Characterization of L5178Y leukemic cells by phagocytosis, cytochemical methods, and membrane marker techniques. Membrane marker techniques, endocytosis, and cytochemical methods were used to characterize L5178Y cells. L5178Y cells were found to have surface membrane immunoglobulin that typifies B-cells, and membrane receptors that are common to both B-cells and monocytes. L5178Y cells also have endocytic and enzymatic activities that are characteristic of monocytes. Thus L5178Y cells have the dual characteristics of B-cells and monocytes.


Regulation of mammary tumor virus production by prolactin in BALB/cfC3H mouse normal mammary epithelial cells in vitro. Hormonal regulation of mammary tumor virus (MTV) production, has been analyzed with normal mammary epithelial cells from chronically infected BALB/cfC3H mice. The effect of prolactin in terms of increased MTV production was not reproducibly observed in cells cultured in tissue culture dishes, whereas the cells grown on floating collagen gels consistently responded to prolactin in a dose-dependent manner. Of the three media tested. Dulbecco's modified Eagle's medium was found to be the best in terms of responsiveness to prolactin and in maximal MTV production. Specificity studies with other pituitary and placental hormones in place of prolactin have shown that both growth hormones and human placental lactogen can replace prolactin, whereas follicle-stimulating hormone, luteinizing hormone, and thyrotrophin were ineffective. Contrary to the mammary tumor systems, where it has been shown that insulin and glucocorticoid can maximally stimulate MTV production, these hormones alone elicited only a small response in the absence of prolactin in normal mammary epithelial cells. Although prolactin alone had very little effect by itself, its presence was necessary (permissive effect) in order for the glucocorticoids to be able to maximally stimulate MTV production in normal cells.


Isolation and some biochemical characteristics of nuclei from AH-66 hepatoma cells. Cetylpyridinium chloride uniquely facilitated the isolation of nuclei from AH-66 hepatoma ascites cells in an isotonic medium without homogenization because of its strong solubilization of their plasma membranes, which were resistant to mechanical shearing with the commonly used nonionic detergents such as Triton X-100, Nonidet P-40, and Tween 80. Virtually all the nuclei in a population of AH-66 cells (10(6)/ml) can be isolated with 0.2% cetylpyridinium chloride. The isolated nuclei were free of adherent cytoplasm, maintained satisfactory morphology, and had high activity of nicotinamide adenine dinucleotide pyrophosphorylase. Two-dimensional polyacrylamide gel electrophoresis of the acid-soluble nuclear proteins of the AH-66 hepatoma nuclei isolated by the cetylpyridinium chloride procedure as well as by the citric acid procedure revealed that Spots Ac and C16-C18 were significantly intense in the gel pattern. Unexpectedly, Spot A10 was absent from the gel pattern of AH-66 hepatoma nuclei.


Relationship between feline leukemia virus antigen expression and viral infectivity in blood, bone marrow, and saliva of cats. Correlation was greater than 90% between feline leukemia virus (FeLV), group-specific antigen (GSA) in leukocytes, and viral infectivity (VI) in serum or plasma from 132 cats infected with either the Rickard strain of FeLV, the Snyder-Theilen strain of feline sarcoma virus, or field strains of FeLV. Detection of GSA in blood cells was at least as sensitive as detection of VI in serum. In 45% of FeLV GSA-positive cats inoculated with FeLV-Rickard strain, VI was detected in saliva. No saliva samples from GSA-negative cats had VI. Sequential bone marrow biopsies from 34 cats inoculated with Snyder-Theilen feline sarcoma virus indicated that the correlation between FeLV GSA in bone marrow cells and blood cells was virtually 100%. FeLV GSA appeared in bone marrow leukocyte precursors 1 week before its appearance in peripheral blood leukocytes in 50% of the cats. The FeLV GSA-positive state was transient (3 to 6 weeks) in 34% of the Snyder-Theilen feline sarcoma virus-inoculated cats.


