Survival with mammary cancer related to the interaction of germinal center hyperplasia and sinus histiocytosis in axillary and internal mammary lymph nodes. In a review of the histologic sections of axillary and internal mammary lymph nodes removed during surgery for invasive ductal carcinoma of the breast, we found that 16 of 17 patients in whom sinus histiocytosis was the dominant lymphoid proliferative reaction are alive with no evidence of cancer 5 or more years after operation. In contrast, 5 of 6 patients in whom germinal center hyperplasia was the only significant reaction found died of cancer in less than 5 years. Patients with both sinus histiocytosis and germinal center hyperplasia in significant amounts had survival that was intermediate; 17 of 25 of these patients are currently alive and apparently free of cancer. In addition, 5 of 6 patients in whom no evidence was found of any lymphoid proliferative reaction and 3 of 3 patients with diffuse cortical hyperplasia in their axillary lymph nodes died of cancer in less than 5 years. Germinal center hyperplasia was associated with nodal metastases anatomically in individual lymph nodes and statistically in the series of cases. The internal mammary lymph nodes of most cases showed less proliferative reaction to tumor than the axillary lymph nodes. The pattern of proliferative reactions in lymph nodes and its correlation with survival after surgery suggest that different immune reactions may either suppress or enhance the growth of carcinoma of the breast.


Nonchromaffin paraganglioma (chemodectoma) of thyroid region. A case of a paraganglioma located over the thyroid cartilage in the midline is reported. The tumor had all the appearances of a carotid body tumor, but its odd location could not be explained on the basis of previously known areas where paraganglionic tissues have been found. Since a paraganglioma has also hitherto been described within the thyroid gland, clinicians and pathologists alike are cautioned that unusually located paragangliomas may be expected in the future.


The unique association of mammary stromal sarcoma with intraductal carcinoma. The unique combination of clinically unsuspected stromal sarcoma and noninfiltrating intraductal mammary carcinoma is presented. This association has not been previously documented. The clinicopathologic features of cystosarcoma phyllodes and mammary stromal sarcoma are reviewed to provide background perspectives to illuminate the unusual aspects of this case.


Current status of estrogen receptors in human breast cancer. In estrogen target tissues and hormone-dependent tumors, the steroid enters the cells and binds to a cytoplasmic protein called the estrogen receptor (ER). The steroid-receptor complex then migrates to the nuclei, where it initiates the biochemicial events characteristic of estrogen stimulation. Since ER is absent in tissues not responsive to estrogen, recent studies have asked whether ER assays in human breast cancer tissue might be used to identify those patients likely to respond to endocrine therapy. Data on 436 clinical trials contributed from a dozen centers around the world now clearly indicate that if a patient's tumor does not contain ER, there is virtually no chance of tumor regression following endocrine therapy. A large number of patients can be thus spared unrewarding major endocrine ablative therapy if ER assays are performed routinely. Of tumors with positiev ER, 55-60% respond to endocrine therapy. This single piece of data, when coupled with available clinical prognostic factors such as menopausal status, disease free interval, site of the dominant lesion, and especially response to previous hormonal therapies, should be practicing oncologist to select or reject endocrine therapy with considerable confidence.


Clinical management of advanced gastrointestinal cancer. Although advanced gastrointestinal cancer is the most commonplace problem encountered by the medical oncologist, this group of diseases has proved exceedingly resistant to past chemotherapy efforts. 5-Fluorouracil (5-FU), accepted by some as standard treatment, had provided only infrequent, incomplete, and fleeting antitumor effects, which are probably more than counterbalanced by its gastrointestinal, mucocutaneous, and hematologic antihost effects. There is no evidence that any manipulation of route or schedule of administration provides any improvement in the therapeutic ratio of 5-FU. There is no evidence that this drug contributes to patient survival when used at any stage of any type of gastrointestinal carcinoma. The search for alternative single drugs to 5-FU has been disappointing. The nitrosoureas and Mitomycin C produce occasional regressions, but they do not match the meager effectiveness of 5-FU; and they, in addition, present the difficult problem of cumulative bone marrow suppression. Recent trials with combination regimens have given some indication that the long stalemate in chemotherapy of gastrointestinal cancer may be breaking. Substantial improvements in frequency of tumor regression have been recorded for gastric carcinoma with combinations of 5-FU and BCNU, 5-FU and methyl CCNU, and 5-FU, Mitomycin C, and cytosine arabinoside; for colorectal carcinoma, with the combination of 5-FU, methyl CCNU, and vincristine; and for carcinoid tumors and islet cell carcinomas, with the combination of 5-FU and Streptozotocin. There are also suggestion that such combination chemotherapy with response rates in the 30 to 50% range may produce increased survival when compared to the untreated patient and patients treated with single-drug regimens. While the accomplishments of chemotherapy for the gastrointestinal cancer patient remain less than spectacular there is nevertheless realistic hope that a respectable contribution can now be made to multidisciplinary efforts applied at a stage of disease with minimal tumor burden.


Future prospects for immunotherapy.

Future prospects for immunotherapy. The use of immunotherapy for the treatment of cancer is in its infancy. Definitive examples currently exist in both experimental animals and in humans that immunotherapy can beneficially affect the natural course of established growing malignant disease. Whether these pioneering examples will be extended so that immunotherapy can have a broad applicability in the treatment of cancer in man in not yet known and is a subject of much current work.


Correction of a murine mammary tumor virus-associated immunological depression by selective immunosuppression with cytosine arabinoside. Mammary tumor virus (MTV) infection has been shown to be associated with a diminished hypersensitive reaction to methylated bovine serum albumin. Since methylated bovine serum albumin-induced hypersensitivity appears to be a mixed [humoral versus cell-mediated immunity (CMI)] reaction, the deficit in reactivity could be caused by, among other things, a direct depression of CMI or an increase in a humoral, blocking component. Assay of oxazolone-induced contact sensitivity and phytohemagglutinin-induced lymphocyte stimulation revealed normal or greater than normal CMI in MTV-positive animals. Treatment of MTV-positive and -negative animals with a regimen of cytosine arabinoside designed to inhibit only humoral immunity and leave CMI intact, corrected the deficit in methylated bovine serum albumin reactivity in MTV-positive mice. Thus, it is suggested that MTV infection may facilitate the production of interfering or blocking humoral immunity.


Failure to detect anti-group-specific murine leukemia virus activity in tetraparental AKR-CBA chimeras. Tetraparental AKR-CBA/H-T6 chimeras were primarily derived and investigated to determine whether factors associated with the tumor resistance of the CBA/H-T6 could overcome the innate lymphoma susceptibility of the AKR. Evidence has since shown that, on comparison with the AKR, lymphomas were not only delayed but were also less common in a group of 18 early embryo aggregation derived AKR-CBA/H-T6 tetraparental chimeras. Evidence here has shown other clear differences between the AKR and AKR-CBA/H-T6 chimeras. Whereas murine group-specific murine leukemia viral antigens were detected in the sera in both situations, immunoabsorption studies showed that, in the AKR, the antigens exist complexed to the corresponding antibodies. The situation in the chimeras was in complete contrast, since here antigens exist as a "free" form. This in turn has led us to suggest that the advantage in respect to tumor immunity in the AKR-CBA/H-T6 chimeras is due to the tolerance to oncogenic virus being maintained. In this situation and in contrast to the AKR, in the absence of "masking" antibody-viral antigenic complexes, "normal" tumor immunity can be effected. It has to be assumed that tolerance to the oncogenic Gross virus in the AKR-CBA/H-T6 chimeras reflects the influence of the CBA component. How this has possibly been achieved is discussed.


Seroepidemiological evidence for horizontal transmission of bovine C-type virus. Thirty colostrum-deprived calves from leukemia-free herds were foster nursed for 10 weeks on cows infected with bovine C-type virus (BLV) from multiple-case herds or on cows from leukemia-free herds. After weaning, the calves were raised in continuous contact with BLV-infected animals of approximately the same ages. Sera collected at 6 to 18 and 43 to 48 months of age were examined for the presence of antibodies to BLV by the immunofluorescent antibody test. At 6 to 18 months of age, only 1 of the 30 calves from leukemia-free herds had a detectable antibody response to BLV. By 43 to 48 months of age the number of antibody-positive animals had risen to 17. The foster dam's herd of origin did not significantly affect the rate of BLV infection. These results indicate that BLV can be horizontally transmitted from infected to noninfected animals.


Quantitation of virus-induced (MLr) and normal (Thy.1.2) cell surface antigens in isolated plasma membranes and the extracellular ascites fluid of mouse leukemia cells. Plasma membranes were isolated by two methods from mouse leukemia cells containing mammary tumor virus-induced (MLr) and normal (Thy.1.2) antigens on their surfaces. A number of chemical components, enzymic activities, and the antigenic contents were determined in subcellular fractions and found to be specifically concentrated in the plasma membrane fractions. The major part of the cellular MLr, in contrast to Thy.1.2, was present in the 105,000 X gmax supernatant of the cell homogenate. This and other results indicate an easy release of the antigen from the plasma membrane. A considerable amount of MLr was also present in the ascites fluid, partly free and partly bound, supposedly in an immune complex that allowed the isolation of three components of similar molecular weights as mammary tumor virus components. The extracellular presence of MLr may illustrate that, by shedding of antigen, the tumor may protect itself against the immunological defense of the host.


DNA synthesis in membrane-denuded nuclei and nuclear fractions from host liver and Morris hepatomas. Incorporation of [3H]TTP into membrane-denuded nuclei and fractions of these nuclei from host liver and Morris hepatomas has been compared. Treatment of sucrose nuclei with Triton X-100 removed 95% of the phospholipids and 15 to 20% of the protein. These membrane-denuded nuclei remained physically stable. The Triton X-100-extracted nuclei incorporated label into their DNA in nuclear-incorporating system similar to sucrose nuclei with their membranes intact. Triton X-100-treated nuclei from hepatoma 7777 incorporated six times more label and those from hepatoma 7800 incorporated three times more label than Triton X-100-treated host liver nuclei. Nuclei from the three sources incorporated more label when exogenous DNA was added to the incubation system, but the difference in incorporation between the hepatoma nuclei and the host liver nuclei disappeared. When Triton X-100-treated nuclei, prepared from a tumor-bearing animal given injections of [3H]thymidine for 10 min were fractionated on sucrose gradients after disruption by high Mg2+ concentration, the fractions from hepatoma 7777 nuclei contained six times as much label as the host liver nuclear fractions. Nuclear fractions prepared from unabeled hepatomas or host livers had DNA polymerase activity. The activity, however, is the same in fractions prepared from hepatoma 7777 or host liver nuclei. It is suggested that the nuclear membrane does not play an important role in nuclear DNA synthesis. It is further suggested that the increased incorporation found with hepatoma nuclei is dependent on a physical or chemical arrangement of components within the nucleus and not solely on different enzyme levels.


