Nonimmunological host defenses: a review. Nonimmunological defenses are very diverse in type. Some are directed against already transformed cells and belong to mechanisms of containment. Others exert a surveillance by preventing or inhibiting initial events of carcinogenesis. Chalones and oncolytic factors in sera and exudates are agents of containment. Under appropriate circumstances, the autoxidation of thiols and the formation of mixed disulfides lead to destruction of tumor cells in vitro and in vivo. Both processes involve the generation of superoxide radicals and of hydrogen peroxide which, in turn, activate the peroxide:peroxidase:halide system. Thiol:disulfide ratios and interchange codetermine the antioxidative activity of cellular membranes, thus bearing on carcinogenesis. Many aliphatic and aromatic antioxidants are endowed with anticarcinogenic properties. The fact that they are inhibitors of free radical processes corroborates the increasingly evident role of free radicals in carcinogenesis. Endogenous antioxidants and exogenous ones in foods are agents of surveillance. Antioxidant activity, linked with the ergastoplasm, points to a homeostatic mechanism that prevents self-accelerating chain reactions from leading to membrane damage or to carcinogenesis. Carcinogens can also be inactiviated by microsomal enzymes belonging to an overall mechanism of detoxification. Activity levels of these systems depend on diet and state of nutrition. They may be naturally very low, but they can be increased with various inducers.


Corticosteroids and phosphatidylcholine biosynthesis in microsomal fractions from L5178Y lymphoma. Microsomal fractions from mouse lymphoma L5178Y and from rat thymocytes were used to follow incorporation of radiolabel from cytidine diphosphate-[methyl-14C]choline into microsomal lipids. Dexamethasone, at concentrations ranging from 2.8 X 10(-6)M to 2.8 X 10(-5)M, partially inhibited this transfer reaction. Microsomes prepared from freshly isolated thymocytes were more sensitive to the effects of dexamethasone showing inhibition at concentrations of steroid as low as 2.8 X 10(-8)M. The inhibitory effect did not depend on the amount of the available endogenous diglycerides and was not related to a possible stimulation of cytidine diphosphate choline transferase back reaction by the steroid. The survey of a broad selection of different steroids revealed a lack of correlation between the known lymphocytolytic properties of steroids and their effects on cytidine diphosphate choline transferase. Dexamethasone was the only steroid of the glucocorticoid group that inhibited this reaction in microsomal fractions of L5178Y lymphoma. The structural requirement for the inhibitory effect was related to the absence of oxygen functions in positions 11 and 17 of the steroid and, possibly, to the presence of both C-20 and C-21 on the side chain.


Histogenesis and growth pattern of 1,2-dimethylhydrazine-induced rat colon adenocarcinoma. The histogenesis and growth pattern of colon adenocarcinoma have been studied using 74 BD IX rats given 20 mg 1,2-dimethylhydrazine hydrochloride per kg, s.c., weekly from Day 11 to their 24th week and serially sacrificed with controls. Modifications in DNA synthesis activity and early tumor changes were sought on histological and radioautographic preparations of normal-appearing colon mucosa. All visible colorectal tumors were analyzed for size, site, and pathology. Chronic dimethylhydrazine treatments resulted in a simultaneous increase in the number of both total and tritiated thymidine-labeled cells in the glands of Lieberkühn. In addition, microscopic carcinomatous foci were observed after the 15th week, and the first macroscopic adenocarcinomas appeared in 24-week-old animals. Their number thereafter exponentially increased with time. A total of 252 macroscopic tumors were obtained, of which 14 were classified as signet ring cell carcinomas and 238 as adenocarcinomas. Among the latter, local invasion could be documented in 230, including the smallest. No benign polypcancer sequence could be demonstrated in this material. The average growth pattern of those adenocarcinomas could be adequately described by a Gompertz curve, with a short initial doubling time (e.g., 7.5 days in 1.0-cu mm tumors) that progressively increases with time (e.g., 59.6 days in 5000-cu mm tumors).


Inhibited growth in vivo of a mouse pregnancy-dependent mammary tumor (TPDMT-4) by an antiestrogen, 2alpha, 3alpha-epithio-5alpha-androstan-17beta-ol (10275-S). TPDMT-4 mammary tumors, characterized by growth during pregnancy and postpartum regression, grew continuously in DDD virgin mice carrying pituitary isografts or 17 beta-estradiol plus progesterone pellets. In the experimental models, effects of 2alpha,3alpha-epithio-5alpha-androstan-17beta-ol(10275-S), testosterone, and ovariectomy were investigated. When tumors implanted with pituitary isografts into the fat pad reached palpable volumes, animals were ovariectomized or received 5 s.c. injections of 100 mug to 1 mg 10275-S or 300 mug testosterone weekly. Tumors regressed immediately after ovariectomy or after the start of 10275-S treatment and after approximately the 10th day of testosterone treatment. Cystadenomatous morphology with strong secretory activity was noticed in treated animals. Tumor growth induced by 17beta-estradiol plus progesterone pellets was inhibited completely or partially by 3 s.c. injections of 50 to 500 mug 10275-S or 100 mug testosterone weekly starting from the day after tumor and 17beta-estradiol plus progesterone pellet implantation.


Cell cycle-related hormone carcinogen interaction during chemical carcinogen induction of nodule-like mammary lesions in organ culture. The immature mammary glands of BALB/c female mice were treated with 7,12-dimethylbenz[a]anthracene (DMBA), 2 micrograms/ml, or 3-methylcholanthrene (10 micrograms/ml) for a 24-hr period at different times during the inital six days of lobuloalveolar growth in hormone-supplemented organ culture. Nodule-like alveolar lesions (NLAL) were detectable in 80% of the glands treated with DMBA (40% in 3-methylcholanthrene-treated glands) in the presence of insulin + prolactin + aldosterone + cortisol in the medium. No NLAL were present in dimethyl sulfoxide-treated control glands cultivated with the same hormones. The hormone combination insulin + prolactin + cortisol was unfavorable for NLAL induction by DMBA, and the combination of aldosterone + insulin + prolactin was only moderately conducive. Thus, the presence of cortisol with insulin + prolactin + aldosterone enhances NLAL incidence of mammary cells by DMBA. The highest incidence was found in glands that were treated with DMBA for 24 hr between the third and fourth day of culture, the period corresponding to the onset of the second wave of DNA synthesis in the gland. Cytotoxicity of DMBA was pronounced between 24 and 48 hr, when a high frequency of cells were in DNA synthesis, and survival of the cells after the cytotoxic effect of DMBA appeared to play a role in NLAL incidence. This suggests that DMBA-induction of NLAL in mammary glands in organ culture involves a complex carcinogen-hormone-cell cycle interaction. We emphasize that, although NLAL morphologically resembles the hyperplastic alveolar nodules of mouse mammary gland in vivo, the abilitity of NLAL to produce typical hyperactive alveolar outgrowth and mammary tumor after transplantation iv vivo remains to be determined.


Inactivation of tumor cell-associated feline oncornavirus for preparation of an infectious virus-free tumor cell immunogen. Ultraviolet (UV) and thermal methods of inactivating the oncogenic potential of C-type particle-producing feline oncornavirus-induced tumor cells were developed. The techniques were evaluated by several parameters for their use in preparation of cellular immunogens. The UV inactivation dose required to reduce the number of focus-forming units per ml by 1 log10 for FL-74 lymphoblastoid cell-associated feline leukemia virus was 44,000 ergs/sq mm, and the thermal inactivation dose required to reduce the number of focus-forming units per ml by 1 log10 at 45 degrees was 16 min. Inactivation of greater than 6 log10 of virus per ml associated with 4 x 10(8) cells required a UV dose of 270,000 ergs/sq mm, 100 min at 45 degrees or 3 min at 56 degrees. All three treatments concomitantly destroyed the replicating potential of FL-74 cells as shown by their inability to propagate under normal growth conditions and to incorporate [3H]thymidine into nuclear DNA. UV inactivation and thermal inactivation at 45 degrees allowed the best retention of feline oncornavirus-associated cell membrane antigen. A 50% loss in antigenic activity was observed as a result of 56 degrees treatment, but this method was the only one that did not destroy the surface structural integrity of FL-74 cells.


Impaired host defense against XC cell-induced tumors in thymectomized and in bursectomized chickens. XC cells originally derived from the tumor of a rat previously inoculated with the Prague strain of Rous sarcoma virus were used to induce tumors in chickens surgically thymectomized or bursectomized in the newly hatched period. Thymectomized chickens had a significantly higher incidence of tumors, larger tumors, and a higher tumor mortality, compared with control chickens, when both groups were given 5 X 10(6) XC cells into the wing webs. Bursectomy could significantly influence the tumor size only. It appeared that the capacity of XC cells to induce tumors and the growth of such tumors were subject to immunological influence, with the thymus playing a major and the bursa of Fabricus a minor role under the conditions used.


Urinary excretion of cyclic guanosine 3':5'-monophosphate and cyclic adenosine 3':5'-monophosphate in rats bearing transplantable liver and kidney tumors. Cyclic guanosine 3':5'-monophosphate (cyclic GMP) and cyclic adenosine 3':5-monophosphate were measured in the urine of normal rats and those bearing transplantable liver and kidney tumors. The level of cyclic GMP ranged from 1.4 to 1.6 mumoles/g urinary creatinine in several strains of rats without tumors. Rats bearing Morris hepatomas 20, 21, 9618A and 9633F and kidney tumor MK2 had urine levels of cyclic GMP from 1.3 to 3.6 mumoles/g creatinine. Rats bearing the fast growing hepatomas 9618A2 and 3924A and Morris kidney tumor MK3 had urinary values of 5.6. 41.9, and 32.7 mumoles cyclic GMP per g creatinine, respectively. Urine levels of cyclic adenosine 3':5'-monophosphate ranged from 10.1 to 19.7 mumoles/g creatinine in all normal and tumor-bearing rats and were not significantly different in any of the groups examined.


Multilaminar endoplasmic reticulum and abnormal mitosis in Hodgkin tumor cells. A multilaminar alteration of endoplasmic reticulum (ER) has been observed in tumor cells of eight patients with Hodgkin's disease and a patient with histiocytic lymphoma. These multilaminar structures are more numerous in dividing cells and thus appear to arise primarily during mitosis. The stacked membranes in the multilaminar structures possibly result from abnormal sticking of organelle membranes, as evidenced in this study of adherence of ER to other elements of ER, nuclear envelope, mitochondria, or lipid droplets. Multilaminar ER was identified in all mitotic tumor cells, a rare mitotic plasma cell, and numerous interphase Hodgkin cells. The paucity of multilaminar ER in normal mitotic cells and its virtual absence for normal interphase cells suggest that this structure represents a pathological alteration in tumor cells from patients with Hodgkin's disease and histiocytic lymphoma. The multilaminar defect of ER is associated with other atypical features of ER in Hodgkin tumor cells, including the excessive length and curving of ER profiles, the collapse of the ER cisternae, and the overall sparsity of this organelle. Other abnormalities observed in mitotic Hodgkin tumor cells include the presence of disorganized microtubules, large cytoplasmic vacuoles, and abnormally clumped chromosomal material and the persistence throughout mitosis of bodies suggestive of nucleoli and of the nuclear bodies of interphase cells.


