Irving S. Johnson

Oncogenicity of the Simian Adenoviruses

Five of 17 adenoviruses of rhesus or cynomolgus monkey origin induced tumors in newborn hamsters. The tumors appeared between 42 and 280 days after subcutaneous inoculation and had the general characteristics of lymphomas. The tumors were specific by cross-complement fixation tests. An adenovirus recovered from Cercopithecus monkeys appeared to be highly oncogenic; all 23 inoculated hamsters developed tumors within 30 to 40 days.

Comparative effects of some cardioactive agents on automaticity of cultured heart cells.

Abstract Cells from mouse hearts, disaggregated with collagenase were cultured in F 12 medium. These cells were viable for approximately 6 months. Using a sensitive monitoring system that allowed recording of both chronotropic and inotropic responses, we studied the effects of several cardioactive agents on the contractility of the cultured cells. The ventricular cells responded to norepinephrine, but not to guanethidine or tyramine. Since both guanethidine and tyramine release norepinephrine from adrenergic nerve endings, the lack of response to guanethidine and tyramine indicate that the heart cells are not innervated by adrenergic nerves. Atrial cells, but not ventricular cells, are responsible to acetylcholine. Dibutyryl cyclic AMP, but not cyclic AMP, elicited a positive inotropic and chronotropic response. Both positive inotropic and chronotropic responses were observed following glucagon administration and these effects were not antagonized by propanolol. Both epinephrine and T 3 elicited a positive inotropic effect but through different mechanisms. Arrhythmia and fibrillation of the cells could be induced by aconitine. The culture method and significance of responsiveness of the cultured cells are discussed.

Biological activities of 3-isoadenosine

Abstract An isomer of adenosine, 3-β- d -ribofuranosyladenine (3-isoadenosine), has been studied in a number of bacterial and mammalian cell systems. While 3-isoadenosine readily supported the growth of the adenine-requiring Escherichia coli B97, it failed to support the growth in tissue culture of a murine cell line rendered dependent on an exogenous purine source by the folic acid antagonist amethopterin. 3-Isoadenosine inhibited the growth of various mammalian cell lines in Eagles medium at levels of 10−4 to 10−6 M and it displayed a cytotoxicity for the lymphoblastic leukemia cell line L5178Y of the same order as that of 6-azauridine. When tested by an agar overlay method, 3-isoadenosine also inhibited the growth of Adeno III virus in tissue culture. In order to investigate the inhibitory activity of 3-isoadenosine, comparative experiments were carried out in the above systems with other nucleoside analogs (psicofuranine, pseudouridine, triacanthine, etc.), and an unsuccessful attempt was made to reverse this inhibition with nucleosides and other complex materials. A daily dose of 3 mg/kg given intraperitoneally to mice for ten days was well tolerated but 6.0 mg/kg was toxic. The final phase of this study was the evaluation of 3-isoadenosine in tumor-bearing and virus-infected animals. The interpretation of the observed biological activity in terms of the underlying biochemical mechanisms has been attempted by comparison of the activities of 3-isoadenosine and of related nucleosides and other agents. This comparison revealed a striking similarity in the characteristics of inhibition of mammalian cells in tissue culture by 3-isoadenosine and by 7-deaza-adenosine.

Extended Production of Insulin by Isolated Rabbit Pancreatic Islets; Evidence for Biosynthesis of Insulin

Summary Methods for prolonged primary suspension cultures of rabbit pancreatic islets were established, and incorporation of carbon-14 labeled amino acids into insulin and other proteins was demonstrated.

Possible utility of the Rous sarcoma for antitumor screening.

This paper is an interim report on a study that is still in progress. Previous reports’sZ have described the utility of a Rous sarcoma system in the detection of antiviral activity of a highly selected group of antibiotic broths as correlated with concurrent tests in a mammalian virus system. In this test 0.2 ml. of arbitrarily selected log dilutions of semipurified Rous sarcoma virus (RSV) * in tissue culture medium 199 containing 10 per cent inactivated horse serum are injected between the 2 layers of skin of the wing web of groups of 14-dayold White Leghorn chicks. This infection results in a localized wing tumor which, if allowed to go unchecked, metastasizes, killing the host, but may be modified by intraperitoneal treatment of the host with various agents. These tumors are easily removed at the end of the experiment and may be weighed to obtain mean tumor weights per bird for comparison with those of salineor water-treated controls. Such comparisons in the case of the group of highly selected antiviral antibiotic broths resulted in dose-response curves and highly reproducible effects; in some cases these approximated 100 per cent inhibition of the tumors. In the present study a second group of antibiotic broths was selected on the basis of possible activity against transplantable murine neoplasms. These broths have given a 30 per cent or greater inhibitionof Sarcoma 180; prolonged life by 20 per cent or more when tested against the P 1534 leukemia; or, in a few rare instances, proved active against both murine tumors. An important difference should be borne in mind concerning the effectiveness of the selection systems for the antiviral and antitumor broths. In the former case, the antiviral broths had repeatedly given evidence of activity against a mammalian virus. The antitumor broths, on the other hand, had been for the most part of a low order of reproducibility when they were at all reproducible, and the effects had never been more than minimal. We did not expect to obtain the same degree of correlation of activity or magnitude of response between the RSV and mouse tumor systems as was obtained between the RSV and mammalian virus systems. We did feel that concurrent tests of the same fermentation lots of these broths in the RSV and murine tumor systems might furnish a preliminary evaluation of the utility of the RSV as a screening tool for agents possessing potential chemotherapeutic activity in experimental malignancy. A far greater number of the broths tested significantly inhibited the Rous tumor, that is, by more than 35 per cent, than did either of the mouse tumors when tested concurrently (TABLE 1) . Slightly more than 50 per cent of the broths were positive by our standards in the Rous test, while approximately 10 per cent still indicated activity in the murine systems. These data may well * Lot CT694 of this virus was prepared and supplied to us by W. Ray Bryan of the National Cancer Institute, Public Health Service, Bethesda, Md.