Inhibition of B-16 melanoma growth in vivo by a synthetic analog of prostaglandin E2. The effect of systemic administration of 16,16-dimethyl prostaglandin E2-methyl ester (di-M-PGE2) on the growth of B-16 melanoma tumors has been studied in C57BL/6J mice. Daily i.p. injection of 5 mu of di-M-PGE2 commencing on the day of tumor inoculation with 10(5) and 10(6) viable cells delayed appearance of tumors; for the smaller tumor inoculum, it also increased median survival among treated mice from 23 to 33 days. Di-M-PGE2 treatment of mice with established tumors caused significant inhibition of tumor growth, as measured by a number of parameters including tumor diameters and volumes. At the time of sacrifice, di-M-PGE2-treated mice had tumors that were an average of 32% smaller (by weight), contained 60% fewer melanoma cells, and had higher concentrations of cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate (+225% and +100%, respectively).


Reduction of streptozotocin toxicity by 3-O-methyl-D-glucose with enhancement of antitumor activity in murine L1210 leukemia. 3-O-Methyl-D-glucose (3-OMG), a nontoxic nonmetabolizable derivative of glucose, is effective in reducing the toxicity of streptozotocin (SZ). In mice the administration of 3-OMG prior to SZ increased the dose that killed 50% of the animals from 240 to 340 mg/kg. Furthermore, the combination of 3-OMG plus nicotinamide (also effective in reducing SZ toxicity) increased the dose that killed 50% of the animals to 540 mg/kg. In L1210 leukemic mice treated with SZ, there was a 2-fold increase in the median survival of animals pretreated with 3-OMG and a 3-fold increase in that of animals pretreated with the combination of 3-OMG and nicotinamide. Neither 3-OMG nor nicotinamide alone enhanced the survival of the leukemic mice. Pretreatment of normal mice with 3-OMG partially prevented the expected fall in hepatic nicotinamide adenine dinucleotide content. This study suggests that 3-OMG, by protecting normal tissue, will permit the administration of larger therapeutic doses of SZ in leukemic L1210 mice. The protective effect of 3-OMG against SZ toxicity appears to be partially mediated through conservation of the nicotinamide adenine dinucleotide content in the tissue.


Inhibition of normal lymphocyte mitogenic reactivity by serum from feline leukemia virus-infected cats. The effect of serum from 12 cats with lymphosarcoma induced by feline leukemia virus (FeLV) on the response of normal cat peripheral blood lymphocytes to phytomitogen-induced blastogenesis was studied. The majority of FeLV sera, when tested at a concentration of 20% of the incubation medium, caused a 40 to 70% reduction in the mean blastogenic response to concanavalin A compared to the response obtained with a similar concentration of normal feline serum. Results with pokeweed mitogen were similar, but the depression in blastogenesis was less than with concanavalin A. Further studies showed that the blastogenic inhibitory activity of FeLV serum (a) was heat labile at 56 degrees for 30 min, (b) could not be overcome by greater concentrations of mitogens, (c) was proportional to the concentration of FeLV serum in the incubation medium, and (d) could not be demonstrated when lymphocytes were preincubated in FeLV serum followed by washing and reincubating in normal feline serum. The results suggested that a substance present in the serum of FeLV-infected cats contributes to altered immunological reactivity during leukemogenesis in the cat.


Hepatocarcinogenic effects of N-nitroso-N-methylurea in guinea pigs. Intragastric administration of N-nitroso-N-methylurea to strain 13 male guinea pigs, at a weekly dosage of 7.5 mg/kg for 15 weeks and then twice weekly for a subsequent 15 weeks, induced high toxicity, as evidenced by weight loss and mortality and a high incidence of malignant neoplasms, over a total observational period of 40 weeks. The neoplasms included hepatic angiosarcomas, cholangiocarcinomas, and generalized lymphoblastic lymphomas.