Regulation of the adenylate cyclase system in transplantable hepatomas. Adenylate cyclase systems were examined in purified membrane preparations from normal rat liver and several Morris hepatomas with differing growth rates. All tumor membrane preparations had lower relative specific activities than did liver preparations. Liver adenylate cyclase was stimulated by fluoride, glucagon and guanyl-5'-yl imidodiphosphate [Gpp(NH)p]. Membranes from two slow-growing hepatomas (hepatomas 20 and 21) contained adenylate cyclase activities which are also stimulated by each of these three modulators. Membrane adenylate cyclases from several fast-growing hepatomas (hepatomas 3924A, 7777, 5123tc, and 9618A2) were marginally stimulated by glucagon but were readily stimulated by fluoride and Gpp(NH)p. Examination of the highly specific binding of 125I-glucagon to the various membrane preparations revealed much less binding in all the tumor membranes than in liver membranes. More detailed kinetic examination of membranes prepared from liver, slow-growing hepatoma 21 (which had reasonable binding to and stimulation by glucagon), and fast-growing hepatoma 3924A (which had marginal binding to and stimulation by glucagon) revealed major differences in rates of cyclic adenosine 3':5'-monophosphate production in the absence and presence of glucagon, Gpp(NH)p, and glucagon plus Gpp(NH)p and in the combined alteration of magnesium:adenosine 5'-triphosphate ratio and temperatures. The different kinetic characteristics in the hepatoma adenylate cyclase systems may be due to different structural characteristics of the tumor membranes or may be due to altered hormonal receptors, catalytic units, or receptor-catalytic unit interrelationships within the tumor membrane.


Inhibition of rat liver RNA polymerases by action of the methylating agents dimethylnitrosamine in vivo and methyl methanesulfonate in vitro. Dimethylnitrosamine maximally inhibits rat liver nuclear RNA synthesis by 50% at a dose of 40 mg/kg of body weight. The inhibition develops during the first 4 hr and persists through the 12th hr. All parenchymal cells of the lever lobule seem to be affected. The decreased RNA synthesis can be accounted for entirely by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified. A similar inhibition of the polymerase activities was demonstrated in the intact nuclei by inactivating the endogenous template with actinomycin D and assaying the polymerases with an added exogenous template, poly(deoxy-adenylate-deoxythymidylate). Chromatin was prepared by two methods differing in the extent to which they remove the endogenous polymerase activity. Each preparation was transcribed with either added Escherichia coli or partially purified rat liver nucleoplasmic RNA polymerase. With either polymerase or chromatin preparation, no inhibition of the template activity of liver nuclear chromatin isolated from the DMN-treated animals was detected. A similar mechanism of inhibition of RNA synthesis was produced by the action of the methylating agent methyl methanesulfonate on whole nuclei in vitro. The dose-dependent inhibition of RNA synthesis could be accounted for by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified from the affected nuclei. Chromatin prepared from the methyl methanesulfonate-treated nuclei had a normal template capacity with either E. coli or rat liver nucleoplasmic RNA polymerase. No preferential methylation of the RNA polymerases by [14C]methyl methanesulfonate could be demonstrated. It is concluded that the action of the two methylating agents on RNA metabolism is similar and that the inhibition of liver nuclear RNA synthesis results from inactivation of the RNA polymerases. At the same time, dimethylnitrosamine and methyl methanesulfonate leave the chromatin template intact, at least quantitatively, for the synthesis of RNA. The implications of such an effect on RNA synthesis are discussed.


Some effects of acute and chronic dosing with aflatoxin B1 on rat liver nuclei. A study has been made of changes occurring in rat liver nuclei as a result of the chronic and acute administration of aflatoxin B1. This has involved resolution of the nuclei into populations of differing ploidy by means of centrifugation in a zonal rotor. Chronic feeding of the toxin to weanling rats prevents the development of the predominant tetraploid hepatocyte nuclear population, which normally takes place during maturation. Increased populations of diploid and octaploid hepatocyte nuclei are observed. Chronic feeding of the toxin to adult animals also causes a reduction in the tetraploid population already established and, again, leads to increased numbers of diploid and octaploid nuclei. Once an abnormal nuclear population has been established by feeding the toxin, it persists until the development of hepatocarcinoma (if a 6-week carcinogenic feeding regimen has been used). The hepatomas have diploid nuclei. Labeling hepatic RNA and DNA in vivo has indicated that feeding the toxin causes a reversion to the immature distribution of DNA synthesis among the nuclear population, with little effect on the pattern of distribution of RNA synthesis. Acute administration of aflatoxin to adult rats also causes a reduction in the size of the tetraploid population, with increased proportions of diploid, octaploid, and higherploidy nuclei. These results are discussed in terms of a dual action of the toxin, antimitotic and necrogenic, and the possible relationship of these to the carcinogenic process.


Electron microscopic study of virus particles in yolk sac and placenta and in hematopoietic organs of newborn C3Hf mice. Examination of yolk sac from a C3Hf and a C3H mouse with the electron microscope revealed the presence of C-type virus particles in the blood islands. Particles were observed budding from the plasma membrane of hemocytoblasts, from erythroblasts, and occasionally from reticulocytes. C-type particles were also found in similar cells in hematopoietic foci in the liver, spleen, and bone marrow of embryos, and they continued to be present in newborn C3Hf mice up to 11 days of age. Particles consistently appeared in the thymus, even in older suckling mice. A comparison is made between the presence of C-type particles in organs of embryonic, newborn, and adult C3Hf mice. C-type particles were not observed in the chorioallantoic placentas from mice that were given injections of mouse leukemia virus (Gross) or from normal noninjected mice; however, intracisternal A-type particles were present in cytotrophoblast cells from these placentas.


Characteristics of progesterone-binding components in neoplastic mammary tissues of the rat. Characteristics of [3H]progesterone-binding components were studied in cell-free preparations of two hormonally responsive tumors: the R3230AC mammary adenocarcinoma and 9,10-dimethyl-1,2-benzanthracene-induced mammary tumor of the rat. Progesterone-binding macromolecules from cytosols of both mammary neoplasms exhibited sedimentation coefficients of 3.5 to 4.0 S on sucrose gradients of either low or high ionic strength. From Scatchard analyses of titration data, apparent dissociation constants of 4 to 6 X 10(-8) M were determined for ligand-binding protein complexes from either tumor. Specific progesterone-binding capacities varied considerably, ranging from 150 to 650 fmoles/mg of cytosol protein. Optimal binding of [3H]progesterone was reached by 2 to 3 hr at 3 degrees, pH 7.4, and then decreased rapidly. Specificity studies indicated that cortisol, corticosterone, and triamcinolone acetonide competed effectively for [3H]progesterone-binding. This suggested that [3H] progesterone was bound largely to a macromolecule distinct from transcortin, which does not bind glucocorticoids containing 9alpha-fluoro groups. Aldosterone, as well as several androgens and estrogens, were weak competitors of binding except at high concentrations. The nature of the inhibition of progesterone-binding sites by triamcinolone acetonide and corticosterone was competitive. Concurrent titrations of [3H]progesterone and [3H]triamcinolone acetonide-binding sites demonstrated that their binding capacities were similar, considering the relative stabilities of the complexes. These results, which indicated that progesterone and glucocorticoids compete for the same binding site, suggest that these hormones may influence mammary gland differentiation and development by a common mechanism.


Phase transition of plasma membranes of rat hepatocyte and hepatoma cells by electron diffraction. The transition temperatures (Tc) of the plasma membranes of Reuber H-35 hepatoma and normal ACl rat hepatocytes were measured by electron diffraction under physiological conditions. Diffraction rings below Tc indicate the existence of solid lipid domains. The Tc of both membranes increase to a plateau value of 17.5 degrees during the storage period of 4 days at 4 degrees, the rate of increase being slower for normal liver membranes. The extrapolations to zero storage suggest an innate difference of Tc for these two membranes. Storage in oxygen-reduced media slows down the initial increase in Tc in normal liver membranes, while the depletion of divalent cations accelerates the increase. Differences in lipid composition are partly responsible for this behavior.


Enhancement of tumor growth following immunization with Bacillus Calmette-Guérin cell walls. The effect of preimmunization with Bacillus Calmette-Guerin cell walls (BCGcw) on tumor growth was studied with the use of transplantable Morris Hepatoma 3924a in inbred ACl rats. There was an increase in tumor incidence, an increase in the i.m. tumor growth rate, and an increase in the average i.m. tumor mass in animals that were preimmunized with BCGcw. The BCGcw-induced enhancement in tumor growth was dependent upon the amount of tumor present at the time of BCGcw immunization. No enhancement occurred in animals immunized with BCGcw 12 days after the inoculation of tumor cells.


The effect of substituents on the carcinogenicity of n-nitrosopyrrolidine in Sprague-Dawley rats. N-Nitrosopyrrolidine and two of its derivatives were prepared and fed in drinking water to Sprague-Dawley rats to compare the effects of substituents on the carcinogenicity of the N-nitrosopyrrolidine molecule. 3,4-Dichloro-N-nitrosopyrrolidine induced esophageal tumors in 13 of 14 animals, olfactory carcinomas in 4, and a hepatocellular tumor in 1. All animals that received this compound were dead at 55 weeks after the start of the experiments. N-Nitrosopyrrolidine induced hepatocellular tumors in 26 of 29 animals and induced 1 olfactory carcinoma. Not all animals in this group were dead until 104 weeks of the experiment. 2,5-Dimethyl-N-nitrosopyrrolidine induced only 2 hepatocellular tumors in 29 animals. The alpha-methyl substitution diminished the liver carcinogenicity, while the beta chlorine substitution affected a different target organ, the esophagus, and greatly reduced the time to death with tumors.