Postreplication repair of alkylation damage to DNA of mammalian cells in culture. Incorporation and alkaline sucrose sedimentation studies of DNA from mouse L-cells have demonstrated the following effects of N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS). Increasing the concentration of both agents increases the number of single-strand breaks or alkali-labile lesions of existing DNA, which affects the incorporation of [3H]thymidine into DNA by reducing its relative rate. DNA that is newly synthesized during the 1st hr in [3H]thymidine after MNU treatment is of lower molecular weight than is existing DNA with alkali-labile lesions in treated cells and is also lower than DNA synthesized in control cells. Such small segments formed in treated cells are elongated and joined to form high-molecular-weight DNA in the subsequent 4-hr chase in thymidine or 5-bromo-2'-deoxyuridine. Near-ultraviolet photolysis selectively degrades 5-bromo-2'-deoxyuridine-elongated DNA to segments that are nearly as small as those before chase. Further, caffeine (2 mM) present during the thymidine chase prevents nascent-strand elongation, although caffeine-insensitive chain growth occurs partly in MNU-alkylated cells. The MMS lesion (single-strand breakage in alkali) in existing DNA also temporarily interrupts replicative synthesis and makes short segments, but their elongation seems insensitive to caffeine. Our results indicate that MNU may produce both caffeine-sensitive interruptions (probably gaps), as ultraviolet damage does, and apurinic site-directed, caffeine-insensitive interruptions in nascent strands, while MMS may cause exclusively the latter. Further evidence for this is the caffeine potentiation of only MNU killing, like ultraviolet killing, of L-cells. The extent of such a specific MNU lesion is estimated to be no more than 4% of the total extent of methylation, predicting that the lesion that is accessible to caffeine-sensitive repair will be a minor product(s) other than N7-methylguanine. Mutagenic and carcinogenic effects of MNU, which are higher than those of MMS, could be ascribed to such a particular MNU lesion(s) and its repair.


Some ultrastructural features of the cell surface after SV40 transformation and somatic hybridization with normal untransformed cells. We have studied the surface properties of four cell lines: normal human fibroblasts (GM-177), SV40 transformed Lesch-Nyhan fibroblasts (LN-SV), mouse macrophages, and somatic cell hybrids between mouse macrophages and human SV40-transformed fibroblasts (LN-SV). The concanavalin A (Con A)-peroxidase method was used to visualize the localization of the Con A binding sites by means of electron microscopy. The mean thickness of the reaction product was calculated and the distribution pattern was visualized by drawing "glycograms," from which the ratio of covered to uncovered areas was measured. Ruthenium red technique was used to study the ability of the cell surface to retain ruthenium red-positive materials after rinsing in phosphate-buffered saline. Before rinsing, there were no differences between thicknesses of the ruthenium red-positive surface layers of normal and virus-transformed Lesch-Nyhan fibroblasts. After rinsing, however, significant differences in the ability to retain ruthenium red-positive material were seen in favor of the SV40-transformed Lesch-Nyhan fibroblasts (54% over 35%). Before rinsing, significant differences in the thickness of the ruthenium red-positive material were found between mouse macrophages and the hybrids between mouse macrophages and SV40-transformed human fibroblasts, with the hybrids expressing a thicker ruthenium red-positive layer than did the parental mouse macrophages. After washing, however, the ability to retain this material was greater in mouse macrophages than in hybrids. The hybrid cell surface resembles that of SV40-transformed human Lesch-Nyhan fibroblasts. The Con A-peroxidase method shows a different distribution of the reaction product. In general, in all four cases the central portions of the cells had less continuous labeling patterns than did their peripheries, while the marginal portions were very often devoid of the reaction product. Normal human fibroblasts had the most continuous surface reaction product, the thickest reaction product, and the greatest Con A:gap ratio. In all four of these measurements, the hybrid cell line expressed great similarity to the parental cell lines, especially to the mouse macrophages.


Sequential histological and histochemical study of the rat liver during aflatoxin B1-induced carcinogenesis. Male Wistar rats were given 50 mug of aflatoxin B1 twice a week for 4 weeks, and thereafter 75 mug twice a week for 10 weeks. Their livers were investigated histologically and histochemically for glycogen, RNA, fat, alkaline and acid phosphatases, adenosine triphosphatase, 5'-nucleotidase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, and alkaline and acid nucleases. No significant lesions occurred before 15 weeks. During this period, the liver was histochemically unchanged except for a periportal decrease of alkaline phosphatase and adenosine triphosphatase. Scattered hepatocytes with a strong glucose-6-phosphatase activity appeared. These changes represent toxic effects of aflatoxin B1 and are irrelevant to carcinogenesis. From 15 weeks onward, three types of liver cell hyperplastic foci and nodules developed. Histologically, and with respect to glycogen, fat, and RNA content, only two of these types were considered as potential precursors of hepatocarcinomas. However, all types exhibited a decrease or absence of the enzymes studied. Both histological and histochemical changes stressed the complex heterogeneity existing between and within hepatic foci and nodules. From 11 months on, hepatocarcinomas developed. The tumors disclosed similar histochemical changes. This similarity further supports the "precarcinomatous" nature of hyperplastic foci and nodules. It appears that focal changes in surface as well as in cytoplasmic and nuclear enzymes are intimately and very early linked to the carcinogenic process. Whether they are fundamental or only represent an epiphenomenon remains unclear.


Concanavalin A agglutination of cells from primary hepatocellular carcinomas and hepatic nodules induced by N-2-fluorenylacetamide. A previous study demonstrated that cells of transplantable hepatocellular carcinomas were agglutinated by the plant lectin concanavalin A, while normal hepatocytes were not. In the present experiments, 95% or more of cells obtained from primary hepatocellular carcinomas which resulted from exposure of rats to N-2-fluorenylacetamide were agglutinated by this lectin. Exposure to this carcinogen also produces grossly visible foci of morphologically and biochemically altered hepatocytes which have been termed hepatic (hyperplastic; premalignant, neoplastic) nodules. Although these hepatocyte aggregates are generally accepted as precursors of the hepatocellular carcinomas, no agglutination was detected when their cells were exposed to concanavalin A. These results indicate that concanavalin A agglutinability is not acquired as a result of tumor transplantation. Furthermore, they suggest that significant alterations must occur in the cells of hepatic nodules prior to the manifestation of malignant behavior.


Microinjection of thymidine kinase and bovine serum albumin into mammalian cells by fusion with red blood cells. A procedure is described by which proteins can be rapidly and efficiently microinjected into large numbers of culture cells. Proteins were first introduced into mammalian red blood cells during hypotonic hemolysis, and the resealed red cells were subsequently fused to culture cells using Sendai virus. In seven experiments, thymidine kinase or 125I-BSA were transferred to culture cells during fusion. Although proteins were used in the present experiments, the microinjection procedure should work equally well for other macromolecules.


The effect of methylene dimethanesulphonate (MDMS) on the conformation of DNA and its dependence on base composition. The interaction of the alkanesulphonate, methylene dimethanesulphonate (MDMS) with DNA has been studied. Thermal denaturation studies on mixtures of MDMS and DNA showed a dose-dependent decrease of the melting temperature midpoint (Tm) of the DNA. In addition, an irreversible decrease in ultraviolet absorption (hypochromism) preceded the hyperchromic shift, the magnitude of the former being linearly related to both the relative concentration of MDMS and the G-C content of the DNA used. Neither the reduction in melting temperature nor the initial UV absorption decrease occurred after dialysis of the reaction mixture. Equimolar proportions of the hydrolysis products of MDMS did not give the same effects as observed with the unhydrolysed agent. A similar hypochromism followed by strand separation occurs when DNA is allowed to stand with MDMS at room temperature, the time of subsequent strand separation being related to the treatment level of the drug. A weak association of MDMS with DNA is considered to be involved resulting in a local compression of the helical structure in the vicinity of the G-C pairs. It is suggested that this conformational change may act as a substrate for repair enzymes in vivo.


The effects of several croton oil constituents on two types of DNA repair and cyclic nucleotide levels in mammalian cells in vitro. The purpose of the present study was to determine the effects of two potent tumor-promoting agents on two DNA repair mechanisms and cyclic nucleotide levels in mammalian cells. Human amnion (AV3) cells were treated with low dose levels of either UV of N-acetoxy-acetylaminofluorene. Subsequently, DNA excision repair as measured by unscheduled DNA synthesis was followed in the absence or presence of non-toxic levels of either 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibenzoate (PDB), both potent tumor promoters, or phorbol, a non-promoter. Neither of these compounds inhibited DNA repair synthesis occurring in response to low doses of the carcinogenic agents. In addition, TPA did not inhibit "post-replication repair" in response to UV irradiation of growing Chinese hamster (V79-4) cells. However, both TPA and PDB did cause rapid dramatic increases in cyclic guanosine monophosphate levels in human amnion cells; phorbol had no effect. Neither of these compounds affected cyclic adenosine monophosphate levels. These results are discussed in the light of a possible mechanism of the action of tumor promoters involving "post-replication repair".


[Presence of autocomplementary RNA with viral specificity in cells infected with herpes virus]. RNA from cells infected with Herpes simplex virus contain a higher percentage of double-stranded RNA than non-infected cells. This percentage increases three-fold upon self-annealing. The complementary RNA sequences were shown to be virus-specific by the following criteria: (1) high melting temperature than double-stranded RNA from non infected cells; (2) higher density in caesium sulphate; (3) specific hybridization with viral DNA.


[Description of the folliculo-stellate cells in the anterior hypophysis of the monkey Macacus irus]. Folliculo-stellar cells are present in the anterior lobe of hypophysis of the monkey Macacus irus. These cells limit strait cavities in which one can observe microvillosites at the apical pole of the cells. The folliculo-stellar cells possess long and thin fibers which insert themselves between different types of granular cells and sometimes extend to the peri-capillar regions. Numerous organelles are present in these folliculo-stellar cells, especially microfilaments.


[Induction of cutaneous or subcutaneous fibroblastic tumors in the Afghan pika (Ochotona rufescens rufescens) by injection or bovine papilloma virus]. Newborn Afghan pikas have been inoculated with bovine papilloma virus via the subcutaneous route. Cutaneous or subcutaneous fibromas and fibrosarcomas were observed after a mean incubation period of nine months. The transmission of these tumors by homograft has been obtained. Bovine papilloma virus antibodies have been demonstrated in most of the animals inoculated at birth. They have not been detected in animals bearing transplanted tumors.