Fetal infection with bovine leukemia virus in sheep. Several sheep fetuses were thymectomized, and their tails were removed at 58 to 65 days of gestation for tissue culture. Bovine leukemia virus (BLV) antigens were detected in serial culture of tissues from fetuses whose dams and sires were both BLV positive. However, no BLV antigens were detected in serial cultures of tissues from fetuses whose dams were negative but whose sire was positive. Precolostral serums from 3 of 16 neonatal lambs, whose sire and dams were both BLV positive, were BLV antibody positive. Thus, BLV may be vertically transmitted from a positive dam to her lamb via the placenta and/or germinal cells but not from sire to lamb.


Detection and evaluation of feline oncornavirus-induced cell surface antigen(s) shed from cells in vitro. A method for preparation of soluble feline oncornavirus-induced cell surface antigens was described. This technique relied upon the natural release of antigen(s) from FL-74 feline lymphoblastoid cells during their maintenance at 37 degrees in serum-deficient medium. When concentrated and clarified spent medium from 4-day cultures was tested for its antigen content by inhibition of humoral cytotoxicity, it was found that this  natural production of soluble antigen provided more feline oncornavirus-associated cell membrane antigen per cell than did a solubilization procedure in which papain was used. The shed antigen preparation was immunogenic in cats, eliciting humoral antibody that was reactive with the surface of FL-74 Cells and feline sarcoma virus-transformed nonproducer mink cells but was not reactive with feline leukemia virus in a virus neutralization assay.


Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells. Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle.


Investigations on the turnover of adrenocortical mitochondria. IX. A stereological study of the effects of chronic treatment with acth on the size and number of mitochondria from adult human adrenocortical cells cultured in vitro. The effects of ACTH on the mitochondria of adult human adrenocortical cells cultured in vitro were investigated by electron microscopic and stereological methods. It was found that ACTH induces increase in the volume of the mitochondrial compartment, which is due to both a hypertrophy and an increase in number of the organelles. The hypothesis that ACTH controls the growth and proliferation of human adrenocortical mitochondria is discussed.


On the occurrence of Merkel cells in the epidermis of teleost fishes. The ultrastructure of a differentiated cell type in the epidermis of two species of teleost fish, Ictalurus melas and Phoxinus phoxinus, is described. This cell type has a synaptic association with nerve fibres, microvillus-like peripheral processes, and membrane-bounded inclusions, which together are the diagnostic features of the Merkel cells of tetrapod vertebrates. Other cytoplasmic features are shared with the epithelial cells. The appearance of the membrane-bounded granules depends on the fixative used; after fixation with glutaraldehyde the granules are of a size and electron-density comparable to that found in tetrapod Merkel cells, but after fixing in osmium tetroxide the granules are inconspicuous.


Coupled transcription-translation of DNA injected into Xenopus oocytes. A previous report from this laboratory showed that purified DNAs are transcribed after injection into the nucleus of Xenopus laevis oocytes (Mertz and Gurdon, 1977). Here we demonstrate that at least some of the RNA synthesized is translated within these injected cells to produce the correct protein products. Injection of Simian Virus 40 DNA into oocytes induces the synthesis of at least two proteins not normally synthesized in these cells. Using two-dimensional polyacrylamide gel electrophoresis and well characterized deletion mutants of SV40 that produce proteins of smaller size, we show that these two proteins are, indeed, the virus-coded proteins VP1 and VP3. Synthesis of VP1 and VP3 is inhibited by alpha-amanitin and, therefore, presumably mediated by a type II RNA polymerase. We also present evidence indicating that a histone-like protein is induced after the injection of a cloned plasmid DNA that codes for the Drosophila melanogaster histone proteins. This in vivo coupled transcription-translation system should be useful for identifying and mapping proteins coded by cloned eucaryotic DNAs.