Cyclic nucleotide phosphodiesterase activity in normal and neoplastic rat mammary cells grown in monolayer culture. The activity of cyclic 3':5'-nucleotide phosphodiesterase (PDE) (EC 3.1.4.17) was measured in cultured normal and neoplastic rat mammary epithelium. Total PDE activity in normal cells was 1.6 to 6 times higher than that in tumor cells over a concentration range of 0.01 to 1 mM cyclic adenosine 3':5'-monophosphate. PDE activity was distributed between the low-speed (4000 x g) particulate and supernatant fractions in both cell lines, with the particulate fraction possessing 60 to 70% of the total. Double reciprocal kinetic plots were nonlinear, suggesting the presence of high- and low-affinity PDE activities. Similar, but not identical biphasic curves obtained from both normal and neoplastic cells suggested that at least two different PDE activities were present in a membrane-bound as well as a soluble form. Apparent Michealis constants for the high-affinity enzyme ranged from 2 to 6 muM; the low-affinity enzyme was 1 mM. In the presence of 10 mM caffeine and at a substrate concentration of 1 muM, PDE activity was inhibited 40 and 80% of basal levels in normal and tumor cells, respectively. In general, the membrane-bound enzyme was inhibited to a greater extent than the soluble, regardless of the cell line examined. Although normal cells exhibited higher PDE activities in terms of total specific activity, when soluble activities were compared at low substrate concentrations, the opposite was the case. At a substrate concentration of 0.01 muM, normal cell, low-Km soluble specific activity was 40% less than comparable tumor cell activity. Our results support the contention that PDE is induced by its own substrate, cyclic adenosine 3':5'-monophosphate. In addition, they suggest that the low cyclic adenosine 3':5'-monophosphate steady-state levels characteristic of malignant cells are maintained by a soluble high-affinity isozyme of PDE.


Transfer RNA methylase activity and capacity during aflatoxin B1-induced hepatocellular carcinogenesis. Transfer RNA methylase (tRNA methylase) activity and capacity were monitored in whole-rat-liver preparations during the induction of hepatocellular carcinomas by an 8-week aflatoxin B1 dosing regimen that produced minimal toxic effects. Significant phases of elevated tRNA methylase capacity occurred at 6 to 9 weeks (20%) and 24 to 29 weeks (40%). No significant change in tRNA methylase activity was noted over the course of the 55-week experiment. Higher aflatoxin B1 doses, producing acute toxic liver damage, resulted in elevated tRNA methylase activity (50%) and capacity (30%) at least as early as 1 week after dosing. Experiments with individual nodular lesions excised from livers of rats continuously fed a diet containing 2 ppm aflatoxin B1 demonstrated similarly elevated tRNA methylase activities and capacities in hyperplastic (preneoplastic)nodules, with and without histological evidence of carcinoma.


A survey on the effect of steroid hormone on type C virus production from cultured murine cells. Dexamethasone stimulates type C virus production induced by iododeoxyuridine (IdUrd) from several murine cell lines: uninfected BALB cells, virally transformed nonproducer K-BALB cells, mouse neuroblastoma N-4 cells, and rat tumor XC cells. Dexamethasone also stimulates virus production from BALB cells newly infected by some murine leukemia or leukemia sarcoma viruses, from a murine myelogenous leukemic cell line (M-1) producing type C virus, from K-BALB(l) cells (K-BALB producing cells previously induced by IdUrd), and from K-BALB cells rescued by Rauscher leukemia virus. However, this stimulatory effect is not universal, since we observed that dexamethasone did not stimulate virus production from BALB cells newly infected by B-tropic virus, from S2CL3 cells producing N-tropic virus (a clone of spontaneously transformed BALB cells), from virus producing normal rat kidney cells, and from a mouse adrenal gland tumor Y-1 cell line chronically producing type C virus. Some estrogenic hormones that do not have any stimulatory effect on virus production from BALB or K-BALB cells induced by IdUrd stimulate virus production from normal rat kidney cells induced by IdUrd. When there is no stimulation of virus production in a cell system by steroid hormones, very often there is some inhibitory activity. Furthermore, we observed that in JLSV10 cells chronically producing Rauscher leukemia virus and in K-BALB cells newly infected by Rauscher leukemia virus, virus production is enhanced by dexamethasone when the cells are still producing a low titer of virus but is not enhanced when the cells are producing a high titer of virus.


Tumor-associated thymidine kinase in the sera of rats with transplanted hepatomas. A cytoplasmic form of thymidine kinase was detected in sera from rats bearing Morris hepatoma 7777. Polyacrylamide electrophoretic properties demonstrate similarity between the serum enzyme extracted from hepatoma tissue. This form of thymidine kinase has also been described in normal human fetal tissue and a variety of human tumor cells. The results suggest that serum isoenzymes of thymidine kinase may be a useful adjunctive test for the presence of certain types of malignant tumors.


Enhancement of infectivity and oncogenicity of a murine leukemia virus in adult mice by X-irradiation. Infection of adult BALB/c mice with murine leukemia virus (MuLV) induces thymic lymphomas histologically indistinguishable from those caused by neonatal infection. X-irradiation permitted early and high levels of viral expression when given before or after MuLV administration and hastened the development of lymphomas. Expression of virus was assayed by using a radioimmune assay for murine p30, a virion core protein. Seventeen to 21 days after injection of MuLV into adult mice, there was 0.3 mug p30 per ml serum, approximately 5 times normal. Seventeen to 21 days after injection of MuLV into X-irradiated (600R) adult mice, there were 2.7 mug p30 per ml serum. The virus produced by infected adult mice was infectious and oncogenic when given to newborn mice.


Nucleotides in embryos in the stages of morula, gastrula, and neurula. Levels of nucleotides and sugar nucleotides in embryos of Bufo arenarum at the stages of morula, gastrula, and neurula have been measured. The total amounts of purine nucleoside diphosphates decreased from morula to gastrula, but increased sharply from gastrula to neurula. The levels of ADP followed this pattern, but those of GDP did not change significantly through the three stages. Purine nucleoside triphosphate levels, which had increased immediately after fertilization, remained almost constant through morula, gastrula, and neurula. As with the purine nucleoside diphosphates, the adenine nucleotide decreased from morula to gastrula, and increased from gastrula to neurula. In contrast, the level of GTP showed a sharp maximum at gastrula. The total pyrimidine nucleoside triphosphate did not change significantly from morula through neurula. As in previous stages of development, only uridine sugar nucleotides were detected. A sharp increase of the galactosyl ester of nucleotides was found at gastrula.


Study on the proliferative state of haemopoietic stem cells (CFU). Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUs) in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rated of endogenous CFUs (endo-CFUs) and exogenous CFUs (exo-CFUs) are identical. After irradiation (650 R) the surviving endo-CFUs begin to proliferate immediately. By contrast exo-CFUs transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosin 3',5'-monophosphate shortly after marrow cell graft, triggers the transplanted CFUs into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFUs become to be increasingly sensitive to hydroxyurea.


Investigations on the turnover of adrenocortical mitochondria. IV. A stereological study of the effect of chronic treatment with ACTH on the size and number of rat zona reticularis mitochondria. The effects of chronic administration of of ACTH (up to 36 consecutive days) on the mitochondria of the zona reticularis of the rat adrenal cortex were investigated by stereologic techniques. It was found that ACTH induces two phases of hypertrophy of mitochondria alternating with two proliferative stages, which are associated with a significant decrease in the average volume of the organelles. It is suggested that, as in the zona fasciculata, ACTH controls the processes of growth and division of mitochondria in the zona reticularis. The mechanism underlying this action of ACTH as well as the differences between the responses to ACTH of the mitochondrial population of the two adrenal zones are discussed in the light of evidence indicating that mitochondria contain a complete genetic apparatus largely independent of nuclear control.


Three muscle fibre types in the axial muscle of axolotl (Ambystoma mexicanum Shaw): a quantitative light- and electron microscopic study. Morphometric analysis by light microscopy of p-phenylene-diamine stained semithin sections of axolotl tail muscle revealed differences in the cross-sectional area of the fibres and in the number of mitochondria and of lipid inclusions per fibre, and indicated the presence of three distinct types of fibres. The tripartition was found to be statistically highly significant. Representative fibres from each group established by light microscopic morphometry were subjected to an ultrastructural morphometric analysis. The volume content of mitochondria amounted to 9.8% of the fibre volume for red, 4.0% for intermediate and 0.8% for white fibres. The myofibrils composed 60%, 70% and 83% in the same fibres. The volume of the sarcotubular system (t-tubuli and sarcoplasmic reticulum) was 2.5% in red, 4.5% in intermediate and 11.7% in white fibres. The three fibre types also demonstrated differences in myofibrillar cross-striation pattern and number of triads. The reliability of the light microscopic morphometry was tested by correlation with EM montages of the representative fibres.


A morphometric study on the pars intermedia of the hypophysis during impairment of the renin-angiotensin-aldosterone system in sodium depleted mice. Fine structural alterations were investigated in cells of the pars intermedia of the pituitary of mice treated for four weeks with (a) a sodium deficient diet, (b) a sodium deficient diet mixed with propranolol (renin-inhibitor), (c) a sodium deficient diet combined with propranolol and amino-glutethimide (corticosterone 18-hydroxylase inhibitor), and (d) a sodium deficient diet combined with propranolol, aminoglutethimide and dexamethasone. The number of secretory granules decreased from 5.0/mum2 in the normal control of 2.4/mum2 in all four experimental groups suggesting that the cells in treated groups had reached an equilibrium in the production and release of secretory granules during the chronic treatments. The number of immature Golgi granules per unit Golgi area was 0.91 in the control, while this value rose to 3.29 (3.62 fold of the control), 4.37 (4.8 fold), 4.94 (5.43 fold) and 5.16 (5.67 fold) respectively in the four experimental groups. In these groups a good correlation was observed between the number of immature granules and the percent volume of rough endoplasmic reticulum (r=0.985, p less than 0.01). The present study suggests that the pars intermedia contains an unidentified pituitary factor (or factors) essential for aldosterone biosynthesis.


Effects of chronic exercise on the ultrastructure of the adrenocortical cells in the rat. Four groups of male rats were exercised for periods of 2, 4, 6, and 8 weeks with controls in each group. As a result of chronic exercise there was an increase in the width of the zona fasciculata of the adrenal cortex. Also, there was an increase in the number and size of the mitochondria, and an increase in the quantity of smooth endoplasmic reticulum, and during the first 4 weeks of exercise an increase in the number of lipid droplets in the zona fasciculata. The close relationship between the smooth endoplasmic reticulum and the mitochondria, and the relationship between the smooth endoplasmic reticulum and the lipid droplets suggests a possible means for a transport mechanism for movement of precursors between these organelles.


