initial dose of 400 mug Con A or daily doses of 50 mug depressed the cell-mediated immune response to tumor cells during the progressive growth of tumors, as determined by a radioisotopic foot-pad assay. The immune depression correlated with an increase in ultimate tumor weight. Similarly, Con A suppressed the antitumor cellular immune response in tumor-immune animals. Immune reactivity returned within 6 days after a single injection of 400 mug Con. Continuous administration 50 mug Con A resulted in a gradual decline in antitumor cellular immune responsiveness, which reached a plateau by the 5th day. Splenic lymphocytes from Con A-treated, immune mice failed to elicit a local adoptive transfer reaction; their immune responsiveness tended to return after incubation with alpha-methyl-D-pyranosyl sugars.


Properties of a chicken lymphoblastoid cell line from Marek's disease tumor.

Properties of a chicken lymphoblastoid cell line (MSB-1) from a Marek's disease tumor were studied. The cell line grew well at 41 degrees C in medium RPMI-1640 supplemented with 10% bovine fetal serum and had a doubling time of 8-12 hours. Cells grown in stationary suspension culture did not attach to the vessel and had the morphology of typical lymphoblasts. At 37 degrees C, the cell line grew initially but ceased to divide after several subcultures. In the subcultures maintained for 48-72 hours, 1-2% of the cells produced Marek's disease virus (MDV)-specific intracellular and mambrane antigens and contained herpesvirus particles when examined by the electron microscope. Cocultivation of these cells with duck or chicken embryo fibroblast cultures resulted in transfer of infection and production of microplaques typical of MDV. Peripheral nerve lesions and lymphoid tumors characteristic of Marek's disease were caused by inoculation of susceptible chicks with MSB-1 cells or duck cells infected with strain BC-1 of MDV recovered from the MSB-1 cell line. No specific tumors were produced at the site of inoculation, and infection was readily transmitted to cagemates. Tumors were also produced in the skeletal muscles and seemed to be largely virus induced. MSB-1 cell line was free of C-type virus particles.


Estrogen-dependent in vitro stimulation of RNA synthesis in hormone-dependent mammary tumors of the rat.

Incubation of estradiol in vitro at 25 degrees C with homogenates of carcinogen-induced mammary tumors of ovariectomized rats stimulated the magnesium-dependent RNA polymerase activity of nuclei of the hormone-dependent (HD) (regressing) tumors, but had no effect on this activity in nuclei of hormone-independent (HI) (growing) tumors. Furthermore, recombination of the nuclei and cytosol fractions of HD and HI tumors indicated that the in vitro effect of estradiol on subsequent tumor nuclear RNA synthesis required the estrogen receptor-containing cytosol but was specific to nuclei of the HD tumor. This constituted the first direct in vitro effect of estrogen on a specific biochemical process in an HD mammary tumor.


Spontaneous lymphoma associated with Herpesvirus saimiri in owl monkeys.

Two cases of lymphoma and one case of lymphoproliferative disease were found in a group of 7 owl monkeys imported into our colony as a single group. Herpesvirus saimiri (HVS) was isolated from the tumor cells of 1 lymphoma by cocultivation and from kidney cell cultures from the monkey with lymphoproliferative disease. Antibody to HVS was found in serum samples from 2 monkeys positive for HVS but not in the sera from the 4 clinically normal monkeys. Antibody to Epstein-Barr virus-infected cells was also found in the serum from the animal with lymphoma.


Virus envelope markers in mammalian tropism of avian RNA tumor viruses.

Pseudotypes of vesicular stomatitis virus were prepared with avian sarcoma viruses and avian leukemia viruses representing five different subgroups. These pseudotypes display a host range restricted to that of the avian tumor virus when assayed on avian cells and are neutralized by subgroup-specific antisera. The efficiency of penetration of mammalian cells was assayed by using these vesicular stomatitis virus pseudotypes. Pseudotypes of avian tumor viruses belonging to subgroup D and of B77 virus were able to plate on mammalian cells with a high efficiency, whereas pseudotypes of other strains were not. The efficiency of penetration of the vesicular stomatitis virus pseudotypes was 10-2-to 10-3-fold higher than the efficiency of transformation of the corresponding avian tumor virus strain assayed on mammalian cells, suggesting that there are postpenetration blocks to the expression of transformation in these cells.


Products of complementation between temperature-sensitive mutants of simian virus 40.

Temperature-sensitive mutants of the D complementation group of simian virus 40 exhibit delayed complementation. Analysis of the thermal stability, kinetic profiles in temperature shift experiments, and progeny of complementation have led to the hypothesis that delayed complementation is not true complementation, but the result of a very low level of leakiness, followed by phenotypic mixing of the progeny D mutants. This hypothesis is consistent with the proposal that D mutants are defective in uncoating. In the course of these experiments, it was observed that fresh medium suppresses the growth of D mutants at the restrictive temperature.


DNA infectivity and the induction of host DNA synthesis with temperature-sensitive mutants of simian virus 40.