Secificity of mouse endocervical cell response progesterone: presence of receptors. The mouse cervical cell response to progesterone, corticosterone, and androgens was studied in vitro and comparatively by grafts on males. Contrary to the exocervical cells which responded more or less to the three steroid hormones, the endocervical cells responded exclusively to progesterone even in an estrogen free system. This result suggests the existence in the mouse endocervical cells of specific receptors to progesterone and indicates that the squamons cells of the uterine cervix have a different response to steroid hormones depending on where these cells are located.


[RNA fragments, in vivo inhibitors of Shope fibroma and vaccinia virus multiplication]. RNA-fragments rich in purine nucleotides and resulting from degradation of ribosomal RNA from E. coli M 500 Sho-R with pancreatic RNase exhibit only in vivo an inhibitory effect on Shope fibroma and vaccinia virus multiplication.


[C-type virus particles in lymphocytes cultured from cows with persistant lymphocytosis. Preliminary results on French cattle stock]. C type virus particles were shown in cultures of lymphocytes originating from cows with persistent lymphocytosis. These observations on French cattle are in complete accordance with data previously obtained in the USA. Viral replication is highly stimulated by the addition of phytohemagglutinin in the culture medium. Several morphological and biological properties, specific to the bovine virus differentiate this virus from C type viruses of other species.


Interaction between cyclic adenosine monophosphate and cyclic gunaosine monophosphate in guinea pig ventricular myocardium. The purpose of this study was to examine the mechanisms underlying adrenergic-cholinergic antagonism in ventricular myocardium. Myocardial contractility, cyclic adenosine monophosphate (AMP) levels, and cyclic guanosine monophosphate (GMP) levels were measure in isolated guinea pig ventricles after treatment with various inotropic agents given alone and simultaneously with acetylcholine. Acetylcholine alone markedly elevated cyclic GMP levels but did not substantially change myocardial contractility. However, the same concentration of acetylcholine significantly attenuated the inotropic effect of isoproterenol and histamine, two drugs that may act by increasing myocardial levels of cyclic AMP. The decrease in the inotropic response to isoproterenol did not appear to be due to a decrease in the generation of cyclic AMP, because cyclic AMP levels were similar in hearts receiving isoproterenol alone and those receiving isoproterenol with acetylcholine. Dibutyryl cyclic GMP also antagonized the intropic action of isoproterenol. Acetylcholine did not alter the inotropic effects of ouabain, an agent that increases myocardial contractility without changing cyclic AMP levels. These results suggest that cyclic GMP mediates the antiadrenergic effects of acetylcholine by specifically antagonizing the inotropic actions of cyclic AMP.


Sleep-stage regulation of ventricular arrhythmias in the unanesthetized pig. In two groups of purebred juvenile Hampshire pigs, left anterior descending coronary occluders were implanted. The pigs were then adapted to the recording chamber until they would manifest a criterion pattern of sleep while in it. In group A, permanent coronary artery occlusions were produced, and the effects of various sleep stages on the resultant cardiac arrhythmias were observed. Sleep intervals during which transitional and slow wave (SW) sleep alternated were correlated with increased arrhythmias relative to the awake state (P less than 0.04). The increase was maximum during sustained periods of SW sleep. Intervals during which rapid eye movement (REM) sleep predominated were correlated with a reduction in arrhythmias. In group B, temporary occlusions were made during both an awake condition and after criterion patterns of either SW or REM sleep. Occlusions after SW sleep reduced the ventricular fibrillation latency compared with that during the awake control state (P less than 0.05), whereas occlusions after REM sleep increased ventricular fibrillation latency (P less than 0.05). We conclude that SW SLEEP, BUT NOT REM sleep, has a deleterious effect on the ischemic myocardium. REM sleep may have a beneficial effect, since it increases ventricular fibrillation latency. Heart rate changes do not appear to be correlated with the effects of either sleep stage.


Histochemical and structural changes in human myocardial cells after cardiopulmonary bypass. The histochemical and ultrastructural effects of extracorporeal circulation and aortic cross-clamping during coronary heart surgery have been examined in drill biopsy samples of the left ventricle in 22 patients. The biopsies were obtained before and after bypass with a DeSoutter drill. Histochemical studies indicated definite differences between control and experimental biopsies, with increased succinic dehydrogenase, cytochrome oxidase, and LDH activity, while phosphorylase A and myosin ATPase activities declined. Furthermore, free phospholipid levels increased, as determined by the acid hematein reaction. Ultrastructural examination demonstrated loss of glycogen, intracellular swelling, and mitochondrial damage, which included loss of matrix density, loss of cristae, and eventual disruption in the postbypass biopsy. These results, which closely resemble the effects of ischemia and reperfusion observed in animal experiments, suggest that the initial insult is a change in membrane permeability regulation.


Morphologic correlates of technetium-99m stannous pyrophosphate imaging of acute myocardial infarcts in dogs. To obtain insight into the mechanism(s) responsible for the direct visualization of acute myocardial infarcts by myocardial scintigraphy with technetium-99m stannous pyrophosphate (99mTc-PYP), scintigraphic and morphologic studies were performed in 22 dogs subjected to occlusion of the proximal left anterior descending coronary artery (LAD). Grossly visible myocardial infarcts occurred in ten of 11 dogs with LAD occlusion for one day, five with LAD occlusion for two days, two with LAD occlusion for seven days and two with LAD occlusion for 13 days. Rare, microscopic foci of necrosis were observed in one dog with LAD occlusion for one day, and no lesions were present in two dogs subjected to temporary LAD occlusion for eight minutes and reflow for 24 hours. In the latter three dogs, 99mTc-PYP myocardial scintigrams were negative. In the 19 dogs with gross infarcts, 99mTc-PYP myocardial scintigrams were strongly positive at one and two days after LAD occlusion, much less positive at seven days and faintly positive at 13 days after occlusion. Positive myocardial scintigrams in most showed "doughnut" patterns, with marked peripheral concentration of radioactivity around central zones of much lower activity. On histologic examination, the one and two-day-old infarcts exhibited subendocardially located central zones and surrounding peripheral zones, both of which showed distinctive histopathological and histochemical features, including the selective occurrence in the peripheral zones of calcified muscle cells with ultrastructurally demonstrable apatite-like crystals in mitochondria. Selective occurrence of high tissue levels of 99mTc-PYP radioactivity also was demonstrated in the peripheral zones of four infarcts. Hearts with older infarcts (seven and 13 days) showed progressive replacement of necrotic myocardium by granulation tissue and progressive reduction in calcium deposits in the areas of damage. The data obtained in this study establish a temporal and topographical relationship between calcium accumulation in acute myocardial infarcts and 99mTc-PYP uptake responsible for scintigraphic detection of the lesions with this radionuclide in dogs subjected to proximal LAD occlusion.


Determination of phosphodiesterase I activity in human blood serum. Phosphodiesterase I (EC 3.1.4.1) activity was detected in normal human blood serum. The enzyme is stable at laboratory temperature for three days, but is inactivated at pH less than 7. The pH for optimum activity increases with the substrate concentration (under the conditions used, from pH 9.0 to 10.2) and, conversely, the Km increases with pH and buffer concentration. The enzyme is inhibited by ethylenediaminetetraacetate but not by phosphate (0.1 mol/liter). We developed a simple quantitative method for its determination, based on hydrolysis of the p-nitrophenyl ester of thymidine 5'-monophosphate and subsequent measurement of the liberated p-nitrophenol at 400 nm in NaOH (0.1 mol/liter). Normal values (mean +/- 2 SD) were determined to be 33 +/- 6.4 U/liter. Preliminary studies indicate that phosphodiesterase I activity is greater than normal in serum of patients with necrotic changes in the liver or kidney or in cases of breast cancer, but not in that of patients with myocardial infarction, bone cancer, lung cancer, or chronic liver cirrhosis.


Iodine organification defect following treatment of thyrotoxicosis with antithyroid drugs. Two patients remained clinically euthyroid following treatment for thyrotoxicosis with antithyroid drugs in spite of persistently elevated thyroid radioiodine uptakes not suppressable by exogenous triiodothyronine. Perchlorate discharge tests showed a defect in the intrathyroidal organification of iodine. Circulating levels of thyroxine were normal. From our study of 105 patients treated for thyrotoxicosis with antithyroid drugs, apparent remission of thyrotoxicosis by this mechanism might occur in up to 2 percent of patients.


Steroidogenesis in a virilizing ovarian tumour. A metabolic study with tissue from a virilizing arrhenoblastoma, using as precursors [7-3H]pregnenolone, [7-3H]17alpha-hydroxypregnenolone,[4-14C]17alpha-hydroxyprogesterone and [4-14C]testosterone, revealed that in spite of a deficient activity of 3beta-hydroxysteroid dehydrogenase-5-isomerase the overall production of testosterone was compensated by an increased activity of a lyase converting [4-14C]17alpha-hydroxyprogesterone to testosterone (via androstenedione) and was comparable to the production obtained by normal ovarian tissue. The masculinizing effects of the tumour in vivo were most probably caused by accumulation of testosterone due to deficiencies in enzymes catabolizing testosterone to 17-ketosteroids and its aromatization to oestrogen. The unique property of the arrhenoblastoma to convert [4-14C]17alpha-hydroxyprogesterone to [4-14C]11-deoxycortisol (Reichstein's compound S) suggests an adrenal origin of the tumour which may explain its limited capacity to aromatize testosterone.


Early diagnosis of congenital adrenal hyperplasia by measurement of 17-hydroxyprogesterone. Plasma 17-hydroxyprogesterone was measured by a simple radioimmunoassay technique in six infants, aged 3 days to 3 months, with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Hormonal levels in this group were compared to those of twenty normal newborns and to those found in sixty samples of umbilical vein blood from normal deliveries. Plasma 17-hydroxyprogesterone concentrations were markedly elevated in all congenital adrenal hyperplasia infants (range 544.7-1837 nmol/l, normal 3.03-19.06) at a time when urinary studies in some of these were either not diagnostic or inconclusive. In one infant whose cord blood was analysed, the level was also greatly raised. The data suggest that early definitive diagnosis of congenital adrenal hyperplasia can be established by measurement of plasma 17-hydroxyprogesterone.


Pathology of osteosarcoma.