A novel method to map transcripts: evidence for homology between an adenovirus mRNA and discrete multiple regions of the viral genome. A method has been devised which permits mapping of transcripts by a two-step hybridization procedure (sandwich hybridization). RNA extracted from cells infected with an adenovirus-SV40 hybrid (Ad2+ND1) was hybridized to restriction endonuclease fragments of adenovirus type 2 (Ad2) DNA immobilized on nitrocellulose filters. RNAs containing both Ad2 and SV40 sequences formed duplexes through their Ad2 sequences, leaving their SV40 sequences as protruding tails. Annealing with 32P-labeled SV40 DNA caused these tails to become labeled, permitting autoradiographic identification of the sequences of Ad2 DNA which are homologous to the RNA. The high sensitivity of this technique, achieved through the use of 32P-labeled RNA of high specific activity, has led to the observation that hybridization of Ad2+ND1 RNA occurs at several locations on the Ad2 genome, in addition to the expected sites of hybridization proximal to the SV40 insertion.


Avian myeloblastosis virus RNA is terminally redundant: implications for the mechanism of retrovirus replication. We have determined the terminal heteropolymeric sequences of AMV RNA by the following procedures: first, RNA sequence determination on the 5' terminal and the poly(A)-linked 3' terminal T1 oligonucleotides, and second, analysis by the Maxam and Gilbert (1977) method of AMV strong stop DNA and of DNA complementary to the poly(A)-linked T1 oligonucleotide, synthesized with reverse transcriptase and (pdT)13 as primer. The structure deduced for the 5' terminal region is (5')7mGpppGmCCAUUCUACCUCUCACCACAUUGGUGUGCACCUGGGUUGAUGGCCGGACCGUCGAUUCCCUGACGACUACGAGCACCUGCAUGAAGCAGAAGGCUUCAU... Two distinct 3' terminal sequences were deduced: GCCAUUCUACCUCUCAAA...AOH and GCCAUUCUACCUCUCACCAAA...AOH. The two termini, differing by a C-C-A sequence, may reflect genetic heterogeneity of the AMV stock or, more probably, may be generated at or after RNA transcription. These results demonstrate a terminal redundancy of the hetero polymeric sequence of 16 and 19 nucleotides, respectively. The terminal redundancy allows for mechanisms which involve transfer of the DNA segment synthesized on the 5' terminal redundant sequence to the 3' terminal redundant sequence.


Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle. We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.


Double viral infection of the mouth in the cancer patient. An autopsy case of renal cancer complicated with multiple oral ulcers is presented. Although serological tests were not available, double viral infection with herpes simplex virus and cytomegalovirus was suspected as causal agents in the retrospective examination. This infection may be a complication in the prolonged stage of cancer patients.


[Changes in the blood cortisol levels during the first week of life in calves (Bos taurus) and lambs (Ovis aries)]. At birth, the plasmatic level of cortisol is very variable and very high in the new-born Calf or the newborn Lamb. This level diminishes within one day and then varies between 10 and 30 ng/ml during the six following days. In the Calf, the secretion of cortisol can be directly stimulated by ACTH or inhibited at the level of the hypothalamic and hypophyseal system with dexamethasone on the day following birth.


[The replication of a xenotropic murine C type virus in some mammalian cells. Definition of a specific antigen]. The use of our mink cell line maintained in vitro infected with the murine xenotropic AT 124 virus, and that of a (W/Fu x bn) f1 rat anti-124 serum allow us to define a new cell surface antigen specific of murine xenotropic type C viruses.


[Expression of an antigen associated with Gross virus on the surfaces of murine cells producing an oncornavirus from the radioleukemia of C57BL/6 mice]. A leukemogenic viral complex was demonstrated in cultures of 13-3 C cell line derived from a C57BL/6, radiation leukemia virus (RadLV-Rs) induced tumor. Both 13-3C and leukemic cells induced in C57BL/6 mice by 13-3C virus carry a cell surface antigen associated with Gross leukemia virus (GCSAa). These findings point to a close similarity between these antigens and those of murine endogenous ecotropic viruses.


[Electrophoretic analysis of lactate NAD oxidoreductase in interspecific hybrids between three species of the genus Sphaeroma: S. monodi, S. Rugicauda and S. hookeri]. The electrophoretic analysis of lactate NAD oxydoreductase in interspecific hybrids between three species of genus Sphaeroma (S. monodi, S. rugicauda and S. hookeri hookeri) leads, for each category of hybrids, to a distribution between the two parental enzymatic patterns and, consequently, shows the absence of an isozyme appropriate to these hybrids.