Host DNA synthesis is induced when CV-1 (monkey kidney) cell cultures are infected at 40 C with wild-type virions or with temperature-sensitive Simian virus 40 mutants of the "early" complementation group A. Host DNA synthesis is not induced when cultures are infected with mutants of the late complementation group D. The simplest explanation for these observations, that induction depends not upon the expression of some early gene function but rather on the presence of an active D protein in the infecting virion, has been examined. Indirect experiments suggest that this explanation is not correct. Moreover, the induction of host DNA synthesis is impaired when cultures are infected with mutants of the A group at 42.5 C rather than 40 C, suggesting that the A function may be responsible for host induction. The inability of D virions to induce host DNA synthesis may reflect their inability to "uncoat" at 40C.


Molecular weight of RNA subunits of Rous sarcoma virus determined by electron microscopy.

Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV). Five days after infection, the medium was replaced at 2-h intervals with phosphate-free Eagle medium containing 50 muCi of [32P]orthophosphate per ml. Virus was collected by centrifugation, and the RNA was extracted and denatured with dimethyl sulfoxide, and the 33S subunit RNA was isolated by sucrose gradient centrifugation. The molecular weight of the RSV subunit RNA was determined by length measurement in the electron microscope, by using bacteriophage MS2 RNA as a length marker. Molecules of between 2.5 and 3.3 mum in length made up over 50% of the subunit RNA preparation. In this paper, we define RSV RNA subunits as that RNA released from the 70S RNA complex by dimethyl sulfoxide treatment, which sediments as a peak at 33S. Assuming the molecular weight of MS2 RNA to be 1.2 times 10-6, we calculate the molecular weight of RSV subunit RNA to be 3.12 times 10-6 plus or minus 0.25 times 10-6.


Electron microscope localization of a protein bound near the origin of simian virus 40 DNA replication.

A salt-stable complex of protein and viral DNA obtained from Simian virus 40 (SV40)-infected monkey cells or mature SV40 virions has a novel structure. When viewed by high resolution electron microscopy, the circular SV40 DNA molecule has bound to it one to three globular protein "knobs". Using ecoRI and hpaII restriction endonucleases, each of which can cleave SV40 DNA once at a known location (10, 11, 12, 14), the bound protein can be localized at 0.7 plus or minis 0.05 on the SV40 DNA physical map (SV40 fractional length, clockwise from the ecoRI endonuclease-cleavage site).


Characterization of human papovavirus BK DNA.

The DNA of the BK virus (BKV) human papovavirus was found to be heterogeneous, consisting of at least four discrete species of DNA. Only the largest of these four species, BKV DNA (i), which has a molecular weight calculated to be 96% that of simian virus 40 (SV40) DNA, was infectious. Homogeneous preparations of BKV DNA were obtained, however, from virions purified after low multiplicity infections of human embryonic kidney cells. BKV DNA (i) was shown to contain a single R-Eco RI and four R-Hind cleavage sites. The R-Eco RI site was localized in the largest R-Hind cleavage fragment. Radiolabeled BKV DNA reassociated slightly faster than SV40 DNA; 20 to 30% polynucleotide sequence homology was demonstrated between the genomes of BKV and SV40 when the reaction was monitored by chromatography on hydroxyapatite.


Studies of polyoma virus DNA: cleavage map of the polyoma virus genome.

A small-plaque polyoma virus, MPC-1, was isolated from a mouse plasmacytoma. The DNA of this polyoma virus was cleaved with a restriction enzyme from Haemophilus influenzae (Hin d), and the molecular weights of the limit products were analyzed by electrophoresis and electron microscopy. The fragments produced by this enzyme have been ordered by analysis of partial digest products. A physical map of the polyoma virus genome was then constructed.


RNA synthesis in cells infected with herpes simplex virus. IX. Evidence for accumulation of abundant symmetric transcripts in nuclei.

RNA extracted from nuclei of 8-h infected cells drove approximately 50% of herpes virus DNA into DNA-RNA hybrid. The same RNA, preannealed under conditions which allowed base pairing to take place, drove only 35% of the DNA into DNA-RNA hybrid; further annealing of the RNA did not diminish the amount of RNA sequences remaining available for subsequent hybridization with DNA. Upon denaturation of the preannealed RNA, the RNA sequences sequestered during preannealing became available again for hybridization with DNA. The base pairing that occurred during preincubation of the RNA was inter-molecular, since it was RNA concentration dependent and was not affected by limited alkaline hydrolysis. The nuclear viral transcripts that remained available for hybridization, after preannealing of the RNA, were subset of the RNA sequences that accumulated in the cytoplasm of infected cells. In addition, a small amount (derived from 5% or less of the viral DNA) of complementary transcripts was detected in the cytoplasm.


Screening procedure for complementation-dependent mutants of vesicular stomatitis virus.

To isolate new types of vesicular stomatitis virus (VSV) mutants, a four-stage screen was developed which identifies and characterizes mutants capable of complementing the defect in the VSV temperature-sensitive mutant tsG11. Two types of mutants of VSV, Indiana serotype, have been found by using the screen; they are new temperature-sensitive mutants which are, of necessity, not in complementation group I and mutants which do not produce plaques under conditions of single infection at 31 C (the normal permissive temperature) and are, therefore, called complementation-dependent mutants. The newly isolated, temperature-sensitive mutants fall into three complementation groups, two of which are congruent with known complementation groups; the newly identified group extends to six the number of complementation groups of VSV Indiana. The nature of the complementation-dependent mutants has not been established, but one was shown to not contain a significant deletion in its nucleic acid.