Tissue distribution and mode of DNA methylation in mice by methyl methanesulphonate and N-methyl-N' -nitro-N-nitrosoguanidine: lack of thymic lymphoma induction and low extent of methylation of target tissue DNA at 0-6 of guanine. The methylating agents methyl methanesulphonate (MMS) and N-methyl N'-nitro-N-nitrosoguanidine (MNNG), administered by single i.p. injection in mice failed to yield thymic lymphoma at doses around 60% of the LD50 values, in contrast to MNUA which gives a high yield of tumours by this route. Comparison of the tissue distribution and mode of DNA methylation by these agents showed a positive correlation with ability to methylate the 0-6 atom of guanine in DNA of the target tissues thymus and bone marrow and tumorigeneis. MMS gave a low yield of this product due to its relatively low Sn1 reactivity but was able to methylate DNA extensively at other sites in the target tissues and other organs examined. MNNG despite its ability to methylate 0-6 of guanine in DNA in vitro to the same relative extent as the potent carcinogen MNUA, methylated DNA of thymus and bone marrow to a very small extent in vivo but was able to methylate DNA in certain other tissues nearer the site of i.p. injection. These findings contrast with the general relatively extensive methylation of 0-6 of guanine in DNA of the target tissues and other organs by N-methyl-N-nitrosourea (MNUA).


An assay for phosphotriester formation in the reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo. Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters invivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl-N-nitrosourea (MNUA) disappear with time (50% in 4-5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl-N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methane-sulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.


Mitochondrial functional changes during hepatic hyperplasia and azo dye carcinogenesis. Resting and active-state respiratory velocities, respiratory control, high amplitude volume changes, and latent ATPase activities were examined in hepatic mitochondria from rats fed 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) for production of liver tumors and from rats in three phases of liver regeneration subsequent to subtotal hepatectomies. Tetrabutylammonium bromide, a lipophilic probe capable of selectively inhibiting phosphorylating oxidation or uncoupling oxidation from phosphorylation, was used to detect subtle alterations in lipophilicity characteristics of the organelles and it was concluded that mitochondria from pre-hyperplastic, hyperplastic, and neoplastic tissues had a higher than normal degree of membrane lipophilicity at specific functional sites. Control of respiration by ADP was markedly augmented in all experimental groups; this behavior, plus depressed sensitivity to swelling agents and energized contraction, were similar in mitochondria from hepatomas and from 3-day regenerating livers. These mitochondrial functions were even more pronounced, however, in cells in pre-hyperplastic states (6 and 16 h subsequent to partial hepatectomy). Many forms of liver damage result in mitochondrial alterations which elevate the capacity for oxidative phosphorylation. Such changes associated with induction of azo dye oncogenesis are mimicked by the degree of hyperplasia in the tissue following the first mitotic wave of regeneration; implications relevant to hepatocarcinogenesis are discussed.


A versatile, flexible synthesis of 1,3-diglycerides and tryglycerides. A flexible method for synthesising 1,30diflycerides and triglycerides is described. Glycidol esters, prepared by a known route from epichlorohydrin and the sodium salt of a fatty acid, were heated with another or with the same fatty acid and a quaternary ammonium salt. This resulted in a fast, mild reaction and higher yields and greater purity of the diglycerides than hitherto obtained in this synthesis. The mixture of 1,3- and 1,2-diglycerides obtained was isomerised by heating while still in the solid phase to 1,3-diglycerides. Triglycerides were prepared from the diglycerides by acylation using a fatty acid chloride and pyridine in hexane.


[Immunologic blood-brain barrier in the polyoma virus--Syrian hamster system]. The authors report premilinary results of an experiment on permeability of the blood-brain barrier (BBB) to anti-tumor virus-induced immunological factors in the polyoma virus/Syrian Hamster system. The animals were protected by subcutaneous or intracranial injections with virus before challenge with polyoma virus transformed cells by both routes. BBB seemed to be permeable to the efferent part of the subcutaneously induced immune reaction. On the contrary, antigenic information introduced in the central nervous system was trapped inside the BBB. Thus the BBB might offer a "one-way" permeability in this system.


Control of protein balance in hypertrophied cardiac muscle. The levels of intracellular proteins are determined by a balance between their rates of synthesis and degradation. During the development of regression of cardiac hypertrophy, both of these rates can be expected to change. Possible control sites of synthetic and degradation processes are discussed in this article. The following experimental results are presented: (1) Cardiac mitochondrial cytochromes accumulate early after imposition of pressure overload, as a result of an increased rate of synthesis and decreased rate of degradation. (2) The half-life of myosin heavy chains (HC) in the steady state was determined from incorporation kinetics, using leucyl-tRNA as precursor, to be 5-6 days. (3) The existence of a pool of newly synthesized myofilaments which are not fully incorporated into the core of myofibrils is indicated by the incorporation data.


Pyridine nucleotide as an indicator of the oxygen requirements for energy-linked functions of mitochondria. The responses of cardiac mitochondria to anoxia may be evaluated in terms of the oxidation-reduction state of the electron carriers and the ability of the mitochondria to function in energy-linked reactions. The previous detailed evaluation of the oxygen requirements for electron transfer in mitochondria is here extended to the oxygen requirements for energy-linked functions. Four functions are evaluated: the energy-dependent reduction of pyridine nucleotide, the phosphorylation of ADP, the retention of Ca2+, and the establishment of a membrane potential. All of these functions are half-maximally activated with 10-20% oxidation of cytochromes c and a + a3. Fifty percent oxidation of pyridine nucleotide is required for these functions. In a normoxic-anoxic titration, an increment of 50% in the reduction of pyridine nucleotide in intact tissue corresponds to the point at which the mitochondria are half-maximally active in energy coupling. Thus, the use of pyridine nucleotide fluorescence as an optimal indicator of tissue oxidation-reduction states has now been extended to the assay of energy-linked functions of mitochondria in situ in cardiac tissue.


Enhanced aldosterone response to angiotensin II in human hypertension. Adrenal and vascular responsiveness to graded doses of angiotensin II (A II) were recorded for seven normal subjects and 12 patients with essential hypertension while in balance on an intake of 200 mEq sodium/100 mEq potassium. Patients with essential hypertension had been previously studied and known to have normal responses of plasma renin activity to sodium restriction and upright posture. A II was administered for 30 minutes at rates of 0.1, 0.3, 1, and 3 ng/kg per minute and plasma aldosterone responses were assessed 20 and 30 minutes later; blood pressure was monitored at intervals of 1 minute during infusion of A II at each rate. A significant increment in plasma aldosterone occurred at an infusion rate of 0.3 ng/kg per minute in patients with hypertension. This change was not seen until the infusion rate reached 1.0 ng/kg per minute in the normotensive control subjects. Even at an A II infusion rate of 1 ng/kg per minute, the increment in plasma aldosterone levels in normotensive subjects (4.2 +/- 0.6 ng/dl) was significantly less (P less than 0.001) than that in patients with essential hypertension (19 +/- 3 ng/dl). In both groups, a significant rise in mean arterial blood pressure occurred at an A II dose of 0.3 ng/kg per minute, but the pressor response of the hypertensive group was significantly greater at the highest infusion rate (3 ng/kg per minute) (P less than 0.05). Thus, enhanced adrenal and pressor responsiveness to infused A II was observed in the hypertensive subjects, suggesting a change in A II receptor affinity.


Receptor binding of angiotensin II and antagonists. Correlation with aldosterone production by isolated canine adrenal glomerulosa cells. The binding properties of the angiotensin II receptors of the adrenal cortex have been studied in isolated cells prepared by collagenase dispersion of the zona glomerulosa of the canine adrenal gland. Such cell preparations are responsive to physiological concentrations of angiotensin II, and permit correlation of binding of angiotensin II and its analogues with aldosterone production in vitro. Uptake of 125I-angiotensin II (5 X 10(-11) M) by glomerulosa cells at 37 degrees C reached a steady state at 45 minutes, with a subsequent plateau for at least 60 minutes. Angiotensin II binding was also dependent upon the hormone and cell concentrations employed during uptake studies. Bound angiotensin II was rapidly dissociated from canine adrenal cells after addition of the unlabeled octapeptide. High affinity sites with equilibrium association constant (Ka) of 3.3 X 10(9) M-1 comprised 25-33% of the receptor population and the remainder of the sites were of lower affinity, 2.5 X 10(8)M-1. Binding of angiotensin II analogues and antagonists was found to be consistent with their biological activities. The analogue most extensively evaluated was [Sar-1]angiotensin II, which exhibited enhanced binding activity when compared to angiotensin II, and had a higher equilibrium association constant by kinetic analysis and direct binding studies. Direct binding of labeled angiotensin II to the adrenal glomerulosa receptor has been correlated with a progressive response in aldosterone production. The steroidogenic response to angiotensin II was maximal when 25% of the receptor population was occupied; this fraction corresponds to the proportion of high affinity receptor sites measured by binding analysis. In addition, inhibition of angiotensin II binding to receptor sites by the competitive antagonist [Sar-1, Ala-8]angiotensin II has been correlated with inhibition of aldosterone production. These findings serve to demonstrate the biological significance of the angiotensin II binding sites of the adrenal cortex, and confirm their role as receptors which mediate the steroidogenic responses to angiotensin II.


The effects of des-Asp1-angiotensin II on blood pressure, plasma aldosterone concentration, and plasma renin activity in the rabbit. To examine the hypothesis that des-Asp-1-angiotensin II (angiotensin III) may be an important mediator of various responses to angiotensin II, both peptides were given sequentially at 25 ng/kg per min by systemic infusion to conscious rabbits, and the effects on blood pressure, plasma aldosterone concentration, and plasma renin activity were compared. The mean arterial pressure increased with angiotensin II infusion an average of 18.2 mm Hg, and with angiotensin III infusion only 3.2 mm Hg. Both peptides, however, produced a six- to sevenfold increase in plasma aldosterone concentration, and a threefold decrease in plasma renin activity. Renal clearance data suggest that the suppression of renin activity was not a consequence of renal hemodynamic change or altered sodium transport, but of direct inhibition through a receptor mediating a short feedback loop. These data in the intact rabbit, taken together with in vitro studies by others, suggest that the responses of these three organ systems to angiotensin peptides are mediated by receptors of varied specificity: those mediating vasoconstriction exhibit a greater response to angiotensin II than to angiotensin III; those mediating the secretion of aldosterone exhibit a lesser response to angiotensin II than to angiotensin III; and those mediating the suppression of renin release do not appear to discriminate between the two peptides.