Pathology of osteosarcoma. Osteosarcoma of bone is a tumor composed of malignant cells that produce osteoid. Some tumors show predominant chondroid or fibromatoid ground substance. All, however, are highly malignant and about 80 per cent produce death with metastases. The roentgenogram affords important evidence for the correct diagnosis of many of them. Differential diagnosis should include consideration of those sarcomas with many benign giant cells and the group of "telangiectatic" osteosarcomas that may contain only small diagnostic areas. Malignant fibrous histiocytoma is now considered as a possible diagnosis for some malignant bone tumors, but the exact criteria for the diagnosis of this condition are still somewhat obscure. Newer modalities of adjunctive treatment, such as resection of pulmonary metastases, chemotherapy, and immunotherapy, give promise of improving the prognosis for osteosarcoma.


Neurovascular complications following tibial osteotomy in children. A case report. Anterior tibial sensory and motor losses following tibial osteotomy in children may result from any of 3 distinguishable etiologies. Peroneal nerve traction does not result in abnormalities of the dorsalis pedis pulse, pain on passive muscle stretch or a tense anterior tibial compartment. An anterior compartmental syndrome may or may not produce an abnormal pulse, but passive muscle stretch is painful and the compartment is tense and tender. Significant occusion of the anterior tibial artery produces diminution of the pulse and painful muscle stretch in the absence of a tense compartment. Certain pre-, intra- and postoperative measures can minimize the chance of these complications and facilitate their detection and treatment. These include: prophylactic fasciotomy, external pin fixation and frequent, thorough examination. If one of these complications arises, dressing should be loosened and the leg returned immediately to its preoperative position. Subsequent therapy is based on continued observation and the specific diagnosis.


Newer knowledge of non-collagenous protein in dentin and cortical bone matrix. The current state of knowledge of the composition of the NCM components of bone and dentin has been summarized at the end of the appropriate sections. It is significant that increasing interest in the chemistry of hard tissue matrices has coincided with the development and refinement of a wide range of separation techniques, resulting in the isolation of an unexpectedly large number of components. The most sophisticated techniques, such as iso-electric focusing and isotachophoresis, give rise to discrete fractions often of very similar composition, particularly in terms of amino acid content. Such components might best be considered in groups, especially should such groups be identified in terms of common immunochemical properties. Dickson has used such an approach in a recent study of the proteins of sheep cortical bone. The Liverpool group has approached the problem on a broad front and thus directed attention to the number and diversity of NCM components, but it will be seen that those studies directed to the isolation of a specific component have invariably revealed the presence of several other fractions, set aside while attention was directed to the component under investigation. It is clear that the major proportion of bone and dentin NCM consists of glycoproteins of the less-acidic and anionic types. Several of the anionic components contain phosphate, levels being higher in those derived from dentin. Glycosaminoglycans, the first class of non-collagenous compounds to be identified in hard tissue matrices, are now known to comprise only about 5-7 per cent of the NCM. Precise details of the glycosaminoglycan fraction of human dentin and considerable information concerning that of bovine bone are now available. The major component in each tissue is chondroitin-4-sulfate, which exists in the form of proteoglycan, the protein moieties of those from bovine bone and human dentin being very different. Although greater interest is currently being shown in the glycoprotein fractions, several studies have been made of the calcium-binding properties of proteoglycan preparations and of individual glycosaminoglycans. Attempts to relate differences in chemical composition and properties to specific bone sites may not prove to be the best approach to the study of the precise chemistry of mineralization. It is clear, from the earlier work of Lindenbaum and Kuettner that mineralization takes place in a very narrow layer which will normally represent only a fraction of a typical zone prepared for analysis...


Electron microscopic observations of 20 human osteosarcomas. Twenty primary osteosarcomas of bone and two osteosarcomas metastatic to the lung were examined by electron microscopy. The tumor cells, whether from an area exhibiting chondroid, osteoid or collagenous matrix, showed common abnormalities in configuration and fine structure. The cells tended to be spindle-shaped with centralized nuclei. The nucleus was enlarged, irregular in shape, and frequently lobulated. The choromatin was dense and arranged around the periphery of the nucleus. There was a large prominent, irregularly shaped nucleolus. Smooth membranes were sparse to absent. The rough endoplasmic reticulum was abundant, disorderly, and tended to be markedly dilated in mature cells. The mitochondria were numerous, variable in size and configuration, and were intimately surrounded by rough endoplasmic reticulum. The collagenous matrix was disorganized with the fibers and bundles randomly oriented and arranged. No structures definitely identifiable as virus-particles were seen. However, microstructures resembling unenveloped nucleocapsids of paramyxo- or related viruses were noted. All fine structure abnormalities could be associated with an increased metabolic rate and/or with abnormal protein and enzyme synthesis. No definitive diagnostic features were found. The fine structure of cell cultures derived from the osteosarcomas in this study was similar to that of the fresh tissue. No virus-like particles were seen in any of these cell lines.


Collagenolytic systems in rheumatoid arthritis. As the proliferative lesion of rheumatoid arthritis becomes polarized and invasion of articular cartilage and subchondral bone begins, it is likely that many mesenchymal cells, including periosteal and perichondral cells, and perhaps even the chondrocytes and osteoblasts themselves can be activated to produce destructive enzymes. Early in the course of RA cartilage proteoglycans are depleted, leaving the remaining collagen more susceptible to mechanical breakdown as well as to enzymatic breakdown. Specific collagenases are released by synovial cells and, in addition, by polymorphonuclear leukocytes. The latter enzyme may account for free collagenase found in synovial fluid, a finding possibly related to saturation of inhibitory proteins by proteases with greater affinity for them, leaving collagenase active. At this time in the course of rheumatoid arthritis, a joint would be under double jeopardy from enzymes released by the invading pannus as well as by collagenase free and active in the synovial fluid. Rapid destruction could occur. Although cartilage collagen has an intrinsic resistance to collagenase conferred by its primary structure and by higher order structure (e.g. intermolecular cross-links), it seems wise to cool down hot joints because increased temperature may increase the rate of collagen degradation and, therefore, cartilage destruction. In addition, superimposed sepsis or acute flares of rheumatoid disease result in enough influx of polymorphonuclear leukocytes into the joints to result in free collagenolytic activity being present. This provides a rationale for frequent aspiration of any joint fluid, septic or otherwise, containing high polymorphonuclear leukocyte counts.


Studies on characterization of the integration sites of avian RNA tumor virus-specific DNA. A sequential hybridization procedure is described which allows the integration sites of viral-specific DNA to be characterized according to their reassociation kinetics. In addition, this approach enables us to estimate the size of the integrated viral DNA. Endogenous virus sequences in normal cells appear to be associated with cell sequences reiterated 1200 times, and each integration unit is approximately equal to one 35S RNA subunit. In AMV-infected cells, the additional AMV-specific DNA sequences reassociate as if they were integrated adjacent to unique cellular DNA or in tandem with the endogenous viral DNA sequences.


A  study of skeletal age and height in girls with idiopathic scoliosis. The height and skeletal age of girls with idiopathic scoliosis were studied. The skeletal age was evaluated by two independent methods and was compared with the chronological age and the corrected height of the patients. With both methods the same tendency was noticed. Early in the adolescence (ages 11 and 12 years) the skeletal development was more advanced in girls with idiopathic scoliosis than expected. Later (ages 16 and 17 years) the reverse condition was found. The standing height, corrected for the deformity, was significantly greater in idiopathic scoliosis patients than in controls of either the same chronological or skeletal age. These observations indicate that girls with adolescent idiopathic scoliosis have a growth pattern differing from normal, and that growth factors are connected to the disease.


Infectious viral DNA in Rous sarcoma virus-transformed nonproducer and producer animal cells. Nonproducer and producer RSV-transformed cells and producer nontransforming virus-infected cells harbor viral DNA specifying the respective avian tumor virus. In nonproducer Rous sarcoma cells, the residing viral DNA is linear, double-stranded and covalently linked to the chromosomal DNA. Both double-stranded and single-stranded forms of RSV DNA transfect chicken cells. The progeny virus is indistinguishable from the DNA parent with respect to the morphological, biological and antigenic properties. Unlike the DNA extracted from nonproducer RSV-transformed mammalian cells, that extracted from producer RSV-transformed chicken cells gives rise, in transfection experiments, to both sarcoma virus and its nontransforming derivative. The DNA from nontransforming virus-infected chicken cells generates only nontransforming viruses. The frequency of nontransforming virus recovery is different from that of sarcoma virus recovery, the latter being a nonlinear function of the amount of transfecting DNA, while the former may suggest a linear relationship. On the other hand, the end-point dilution of transfecting DNA for sarcoma virus recovery is approximately the same as that for nontransforming virus recovery. The following is assumed: Sarcoma viruses and nontransforming derivatives are recovered from two different species of viral DNA which carry or lack, respectively, the transforming genetic material. The size of double-stranded viral DNA is substantially smaller than 20 times 10(6) daltons. In transfection experiments, the sarcoma virus DNA is first integrated into the host cell genome before being expressed, while the nontransforming viral DNA apparently bypasses the integration step. The latter DNA generates the progeny virus when taken up, carried, and transcribed in a permissive cell.


Infectious DNA from cells infected with Rous sarcoma virus, reticuloendotheliosis virus or Rous-associated virus-O. We have described an efficient and quantitative assay for infectious DNA of the avian ribodeoxyviruses and have applied this method to study the possible existence of infectious viral DNAs in uninfected cells. Infectious DNA from cells infected with RSV or REV consisted of a single unit of DNA with a specific infectivity of 10(-5)-10(-6). The minimum molecular weight of RSV DNA required for infection of chicken cells was about 6 million, while the minimum molecular weight of infectious REV DNA was about 20 million. This difference may reflect complementation of the RSV DNA by endogenous avian leukosis virus-related DNA in uninfected chicken cells. Uninfected chicken cells do not contain infectious DNA for an REV, nor for a strongly transforming or a nontransforming avian leukosis virus.


AMV RNA transcription in cell-free systems and properties of in vitro chromatin-directed RNA synthesis. In this report we have presented evidence that viral sequences in the genome of AMV-infected myeloblasts can be transcribed in vitro. The RNA products synthesized in either nuclei isolated from these cells or by eukaryotic RNA polymerase B from the isolated chromatin contained approximately 1% virus-specific sequences. This result, which is in agreement with the fraction of viral RNA in infected cells (Garapin et al. 1971), is higher than expected from a random transcription of the genome, and thus shows that a degree of selectivity in transcription is maintained in both systems. The inhibition of synthesis of viral sequences in nuclei by alpha-amanitin as well as the finding that RNA polymerase B catalyzed the synthesis of viral sequences from chromatin support the hypothesis that the expression of viral information is mediated by nucleoplasmic RNA polymerase. An investigation of the properties of the chromatin-directed products led to the suggestion that RNA synthesis in vitro was initiated on single-stranded or denatured regions of the template; a limiting factor in the synthesis of large molecular weight RNA from isolated chromatin appeared to be the extent of the denatured region available to the enzyme. These findings are consistent with the suggestion that gene activation in eukaryotic organisms results from the unwinding of segments of chromatin DNA (Crick 1971).