Temperature-sensitive virus derived from BHK cells persistently infected with HVJ (Sendai virus).

BHK-HVJ cells, a cell line of baby hamster kidney cells persistantly infected with HVJ (Sendai virus), started to produce infectious virus by shifting down the incubation temperature from 38 to 32 C. The virus derived from BHK-HVJ cells, designated as HJV-pB, was effectively neutralized with antibody against wild-type virus (HVJ-W) which was used for the establishment of BHK-HVJ cells. HVJ-pB replicated in eggs at 32 C, but not at 38 C, while HVJ-W grew equally well at both temperatures. When BHK cells infected with HVJ-PB were incubated at 38 C, production of infectious virus, hemagglutinin, and neuraminidase was markedly restrained, whereas a considerable amount of viral nucleocapisid and envelope antigens was detected in the cells by complement fixation tests. These viral activities became detectable immediately after temperature shift-down from 38 to 32 C even at the later stage of infection. HVJ-pB was indistinguishable from HJV-W with respect to particle size, density, and morphological characteristics, but appeared to possess a higher neuraminidase activity and was inactivated more rapidly at 50 C than HVJ-W. HVJ-pB was less cytocidal and could easily cause latent infection in BHK and mouse L cells.


Cycloheximide induction of xenotropic type C virus from synchronized mouse cells: metabolic requirements for virus activation.

The information for type C RNA viruses is genetically transmitted within the cellular DNA of the normal mouse cell. These viruses can be induced after exposure of cells to two classes of chemicals, inhibitors of protein synthesis and halogenated pyrimidines. The metabolic requirements for activation of one endogenous virus of BALB/c mouse cells by representatives of each class of drugs were studies. Cycloheximide and iododeoxyuridine each induce virus efficiently from cultures in exponential growth but are inactive on cells in stationary phase. However, cells are maximally sensitive to the actions of each drug at different times within the cell cycle. Further, virus induction in response to each is differentially inhibited under conditions of simultaneous cell exposure to inhibitors of DNA or RNA synthesis. The results provide support for the concept that inhibitors of protein synthesis and halogenated pyrimidines act by different mechanisms to induce type C virus release.


The 5' nucleotide sequence of vesicular stomatitis viral RNA.

The 5' nucleotide sequence of vesicular stomatitis viral RNA is pppApCpGp....


Analysis of uridine incorporation in chicken embryo cells infected by vesicular stomatitis virus and its temperature-sensitive mutants: uridine transport.

The shut-off of RNA synthesis in chicken embryo cells, after infection with vesicular stomatitis virus, is partially due to a reduced capacity of the infected cells to transport uridine. Permeability to uridine decreases exponentially after infection. This loss of ability to transport uridine may be caused either by structural components of the input virions or may result from the expression of the viral gene products. In the latter case, only minor levels of viral transcription is sufficient to modify cellular permeability, since, even at low multiplicities, RNA minus temperature-sensitive (ts) mutants of vesicular stomatitis virus bring about a significant diminution of uridine incorporation in cells infected under nonpermissive conditions. Experiments with mutants of group III suggest that the M protein of the viral envelope may play a role in the sequence of events that modifies uridine transport. In addition to this cause of the diminution of incorporation of uridine by infected cells, another mechanism is noted which requires protein synthesis.


In vitro polyoma DNA synthesis: requirement for cytoplasmic factors.

Purified nuclei from polyoma-infected mouse (3T3) cells were found to be greatly reduced in their ability to synthesize viral DNA in vitro when compared with a crude system consisting of an unfractionated hypotonic lysate of the infected cells. The synthetic capacity of the nuclei could be fully reconstituted when a high-speed cytoplasmic supernatant was added back to them. Cytosols from uninfected mouse, monkey, and hamster cells were equally as effective in stimulating purified nuclei as that of virus-infected mouse cells. Optimal complementation required high concentrations of the cytosol, and most of the complementing activity was destroyed by heating to 60 C. Dialysis had no effect on the activity. Analysis of the viral DNA synthesized in purified nuclei showed an accumulation of Okazaki-type short DNA chains, which could be chased into viral progeny DNA strands if cytosol was added back to the nuclei. Kinetic analysis of the pulse-labeling pattern of viral replicative DNA showed a strong dependence of the extension of viral progeny strands and of the processing of Okazaki-type fragments on the amount of cytosol present during the reaction. It is suggested that the cytoplasmic DNA polymerase might be one of the active components in the cytosol, but most likely not the only one.


Temporal appearance of bacteriophage T4-modified valyl tRNA synthetase in Escherichia coli.

Bacteriophage T4-induced modification of Escherichia coli vlayl-tRNA synthetase (EC 6.1.1.9) requires: synthesis of a phage-gene specified tau factor, addition of the factor to host valyl-tRNA synthetase to produce a urea-stable enzyme, and interaction of the modified enzyme with tRNA to produce a more rapidly sedimenting valyl-tRNA synthetase activity on sucrose density gradients. This report demonstrates that the coincident, chloramphenicol-sensitive appearance of urea-stable and rapidly sedimenting valyl-tRNA synthetase activity are immediate early phage functions. It implies that once the tau factor is synthesized, further interactions are stoichiometric rather than catalytic. The potential for valyl-tRNA synthetase modification accumylates when E. coli is infected with T4 PHAGE IN THE PRESENCE OF CHLORAMPHINICOL AND IS EXPRESSED DURING THE RESUMPTION OF PROTEIN SYNTHESIS WHEREAS FURTHER RNA synthesis is inhibited by rifampicin. The modification phenomenon occurs similarly in several strains of E. coli and represents a novel virus-host interaction.