[Compared expression of murine plasmacytoma associated virus in tumor cells and in mouse embryo cells]. A DNA complementary to the viral genome of C-type particles produced by a Mouse myeloma derived cell line (MF2 cell line) was synthesized. This cDNA was used as a probe to study the viral genome expression among the total RNA and the poly (A)-rich RNA extracted from the MF2 and Balb/c embryonic cells. As evidenced by molecular hybridization experiments, the presence of at least one endogenous Balb/c virus in the MF2 virus stocks is suggested. In the productive cells, the viral RNA sequences are expressed in the poly (A)-rich RNA fraction.


[Sensitivity of transformed EHSVi fibroblasts to the antibody-complement lytic system. Cooperation between the lytic units, and existence of a cellular defense]. The sensitivity of transformed EHSVi fibroblasts to the antibody-complement lytic system was evaluated, at various antibody concentrations (Ac), in conditions in which a variation of the lytic activity proportional to Ac3, 1 may be observed. This points out, not the expected cooperation between IgG molecules in activating C1 within a definite lytic unit, but a cooperation between the lytic units themselves, and therefore the existence of a cell defence.


[3'5'-Cyclic AMP phosphodiesterase activity in the internal and external layers of human myometrium at the end of pregnancy]. The hydrolysis of cAMP by phosphodiesterase was studied in whole homogenate from human myometrium at the end of the pregnancy before onset of labour. Tissue samples were taken from outer and inner layers of placental and anti-placental sites. Kinetic analysis shows in every case two apparent Km values for low and high cAMP concentrations in the order of 1 x 10(-5) M and 1 x 10(-4) M. On the other hand Vmax values are lower for the enzyme isolated from the placental site than for the one isolated from the anti-placental area. In the 4 zones studied, an appreciable proportion of the low Km enzyme is present.


[Heterotransplantation of human glioblastoma in the brain of newborn mice]. 7 human multiform glioblastoma have been transplanted into the brain of 100 new born Mice. Electron microscopy and chromosome analysis confirm the growth of a human tumor showing malignant features of glioblastoma.


[Comparative activity of endorphins on the opiate receptors]. Displacement of naloxone from membranes of Rat brain by alpha, beta and gamma-endorphins with and without Na+ in the incubating medium has been studied. beta-endorphin shows a higher affinity for the opiate receptors and a stronger agonist property than morphine. alpha and gamma-endorphins have a much lower affinity than morphine and a marked antagonist characteristic. This study suggests the possibility of naturally occurring antagonists of the opiate receptors.


Estrogens and experimental atherosclerosis in the baboon (Papio cynocephalus). One hundred twenty-six adult female baboons (Papio cynocephalus) were hysterectomized and all except 18 were ovariectomized. The animals were fed a moderately atherogenic diet (40% calories form hydrogenated vegetable oil, 1.5 gm cholesterol/kcal) for two years. Ovariectomized-hysterectomized animals received estrone sulfate, ethynyl estradiol, or diethylstilbestrol orally in daily doses similar to those given humans. An ovariectomized-hysterectomized group and a hysterectomized group received no drug. The average total serum cholesterol concentration rose from 136 mg/dl to 223 mg/dl and declined to 186 mg/dl. Concentrations of cholesterol, triglyceride, and phospholipid in whole serum, low density lipoproteins and high density lipoproteins showed no consistent statistically significant differences among the groups. Triglyceride and phospholipid concentrations were higher in the estrogen-treated and intact-ovary groups than in the ovariectomized nonestrogen treated group, but not all pairwise comparisons were statistically significant. There were no consistent statistically significant differences in atherosclerotic lesions among the groups. Neither ovariectomy nor estrogen replacement influence diet-induced experimental atherosclerosis in the baboon within two years.