Changes in cardiovascular and adrenal cortical responses to angiotensin III induced by sodium deprivation in the rat. The restriction of dietary sodium intake is known to depress the cardiovascular responses to angiotensin II and increase the sensitivity of the adrenal zona glomerulosa to this steroidogenic octapeptide. In sodium-repleted animals, angiotensin III is a weak pressor substance and a potent stimulant of aldosterone biosynthesis. The effect of a low sodium diet on vascular and steroidogenic responses to angiotensin II and angiotensin III was investigated. In nephrectomized rats, angiotensin III had one-third of the pressor activity relative to angiotensin II when either normal or sodium-deprived animals were compared. When administered subcutaneously (sc) to rats, angiotensin II and III induced comparable steroidogenic responses, whereas only angiotensin II significantly elevated blood pressure. The comparison of cell suspensions from control adrenals with suspensions of adrenals from sodium-deprived animals showed that the zona glomerulosa from rats on low sodium diets had increased wet weight (20%), cell protein (25%), and basal steroidogenic rats (45%). Adrenocorticotropic hormone (ACTH)-induced responses in adrenal cells from low sodium animals were about twice the responses of cells from normal adrenals. Angiotensin II and III stimulated the cortex at a threshold concentration of 5 X 10(-10) M and induced a maximum response at about 5 X 10(-8) M in cells prepared from normal rat adrenals. In cells dispersed from adrenal capsules of sodium-deprived rats, the maximal steroidogenic response to angiotensin II occurred at 3 X 10(-8) M, whereas angiotensin III was maximal at 1 X 10(-9) M. Aldosterone synthesis induced by both peptides was increased approximately 45% in adrenal cells from low salt rats. At 0.9 mumol/kg, sc, Sar-1, Ile-8-angiotensin II antagonized cardiovascular responses to angiotensin II and did not alter aldosterone in the sodium-deprived rat. In contrast, treatment with Ile-7-angiotensin III blocked the adrenal cortex but not the vascular actions of angiotensin II. These data are consistent with a role for angiotensin III in the renin-angiotensin-aldosterone response to sodium deprivation.


Sympathetic nerve activity: role in regulation of blood pressure in the spontaenously hypertensive rat. Sympathetic nerve activity (SNA) and high pressure baroreceptor regulation of SNA were studied in the Okamoto strain of spontaneously hypertensive rat (SHR). Mean arterial pressure (MAP) and SNA were not significantly affected by anesthesia with low doses of pentobarbital (20-25 mg/kg). Thus, most of these studies were performed in anesthetized rats. SNA in visceral sympathetic nerves increased rapidly with age up to 24 weeks and slowly thereafter. MAP increased with SNA, following the same time course. Both SNA and MAP in SHR were significantly greater than that found in normotensive Wistar control rats of comparable ages. Abolition of ganglionic transmission with hexamethonium in both SHR and normotensive controls reduced postganglionic SNA and MAP to comparable levels. In SHR less than 16 weeks old, increased baroreceptor stimulation effectively inhibited SNA with the same sensitivity as found in Wistar control rats. However, older SHR appeared to lose their ability to completely inhibit SNA during induced hypertension, whereas in Wistar control rats as old as 52 weeks, elevation of blood pressure to 165.3 +/- 2.3 mm Hg completely suppressed SNA. These results suggest that SNA may play an important role in the development and maintenance of hypertension in SHR, and that central sympathetic centers, uninhibited by baroreceptor afferents, become active during the development of hypertension in the SHR.


Plasma cyclic adenosine 3':5'-monophosphate response to isoproterenol and glucagon in hyperkinetic borderline (labile) hypertension. The hemodynamic changes observed in patients with the "hyperkinetic" form of borderline (labile) essential hypertension (BEH) could be related to the hyperresponsiveness of cardiac beta-adrenergic receptors to catecholamines. The isoproterenol-induced increase in plasma cyclic adenosine 3':5'-monophosphate (cAMP) reflects the response of adenylate cyclase to beta-adrenergic stimulation, whereas a non-beta-receptor-mediated increase occurs with the administration of glucagon. Both substances were infused into 13 control subjects and 14 patients with the hyperkinetic form of BEH before and after propranolol administration. Baseline plasma cAMP concentrations were comparable in both groups. After 30 minutes of isoproterenol infusion (20 ng/kg per min) a significantly higher increase in plasma cAMP and heart rate and a smaller decrease in diastolic blood pressure were seen in this type of BEH than in control subjects. The increase in plasma cAMP and in heart rate correlated positively when all subjects were considered together. Propranolol abolished hemodynamic and humoral responses to a similar degree in both groups. The plasma cAMP responses to glucagon (200 ng/kg per min) were slightly lower in our patients with BEH than in control subjects and were not suppressed by propranolol. The data are compatible with a hyperreactivity of the beta-adrenergic receptors or of the adenylate cyclase or both in hyperkinetic BEH and could correspond to the previously observed exaggerated beta-adrenergic drive to the heart in this type of hypertension. The non-beta-receptor-mediated rise in plasma cAMP (glucagon), however, remains comparable in control subjects and BEH.


Evidence that des-Asp1 angiotensin II mediates the renin-angiotensin response. Studies were undertaken to compare and evaluate the influence of angiotensin II and its heptapeptide fragments, des-Asp-1-angiotensin II, at various receptor sites for angiotensin in both dogs and rats. Receptor sites evaluated were those which are found in the glomerulosa, reticularis, and fasiculata of the adrenal cortex, in the renal arterioles and the juxtaglomerular cells of the kidney, and in the peripheral arterioles. Both peptides produced similar changes in the steriod secretion profiles for aldosterones, corticosterone, and cortisol in the dog. In the rat, both peptides similarly increased aldosterone and corticosterone secretion; however, a larger dose of the competitive antagonist Sar-1,Ala-8-angiotensin II was required to block the steroid response to the heptapeptide. This finding suggests that receptor affinity for des-Asp-1-angiotensin II may be greater than its affinity for angiotensin II. Both peptides also decreased renin secretion and renal blood flow similarly in the dog. The pressor response to the heptapeptide was only about one-half the pressor response to angiotensin II in both the rat and dog studies. Collectively, these observations in dogs and rats suggest that des-Asp-1-angiotensin II may mediate the response to the renin-angiotensin system at both adrenal and renal receptors.


Studies on the serum lipoprotein composition in 50-year-old men. A suggestion of chemical criteria for diagnosis of hyperlipoproteinaemia type III (Broad-beta disease). The serum lipoprotein (LP) composition in hyperlipoproteinaemia (HLP) type III, when studied in a population of 50-year-old men, was characterized by a high cholesterol/triglyceride ratio in the very low density lipoprotien (VLDL) fraction, a high low density lipoprotein (LDL) triglyceride concentration and a low LDL cholesterol/triglyceride ratio. No one of these three variables, however, was found to be a suitable single parameter for diagnosis for HLP type III because of an overlap between HLP type III and other LP patterns with regard to the studied variables. In an effort to find a parameter with a better discriminative capacity a "III-index" was invented.


Effects of growth hormone release-inhibiting hormone and bromocryptine (CB 154) in states of abnormal pituitary-adrenal function. Growth hormone release-inhibiting hormone (GHR-IH) was administered to five patients with abnormal pituitary-adrenal function. There was immediate suppression by about 50% of ACTH levels in two patients who had undergone bilateral adrenalectomy because of Cushing's disease; one of them had Nelson's syndrome. Bromocryptine (2-5 mg) suppressed ACTH levels by 62-67% for more than 6 h in these patients. GHR-IH did not significantly influence cortisol secretion by an adrenal carcinoma and only slight changes were seen in two patients with Addison's disease. The possible therapeutic implications are discussed.


Circulating antibody to renal collecting ducts in patients with hepatoma or renal-cell carcinoma. An anti-kidney antibody was demonstrated by the indirect immunofluorescence method in the serum of patients with primary tumours of the liver or kidney. The distribution of fluorescence in rabbit kidney was consistent with that of antibody to collecting ducts. The anti-collecting duct antibody (anti-CDA) could be absorbed from serum by normal adult rabbit or human kidney tissue and by one of three specimens of renal-cell carcinoma tissue. Anti-CDA differed from anti-mitochondrial antibodies and from anti-liver/kidney microsomal antibody in the pattern of fluorescent staining obtained with rabbit kidney. Two-hundred sera from patients with cancer and other diseases and forty-three from healthy hospital personnel were tested for anti-CDA. Eleven of the twenty-five positive sera were from patients with primary cancer of the liver or urinary tract, and all but six of the remainder were from patients with tumours involving the liver or with liver disorders that may be associated with nodular hyperplasia or tumour.


Cell-mediated immunity to microbial antigens in mother and child. We have looked at evidence for the transplacental passage of cell-mediated immunity in man. The lymphocyte response of mother-neonate pairs was assessed to non-specific mitogens, allogeneic cells, and to microbial antigens. Although six mothers were known to be chronic carriers of Herpes simplex virus only one of their neonates responded to this viral antigen. On two occasions a lymphocyte response was induced in cord blood by antigens to which the mother did not respond. This paradox remains unexplained. We suggest that the relative immunity of the neonate to some infectious diseases does not depend on the transplacental passage of cellular immunity.


Evidence for suppressor cell activity associated with induction of Herpesvirus saimiri-induced lymphoma. Peripheral blood lymphocytes (PBL) from Herpesvirus saimiri (HVS) infected owl monkeys lose their ability to respond to PHA during the development of lymphoma. In this study, five HVS-infected owl monkeys were examined for their ability to respond to PHA and to confer this loss of PHA reactivity to normal lymphocytes. Four of the monkeys developed HVS-induced disease and lost their ability to respond to PHA. Although less affected, reduced responses were also found with PWM. The animals which developed disease and suppressed PHA responses were found to be able to confer this loss of PHA reactivity to normal lymphocytes. One HVS-infected monkey did not develop disease or loss of PHA reactivity and did not confer a loss of PHA reactivity to normal lymphocytes. In the four affected monkeys, the conference of suppression to PHA was not accompanied by a concomitant reduction in response to PWM by the normal lymphocytes, demonstrating some specificity in the suppressor cell activity. Thus, the presence of suppressor cell activity appears to be associated with the inhibition of T-cell function and possibly the eventual development of lymphoma.


Effects of marijuana extract and tetrahydrocannabinol on electroencephalographic sleep patterns. Marijuana extract, given in daily doses containing 70 to 210 mg delta-9-tetrahydrocannabinol (THC), induced effects on sleep that were virtually identical to those produced by the same doses of relatively pure (96%) THC. Both drugs reduced eye movements density with some tolerance developing to this effect. Stage 4 tendend to increase with drug administration. Abrupt withdrawal led to extremely high densities of eye movement, increased rapid eye movement (REM) durations, and a sharp but transient fall in stage 4 to baseline levels. These effects may be useful in the elucidation of the pharmacology of sleep. The effects on sleep of THC administration (but not withdrawal) closely resemble those induced by lithium. For this reason, we suggest further studies of THC in affective disorders. Evidence available thus far suggests that THC produces dysphoric symptoms in unipolar but not in bipolar depressed patients; these differences in response may prove of diagnostic value. An adequate therapeutic trial of THC in bipolar depressed patients has not yet been carried out.