Metabolites from animal and plant cell culture.

Publisher Summary This chapter compares transformation of organic compounds in cell cultures of higher plant and animal origin. It includes reports of any metabolites that have been produced by cell cultures, with the exception of cells from insects, invertebrates, and cold-blooded vertebrates. Although animal cells presumably contain all genetic material necessary for totipotentiality, a marked difference between animal and plant cells is noted while demonstrating this phenomenon in culture. Exogenous DNA may alter synthesis of animal cellular products by induction. A large number of proteins have been produced by many types of both plant and animal cells. A number of polysaccharides and glycoproteins, as well as other high molecular weight materials, are produced in the culture of both plant and animal cells. These include the hydroxyproline-containing proteins such as collagen. The chapter closes with the future applications of plant and animal cell cultures. It states that a great deal research on animal cells are toward establishment of long-term continuous cell strains.

OBSERVATIONS ON ANTIVIRAL SCREENING

The organization of this meeting is indicative of a change in scientific opinion during the past few years. I think it is fair to state that the majority opinion of the informed scientific community, prior to 1959, would be that no drug would ever be useful in the treatment of an acute virus infection. Fortunately, for the purposes of this meeting, there were some who took exception to this view. This change of opinion was, in my view, greatly stimulated by at least four factors. These were: ( 1 ) the increasing evidence for a causal relationship, of viruses and malignant disease; (2) the recent successful clinical experience by systemic therapy with at least two compounds, which presumably would not exert genetic effects; (3 ) the flood of new information related to the biochemical processes of viral replication at the molecular level including the evidence for enzymes specifically induced by the viral infection; and (4) the great multiplicity of infectious agents concerned in the upper respiratory infections with the concommitant problems of low yields of many of them in tissue culture, their generally poor antigenicity, and the difficult although not insurmountable problems concerned with their concentration into polyvalent vaccines. I would emphasize that improved technology could solve some of the problems of a vaccine approach in this area, but a drug active in the same clinical area would be of considerable interest. The problem discussed in this session of the conference is the detection and evaluation of potential antiviral drugs. There are two sources available and two approaches to this problem. The two sources are obviously fermentation products and synthetic compounds with the possible addition of plant extracts. The two approaches are empirical “blind screening” or molecular modification of existing “active” compounds. We have tried to utilize all of these sources and approaches in the past few months on a small scale. The papers by DeLong and by Stone are based on findings via the empirical approach, and Dr. Gerzon’s discussion concerns work via the molecular modification approach. We have been using essentially a five-virus primary screen and have worked in v ivo for detection purposes as much as possible (TABLE 1) . Like Dr. Ehrlich, we felt that it was desirable to have a spectrum of viruses represented and a “model system” as well. Dr. Ehrlich has described some pitfalls of in virro antiviral screening which includes the isolation of tissue culture “actives” which are known antibiotics with little or no in v ivo activity. These pitfalls could be met by testing fermentations previously selected for a lack of antimicrobial activity or by screening in v ivo where most of these uninteresting materials are ineffective and would not be detected. Screening in v ivo obviously limits the number of materials which can be examined, but “positives,” when found, are less apt to be false positives. I t is our experience and, I think that of others, that most antiviral compounds active in v ivo are active in vitro, but the reverse is not necessarily true. If I interpret Dr. Buthala’s data correctly, over 85 per cent of the in v ivo active materials are active in virro. One must weigh the advantages of fewer false positives against large numbers of false leads and the time spent on running them down and proving them to be false. In virro systems for assays and mechanism studies of existing in v i w leads are a necessity. Our experience with in v i vo screen-

Fractionation by zonal centrifugation of brain of normal rats and rats treated with morphine

Abstract The brains of normal or treated rats were homogenized by explosive nitrogen decompression. The homogenate was fractionated using a sucrose-Ficoll gradient in a B-XXIX zonal centrifuge rotor. Three characteristic peaks of subcellular particulate were routinely obtained from normal rat brains. Brains from rats treated with morphine routinely contained only two of the particulate peaks. The location of peak activities of acetylcholin-esterase and acid phosphatase were shifted in morphine-treated animals.