Alteration of capsid proteins of coxsackievirus A13 by low ionic concentrations.

Several group A coxsackieviruses (A13, 15, 18, and 21), but not polioviruses or group B coxsackieviruses, are rapidly inactivated in low ionic strength solutions at neutral pH. The extent of inactivation is dependent upon temperature and molarity. Virions inactivated in this manner contain a normal complement of infectious RNA which remains in a state resistant to the action of ribonuclease. However, more than 95% of the virus particles are unable to attach to susceptible cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that coxsackievirus A13 virions contain five structural polypeptides (VP1, VP2a, VP2b, VP3, and VP4). Electrophoretic analysis indicates that inactivation of coxsackievirus A13 in low ionic strength solutions is due to the specific loss of the smallest polypeptide VP4 from the virus particle. These results suggest that adsorption of coxsackievirus A13 to receptors on susceptible cells is dependent upon the presence of the capsid protein VP4.


Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine.

Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.


Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny strands of the replicative intermediates of polyoma DNA. When high concentrations of such nuclei were incubated, short DNA fragments were formed and subsequently added onto growing progeny strands. When nuclei were repeatedly washed with buffer containing detergent and then incubated at low concentrations. DNA synthesis was decreased. In particular, the joining process was reduced, resulting in an accumulation of short DNA fragments. All aspects of the synthetic capacity of the nuclei were restored by addition of cytoplasmic extract. Additions of purified enzymes (polynucleotide ligase from calf thymus or Escherichia coli together with E. coli DNA polymerase I) increased the joining function of the nuclei. The system can be used for the identification of the enzymatic steps concerned with polyoma DNA replication.


Early events after infection of Escherichia coli by bacteriophage T5. Induction of a 5'-nucleotidase activity and excretion of free bases.

Thymine-containing compounds, produced degradation of Escherichia coli DNA after infection of the cells with bacteriophage T5, did not accumulate in the cell but were excreted into the medium as the DNA was degraded. The ultimate degradation product was extracellular thymine that was not reutilized when T5 DNA synthesis began. This excretion of thymine may have been due in part to the induction of 5'-nucleotidase activity within 3 min after T5 infection. The level of this activity reached a maximum between 4 to 6 min after infection and then rapidly declined to its preinfection level by 10 to 15 min after infection. Chloramphenicol added before or soon after infection prevented the appearance of the nucleotidase. The induced nucleotidase activity was active not only on dTMP but also on dAMP, dGMP, and dCMP.


Physiological characterization of temperature-sensitive mutants of mengovirus.

Twenty-four temperature-sensitive mutants of mengovirus were characterized physiologically with respect to phenotype. The mutants were separated into four classes on the basis of viral RNA synthesis. L-67-S cells infected with five of the mutants synthesized little viral RNA at 39.5 C. These mutants are designated RNA-. One mutant is designated RNA* since its RNA synthesis is altered at both 39.5 and 31.5 C. The other mutants were divided into two groups,  RNA plus or minus (25 TO 49% of wild-type RNA synthesis) and RNA plus (50 to 100% of wild-type RNA synthesis). The time of expression of the mutation in the RNA- mutants was estimated from the results of reciprocal temperature-shift experiments. The mutatation in ts12 appears to be expressed at the time RNA synthesis normally begins. The defect in three of the mutants was expressed 1 to 2 h before RNA synthesis is normally detectable. Protein synthesis is required before RNA synthesis begins when the cells are shifted from 39.5 to 31.5 C. The RNA polymerase synthesized by cells infected with these RNA- mutants at 31.5 C was stable and fully active when assayed at 39.5 C in vitro. The sedimentation profiles of the viral RNA synthesized by cells infected with RNA plus and RNA plus or minus mutants are similar to wild-type profiles with the exception of ts148. Cells infected with this RNA plus or minus mutant synthesize RNA that sediments in a sucrose gradient like replicative-intermediate RNA, but little mature viral RNA is evident. The results of step-up experiments indicate that the temperature-sensitive period for the majority of the RNA plus and RNA plus and minus mutants extends through most of the replicative cycle. The temperature-sensitive defect of four of the mutants, however, was expressed in the first hour, suggesting that some undefined early function is required for the eventual maturation of mengovirus. The virions of three of the RNA- mutants were more thermolabile than wild-type virions. Five of the RNA plus and RNA plus or minus mutants were also thermolabile. Genetic complementation at a significant level was not detectable in mixed infections of the mutants described.


Human cytomegalovirus stimulates host cell RNA synthesis.

Human cytomegalovirus infection of human fibroblast cells (WI-38) induced cellular RNA synthesis. The RNA synthesis in infected cultures preceded the synthesis of viral DNA and progeny virus by approximately 24 h. RNA species synthesized in infected cells included ribosomal 28S and 18S; and 4S transfer RNA; all were markedly increased in comparison to uninfected cells. This induction of host cell RNA synthesis was dependent upon a protein(s) that was synthesized during the early stages of infection.