Hydroxyproline-containing structural glycopeptide fractions from subacute inflammation connective tissue. Hydroxyproline-containing structural glycopeptide fractions were isolated from collagenase-digested neutral salt-insoluble collagen of five-day sponge-implant connective tissue of the rat. The glycopeptide fractions characterized migrate as a single, strongly anionic band on disc gel electrophoresis at pH 9.5, are eluted on gel filtration as a small molecular weight peak, approximately 2000, and are resolved into thirteen glycopeptide fractions by DEAE-cellulose chromatography. Amino acid analyses of some of these fractions indicate a similarity in composition, the principal ones being aspartic and glutamic acids, serine, glycine, alanine, valine, proline and hydroxyproline. Three neutral carbohydrates, glucose, mannose and xylose, in different relative proportions and hexosamine are also present in the fractions. Amino-terminal amino acid determinations indicate a microheterogeneity of the glycopeptides. The electrophoretic behaviour and non-diffusibility of the small molecular weight glycopeptides suggest an intimate association between acidic hydroxyproline-containing peptides and carbohydrate components of developing connective tissue.


Diffuse interstitial fibrosing pneumonitis and adenovirus infection. With electron microscopy, adenovirus particles, which were almost round, variable in density, and 60-80 mmu in diameter, were observed in the nuclei of type II alveolar epithelial cells, infiltrated plasma cells and alveolar macrophages of a man with chronic interstitial fibrosing pneumonitis. The immunofluorescent technique also suggested adenovirus infection.


Effect of antibiotics on the phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes. The effect of antibiotics on phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes was studied. Carbenicillin and sulbenicillin, when added to an incubation medium at a concentration as low as 1/16 MIC, increased phagocytosis and killing of P. aeruginosa by PMN. Meanwhile, gentamicin and 3',4'-dideoxykanamycin B gave no influence on the PMN activity, and polymyxin B and colistin enhanced the activity only at MIC. The PMN activity was not facilitated even when the cells of P. aeruginosa had been pretreated with antibiotics. The bactericidal activity of PMN decreased after sonification, but was restored following addition of carbenicillin.


Growth inhibition of cells in cultures and of vaccinia virus infected HeLa cells by derivatives of trifluorothymidine. The effects of 5-trifluoromethyluracil (F3Thy), 5-trifluoromethyl-2'-deoxyuridine (F3dThd), F3dThd-5'-P, and F3dThd-5'-methylphosphonate on the growth of HeLa cells, Novikoff hepatoma cells, L5178Y mouse leukemia cells, and on the replication of vaccinia virus in HeLa cells have been determined. F3Thy and F3dThd-5'-methylophosphonate were approximately 500-fold and 100-fold less effective, respectively, than F3dThd or F3dThd-5'-P in their inhibition of these cells. F3dThd and F3dThd-5'-P are potent inhibitors of vaccinia viral replication of HeLa cells. The nucleoside and nucleotide were 1,000-fold more inhibitory than the free base. F3dThd was nost inhibitory when added between 1 and 2 h post-infection; however, it was also somewhat inhibitory when added at later times.


Chromosome-associated paracrystalline nuclear inclusions in the spermatocytes of a pulmonate snail, Planorbarius corneus L. Chromosome-associated paracrystalloids are regularly found in the spermatocytes of snails which were reared in the laboratory. They seem to be largely specific for the male gametocytes as they have been observed only in few cases in the oocytes. It is likely that paracrystalloids are formed during pachytene at the site of large heterochromatic knobs which originate by fusion of heterochromatic terminal segments of some bivalents. During diplotene they are always connected with the telomeres of three or four bivalents, thus forming a large trefoil-like structure. During metaphase I paracrystalloids are shed off from the chromosomes and transferred to the cytoplasm. In early spermatids they are found again in the nuclei, where they "fade away" during spermiogenesis. Histochemically they consist of basic proteins, which are probably crystallized in the cubic system. Radioactive labeling of the structure could not be achieved, neither by 3H-uridine or thymidine, nor by amino acids. The functional significance of this peculiar structure in unknown. Certain features justify a comparison with synaptonemal polycomplexes.


[Estimation of free cortisol in urine (author's transl)]. Radio-immunological estimation of free cortisol in urine in a group of normal persons showed a median daily excretion of 42,5 +/- 22,8 mug (n = 31) using radio-loss-labelling, extraction, and thin layer chromatography. After simple extraction the median value was 54.1 +/- 15.3 mug/d (n = 10). Radioimmunological estimation in simple urine extract seems to be sufficient for routine clinical purposes. The estimation of free cortisol in urine permits a better differentiation between normal adrenal function and adrenal hyperactivity than the determination of 17-hydroxycorticosteroids (17-OHCS). This is shown by comparison of 1) the frequency distribution of the basal values of 17-OHCS and free cortisol in normal persons and in Cushing's disease, 2) the suppression effect of dexamethasone on 17-OHCS and free cortisol in Cushing patients, and 3) the stimulatory effect of ACTH on 17-OHCS and free cortisol in normal persons.


Production of oestradiol, camp and 125I-hcg binding by rat ovaries during the reproductive cycle. Plasma concentration of oestradiol in female rats is dynamically altered in the reproductive cycle, depending on the biosynthesis and secretion of oestradiol secretion by the ovaries. The present study has followed oestradiol and cAMP concentration in the plasma and ovaries, and 125I-HCG binding during the pro-oestrus, oestrus and di-oestrus. Oestradiol concentration in plasma and its production by the ovaries were found to be highest in the pro-oestrus and lowest in the oestrus. These changes depend on the production and secretion of gonadotrophins. The mediator of gonadotropin action is cAMP whose ovarian concentration is lowest in the oestrus. LH stimulates the cAMP synthesis in the ovaries during the cyclic stages, without any significant difference being apparent between the various stages. 125I-HCG binding is highest in the di-oestrus stage, when concentration of endogenous gonadotrophins is low.


A modification of the Detter and Klingmüller's method for urinary alpha- and beta-pregnanediol and pregnanetriol determination: excretion patterns in men and in women during the normal menstrual cycle. This report describes the modification of Detter and Klingmüller's method for the determination of urinary alpha- and beta-pregnanediol and pregnanetriol by means of thin -layer chromatography. The modifications were as follows: 1. The addition of formol prior to hydrolysis to prevent pigments penetration into urinary extracts. 2. The use of Kieselgel HF 254+366 nach Stahl (E. Merck, Darmstadt) which allows to localize the spots under UV-light at 366 nm without the use of colour reagents. The results concerning accuracy, sensitivity, precision and specificity in the modification described are profitable. Using this method in 7 healthy women (aged 28-37) with normal menstrual cycles the urinary excretion of alpha- and beta-pregnanediol and pregnanetriol were evaluated every second day (starting the 6th day of the cycle). The maximum of excretion of alpha- and beta-pregnandiol appeared on day 20 of the cycle, with the mean (+/- S.D.) 1.27 +/- 0.37 mg/24hr and 2.97 +/- 0.80 mg/24hr for alpha- and beta-pregnanediol, respectively. Mean values of pregnantriol were at the same level and ranged from 0.25 to 1.42 mg/24 hr. Single determination of these compounds in 8 healthy men (aged 19-42) revealed the mean excretion values (+/- S.D.) 0.96 +/- 0.17 mg/24 hr, 1.24 +/- 0.40 mg/24 hr, 1.12 +/- 0.65 mg/24 hr for alpha- and beta-pregnanediol and pregnanetriol, respectively.


ACTH and growth hormone relationship in fetal rat adrenal steroidogenesis in vitro. The effects of growth hormone and ACTH, alone or in combination, on fetal rat adrenal steroidogenesis in vitro were examined on the last day of intrauterine development. ACTH increased, while growth hormone did not affect fetal adrenal weight. ACTH increased fetal rat adrenal steroidogenesis, hydroxylation of 4-14C-progesterone to corticosterone, 18-hydroxy-11-deoxycorticosterone, 11-hydroxycorticosterone and aldosterone. Growth hormone alone had no effect on fetal adrenal steroidogenesis. ACTH and growth hormone administered together increased the conversion of progesterone to the above mentioned steroids to a greater extent than ACTH alone. The results indicate that growth hormone may participate in the fetal rat adrenal steroidogenesis potentiating the effects of fetal pituitary ACTH.


The fate of serotonin released from the intrathyroidal mast cells in albino rat. After the administration of 500 muC of 3H-5-HT the mast cells were degranulated by 1500 IU of PO B. The grains were found within the follicular cells and in the vicinity and wall of the arterioles. These findings support both the direct and indirect (vascular) mechanism of action exerted by serotonin on the thyroid function.


Investigations on isolated islets of Langerhans in vitro. XIII. Experiments concerning the preparation conditions with collagenase. During the digestion of pancreatic pieces with collagenase for prepartion of isolated islets the enzymes in incubation medium (collangenolytic and/or proteolytic) can alter the secretion behavior of A- and B-cells. Insulin release after such an enzymatic attack is characterized by an enhanced basal secretion and a diminished and delayed glucose response. Overdigestion results in a decreased glucagon secretion in response to arginine, a diminished insulin content, and a decreased thiol-protein-disulfide-oxidoreductase activity of the islets. Increased albumin concentrations did not prevent the collagenase effect.


Effects of starvation and Ca++ on glucose-induced accumulation of cyclic 3',5'-AMP in pancreatic islets. Exposure to glucose in the presence of 3-isobutyl-1-methylxanthine leads to accumulation of cAMP in islets microdissected from ob/ob mice. This process is dependent on extracellular Ca++ but differs markedly from the glucose action on insulin release in the same in vitro system in disappearing after 18 h of starvation.


Adenyl cyclase activity of mouse liver membranes after incubation with endotoxin and epinephrine. Adenyl cyclase activity in isolated mouse liver cell membranes was stimulated two-fold by endotoxin. Furthermore, endotoxin inhibited epinephrine induction of adenyl cyclase activity, apparently through interruption of the phospholipid moiety of the enzyme complex.


Reinterpretation of the ultrastructure of cartilage matrix. The epiphyseal cartilage from new-born mouse was treated with collagenase in two ways: either before fixation or after glutaraldehyde fixation. The electron dense granules of the matrix were not seen in the micrographs of cartilage treated with collagenase before fixation. It is concluded that collagen plays a definite role in the formation of the granules at the time of tissue fixation and that the granules are fixation artifacts.


Effect of ACTH on the zona reticularis of the rat adrenal cortex: an ultrastructural stereologic study. The effects of ACTH on the rat adrenal zona reticularis were investigated by stereologic methods. It was found that the zona reticularis cell responsiveness to ACTH is similar to that of the zona fasciculata elements. This excludes that the zona reticularis of the adult rat can only function as the site of destruction of worn-out elements migrating from the adrenal outer zones.