Qualitative and quantitative studies of AKR-type murine leukemia virus sequences in mouse DNA. Utilizing a single-stranded [3H]DNA probe highly representative of AKR viral 70S RNA, we have performed association kinetics experiments with cellular DNA in vast excess from 3 high-, 5 low- and 4 non-virus-yielding mouse strains. Our hybridization studies indicate that in the strains so far tested, the complete genome of the AKR-type MLV is present in the DNA of the embryos of both high- and low-virus-yielding mouse strains, while DNA of non-virus strains contains only a part of the genome. Furthermore, at least two populations of virus-specific DNA sequences can be identified (more abundant and less abundant species) according to their rate of association. Low-virus-yielding mouse strains contain a smaller number (1-2 copies) of the less abundant species, and thus a lower number of complete viral genome than do high-virus strains (3-4 copies). Non-virus-yielding strains are lacking these less abundant sequences in their genome. DNA from wild Mus musculus also contained viral sequences, the sample tested showing association kinetics identical to the non-virus-producing strains. Thus there is a good correlation between completeness of the AKR-type MLV genome in cellular DNA and the capacity of the cells to release AKR-type MLV. Mice of a non-virus-yielding strain made partially congenic for the AKR virus-inducing locus Akv-1 contained the complete virus genome, confirming that this locus consists of structural genes of the virus.


Viral gene functions expressed and detected by temperature-sensitive mutants of herpes simplex virus. The expression of HSV-specific gene functions by 22 ts mutants of HSV-1 in 15 complementation groups and 8 ts mutants of HSV-2 in 7 complementation groups has been studied at the nonpermissive temperature. Four cistrons of HSV-1 and three cistrons of HSV-2 with defects in viral DNA and DAN polymerase synthesis were identified. DNA-mutants of HSV-1 revealed a greater alteration in HSV polypeptide synthesis and viral assembly than DNA- mutants of HSV-2. Mutants with apparent defects in structural proteins have been identified for both HSV-1 and HSV-2 as demonstrated by their increased themolability. The general organization of the provisional HSV-1 and HSV-2 linkage maps revealed a similarity in the arrangement of functionally related cistrons, with DNA- mutants being located on the left-hand side of each map and mutants with phenotypic properties similar to those of the wild-type virus, on the right-hand side. An early polypeptide of HSV (VP175, MW 175,000) was found to accumulate in cells infected at the nonpermissive temperature withts mutants of HSV-1 in complementation group B. The VP175 polypeptide was isolated from such cells by a combination of SDS-preparative and analytical disc gel electrophoresis. Antiserum prepared to this polypeptide was found to descriminate between HSV-1 and HSV-2 by immunofluorescence. On the other hand, type-specific gene functions of HSV-1 and HSV-2 were not demonstrated through intertypic complementation and recombination tests with heterologous mutant pairs, whereas mutually exchangeable or common gene functions were readily identified. Eight ts mutants of HSV-2 were used to detect functional HSV genes in hamster embryo cells transformed by HSV-2. Normal hamster cells and SV40-transformed hamster cells failed to support the replication of the mutants at the nonpermissive temperature. However, the replication of two mutants, defective in late functions, was significantly enhanced in two independently derived HSV-2-transformed cell lines. Thus functional HSV genetic information was detected in HSV-2-transformed cells through the use of ts mutants. Moreover, it appears that the information present in both cell lines is not only specific but involves late HSV functions.


Transformation of cells by herpes simplex virus--fact or fantasy? The replication of herpes simplex virus types 1 and 2 is defective in XC cells. One lesion is the failure of viral proteins to be transported to the cell nucleus. The resistance of XC cells to the toxic effects of herpes simplex virus is apparently not due directly to the presence of RNA tumor virus in these cells. Other cell lines transformed by RNA tumor viruses exhibited no comparable resistance. The nature of the association between HSV and cell for a number of generations after first selecting "transformants" is not yet established; on prolonged subculture, much of the original HSV information fails to be expressed. Not more than approximately to 0.1 viral genomes per cell remains permanently associated with the host DNA. The "transformants" retain the capacity to synthesize HSV-specific polypeptides, as determined by radioimmunoprecipitation. The "transformants" have a number of characteristics which distinguish them from parental cells, and such characteristics in the light of the immunological data suggest that XC cells exposed to HSV can permanently retain HSV-specific information.


Plasmid DNA as a possible state of Epstein-Barr virus genomes in nonproductive cells. It has been shown that the EBV DNA in Raji cells is located in chromosomes and that most of this EBV DNA can be separated from high molecular weight cell DNA both in alkaline and neutral glycerol gradients. The data indicate that the latent EBV DNA may exist possibly as a plasmid DNA. As 50 genomes of EBV DNA are equal to 0.1% of the total cell DNA, it may be quite conceivable that such an amount of DNA should remain in a non-integrated form. Chromosomal location may be a requisite for any DNA to persist in the nucleus of cells and to be divided into daughter cells evenly. As the present studies measured only the status of the major portion of EBV genomes in Raji cells, a possibility still remains that a few genomes of EBV may be linearly integrated into cell DNA and these may be responsible for cellular transformation. Another possibility that a small piece of cellular DNA might be attached to EBV DNA in Raji cells, which might serve as a control signal for replication and/or transcription of the latent virust DNA, has also not been ruled out. Even though EBV DNA is not integrated covalently into cell DNA, the association of the virus genomes with cells is very stable and the number of EBV genomes remains unchanged throughout many cell generations. In synchronized cells, all EBV genomes replicate simultaneously at an early S phase before maximum synthesis of cell DNA replication occurs (Hamper et al. 1974). This indicates that EBV DNA in a plasmid state is strictly under a cellular control mechanism. How this latent virus DNA is controlled in cells will be examined in future studies.


35S a and b RNA subunits of avian RNA tumor virus strains cloned and passaged in chick and duck cells. The RNA of transforming and nontransforming avian RNA tumor viruses was heat denatured and examined for its 35S subunit composition by polyacrylamide gel electrophoresis (gel slicing and/or autoradiography). The RNA of virus strains capable of transforming chick embryo fibroblasts in vitro always contained a subunits: the RNA of Pr-RSV of subgroups A, B or C was composed only of a subunits, even after prolonged passage in chick cells, whereas the RNA of SR-RSV-A, SR-RSV-B, SR-RSV-D(H) and B77 also contained b subunits in varying amounts, even when examined as early as possible (8-10 days) after cloning. The b subunit content did not correlate with length of time after cloning. Nontransforming viruses such as RAV-2 and nontransforming derivatives of transforming viruses such as NT-SR-RSV-A, SR-RSV-B AV-3, NC-SRV-D(H), NC-SRV-D(H)(U4), NC-B77-42 and NC-B77-43 contained only b subunits. When B77 virus grown in chick cells was passaged in duck embryo fibroblasts, the ratio of a/b subunits in its RNA did not change markedly. However, when it was cloned in duck embryo fibroblasts, the transformed clones produced virus that contained almost exclusively b subunits. On transfer to chick cells, a subunits once again appeared, and the a/b subunit ratio was soon similar to that of the original B77. B77 virus produced by cloned, transformed duck embryo fibroblasts was found to contain a large excess of nontransforming virus particles.


A biochemical and genetic analysis of mammalian RNA-containing sarcoma viruses. Current studies have shown that all mammalian sarcoma-producing viruses, whether isolated from laboratory experiments of naturally occurring tumors, are deletion mutants of replicating mammalian type C viruses. Nonproducer cells transformed by any of these sarcoma viruses contain RNA homologous to mammalian leukemia viruses, even though the cells contain no known proteins currently coded for by the mammalian leukemia virus. This mammalian leukemia virus information (FT-) is a genetically stable part of the mammalian sarcoma viruses (FT+). Second, another component in the Kirsten and Harvey sarcoma viruses can be identified in addition to this leukemia virus information for the homologous leukemia virus; at least part of the additional information came from rat type C viruses from the animals in which the sarcoma viruses were isolated. This indicates that these two mammalian sarcoma viruses are recombinants between mouse leukemia virus and genetic information in rat cells and suggests that the process of formation of the sarcoma virus is analogous to transduction of information in bacteriophage. Third, the Kirsten sarcoma virus seems to have a third component in it separate from either the mouse leukemia virus or rat leukemia virus information. Fourth, and FT+ leukemia virus isolated from mice, the Abelson leukemia virus which causes as B-cell leukemia, is also defective and can be shown to have information homologous to Moloney leukemia virus. Fifth, in the feline sarcoma virus, feline leukemia information can be detected, but information for the other cat virus, RD-114, cannot be detected. Finally, mutants of Kirsten sarcoma virus temperature sensitive for the maintenance of transformation have been isolated, and out of ten such mutants, thus far no complementation has been observed.


Genetic recombination among temperature-sensitive mutnats of Rous sarcoma virus. Genetic recombination of RSV has been studied, using ts mutations in both initiation and maintenance of transformation as markers. The progeny of a single cycle of mixed infection appears to contain no recombinants, but yields heterozygous particles or viral clumps. On subsequent cycles of infection some of these heterozygotes/clumps persist, but they also segregate recombinant viruses. Some of the markers in these recombinants show evidence of linkage and thus probably recombine by intramolecular exchanges. Studies of td ts mutants (the class most frequently isolated from stock virus) show that recombination between them occurs at a level sufficient to explain cooperative transformation by these viruses. Furthermors, this genetic recombination is probably a necessary prerequisite for cooperative transformation since complementation between the mutants is absent or inefficient. The simplest explanation for this apparent lack of complementation is that the td ts mutants are all derived by lesions in the same cistron.