In vitro synthesis of DNA in nuclei isolated from human lung cells infected with herpes simplex type II virus.

An isolated nuclei system prepared from herpes type II- and mock-infected human embryonic lung cells is able to synthesize cellular and viral DNA in the same proportion as in vivo at various times after infection. Incorporation of (3H)TTP in the in vitro reaction mixture requires Mg2 plus and ATP. Overall in vitro DNA synthesis in nuclei isolated from herpes-infected cells is semiconservative as demonstrated by bromodeoxyuridine-substituted DNA density-transfer experiments, but exhibits a significant fraction of repair-type replication. Relative rates of total DNA synthesis in vitro and in vivo are the same any time after infection. Isolated nuclei synthesize cell and viral DNA for a length of time and at a rate dependent upon the incubation temperature, but there are differences in the length of time of linear in vitro DNA synthesis between herpes- and mock-infected cells. The temperature optima for in vitro DNA synthesis differ significantly for herpes- and mock-infected cells, and are the same for cells abortively infected with herpes type II as for mock-infected cells.


Genetic transmission of endogenous N- and B-tropic murine leukemia viruses in low-leukemic strain C57BL/6.

Spontaneous expression of endogenous N- and B-tropic murine leukemia viruses was stu1bb), DDD (Fuv-1nn), DDD-Fvr (fv-1nn), (DDD or DDD-Fvr times C57BL/6)F1, and 16 partially inbredlines with either the Fv-1nn or Fv-1bb genotype, which had been established from hybrids between C57BL/6 and DDD-Fvr. When tested at middle age, virus-positive mice were found in C57BL/6, F1 hybrids, and 9 out of 16 partially inbred lines. N-tropic viruses were isolated from Fv-1nn, Fv-1bb mice, whereas B-tropic viruses, except for one isolate, were from Fv-1bb mice only. C57BL/6 mice were positive for both N- and B-tropic viruses, whereas DDD-Fvr mice were negative. With respect to the Fv-1 genotype and the presence of endogenous murine leukemia viruses, the partially inbred lines were grouped into five types: (i) Fv-1bb, both N- and B-tropic virus positive, like C57BL/6; (ii) Fv-1nn, virus negative, like DDD-Fvr; (iii) Fv-1bb, virus negative; (iv) Fv-1nn, only N-tropic virus positive; and (v) less convincingly, Fv-1bb, only B-tropic virus positive. These findings indicate that the transmission of N- and B-tropic viruses in C57BL/6 is genetically controlled and that the expression of B-tropic virus, but not of N-tropic virus, is closely associated with the Fv-1 genotype.


Scanning electron microscopic studies of virus-infected cells. I. Cytopathic effects and maturation of vesicular stomatitis virus in L2 cells.

L2 cells infected with vesicular stomatitis virus under single-cycle conditions have been studied by scanning electron microscopy after preparation by the critical point drying technique. Three dimensional images of intact cells show bullet-shaped vesicular stomatitis virus virions budding singly and in radiating clusters both from the plasma membrane between cellular microvilli and from the sides of microvilli. Virus-induced cytopathic effects observed by scanning electron microscopy include intermeshing of microvilli, loss of filipodia which attach cells to the substrate, and rounding up and detachment of infected cells from the substrate.


Inhibition of bacteriophage PBS2 replication in Bacillus subtilis by phleomycin.

Phleomycin is an effective inhibitor of the replication of Bacillus subtilis bacteriophage PBS2, whose DNA contains uracil instead of thymine. Phleomycin does not affect the induction of the known phage enzymes involved in deoxyribonucleotide metabolism. But phage DNA synthesis is severely inhibited by phleomycin, and late virion protein synthesis is eliminated. These effects appear to result from a phleomycin-induced degradation of the parental phage DNA. Similar inhibitory and degradative effects on DNA are seen in phleomyinc-treated, uninfected cells. This system is unaffected by the related antibiotic, bleomycin.


Structural proteins of simian virus 40. I. Histone characteristics of low-molecular-weight polypeptides.

The DNA-associated polypeptides of simian virus 40 (SV40), VP4 (mol wt 14,000), VP5 (mol wt 12,000), and VP6 (mol wt 11,000), have several properties characteristic of cell histones. After extraction from purified SV40 with dilute acids, these three polypeptides co-electrophoresed on low pH polyacrylamide gels with monkey-kidney cell histones F3, F2b, and F2a1. No virus polypeptide co-electrophoresed with histone F1. Polypeptides VP4, 5, and 6 lacked tryptophan, and only VP4 contained cysteine, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of virus labeled in vivo with (3H)lysine and either (14C)tryptophan or (35S)cystine. All of the capsid polypeptides VP1, 2, and 3 contained tryptophan whereas only VP1 and 2 contained cysteine. In addition, VP4, 5, and 6 are rich in arginine and lysine when compared with virus labeled with a mixture of amino acids. Analysis of virus grown in cells labeled prior to infection showed that VP4, 5 and 6 were labeled fivefold greater than the major capsid polypeptide, VP1, which indicates that they were partially derived from preexisting cell histones. Based on these data and on previously determined molecular weight estimates, we conclude that VP4, 5, and 6 are histones F3, F2b, and F2a1, respectively, although the possibility that SV40 contains a small amount of F2a2 could not be excluded.