Monolayer cultures of normal adult rat adrenocortical cells: steroidogenic responses to nucleotides, bacterial toxins and antimicrotubular agents. Monolayer cultures of normal rat adrenocortical cells were treated with agents which stimulate steroidogenesis by Y-1 adrenal tumour cells. Choleragen was active, whereas cyclic nucleotides other than cyclic AMP, bacterial endotoxins and antimicrotubular agents were inactive.


Regulatory properties of the citrate synthase from Rhodospirillum rubrum. Citrate synthase, purified 600-fold from Rhodospirillum rubrum, is activated by KCl and inhibited by ATP and NADH; the effect of the latter inhibitor is completely counteracted by AMP and partially counteracted by KCl.


Increase in membrane conductance by adrenaline in parotid acinar cells. It is shown that excitation of the alpha- or beta-adrenoceptors in mouse parotid acinar cells causes a marked reduction of surface cell membrane resistance. The alpha-adrenoceptor induced membrane effect is an increase in K conductance. The beta-adrenoceptor induced membrane effect does not seem to be mediated by cyclic AMP.


Tumor incidence in Visna virus inoculated mice. Mice (female Swiss albino) inoculated when newborn with Visna virus had tumors in 77% of cases when examined 8-12 months later. The tumors were mainly of the mammary carcinoma type. The tumor incidence in non-infected control animals was only 20%. In contrast, no increased incidence of tumors was observed among Visna virus-inoculated inbred mice (BALB/c, CBA and DBA) with low incidence of spontaneous mammary carcinoma.


Chromatographic conditions in the expression of corticosteroid receptor specificity. It is shown that cytosol preparations bound with various concentrations of a steroid are necessary to reveal physicochemically distinct, heterogeneous and polymorphic receptors present in the hormone specific target organ, that these cannot be fully appreciated in one-shot experiments at suboptimal steroids levels, and that they escape detection by equilibrium binding and Scatchard analysis alone.


Changes in the protein conformation of human spermatozoal membranes after treatment with cyclic adenosine 3':5'-monophosphate and human follicular fluid. Infrared spectra in the amide I and amide II regions of acrosomal membranes isolated from ejaculated human spermatozoa indicate the presence of a high proportion of the constitutive proteins in the most stable protein configuration, the antiparallel beta-conformation. Since the infrared spectra obtained with the membranes suspended in D2O or after extraction of the lipid components do not show any significant change, it can be postulated that the antiparallel pleated sheet conformation of human spermatozoal membrane proteins is independent of the hydrated state and of the lipid constitution of the membrane.


[Membrane defect as a basic disorder of the growth control mechanism in psoriasis pathogenetic concept and therapeutic consequences (author's transl)]. The system glycocalyz - membrane bound enzymes - cAMP may be regarded as a regulatory mechanism for the control of epidermal growth. The glycocalyz of the epithelial cell membrane is obviously a receptor and accumulator for agents stimulating adenyl cyclase and other membrane-bound enzymes of energy utilisation; cAMP, on the other hand, serves as an intracellular 'mediator' between proliferative stimuli and the DNA of the keratinocytes. Numerous cytological, cytochemical, and enzymatic biochemical studies indicate that this mechanism is profoundly disturbed in psoriasis, particularly at the membrane-bound stage.


Enzymatic activities in mitochondria isolated from the rat brain during development. In an attempt to discern whether mitochondrial changes during ontogeny are due to increases in number of mitochondria or to increases in number plus changes in their organization, we investigated the enzymatic markers of the inner and outer membranes of mitochondria from cerebral cortex and subcortical material in young and adult rats. We conclude from the data gathered that significant changes do occur in mitochondrial organization, that gradual maturation occurs in the inner membrane during development, especially in the cerebral cortex and considerably less so in the subcortical structures. We found no such changes in the sturucture of the outer membrane during the periods of development examined.


Insulin receptors in cultured human fibroblasts. In order to study human insulin resistance, we have first characterized the interaction of insulin with specific insulin receptors in cultures of normal human fibroblasts. 125 I-insulin bound rapidly to human fibroblasts in suspension at 15 degrees,  achieving steady state between one and three hours. Insulin was not degraded during the binding assays. In competitive binding experiments, 2 ng/ml. (3.3 X 10(-10) M) of unlabeled insulin reduced 125 I-insulin binding by 50 per cent. Insulin analogues competed for binding in proportion to their biologic potencies. A curvilinear Scatchard plot was obtained, suggesting the existence of negatively cooperative site-site interactions among the insulin receptors. This was confirmed directly by studies of the dissociation kinetics. The high affinity, specificity, and negative cooperativity of the fibroblast insulin receptor closely resembles the properties of other human insulin receptors. The cultuted human  fibroblast should prove a useful tissue for the study of insulin-resistant states in man.


Cyclic  nucleotides in pancreatic islets. Tolbutamide- and arginine-induced insulin release. With the use of isolated rat islet perfusion, levels of the islet cyclic adenosine 3' ,5' -monophosphate (cAMP) were compared with dynamic insulin secretion induced by tolbutamide and arginine. Tolbutamide elevated islet cAMP rapidly and augmented both glucose-induced islet cAMP levels and insulin secretion; arginine, however, did not elevate islet cAMP but did enhance glucose-induced insulin secretion. Since the latter result could have been modulated by cyclic guanosine 3' ,5' -monophosphate, this cyclic nucleotide was also measured and found to remain unchanged during stimulation of insulin secretion by arginine and a combination or arginine and glucose. Thus, the action of tolbutamide appears to  be modulated in part by cAMP, whereas arginine appears to augment insulin secretion independently of cyclic nucleotides.


Some considerations in the preparation of raioiodoinsulin for radioimmunoassay and receptor assay. 125I-insulins, prepared by iodination with chloramine T in marked excess or by stepwise, stoichiometric addition of the oxidizing agent, were compared with respect both to their molecular distribution of iodine and to their suitability for use in a cultured lymphocyte receptor assay. Iodination of insulin in aqueous solution results in the same distribution of iodine atoms, independent of experimental method and dependent only on the average iodine number. This distribution can be calculated on the basis of a Monte Carlo simulation, For insulin iodinated at an average of 0.8 I atoms per molecule, approximately 50 per cent of the radioactivity is in other than monoiodoninsulin. Purification methods that separate on the basis of charge, such as starch-gel electrophoresis, are then required, to obtain monoiodoinsulin. More highly iodinated insulin  do bind to the lymphocyte receptor, although, as in radioimmunoassay, the overiodinated species are less satisfactory for use as tracers. The shelf life of iodinated insulin appears to be related better to the average iodine content than to any other factor, presumably because of decay catastrophe. There is no evidence to suggest that exposure to chloramine T in marked excess for a few seconds is deleterious to insulin.


Diabetes mellitus in the guinea pig.

Diabetes mellitus in the guinea pig. Spontaneous diabetes mellitus has been documented in a colony of guinea pigs. The contagious nature of the disease has been verified, but the nature of the infectious agent is not known. Animals from the original colony or animals exposed to the colony with normal glucose tolerance tests (GTT) became diabetic, as evidenced by elevated one- and four-hour GTT values, and in most cases have significant glycosuria. The severity of pathologic changes in the pancreatic islets parallel, in general, the severity of the clinical symptoms (glycosuria and abnormal GTT). Those animals with severe glycosuria and elevated FBS as well as one- and four-hour GTT values had the most pronounced degranulation and most prominent cytoplasmic inclusions in islet B cells. The severity of scarring in the islets can be correlated with the duration of the overt diabetic state. The other clinical parameters of note were elevated serum triglycerides, normal serum but elevated aortic cholesterol, and absence of ketonemia or ketonuria. The reproductive capacity of diabetic females was compromised. While the clinical manifestations are mild or variable, the presence of significant islet pathology is reminiscent of human juvenile diabetes mellitus. These findings lend support to the concept that infectious and/or immune mechanisms could be operative in the etiology and pathogenesis of human diabetes mellitus.


Preparative action of carbon tetrachloride in liver tumorigenesis by a single application of N-butylnitrosourea in male ICR/JCL strain mice. A dose of 10 or 20 mg of N-butylnitrosourea (BNU) dissolved in 50% ethanol was administered by a gastric tube to 6-week-old male ICR/JCL strain mice and they were sacrificed 15 months later. One of 18 animals developed a hepatoma, but none of the mice given CCl4 in 0.1 ml of 50% oliver oil subcutaneously at the right thigh developed hepatoma. However, a marked enhancement of hepatoma induction was observed in mice injected with CCl4 one day before the single intragastric administration of BNU, with 12 out of 28 mice developing one or more hepatomas (average 3.2/mouse) ranging in diameter from 0.5 to 1.5 cm. By extending the administration interval between CCl4 and BNU to 1 week or 1 month, or by reversing the order of administration, the hepatotumorigenic action was virtually lost. There was no occurrence of hepatoma but a predominant development of leukemia, of either thymic or nonthymic origin, was observed in mice of younger age treated with CCl4 one day before continuous oral administration of BNU (1 mg/day/mouse). It is thus concluded that the preparative (cocarcinogenic) action of CCl4 is indispensable for hepatotumorigenesis with a single large dose of BNU.


Lack of correlation between the presence of estrogen receptor and histological type of mammary cancer induced by 7,12-dimethylbenz[alpha]anthracene. The relation between the histological type and the presence of estrogen receptor or response to oophorectomy was investigated in the mammary cancer induced by 7,12-dimethylbenz[alpha]anthracene in Spargue-Dawley rats. The estrogen receptor was present in 75.0% (3/4) of poorly differentiated cancer and in 62.5% (10/16) of well-differentiated cancer. The histological degree of differentiation or grading of the rat mammary cancer did not affect the presence of the estrogen receptor or response to oophorectomy. In contrast, there was clearly a good correlation between the presence of the receptor and response to oophorectomy.


Intracisternal A particles in transplantable reticulum cell sarcoma of rats. Electron microscopic examination on three lines of transplantable reticulum cell sarcomas, induced spontaneously in the mesenteric lymph nodes of inbred strain ACI rats, proved the presence of intracisternal A particles in all the three tumor lines. The doughnut-shaped particles measured 50 to 70 nm and were seen to bud from the membranes of rough-surfaced endoplasmic reticulum. Frequency of appearance of these particles varied with the tumor line.