Breast carcinoma in children and adolescents. Seventy-four cases of breast cancer in children and adolescents are reviewed. No predisposing factors were identified. A secretory type of mammary carcinoma characteristically seen in this age group has an excellent prognosis. Other types of mammary carcinoma which also occur in children may have a less favorable prognosis. Persistent breast masses in children and adolescents should be biopsied unless cystic, and should be treated as would carcinoma in adults, keeping in mind the favorable prognosis in so-called secretory carcinoma.


Measurement of plasma lipoproteins by electrophoresis on polyacrylamide gel. Results of densitometric analysis of lipoprotein peaks after electrophoresis on polyacrylamide gel and values obtained by the classical method of ultracentrifugation/precipitation correlate well. The correlation coefficients for high-density, low-density, and very-low-density lipoproteins are 0.85, 0.99, and 0.99, respectively. Thus the densitometric method for lipoprotein analysis appears to be suitable for routine clinical application.


Creatine kinase in serum: 3. Further study of adenylate kinase inhibitors. In search of an appropriate inhibitor to suppress the interference of adenylate kinase with the creatine kinase assay, we found that the combination diadenosine pentaphosphate (10 mumol/liter) and AMP (5 mmol/liter) is a better inhibitor than is fluoride (25 mmol/liter). The latter inhibits adenylate kinase uncompetitively and weakly (Ki = 2.5 mmol/liter), and must be incorporated in the starting reagent, and at 30 degrees C it becoms fully effective only after a lag phase of 6 min. In this concentration, fluoride inhibits adenylate kinase from erythrocytes, muscle, liver or platelets by 94, 92, 88, and 87%, respectively, and creatine kinase by 8%. Bromide and chloride also inhibit creatine kinase. Attempts to replace AMP by a specific inhibitor of liver adenylate kinase failed. Homologs of diadenosine pentaphosphate with either fewer or more phosphoryl groups in the polyphosphate bridge inhibited even more weakly than did the pentaphosphate. Platelets can significantly contribute to adenylate kinase activity in serum. The inhibitor combination inhibited adenylate kinase from platelets by 90%.


Plasma lipoprotein changes in experimental cholestasis in the dog. Plasma lipoproteins were compared before and after surgical induction of extrahepatic biliary obstruction in dogs by immunochemical techniques, zonal ultracentrifugation and gel filtration. A considerable increase in LDL was found one week after operation, with a zonal ultracentrifugal pattern which was more complex than that found before operation. The lipid composition was characterized by an increase of phospholipids and a decrease of triglycerides in cholestatic LDL fractions. None of the fractions displayed the inverse cholesterol-cholesterol ester ratio, characteristic for human LP-X. The apoprotein composition of cholestatic LDL was also markedly changed. Apoprotein A I, already present in the normal LDL2 subclass, increased and two apoproteins with apparent molecular weight of 35000-40000 appeared in considerable amounts. The amount of HDL was decreased during cholestasis without any appreciable changes in composition.


Alterations in serum prolactin heterogeneity by provocative tests in a patient with a pituitary tumour. The proportion of serum prolactin heterogeneity were investigated in a patient with a pituitary adenoma producing both prolactin and growth hormone. Glucose tolerance (GTT) and insulin tolerance (ITT) tests were performed alone and in combination with chlorpromazine both before and after hypophysectomy. Serum prolactin multiple forms were separated by Sephadex G-100 column chromatography at 3 degrees C. None of the provocative tests altered the already elevated serum prolactin either before (300 ng/ml) or after (100 ng/ml)hypophysectomy. Chlorpromazine or ITT did not alter the proportion of prolactin heterogeneity; GTT, whether in conjunction with chlorpromazine or alone and both before and after hypophysectomy, increased the proportions of the larger molecular species. In five tests in which the GTT was not performed the prolactin heterogeneity was as follows: 'void volume', 4.9%; 'big', 13.1%; 'little', 82.1%; in those experiments in which the GTT was performed the proportions of prolactin heterogeneity were: 'void volume', 13.5%, 'big', 19.2%; 'little', 67.3%.