Polyoma virus T antigen. I. Synthesis of modified heat-labile T angiten in cells transformed with the ts-a mutant.

In mouse cells transformed with the ts-a mutant of polyoma virus (ts-a-3T3), only low amounts of the virus-specific T antigen were synthesized at high temperature (39 C). After a shift-down to the permissive temperature (31 C), these cells exhibited the same level of T-antigen production as wild-type polyoma transformants. The T antigen produced by ts-a-transformed cells was inactivated at 39 C in vitro at a faster rate than that produced by wild-type-transformed cells. These observations indicate that T antigen is, or includes, a virus-coded peptide.


Malignant transformation of hamster kidney cells by BK virus.

Primary hamster kidney cells were transformed by BK virus, a new human papovavirus. Transformed (HKBK) cell produced BK virus T antigen and induced tumors in hamsters that developed antibodies to BK virus T antigen. BK virus was rescued from HKBK cells by Sendai virus-assisted fusion with permissive cells. One out of six cell lines derived from HKBK cell-induced tumors showed the same characteristics as HKBK cells.


Action of interferon in enucleated cells.

Interferon induces protection of enucleated BSC-1 cells against infectious vesicular stomatitis virus production if cells are treated before, but not after, enucleation.


Posttranscriptional selection of simian virus 40-specific RNA.

Analysis of the viral-specific RNA in simian virus 40(SV40)-infected monkey kidney cells indicated the extensive transcription of both DNA strands. These symmetrically transcribed sequences were localized in the nucleus of infected cells, whereas only the "true" early and late SV40 transcripts were found in the cytoplasm. These results suggest that selective posttranscriptional degradation and/or transport occurs after transcription of the viral DNA. On the basis of hybridization experiments with cytoplasmic RNA and the separated strands of the SV40 Hin fragments, the early SV40 region appears to include all of Hin fragments A, H, I, and B (48% of the genome), whereas the late region is represented in Hin fragments C, D, E, K, F, J, and G (52% of the genome).


Proteins in intracellular simian virus 40 nucleoportein complexes: comparison with simian virus 40 core proteins.

Intracellular nucleoprotein complexes containing SV40 supercoiled DNA were purified from cell lysates by chromatography on hydroxyapatite columns followed by velocity sedimentation through sucrose gradients. The major protein components from purified complexes were identified as histone-like proteins. When analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, complex proteins comigrated with viral core polypeptides VP4, VP5, VP6, and VP7. (3H) tryptophan was not detected in polypeptides from intracellular complexes or in the histone components from purified SV40 virus. However, a large amount of (3H) tryptophan was found in the viral polypeptide VP3 relative to that incorporated into the capsid polypeptides VP1 and VP2. Intracellular complexes contain 30 to 40% more protein than viral cores prepared by alkali dissociation of intact virus, but when complexes were exposed to the same alkaline conditions, protein also was removed from complexes and they subsequently co-sedimented with and had the same buoyant density as viral cores. The composition and physical similarities of nucleoprotein complex and viral cores indicate that complexes may have a role in the assembly of virions.


Quantitative assay for transformation of 3T3 cells by herpes simplex virus type 2.

The interaction of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) with Swiss/3T3 cells was investigated. Virus-induced cytopathic effects developed in the absence of production of infectious virus. HSV-2 inactivated with UV light (2, 4, 6, and 8 min) also induced cell death in the absence of virus replication. Cell death was not detectable after infection by HSV-2 that had been inactivated by UV irradiation for 10, 12, and 14 min. 3T3 cells infected with UV-inactivated virus (10 and 12 min) continued to replicate past the contact-inhibited monolayer normally associated with these cells. Infection of 3T3 cells with UV-irradiated USV-2 also induced the development of transformed foci. Transformed cells with an epithelioid of fibroblastoid morphology were identified and isolated. All HSV-2-transformed cell lines contained HSV-2-specific antigens detectable by immunofluorescence techniques. The maximum frequency of HSV-2-induced transformation was 3 times 105 PFU per transformed focus, and the observed transformation could be inhibited by pretreatment of the virus with specific antiserum. No type C particles were detected within five cell culture passages after transformation by HSV-2. Type C virus particles were detected after 10 cell culture passages of the HSV-2-transformed cell lines.


Stimulation of non-histone chromosomal protein synthesis in simian virus 40-infected simian cells.

The pattern of synthesis of non-histone chromosomal proteins in simian virus (SV) 40-infected African green monkey kidney cells was analyzed by polyacryl-amide gel electrophoresis to see whether the changes in chromosomal protein metabolism are involved in the viral-induced synthesis of cellular DNA and mRNA. During the prereplicative phase of infection, the rate of histone synthesis was decreased until 15 h postinfection, whereas that of non-histone protein synthesis was increased after 5 h postinfection and reached a maximum at 10 to 15 h postinfection when viral-induced synthesis of cellular DNA and mRNA began to be observed. Stimulation of non-histone protein synthesis was also observed in the infected cells treated with cytosine arabinoside and was dependent on the multiplicity of infection. Stimulation occurred in almost all species of non-histone proteins. These results suggest that the stimulation of non-histone protein synthesis is caused by an early SV40 function and occurs prior to the viral-induced synthesis of cellular DNA and mRNA. During the replicative phase of infection, a marked increase in the rate of synthesis was observed in the non-histone proteins with molecular weights of about 48,000, 35,000, and 23,000, which were subsequently found to be SV40 capsid proteins.