Formation of reiterated simian virus 40 DNA. We have described studies on the biological fate of a minicircular DNA molecule that is a specific, complex deletion mutant of SV40. When the minicircular DNA alone was used to infect monkey cells, its replication was not detected. However, after infection with the minicircles and SV40 DNA together, incorporation of (3H)thymidine into both species of viral DNA was demonstrated. This finding suggests that circular, duplex viral DNA segments, much smaller than SV40 DNA, are able to be replicated in vivo. Furthermore, 26% of the (3H)thymidine-labeled, superhelical DNA sedimented more rapidly than SV40 DNA I (21S) in neutral sucrose gradients (22S-32S). A similar amount of this rapidly sedimenting DNA was also detected when intact DAR DNA containing the triplication mutant was tested. Cleavage of the purified, rapidly sedimenting DNA with R.EcoRI produced 10.4S segments (one-third the size of unit-length SV40) in addition to full-length linears (14.5S) and a new cleavage product (16.7S). Cleavage of the 21S DNA I molecules also produced 10.4S DNA. These results indicate that the minicircular molecules are amplified in vivo, yielding not only the original triplication mutant but also a heterogeneous population of oligomers in which the 10.4S segment has been reiterated as many as 6 to 9 times. Our studies support the model proposed by Khoury et al. (1974) for the generation of the original DAR triplication mutant. In our experiments, cells were infected with a minicircular DNA molecule formed in vitro, which then served as a precursor in vivo in the formation of trimers and higher oligomers, as predicted by the proposed model. The DAR triplication mutant first appeared after the third passage in primary monkey kidney cells and rapidly became the predominant species in later passages (Fareed et al. 1974)...


Stepwise relaxation of supercoiled SV40 DNA. We have shown in this paper that agarose gel electrophoresis can be used to examine the tertiary structure of supercoiled SV40 DNA. Differences as small as one superhelical turn can be detected by this method. When used as an assay, it enabled us to purify from human tissue culture cells a protein which acts on supercoiled DNA, leading to a gradual removal of its superhelical turns. Using in turn the purified DNA relaxing protein to generate reaction intermediates of DNAs with different numbers of superhelical turns, we deduced that native SV40 DNA contains a minimum of 20 physical superhelical turns. The upper limit of this number is less accurately defined, but we think that it is not higher than 24. These numbers agree within very close limits with the numbers determined previously by various indirect physical methods.


Transformation of primate and rodent cells by temperature-sensitive mutants of SV40. Cells of mouse, hamster, human and monkey origin were transformed by wild-type SV40 and by temperature-sensitive mutants defective in gene A. The mutant-transformed cells appear to be temperature sensitive for maintenance of transformation. Evidence of reversion of the transformed phenotype of nonpermissive temperatures was obtained in experiments which assayed saturation density, colony formation, and uptake of 2-deoxy-D-glucose. In addition, S antigen modulated in shift experiments while T antigen did not. Based on these results, a model is presented for the mechanism of transformation by SV40.


SV40: T antigen, the A function and transformation. T antigen, a protein found in the nuclei of SV40-transformed and -infected cells, can be purified and characterized. Our data suggest that T antigen in transformed cells is found at least in part in a large 22S complex, suggesting that this 22S form may contain T antigen complexed with other viral or host proteins. We prefer the assumption that T antigen is correlated with the A function, although a definitive in vitro proof for this hypothesis is still missing. Mutants in the A function most likely interfere with the maintenance of transformation, a result inviting the interpretation that the A function may indeed govern DNA synthesis of the transformed cell or influence the cell's ability to pass from G1 (or G0) to S. Thus transformation by SV40 may be a direct consequence of the introduction of the SV40 replicon and the presence of its initiator function which is used by the transformed cell.


Cytosine arabinoside- and interferon-mediated control of polyoma and SV40 genome expression. By metabolic DNA inhibitors such as araC, viral as well as host DNA replication is suppressed in polyoma- and SV40-infected cells. The interruption of the current viral DNA replication has no effect on the current transcription of the late viral genes. The persistence of the late transcription indicates that the onset, but not the persistence, of the viral DNA replication is a prerequisite for the persistence of the late polyoma and SV40 genome transcription. Pretreatment of monkey kidney cells with poly(I):-poly(C) nearly completely inhibits the SV40 T antigen formation; the early SV40 RNA formation is suppressed far less. This type of SV40 genome control favors the concept of a primary action of poly(I):poly(C)-mediated interference on SV40 translation.


Genetics of cell transformation by simian virus 40. The results described in this paper indicate that the integration of the SV40 genome into human chromosome 7 results in the transformation of the human cells and in the expression of SV40-induced antigens. If integration of the SV40 genome in human chromosomes other than 7 ever occurs, it does not result in cell transformation and the expression of the virus-induced antigens. The expression of the SV40 T antigen in different monkey cells transformed by an adeno 7-SV40 hybrid is also related to a specific monkey chromosome. Somatic cell hybrids between normal nondividing mouse cells and SV40-transformed human cells behave as transformed cells and contain, without exception, the human chromosome 7 carrying the SV40 genome. Since no segregation into SV40 T antigen-negative hybrid clones was observed in these hybrids, it is inferred that the presence of the human chromosome 7 carrying the SV40 genome in the hybrids is mandatory for cell division.


Infection of mouse blastocysts with SV40 DNA: normal development of the infected embryos and persistence of SV40-specific DNA sequences in the adult animals. In SV40-transformed culture cells, viral-specific sequences have been found to be covalently linked to host sequences (Sambrook et al. 1968). The most appealing interpretation to explain the presence of SV40-specific sequences in adult mice following infection at the preimplantation stage would be to assume that the viral DNA was integrated at this early stage of development into the host genome and was thus conserved during further development. However, our results do not exclude an extrachromosomal existence of the SV40 genome, for example, as an independently replicating plasmid or as a lytic infection in a few permissive cells. So far our attempts to demonstrate autonomous SV40 DNA replication in early mouse embryos have been unsuccessful. We plan to investigate whether the SV40-specific information can be genetically transmitted from the infected mice to their offspring; chromosomal integration would be proven if transmission of SV40 DNA occurred in accordance with simple Mendelian expectations. The injection of mouse blastocysts with purified SV40 DNA did not detectably interfere with normal development of the embryos to healthy adult mice, which were still tumor-free at one year of age. This was not due to the trivial possibility that the viral DNA did not successfully infect and was eliminated from the injected embryos, as virus-specific DNA sequences were detected in 40% of the infected year-old animals, or in about 25% of DNA preparations extracted from some of their tissues (Table 1). It is nevertheless possible that the animals may not have been old enough to exhibit tumorigenesis of SV40 origin; to test this possibility, the experiment will have to be repeated for longer survival periods. The absence of any obvious signs of expression of viral genetic functions, i.e., tumor formation, up to one year of age of the host is reminiscent of the "cryptic transformants" described earlier (Smith et al. 1972) which harbor SV40 information but behave essentially like normal untransformed cells. Whether transcription or translation of the virus gene can occur in infected mice is presently an open question. Testing for expression of an integrated viral genome in diverse differentiated tissues may provide a useful model system to study the regulation of differentiation. These matters are currently being investigated.


Studies of hamster cells transformed by adenovirus 2 and the nondefective Ad2-SV40 hybrids. Adenovirus 2 does not produce tumors when injected into newborn hamsters, and this failure is not corrected by the acquisition of the portion of the SV40 genome present in any of the nondefective Ad2-SV40 hybrid viruses. However, nonhybrid Ad2 will transform hamster cells in vitro, and these transformed cells will produce tumors when injected into newborn hamsters. This finding, taken in conjunction with other studies (Graham et al., this volume; McAllister et al., 1969; McDougall et al., this volume; Williams 1973), suggests that the categorization of Ad2 and Ad5 as "nononcogenic" viruses is a reflection of host response rather than an intrinsic property of the viruses. However, transformation of hamster cells by Ad2 is inefficient, requiring 10(7)-10(8) PFU to produce a focus of transformed cells. Transformation of hamster kidney cells by the nondefective hybrids cannot be associated with portions of the SV40 genome. During the transformation process, complex interactions must occur between the hamster cell genome and the viral genome of Ad2 or the nondefective hybrids. Thus while it appears that there is very little Ad2 DNA in one nonhybrid Ad2-transformed cell line, there is a larger amount of Ad2 DNA in all of the hybrid virus-transformed cell lines. Moreover, while one Ad2+ND2-transformed line has apparently lost the SV40 portion of the viral genome, it is present and transcribed in another line transformed by the same virus. Finally, in the lines of nondefective hybrid-transformed cells examined thus far, there is extensive transcription from the Ad2 H strand. This pattern of transcription differs from transcription of the viral genome in most hybrid Ad2-transformed hamster and rat cells in which L-strand transcripion predominates.


Characterization of defective simian virus 40 DNA: comparison between large-plaque and small-plaque types. We characterized defective DNA molecules of large-plaque and small-plaque SV40 produced and accumulated during serial undiluted passages. The electron microscope heteroduplex method revealed that defective SV40 DNA contains deletions, insertions and substitutions. The majority of the large-plaque defective molecules had a simple deletion. The deletions were heterogeneous in size, and the sites of deletion appeared to be mostly overlapping. The majority of the small-plaque defective molecules had a deletion, and more than half of the deletion molecules also had an insertion at a separate site. The small-plaque SV40 genome appeared to have more sites for deletion to occur than large-plaque SV40, and the deletions have occurred at more than two sites. The change of a local nucleo tide sequence resulting from mutation may be related to the difference between the two plaque types.


Endotracheal antibiotics for the prevention of tracheobronchial infections in tracheotomized unconscious patients. A comparative study of gentamicin and aminosidin-polymyxin B combination. Endotracheal administration of gentamicin has been compared to the endotracheal administration of aminosidin plus polymyxin B as a preventive measure against tracheobronchial infections in 25 and 22 tracheotomized patients respectively who had been admitted to a neurosurgical unit. Both series were comparable as far as underlying disease, duration of hospitalization, surgical therapy. Both regimens were similarly effective from the bacteriologic and clinical points of view. Both regimens were similarly effective in preventing colonization of bronchial secretions by potential pathogens and were associated with a similar frequency of infectious episodes (eight in each group). The use of aminosidin-polymyxin B combination was associated with a lower incidence of emergence of gentamicin resistant strains, but the endotracheal administration of gentamicin was better tolerated than that of the combination. It is concluded that the combination of aminosidin-polymyxin is a useful alternative to gentamicin for the prevention of bronchopulmonary infections in unconscious tracheotomized patients.


Acquired tracheomalacia: etiology and differential diagnosis. The noninvasive diagnosis of impaired mechanical integrity or compliance of the trachea is most accurately made by fluoroscopic observation recorded on video tape or cineradiography, with or without benefit of artificial contrast media (contrast tracheography). In particular, localized buckling, collapse or dilatation indicative of focal tracheomalacia may thus be diagnosed in vivo and correlated with diseases of the central airways. Congenital tracheomalacia is a rare, bu, well described entity. Cases of acquired tracheomalacia occur with increasing frequency, but are often not clearly recognized. We contrast the dynamic behavior of the normal trachea with the abnormal dynamics characteristic of focal tracheomalacia. Such lesions may result from trauma, surgical procedures, chronic irritation, inflammation, mechanical changes, or malignancy.