Possible retention of the estrogen-binding capacity after endocrine ablation therapy in the rat and human breast cancer. The estrogen-binding capacity of the regrowing tumors after endocrine ablation surgery was estimated in the rat and human breast cancer. All of 4 tumors that had not had the estrogen receptor before oophorectomy did not show the estrogen-binding capacity after the procedure, in the mammary cancer in the Sprague-Dawley rat induced by 7,12-dimethylbenz[a]anthracene, whereas all the 4 tumors that had the receptor before the procedure showed the binding capacity after oophorectomy. In 4 patients who had been subjected to the major endocrine ablation therapy because of metastatic breast cancer with effective response, the secondary metastatic deposits of cancer showed the estrogen-binding capacity long after the endocrine ablation. The fact that regrowing tumors after complete or partial regression of initial tumors by endocrine ablation retain the estrogen receptor seems to be contradictory to the classical concept of autonomy in recurrence of the breast cancer after hormonal manipulation.


Histological grading, histochemistry, and electron microscopy of scirrhous carcinoma of the breast. Twenty-six scirrhous carcinomas of the breast were divided into three histological grades of malignancy; low (Grade I), intermediate (Grade II), and high (Grade III), and the correlation of the grades with histochemical and electron microscopic findings in both tumor cells and host tissues was examined. The tumor cells contained increased amounts of lysosomal acid phosphatase and beta-glucuronidase. This increase was most marked in Grade I and II tumors and the increase was consistent with lysosome-like fine structures. Both intracytoplasmic lumina and microvilli against stroma were characteristic findings of carcinoma cells and they were mostly found in Grade I and II tumors. Segments of intact basal laminae and myoepithelial cells were also found in Grade I and II carcinomas. The stroma contained moderately increased amounts of intracellular acid phosphatase and beta-glucuronidase, independent of tumor grade. The stroma also contained a large amount of acid mucopolysaccharide ground substance, irrespective of the three grades. There was a striking difference in the ultrastructural organization of the stroma between normal and neoplastic tissues. Although fragmented elastic fibers and increased amount of acid mucopolysaccharide granules, and macrophages rich in phagolysosomes were prominent fine structures of the stroma of carcinomas, there was no apparent difference in them among the three grades of malignancy.


Electron microscopy of C-particles and small vesicles in a transplantable rat ascites hepatoma-66F. Transplantable rat ascites hepatoma AH-66F was examined by an electron microscope.  C-particles were numerously observed in the intercellular spaces and on the cell surface. The size of these particles varied from 90 to 200 nm in diameter. Small vesicles were also observed. These vesicles ranged from 40 to 50 nm in diameter. Most of them were located near the group of C-particles. Occasionally, small vesicles were observed in the form of a rosette around the C-particles.


Influence of sex hormones on kidney tumors induced in rats with N-butylnitrosourea. A total 39 kidney tumors were induced by a continuous oral administration of N-butylnitrosourea (BNU) in 33 out of 204 rats of W/Fu strain and of 41 (W/Fu X ACI/N)F1 rats. No spontaneous renal tumors were observed among 66 males and 109 females of W/Fu rats which survived beyond the age of 19 months. Histologically, renal cell and mesenchymal types were commonly observed; 24 cases belonging to the former and 11 cases to the latter. Two cases of nephroblastoma were also encountered. There was no sex difference in renal tumorigenesis with BNU as a whole. Castration in both sexes was apparently inhibitory for kidney tumor development. Estrogenization of castrated rats either by syngeneic ovary graft or by repeated injections of estradiol benzoate enhanced tumor induction with BNU. Progesterone was not effective in restoring the tumor incidence in castrated rats. Distribution of histological types of tumors thus induced differed among the hosts with different hormonal conditions; in males the majority was renal cell type, whereas almost all mesenchymal tumor and nephroblastoma cases were found in intact females or estrogenized rats. BNU induced a variety of tumors in several organs including cerebral hemisphere, peripheral nerves, mammary glands, hematopoietic system, digestive tracts, and so on. However, such concurrence did not affect the development of renal tumors in the present study.


Collagenolytic enzyme in solid Tawa sarcoma. In order to demonstrate a collagenolytic enzyme in a tumor, solid Tawa sarcoma was subjected to a tissue culture technique. Salt- and acid-soluble collagen used as substrate was extracted from the rat skin and tail tendon. The tumor mass used was obtained on the 5th, 8th, and 11th days after subcutaneous transplantation of ascites Tawa sarcoma cells. Each tissue fragment in the inner and outer layers of the tumor mass was incubated on collagen gel at 37 degrees for 4 days, and collagenolytic activity was determined by comparing the relative content of hydroxyproline in the attacked collagen which was separated by means of centrifugation. All fragments were found to possess collagenolytic activity with some variation. Higher activity was observed in the outer layer than in the inner layer. Tumor fragments were cultured, and the collagenolytic enzyme was isolated from the culture medium and concentrated by ammonium sulfate precipitation and acrylamide disc electrophoresis. Collagenolytic enzyme activity was examined for its mode of attack on native collagen.


Cimetidine inhibits caffeine-stimulated gastric acid secretion in man. In animals, gastric acid secretion stimulated by the methyl xanthine, theophylline, was not inhibited by histamine H2-receptor antagonists. In this study the effect in man of the H2-receptor antagonist, cimetidine, on gastric acid secretion stimulated by the methyl xanthine, caffeine, was examined. Caffeine was given intravenously for 2 hr in a dose of 9 mg per kg-hr to 5 patients with duodenal ulcer and 5 normal subjects. Three hundred milligrams of cimetidine administered orally 30 min before the start of the caffeine infusion completely abolished the acid secretory response in all subjects, decreasing acid secretion to less than basal rates. It is concluded that in man the H2-receptor antagonist, cimetidine, abolished the acid secretory response to the methyl xanthine, caffeine.


Activation of intestinal mucosal adenylate cyclase by Shigella dysenteriae I enterotoxin. Because the mechanism whereby Shigella dysenteriae I enterotoxin induces intestinal secretion is unclear, the effect of this toxin on adenylate cyclase activity in rabbit ileal mucosa was studied under various in vitro and in vivo conditions. Activation of adenylate cyclase by Shigella enterotoxin was observed only when substrate (ATP) concentrations above the Km of adenylate cyclase were employed. These concentrations of ATP are greater than those required to demonstrate activation of adenylate cyclase by cholera toxin. Under optimal assay conditions, doses of Shigella toxin between 5.4 and 900 mug of toxin protein and in vivo incubation times between 6 and 18 hr all increased adenylate cyclase activity by about 100%. Shigella toxin produced significant but highly variable increases in mucosal cyclic AMP concentrations, which were less that the rises seen with a comparable dose of cholera toxin. This variability in cyclic AMP response to Shigella toxin and the disparity between Shigella and cholera toxins' effects on mucosal cyclic AMP are probably the result of the different kinetics of adenylate cyclase activated by these enterotoxins. Mucosal Na-K-ATPase activity was unaffected by Shigella toxin. These observations suggest that alterations in fluid and electrolyte transport induced by Shigella enterotoxin may, in part, be mediated by the adenylate cyclase-cyclic AMP system.


[Reasons for development of chromosome aberrations in cells of a culture of human embryonal fibroblasts infected with herpes simplex virus]. The object of this investigation was the role of lysosomic and virus-induced aberrations in cells of the culture of embryonic human fibroblasts infected with herpes simplex virus. It was established that the frequency of cells with chromosomal abnormalities in infected cultures in 4 and 9 hours after infection was 15,2% and 23,7% respectively, being 2% in the control culture. Stablization of lysosomal membranes with hydrocortisone or the inhibition of lysosome enzymes by trypan blue resulted in the decrease of the proportion of aberrant cells down to the control level in 4 hours after the infection, while in 9 hours after the infection it was higher, than in the control, although significantly lower, than in the cultures infected with the virus, but not subjected to any treatment suppressing the mutagenic effect of the virus. The suppression of the transcription of the viral genome by actinomycin D did not decrease the proportion of aberrant cells in 4 hours after the infection, while in 9 hours after the infection it was significantly lower, than in the infected cultures not treated with actinomycin D, but higher, than in the control. Simultaneous suppression of the effect of virus-induced enzymes by actinomycin D and lysosome enzymes by hydrocortisone or trypan blue decreased efficiently the proportion of aberrant cells down to the control level both after 4 and after 9 hours following the infection. Apparently chromosome aberrations in embryonic human fibroblasts infected with herpes simplex virus are caused both by lysosomal and by virus-induced enzymes. Possibly in different virus-cell systems the relative role of these two factors can be considerably different from one another.


[Mutagenesis under the influence of simian vacuolating virus 40 (SV40). I. Induction of chromosome aberrations in Chinese hamster cells during the first days following infection]. Chromosome aberrations were studied in cultured chinese hamster cells on the 1--4th days after infection with Simian Virus 40. In the first and second mitoses after infection a statistically significant increase of the percentage of aberrant metaphases was observed (up to 22% as compared to 8--10% in intact cells). Already after 3 days following treatment the percentage of aberrant metaphases decreased sharply, reaching the control level. The virus induced a significant increase of the frequency of chromosome and chromatid breaks, as well as the appearance of fragments of an unknown origin. Chromosome breaks were distributed randomly among 5 morphologically distinct chromosome groups, according to the comparative length of respective chromosomes. An increase of the frequency of gaps and coiling deficiencies was also observed in virus-infected cultures. An earlier appearance of the first mitosis was observed in virus-treated cultures. It is suggested that the mutagenic effect of SV40 in the cells studied may be related to mechanisms controlling the integration of viral genomes into the cell chromosomes.


[Mutagenesis under the influence of simian virus 40 (SV40). II. Induct?ION OF MUTATIONS RESULTING IN RESISTANCE TO PURINE BASE ANALOGS IN HUMAN AND Chinese hamster cells]. Induction of gene mutations by SV40 was studied in aneuploid human and Chinese hamster cell lines. It is shown that SV40 penetrates into the cells studied and induces the T-antigen synthesis. The efficiency of the infection in Chinese hamster cells was tested additionally by the ability of infected cells to form colonies in the medium lacking serum growth factor. The maximal number of colony-forming cells was detected 24 hours after the infection. By culturing cells in factor-free medium a Chinese hamster cell subline was isolated, which proved to synthetize T-antigen within 60 days after the virus treatment. This is regarded as an indirect proof of the integration of viral genome into the cell genome. The increased frequency of mutants resistant to 60 mkg/ml of 8-azaguanine (human cells) and to 15 mkg/ml of 6-mercaptopurine (hamster cells) was observed on the 1--4th days following the infection. The analysis of the results of all experiments taken together revealed that the induction is highly significant according to Wilcoxon (greater than 0.99). The resistance of the isolated clones proved to be stable after the prolonged culturing under non-selective conditions. It is suggested that viral genome integration, gene mutations and chromosome aberrations may have common molecular mechanisms. The role of gene mutations in virus-induced carcinogensis and tumour progression is discussed.