Induction of human cell DNA synthesis by herpes simplex virus type 2.

The effect of herpes simplex virus type 2 (HSV-2) infection on the synthesis of DNA in human embryonic fibroblast cells was determined at temperatures permissive (37 C) and nonpermissive (42 C) for virus multiplication. During incubation of HSV-2 infected cultures at 42 C for 2 to 4 days or after shift-down from 42 to 37 C, incorporation of (3H)TdR into total DNA was increased 2-to 30-fold as compared with mock-infected cultures. Analysis of the (3H)DNA suggested that host cell DNA synthesis was induced by HSV-2 infection. Induction of host cell DNA synthesis by HSV-2 also occurred in cells arrested in DNA replication by low serum concentration. The three strains of HSV-2 tested were capable of stimulating cellular DNA synthesis. Virus inactivated by UV irradiation, heat, or neutral red dye and light did not induce cellular DNA synthesis, suggesting that an active viral genome is necessary for induction.


Phosphoproteins: structural components of oncornaviruses.

Oncornaviruses, which contain a virion-associated protein kinase, were found to possess phosphoproteins as virion structural components. One major phosphoprotein common to strains of laboratory and wild mouse oncornaviruses and a strain of feline leukemia virus was shown to be a polypeptide of about 12, 000 mol wt. In addition to this, the Kirsten strain of murine sarcoma virus contained a second major phosphoprotein of about 10, 000 mol wt, and mouse erythroblastosis virus contained a second major phosphoprotein that was either identical to or comigrated with the virion glycoprotein of about 74, 000 mol wt. The major phosphoprotein of RD-114 virus was found to be of about 16, 000 mol wt. The major phosphoamino acid of the 12, 000-mol wt polypeptide of the mouse erythroblastosis virus was identified as phosphoserine, and that of the 16, 000-mol wt polypeptide of the RD-114 virus was identified as phosphothreonine.


Interspersion of sequences in avian myeloblastosis virus rna that rapidly hybridize with leukemic chicken cell DNA.

Liquid hybridization of progressively smaller fragments (35S, 27S, 15.5S, 12.5S, and 8S) of poly(A)-selected avian myeloblastosis virus RNA with excess DNA from leukemic chicken myeloblasts revealed that all sizes of RNA contained sequences complementary to both slowly and rapidly hybridizing cellular DNA sequences. Apparently, the RNA sequences which hybridize rapidly with excesses of cellular DNA are not restricted to any one region of the avian myeloblastosis virus 35S RNA. Instead, they appear to be randomly distributed over the entire 35S avian myeloblastosis virus RNA molecule with some positioned within 200 nucleotides of the poly(A) tract at the 3' end of the RNA.


DNA of Epstein-Barr virus. I. Comparative studies of the DNA of Epstein-Barr virus from HR-1 and B95-8 cells: size, structure, and relatedness.

We have compared the properties of the DNA of Epstein-Barr virus (EBV) purified from HR-1 (EBV HR-1 DNA) and B95-8 (EBV B95-8 DNA) continuous lymphoblast cultures. Our data indicate that (i) the S suc of native EBV DNA relative to T4D DNA is 55S. Using the modified Burgi-Hershey relationship (5), we estimate the molecular weight of native EBV DNA is 101 (plus or minus the molecular weight of native FBV DNA by measurement of the length of 3) times 106. Estimation of the molecule relative to form II PM2 DNA yields a value of 105 (plus or minus 3) times 106. (ii) After alkali denaturation, less than 50% of EBV DNA sediments as a single band in alkaline sucrose gradients in the region expected for DNA of 50 times 406 daltons. (iii) Intact EBV HR-1 and EBV B 95-8 DNAs band at 1.718 g/cm3 and a smaller band (approximately 25% of the DNA) AT 1.720 G/CM3. (IV) EBV HR-1 DNA possesses greater than 97% of the sequences of EBV B95-8 DNA. Hybrid DNA molecules formed between (3H)EBV HR-1 DNA and EBV HR-1 DNA or EBV B95-8 DNA had identical thermal stability. EBV B95-8 DNA lacks approximately 15% of the DNA sequences of EBV HR-1 DNA. We interpret these data to mean that EBV B95-8 is derived from a parental EBV through loss of genetic complexity. This defect may be linked to the ability of EBV B95-8 to "transform" lymphocytes invitro.


Simian virus 40 functions required for the establishment and maintenance of malignant transformation.

Members of the five classes of temperature-sensitive simian virus 40 mutants were tested for their ability to transform Chinese hamster lung cells. Two criteria for transformation were used: the ability to form clones in medium with low serum concentrations and the ability to overgrow a monolayer. Only A mutants failed to transform at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. However, both A and D mutants failed to transform at the restrictive temperature when confluent monolayers and depleted medium were used. When transformed clones were selected, purified by recloning, and examined at both temperatures, only cell lines induced by A mutants lost the transformed phenotype at the higher temperature. Thus, A function is required for maintenance of the transformed phenotype in Chinese hamster lung cells. A function is known to be required for the initiation of viral DNA synthesis in permissive cells. Therefore, transformation may be a consequence of the introduction into a cell of the capacity for aberrant initiation of DNA replication.