Fibrous histiocytoma of the trachea.

Fibrous histiocytoma of the trachea. A histologically benign but locally infiltrating tumor of histiocytes, fibrous histiocytoma, was found in the trachea of a 26-year-old man. Partial resection of the trachea afforded the successful removal of the growth with no evidence of recurrence or metastases 27 months after surgery. Pathology and prognostic aspects of the tumor are discussed.


Evaluation of different antibiotic actions combined with rifampicin. In vitro synergism against Pseudomonas and Proteus. Associations of penicillin, streptomycin, tetracycline and polymyxin B with rifampicin were tested for synergism against Pseudomonas and Proteus pathological strains. The combination of rifampicin and polymyxin B was clearly synergistic for all strains as it was evaluated by isobolograms and killing curvesmthe bactericidal action of this association, without any cross-resistance between its components, may eradicate gram-negative bacteria infections with marked polyvalent drug resistances.


Studies on the additive effect of polymyxin B and the bactericidal activity of human serum against Serratia marcescens. Two of twelve examined S. marcescens strains were promptly killed by 80% (v/v) fresh human serum (within 20 min), analogous to  a serum-sensitive control strain of Escherichia coli; ten strains, however, were killed by fresh serum only after extended incubation (2-4 h). The combination of therapeutically achievable concentrations of polymyxin B (range 5 to 1.25 mug/ml) and fresh, but not heat-inactivated human serum was found to exert an accelerated, additive effect against 9 of 10 'delayed serum-sensitive' isolates of S. marcescens, an organism that is characterized by intrinsic resistance against polymyxins. The combination of 80% (v/v) fresh, defibrinated human blood and polymyxin B likewise resulted in an additive effect. Polymyxin B treatment of S. marcescens strains caused a prompt, marked, though reversible bile salt susceptibility of the cells; in contrast, the effect induced by fresh serum was slight and not apparent until several hours after exposure.


In vitro and in vivo effect of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) on types 1 and 3 parainfulenza virus infections. 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) had significant in vitro activity against type 1 parainfluenza (Sendai) and type 3 parainogenic (HA-1) viruses. Activity was manifested as inhibition of both viral cytopathogenic effect and of recoverable virus or viral hemagglutinin titer. The minimum Sendai virus inhibitory concentration was determined to be approximately 3.2 mug/ml. Previous studies had determined the minimum concentration inhibiting HA-1 virus was approximately 1-10 mug/ml. The effect of time of addition of ribavirin to virus-infected cells was determined; maximal activity was seen when the drug added just prior to either virus or within 4-8 h after each virus, although anti-Senadi viral effects were still apparent when ribavirin was added as late as 24 h after the virus. Ribavirin had no effect on adsorption of HA-1 or Sendai virus to cells. Lethal Sendai virus infections of mice were significantly inhibited by multiple intraperitoneal ribavirin treatment, starting either 4 h before or up to 24 h after virus inoculation. Therapy starting 48, 72 or 96 h after virus exposure had a moderate degree of efficacy. Treatment using an aerosol chamber also was of moderate effectiveness, although the procedure was considered traumatic to the animals. A nonlethal, principally upper respiratory tract infection of hamsters induced by the HA-1 virus was inhibited by ribavirin therapy. Treatment administered intraperitoneally, per os or by aerosol chamber resulted in reduced 23-day antibody titers to the virus, presumably because of reduction of virus in the animal. In a separate experiment, intraperitoneal ribavirin therapy resulted in a 1 log10 or less reduction in virus titer in nasal washings from HA-1 virus-infected hamsters, whereas, when the drug was administered intranasally in a dry powder aerosol spray, nasal virus titers were reduced up to 2 log10 and a moderate virus-induced lung consolidation was completely inhibited.


Antiviral activity of a pyrazino-pyrazine derivative. The 2,3-dihydroxy-6-bromo-pyrazino-[2,3-beta]-pyrazine is a substance selected during the antiviral screening of pyrazino-pyrazine derivatives. The compound shows antiviral activity in vitro against measles, NDV, some influenza viruses and against herpes simplex and zoster, infectious canine hepatitis and vaccinia viruses. It had no effect on ECHO 9 virus. Therapeutic trials showed activity also on herpetic keratoconjunctivitis experimentally induced in rabbits.


[Persistence of latex-agglutination inhibiting and neutralizing antibodies against enterovirus in the healthy population (author's transl)]. Sera of 122 persons were examined by the neutralization and latex-agglutination inhibition tests for antibodies against Polio type I, Coxsackie virus type A 9 and B 4, as well as Echo virus type 9. With increasing age there was a titre difference between latex-agglutination inhibiting and neutralizing antibodies, due to the earlier fall in the former. These antibodies, contrary to neutralizing ones, were in the lower titre region. The results confirmed the usefulness of the latex-agglutination inhibition test in the diagnosis of acute enterovirus infections.


[Carcinoma of the liver in a child after seven-year complete remission of acute lymphoblastic leukaemia(author's transl)]. A malignant hepatoma occurred in a 12-year-old girl who eight years previously had developed an acute lymphoblastic leukaemia which for eight years had been in complete haematological remission. Fourteen months after the last re-induction treatment period had been discontinued, but while on methotrexate and 6-mercaptopurine maintenance, a hepatocellular liver carcinoma developed of which the patient died after a fulminating course, still in complete haematological remission. As far as is known, no direct carcinogenic effect can be ascribed to the two antimetabolites, but it must be assumed that these two drugs, taken by the patient for over seven years, led to cirrhosis of the liver whose malignant transformation was significantly influenced by the immunosuppressive effects of methotrexate and 6-mercaptopurine, given as maintenance therapy according to protocol 02 LA 64, Paris.


[Inhibition of ethanol exidation on prolonged fasting in man: reversibility on fructose infusion(author's transl)]. Fasting for over 36 hours was found to inhibit ethanol metabolism in healthy volunteers by 43% (P less than 0.01) compared with values obtained after 12 hours' fasting. Ethanol oxidation was not inhibited by prolonged fasting when it was stimulated by intravenous fructose. The explanation for this effect is thought to lie in the inhibition of alcohol oxidation mainly by limiting the rate of NADH-reoxidation in the liver.


Corticosterone-induced changes in hypothalamic corticotropin-releasing factor (CRF) content after stress. Central and peripheral humoral responses of the adrenocortical system were measured for 2 h after the application of several stimuli. Two min after the onset of the stresses of sham-adrenalectomy or laparotomy with intestinal traction there was a 4-6 fold increase in hypothalamic CRF content as compared to control content, This is the usual CRF response to stress. In contrast, after adrenalectomy or manipulation of the pedicles of adrenal glands; CRF content at 2 min was only slightly increased above baseline values. This finding suggests that touching the adrenal vascular and nervous supply results in a direct neural input to the hypothalamus that is qualitatively different from most other stimuli. At times later than 2 min after stress, whem plasma corticosterone levels rise in the intact rat, the patterns of CRF and ACTH responses that were observed after adrenalectomy were determined by whether corticosterone replacement therapy was given. Without corticosterone replacement, the CRF and ACTH responses to adrenalectomy resembled those of laparotomy with intestinal traction. When corticosterone was administered 2 and 40 min after adrenalectomy, the CRF and ACTH responses resembled those of sham-adrenalectomy. At 20 min, CRF content was low after laparotomy with intestinal traction or adrenalectomy and high after shan-adrenalectomy or adrenalectomy with corticosterone replacement. Plasma ACTH peaked by 20 min, and remained high for 2 h after the first 2 stimuli, and was significantly decreased from the 20 min peak by 40 min after application of the latter stimuli. CRF content increased to a second peak 80 min after laparotomy with intestinal traction or adrenalectomy. This rise in CRF must represent increased formation of the releasing factor because ACTH levels were elevated and constant for the preceding 60 min. After sham-adrenalectomy or adrenalectomy with corticosterone replacement, CRF content and ACTH are low at 80 min. Measurement of circulating ACTH levels in conjunction with CRF content after these stimuli have yielded sufficient information to assign  mechanisms of altered synthesis and secretion to explain the observed changes in CRF content. Corticosterone damps the adrenocortical system response to the stimuli of sham-adrenalectomy or adrenalectomy with corticosterone replacement by two mechanisms. Firstly, it acts to inhibit CRF secretion probably via rate-sensitive feedback. Secondly, it acts to inhibit the second wave of CRF formation that is observed 80 min after stress is applied, probably via the proportional feedback mechanism.


Competitive binding activity of angiotensin II analogues in an adrenal cortex radioligand-receptor assay;. The competitive binding activities of a number of angiotensin analogues were determined in a radioligand-receptor assay employing bovine adrenal crotex homogenate and [125I] iodoangiotensin II. This assay system has been shown to provide a precise and convenient method for evaluation and comparison of the binding-inhibition potencies of angiotensin II derivatives with agonist and antagonist activities. Agonist analogues of angiotensin II showed competitive binding activities in proportion to their known biological activities upon aldosterone production by the adrenal zona glomerulosa. Thus, the [Des-Asp1] heptapeptide of angiotensin II was equipotent with the native octapeptide in terms of binding-inhibition activity, and the [Sar1] derivative of angiotensin II was more potent than the native peptide. By contrast, the [Des-Asp1, Des-Arg2] hexapeptide and the [Des-Phe8] heptapeptide showed less than 1% of the activity of angiotensin II. Angiotensin II antagonists formed by C-terminal substitution of the octapeptide with isoleucine or alanine were also found to exhibit binding-inhibition activities in proportion to their known potencies as antagonists of the action of angiotensin II upon smooth muscle. Certain antagonists, such as [1-guanidoacetic, 8-isoleucine]-angiotensin II and [1-sarcosine, 8-isoleucine]-angiotensin II displayed significantly greater binding-inhibition potency than E1Asp1, Ileu5,-angiotensin II in the adrenal receptor system. Several, such as [Sar1, Ala8]-angiotensin II and [MeAla1, Ileu8]-antiotensin II were approximately equipotent with angiotensin II, while others such as [1-succinic acid, 8-alanine]-angiotensin II and [D-Ileu8]-angiotensin II showed no significant binding-inhibition activity. The relative binding-inhibition potencies of the antagonist peptides showed a general correlation with the pA2 values of the same components determined upon the smooth muscle response of the isolated aortic strip. The binding-inhibition potency of angiotensin II antagonists was also strongly influenced by the charge of the N-terminal residue, with enhancement of activity by more basic substituents and abolition of activity by highly acidic residues. The influence of the basicity of the N-terminus upon receptor binding was also observed with the agonist analogue [Sar1]-angiotensin II, which showed a 2- to 3-fold increase in binding-inhibition potency in comparison to native angiotensin II. The significant enhancement of binding-inhibition potency by N-terminal sarcosine substitution is attributable to higher affinity of the modified peptide for the angiotensin II receptor site, and is consistent with the increased activity of [Sar1]-angiotensin II upon smooth muscle and aldosterone production in vitro. The determination of binding-inhibition activity in the adrenal radioligand receptor assay provides a valid and convenient method for analysis of the role of binding affinity in the actions of competitive antagonists upon the responses of target cells to angiotensin II.