Studies on the metabolic defect in Broad-beta disease (hyperlipoproteinaemia type III). The apoprotein composition of the main lipoprotein fractions (VLDL, LDL-1, LDL-2 and HDL) was studied initially in 15 patients with Broad-beta disease. Analytical isoelectric focusing of urea-soluble apo-VLDL and apo LSL-1 demonstrated a variant pattern of the polymorphic Apoprotein E with a deficient Apo E-III band in all patients. The Apo E-III deficiency pattern was seen in only six out of 304 hyperlipidaemic controls. These six Apo E-III deficient controls had characteristic signs of Broad-beta disease, and thus represented patients not previously recognized as having the disorder. The Apo E focusing patterns were constant on repeated examinations and were stable under different metabolic conditions. The data show that Apo E-III deficiency in VLDL is a specific qualitative marker for Broad-beta disease, allowing an unequivocal diagnosis that had not been possible previously. Indirect evidence suggests that Apo E-III deficiency is the basic lipoprotein abnormality underlying the familial dyslipoproteinaemia.


Species difference in the beta1/beta2-adrenoceptor selectivity of SM220CL in the cat and guinea-pig. The beta-receptor stimulant effects of Sm220Cl, dl-N-(1,1-dimethyl-3-phenylpropyl)-2-hydroxy-2-(3,4-dihydroxy-2-methoxyphenyl)ethylamine, and (-)-isoprenaline have been compared in isolated atrial (beta1) and tracheal (beta2) preparations from guinea-pigs and cats. 2. The compounds were also tested for their ability to increase the heart rate (beta1), reduce serotonin-induced increases in pulmonary resistance (beta2), and decrease soleus muscle contractility (beta2) in vivo in the two species. 3. In all experiments cumulative concentration or dose-effect curves were established, EC50 or ED50 values obtained and molar activity-ratios (Sm220Cl: (-)-isoprenaline) calculated. 4. Calculated selectivity ratios [activity-ratio (heart):activity-ratio (bronchial smooth muscle)] from the in vitro experiments showed that Sm220Cl possessed beta2-receptor selectivity. This was more marked in guinea-pig than in cat preparations. 5. In the anaesthetized animals this species difference was more apparent; in cats Sm220Cl was non-selective in its actions for beta1- and beta2-receptor mediated responses, while marked beta2-receptor selectivity was obtained in the guinea-pig. 6. Since in both species the activity-ratios for beta2-receptor mediated actions are similar, the differences in the beta1/beta2-receptor selectivity of Sm220Cl are caused by the divergent cardiac effects produced by the drug.


Some myocardial factors of biological oxidation in experimental myocardial infarction. In 175 dogs myocardial infarction was produced by high ligation of descending branch of left coronary artery. At various intervals after the intervention (1, 3, 5, 10, 30, 180 days), the activities and levels of NAD, NADH, FAD, riboflavin, cytochrome C, myoglobin, some NAD-dependent Krebs cycle enzymes, and mitochondrial succinate dehydrogenase and cytochrome oxidase were determined in the infarcted zone. It was found that in the infarcted zone there occurred substantial disturbances of various links constituting the tissue oxidative chain, in the stages of substrate dehydrogenation, electron transport to oxygen molecule, and myocardial oxygen uptake. The greatest disturbances took place in the systems of NAD and NAD-dependent enzymes, whereas the succinate oxidation system sustained substantially lesser damage. The decrease inlevels of flavonoids, which was likewise observed, participated also in the mechanism inhibiting succinate dehydrogenase. The cytochrome system activity was limited by the level of cytochrome C, whose deep decrease persisted considerably long in the infarcted zone. A certain role in disturbances of oxidative processes may have been played by the decreased concentration of myoglobin, an important myocardial reservoir of oxygen.


Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells. The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.