Function of simian virus 40 gene A in transforming infection.

In productive infection by simian virus 40, the A gene is known to regulate the initiation of viral DNA replication and to control the synthesis of late viral RNA. The function of the A gene in transforming infection was investigated by the infection of a variety of cell species with six independently isolated temperature-sensitive mutants belonging to the A complementation group. The A mutants failed to initiate the stable transformation of cells during continuous infection at the restrictive temperature. After the establishment of transformation at the permissive temperature and a subsequent shift to the restrictive temperature to block the A function, however, two distinct virus-cell interactions were identified. In one case, the increased colony-forming capacity of transformed cells remained stable after the temperature shift. In the other case, the temperature shift decreased the capacity of transformed cells to form colonies to the level of untransformed control cells. The outcome of the virus-cell interaction depended both on the nature of the A mutation in a given cell species and on the species of the cell transformed by a given mutant. These findings suggest that the transformation process may require two distinct events, each related to A gene expression.


Role of simian virus 40 gene A function in maintenance of transformation.

Mouse, hamster, and human cells were transformed at the permissive temperature by mutants from simian virus 40 (SV40) complementation group A in order to ascertain the role of the gene A function in transformation. The following parameters of transformation were monitored with the transformed cells under permissive and nonpermissive conditions: morphology; saturation density; colony formation on plastic, on cell monolayers, and in soft agar; uptake of hexose; and the expression of SV40 tumor (T) and surface (S) antigens. Cells transformed by the temperature-sensitive (ts) mutants exhibited the phenotype of transformed cells at the nonrestrictive temperature for all of the parameters studied. However, when grown at the restrictive temperature, they were phenotypically similar to normal, untransformed cells. Growth curves showed that the (ts) A mutant-transformed cells exhibited the growth characteristics of wild-type virus-transformed cells at the permissive temperature and resembled normal cells when placed under restrictive conditions. There were 3-to 51-fold reductions in the levels of saturation density, colony formation, and uptake of hexose when the mutant-transformed cells were the elevated temperature as compared to when they were grown at the permissive temperature. Mutant-transformed cells from the nonpermissive temperature were able to produce transformed foci when shifted down to permissive conditions, indicating that the phenotypically reverted cells were still viable and that the reversion was a reversible event. SV40 T antigen was present in the cells at both temperatures, but S antigen was not detected in cells maintained at the nonpremissive temperature. All of the wild-type virus-transformed cells exhbited a transformed cells exhibited a transformed phenotype when grown under either restrictive or nonrestrictive conditions. Thers results indicate that the SV40 group A mutant-transformed cells are temperature sensitive for the maintenance of growth properties characteristics of transformation. Virus rescued from the mutant-transformed cells by the transfection method was ts, suggesting that the SV40 gene A function, rather than a cellular one, is responsible for the ts behavior of the cells.


Simian virus 40 gene A function and maintenance of transformation.

Transformants have been isolated after infection of rat embryo cells at 33 C with either wild-type simian virus 40 or with the temperature-sensitive gene A mutants, tsA7 and tsA28. Examination of properties usually associated with transformation such as growth in 1% serum, growth rate, saturation density, and morphology show that these properties are temperature dependent in the tsA transformants characterized, but are not temperature dependent in the wild-type transformants that have been examined. In the most thoroughly characterized tsA transformants the expression of T antigen also appears to be temperature dependent. These data suggest that an active A function is required for the maintenance of transformation in these cells. In the lytic cycle, the A function is involved in the initiation of DNA synthesis. Thus transformation by simian virus 40 may be the direct consequence of the introduction of the simian virus 40 replicon and the presence of its DNA initiator function, which causes the cell to express a transformed phenotype.


Formation of nucleoprotein complexes between polyoma empty capsides and DNA.

Purified polyoma empty capsids and polyoma type I DNA interact in a cell-free system to form nucleoprotein complexes. Complexes that consist of one, two, three, and four empty capsids per DNA molecule have been detected. Polyoma virions or capsomers do not react with added DNA to form such complexes.


Detection of a virus-specific antigen on the surface of herpes simplex virus-transformed cells.

A virion-associated antigen, CP-1, was detected on the surface of hamster cells after transformation by inactivated herpes simplex virus type 1. Presence of the CP-1 antigen on the cells correlates with the synthesis of antibody by tumorbearing hamsters capable of neutralizing both herpes simplex virus types 1 and 2.


Ectopic production of erythropoietin in Wilms tumor patients in relation to clinical stage and disease activity.

Measurement of erythropoietin levels was achieved in urine, plasma and tumor extract specimens in 27 Wilms tumor patients. Good correlation of the stage of the disease, the clinical judgment of inactive or responding tumor foci and erythropoietin levels was noted. Erythropoietin levels are believed to reflect ectopic hormone production by the primary or secondary tumor. Since the erythropoietin hormone assayed in these and other Wilms tumor patients was not associated with polycythemia, it is believed that Wilms tumor-associated erythropoietin differs in some manner from the hormone produced normally in man and possible in other states associated with secondary polycythemia. Etythropoietin assay with further purification and technical development can be of additional widespread assistance in the clinical classification and subsequent management of the tumor state. Our study confirms this biological potential.