IN VIVO AND IN VITRO INHIBITION OF HEPATITIS VIRUSES BY STREPTOTHRICIN

In an in vivo antiviral screening procedure recently instituted in our laboratories, an attempt was made to include a spectrum of human viral types for which a comparable small animal infection was available. In order to widen the spectrum, it was desirable to include a hepatitis-causing virus. The recent .reports of viral isolations from human hepatitis-both infectious and serum hepatitis-have indicated that major progress is being made in demonstrating the etiological agents in these However, none of the agents has been clearly shown to cause infections in small animals. On the other hand, a number of isolations have been made from hepatitis in mice and have been the subjects of numerous st~dies.~-lO Accordingly, it was decided to screen antibiotic broths in vivo against a mouse infection. The MHV3 strain, isolated by Dick et aL8 was selected. Subsequently, any active agents were to be tested against a human isolate. For the latter, the A-1 virus of O’Malley et a1.I was selected. In the course of the investigations, a broth filtrate repeatedly showed activity against the MHV3 virus. This observation and subsequent studies are the subject of this report.

Summary of Discussion

Following Dr. Lacys film on the secretion of insulin and morphology of |3 cells of islets, Dr. Werner Creutzfeldt pointed out that the characteristic shape of (3 cell granules in various species is different—e.g. needleshaped in dogs, round in other species, etc. He raised the questions whether submicroscopic morphology of granules could be related to different amino acid sequences of the insulin they contained, which might result in different crystalline arrangements, and whether other factors might also be involved. Dr. Creutzfeldt projected electron micrographs of several human insulin-producing (3 cell tumors. He classified these into four types on the basis of granule morphology: 1. (3 cell tumors with typical multishaped human /? granules. These are aldehyde-thionin positive. 2. j3 cell tumors with atypical /? granules. Most of these granules have only tight membranes and no membranous sac. Therefore they resemble a granules but are aldehyde-thionin positive. 3./? cell tumors of mixed types which contain both types of granules. 4. jB cell tumors which contain no f3 granules and are aldehyde-thionin negative. Biochemically, there was little difference in insulin or proinsulin content of these tumors except that the level of proinsulin was 10-20 per cent, which is two to fourfold higher than that found in normal human pancreas. All except the tumors without granulation respond to diazoxide treatment. Dr. Creutzfeldt suggested that diazoxide might block the secretion of insulin from the storage form by blocking emiocytosis and granule dissolution but not insulin secretion directly. A paper by Orci has suggested two different mechanisms of insulin secretion, namely, emiocytosis and the intracellular dissolution of granules. As evidence for this line of reasoning, G cells in the antrum mucosa secrete gastrin only by dissolving granules within the cell. There has never been any evidence for secretion by emiocytosis in these cells. Secretin is released by distention and also by a change in pH. Generally speaking, both methods of secretion are possible or have been observed in endocrine systems. Dr. Creutzfeldt further commented on the enormous amount of gastrin within the D cells of the islets as shown by Greider and Lacys immunofluorescent antibody staining technics. He had found it difficult, however, to extract gastrin from human pancreas and questioned the specificity of the immunoreactive cells and the antibodies. Morphologically, the G cell of the antrum is quite different from the D cell of the pancreas. Therefore, Dr. Creutzfeldt felt it was too early to add a gastrin-secreting cell as a normal physiological function of the islet of Langerhans in man. Dr. Paul E. Lacy responded to some of the questions posed by Dr. Creutzfeldt, stating that in order to resolve the question of whether different granular structure in various species reflected different amino acid sequence or crystalline arrangement, pure granule fractions would be required. It is difficult to achieve this with present technics because a and /? granules occur in the same isolated fractions. Moreover, the granules within a single /? cell appear heterogeneous when examined by electron microscopy. Dr. Lacy pointed out that Orci has demonstrated by freeze-etching that emiocytosis is the major mechanism of secretion. In a normal /? cell there probably is no other mechanism of secretion, but in a partially degranulated or chronically stimulated cell there might be other mechanisms of secretion as yet undefined. Concerning gastrin in the pancreas, Greider and McCuigan have demonstrated cells in isolated human islets and in intact human pancreas which react with gastrin antibodies labeled with fluoroscein and have applied the necessary controls. There has been no reaction with other species (rat, guinea pig, etc.). Because positive identification of gastrin in D cells requires peroxidase-labeled antibodies coupled with electron microscopy, Dr. Lacy acknowledged the possible prematurity of the D cell as a gastrin secretor.