Degradation of thyrotropin-releasing hormone by the GH3 strain of pituitary cells in culture. Thyrotropin-releasing hormone (TRH), pGlu-His-ProNH2, binds within 1 h to specific receptors on the GH3 strain of pituitary cells. When GH3 cells were incubated for 2 days with 3 nM [2,3-3H-Pro]TRH, an increasing fraction of the total cellular radioactivity (7% after 1 h, 81% after 43 h) was associated covalently with proteins as determined by dialysis, acid precipitation, and gel filtration; this fraction corresponded to label which could not be displaced from intact GH3 cells by the addition of excess unlabeled TRH. R5 and GH12C1 cells, strains which lack TRH receptors, accumulated 16 or 23%, respectively, as much label from [2,3-3H-Pro]TRH as did GH3 cells in 24 h. After 24 h of incubation with [2,3-3H-Pro]TRH and [14C-His]TRH, the ratio of 14C/3H in GH3 cells was the same as in the culture medium, indicating that the intact tripeptide was taken up by the cells. After 24-48 h of incubation with [2,3-3H-Pro]TRH, GH3 proteins appeared to be labeled randomly as surmised by fractionation on Sephadex G-100, DEAE cellulose, Sepharose 4B and sucrose density gradients. In cultures treated with cycloheximide (10 mug/ml) or proline (6.3 mM) the initial binding of [2,3-3H-Pro]TRH to receptors, measured after 1 h, was 97% or 102% of control. However, the incorporation of label from [2,3-3H-Pro]TRH into an acid-precipitable product after 22 h was inhibited by 81 and 74% by cycloheximide (1 mug/ml) and proline (2.5 mM). Formation of [2,3-3H] proline from [2,3-?3H-Pro] TRH was demonstrated by thin layer chromatography; the percentage of non-protein radioactivity with an Rf of proline increased from 20 to 80% in GH3 cells incubated 1 or 24 h with [2,3-3H-Pro]TRH. We conclude that after binding to receptors on GH3 cells, TRH is slowly metabolized to its constituent amino acids, and the products [2,3-3H]proline or [14C]histidine are incorporated into newly synthesized proteins.


The significance of calcium for glucose stimulation of insulin release. The significance of Ca++ for glucose stimulation of insulin release was studied in an in vitro system with beta-cell-rich pancreatic islets microdissected from oh/ob-mice. There was only a slight depression of cAMP in islets exposed to the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine after withdrawal of Ca++ from the incubation medium. The lack of a stimulatory effect of glucose noted in the absence of extracellular Ca++ is therefore probably accounted for by factors other than impaired adenylate cyclase activity. A rise of extracellular Ca++ above the concentration necessary for obtaining the optimal secretagogic effect of glucose resulted in inhibition of the glucose-stimulated insulin release, leaving basal secretions and islet contents of cAMP unaffected. Evidence was provided in support of the idea that H+ completes for Ca++ in glucose stimulation of insulin release. Both the rate of basal insulin release and that seen after stimulation with glucose were diminished by about 50% after introducing 0.2 mM La+++ in the incubation medium. These observations emphasize the significant role of Ca++ in the regulation of insulin secretion, suggesting that not only a decrease but also an increase of the functionally important intracellular pool(s) of Ca++ can result in a diminished response to glucose.


The effect of castration and testosterone replacement on specific proteins and androgen levels of the rat epididymis. The normal weight increase of the epididymis during sexual maturation and its maintenance through adulthood were found to be dependent on the provision of androgens. Binding of [3H]dihydrotestosterone (DHT) to the epididymal 8S cytoplasmic receptor gradually decreased after castration to become undetectable after 25 days. Binding to the androgen binding protein (ABP) was absent 4 days after castration and was not reinduced by 3 weeks of testosterone (T) administration. Unilateral castration for periods of up to 27 days showed the disappearance of ABP with preservation of the 8S receptor on the castrated side, indicating a testicular source for ABP and the epididymal origin of the 8S receptor. The tissue concentrations of T and DHT in the epididymis became undetectable 30 days after castration and were restored to normal values by administration of testosterone in large doses (1.5 mg/100 g BW). Similar results were obtained in rats castrated at 10 days of age and injected with testosterone until 60 days old. The ratio DHT/T was depressed in the castrate and increased with testosterone treatment. The protein content of the epididymis (mg of protein/g wet weight) was also found to be influenced by androgens. Our results show evidence of some mechanisms involved in the trophic effect of androgens upon the epididymis and suggest the possible androgenic control of epididymal 5alpha-reductase activity. They also indicate that a testicular factor is required for the maintenance of the 8S cytoplasmic androgen receptor. It is not known whether this factor is testosterone or some other testicular secretion.


Regulation of in vitro insulin release from a transplantable Syrian hamster insulinoma. An insulin-producing islet cell tumor of the Syrian hamster has been studied in vitro for its capacity to respond to known stimuli of insulin release. Insulin secretion during short term incubation and perifusion of fragments of tumor was detected by radioimmunoassay. Insulin release was increased 2-4 fold by 40 mM potassium in the presence of calcium, glucose (22 mM), glucagon (0.3-3.0 muM), N6,02'-dibutyryl adenosine 3',5'-monophosphate (cAMP; 6mM), and theophylline (10 mM). Concentrations of glucagon that induced insulin release were also effective in activating adenylate cyclase in the membranes of tumor cells. Thus, this tumor appears to possess a cAMP-mediated mechanism for insulin release. Somatostatin (0.8-25 mum) inhibited glucagon-induced insulin release without altering basal or glucagon stimulated adenylate cyclase activity. It would appear that inhibition of glucagon induced insulin release by somatostatin is not mediated by adenylate cyclase. We propose that insulin release by this tumor is sufficiently similar to that found in normal islets so as to make it a suitable model for biochemical studies that require large quantities of homogeneous tissue.


Renal receptors for calcitonin: coordinate occurrence with calcitonin-activated adenylate cyclase. High affinity binding of 125I-labeled salmon calcitonin ([125I]SCT) and calcitonin-activated adenylate cyclase were detectable in renal plasma membranes from the rat. Addition of 5'-guanylyl-imidodiphosphate lowered the threshold for enzyme activation by peptide hormones. Renal plasma membranes from man, dog and cow contained little or no calcitonin-sensitive adenylate cyclase and showed no high affinity binding of [125I]SCT. High affinity binding sites were distributed during membrane fractionation in fractions where the specific activity of hormone-sensitive adenylate cyclase was greatest. Calcitonin binding and activation of the adenylate cyclase enzyme occurred at similar hormone concentrations. The relative potencies of calcitonin analogues were similar whether measured by competition for high affinity binding sites or by effect on adenylate cyclase. Low concentrations of Lubrol-PX, a nonionic detergent, did not affect catalytic function of the enzyme determined in the presence of sodium fluoride but caused parallel loss of high affinity [125I]SCT binding and hormonal sensitivity of the enzyme. This observation provided further evidence that interaction of calcitonin with specific receptors (identified with [125I]SCT binding) is essential for calcitonin activation of adenylate cyclase, but showed that catalytic activity of enzyme does not require functioning hormone receptors.


Antibodies to Epstein-Barr virus in subacute sclerosing panencephalitis patients. Blood samples from 45 cases of subacute sclerosing panencephalitis in Israel were tested for the presence of antibodies to Epstein-Barr virus capsid antigen and early antigen by the indirect immunofluorescence method. Significantly higher titers were found in patients as compared to healthy controls with higher titers especially in Jews of Asian-African origin. Mean geometric titer was 1:42.8 in this group as compared to 1:10.8 in the control group. Antibodies to Epstein-Barr Virus early antigen were found in 50% of the patients and in none of the controls tested. Elevated titers of antibodies to Epstein-Barr Virus antigens seem to be related to impaired immunological mechanisms in subacute sclerosing panencephalitis patients, especially of a certain ethnic group.


Change of the cytochrome oxidase level during exponential growth in Neurospora crassa. In Neurospora cells growing in various media, the specific activity of cytochrome oxidase increases very markedly during early exponential growth, reaching a maximum after 4-5 duplication times, then it slowly declines.


Absence of effects of dibutyryl cyclic guanosine 3',5'-monophosphate on release of alpha-amylase, 45Ca efflux, and protein synthesis in rat pancreas in vitro. Dibutyryl cyclic GMP did not affect basal, or carbachol stimulated secretion of alpha-amylase from rat pancreas. The nucleotide did not have a significant effect on 45Ca release from the pancreas nor did it alter the response to carbachol. The dibutyryl analogue of cyclic GMP did not duplicate or alter the inhibitory effect of carbachol on 3H-leucine incorporation into pancreatic trichloroacetic acid-precipitable protein.


Microsomal metabolism as a determinant of aflatoxin toxicity. The evidences to incriminate the possible role of microsomal drug metabolizing system in aflatoxin toxicity in rat have been reviewed. It is suggested that an effective drug metabolism results in decreased acute toxicity and the products of metabolism are more carcinogenic than the native toxin to the rat liver.


Fetal calf serum alters cyclic adenosine 3',5'-monophosphate's effect on heme synthesis in vitro. An investigation of the effect of cAMP on heme synthesis of rat bone marrow cells revealed that at 10(-2) M this cyclic nucleotide inhibits heme synthesis and that optimum stimulation occurs at 10(-4) M. Some unidentified constituent of fetal calf serum in the culture medium modifies the direction and degree of cAMP's effect.


Appearance of sex hormone receptors in frog (Rana esculenta) tadpole skin during metamorphosis. In tadpole skin of Rana esculenta, a specific testosterone receptor was detected during the climax in both males and females. The Kass ranged between 1 and 2.79 x 10(9)M-1.