Gene mapping in Mus musculus by interspecific cell hybridization: assignment of the genes for tripeptidase-1 to chromosome 10, dipeptidase-2 to chromosome 18, acid phosphatase-1 to chromosome 12, and adenylate kinase-1 to chromosome 2. Chinese hamster X mouse somatic cell hybrids segregating mouse chromosomes were examined for their mouse chromosome content using trypsin-Giemsa (GTG) banding and Hoechst 33258 staining techniques. Simultaneously, they were scored for the presence of 24 mouse enzymes. The results confirm the assignments of 11 genes previously mapped by sexual genetics: Dip-1 and Id-1 to chromosome 1; Pgm-2 and Pgd to 4; Pgm-1 to 5; Gpi-1 to 7; Gr-1 to 8; Mpi-1 and Mod-1 to 9; Np-1 and Es-10 to 14. They also confirm chromosomally the assignments of 3 genes that were made by other somatic cell genetic studies: Aprt to 8; Hprt and alpha-gal to the X chromosome. But most importantly, four enzyme loci are assigned to four chromosomes that until now were not known to carry a biochemical marker which is expressed in cultured cells: Trip-1 to 10; Dip-2 to 18; Acp-1 to 12; and Ak-1 to 2. Cytogenetic examination of clones showing discordant segregation of HPRT and A-GAL, suggested the assignment of alpha-gal to region XE leads to XF of the mouse X chromosome. The cytologic studies provide a comparison between data from sexual genetics and somatic cell hybrids and validate hybrid cell techniques. They provide evidence of the reliability of scoring chromosomes by GTG and Hoechst staining and stress the importance of identifying clones with multiple chromosome rearrangements. Striking examples of norandom segregation of mouse chromosomes were observed in these hybrids with preferential retention of 15 and segregation of 11 and the Y chromosome.


Dysrhythmias and sudden death in acromegalic heart disease. A clinicopathologic study. A clinicopathologic investigation was carried out in a patient with acromegaly from acidophylic adenoma of the hypophysis. Pronounced cardiomegaly and disturbances in impulse formation and conduction led to sudden death. The problem of acromegalic cardiomyopathy has been reviewed. Particular attention has been focused on the histopathologic findings in the working and specific myocardium, and in the intrinsic nervous supply to the sinoatrial node, which were correlated with the electrocardiographic features of atrioventricular conduction delay and sick sinus syndrome.


Infectious mononucleosis and fatal myocarditis. Mycocarditis is an uncommon manifestation and, very rarely, a lethal complication of infectious mononucleosis. A 14-year-old girl initially had exudative pharyngitis and splenomegaly and developed refractory ventricular fibrillation. The diagnosis of infectious mononucleosis was confirmed by both a strongly positive heterophil antibody test and a high titer of Epstein-Barr virus. Pathologic studies demonstrated extensive histiocytic and lypmhocytic infiltration of the myocardium.


In vitro stimulation of thyroid ornithine decarboxylase activity and polyamines by thyrotropin. Thyrotropin (TSH) stimulation of ornithine decarboxylase (ODC) activity and polyamine levels was studied in vitro in rat thyroids. The elevation in ODC activity was related to the concentration of TSH in the incubation medium with peak activity at a concentration of 25mU/ml. ODC activity with 50 mU/ml of TSH was 3 to 5-fold higher than control activity at 5 h of incubation; this stimulation was enhanced by the addition of 0.5 mM 3-isobutyl-l-methyl xanthine (MIX), a phosphodiesterase inhibitor. Dibutyryl cyclic AMP (DbcAMP) also stimulated ODC activity with a dose response up to 2.0 mm. The increase in ODC activity with TSH and MIX was prevented by incubation with actinomycin D (10 microgram/ml) or puromycin (0.2 mM). Putrescine concentrations in rat thyroids rose to three times basal levels after 6 h of incubation with TSH and MIX; no significant elevation in spermidine or spermine was observed after up to 7 h incubation. The increase in tissue putrescine preceded a rise in [3H]uridine incorporation into acid-soluble material that occurred at 7 h. The results suggest that stimulation of thyroid ODC activity by TSH is mediated by a cyclic AMP; the data further are consistent with a role for polyamines in the control of RNA synthesis in the thyroid.


Somatostatin inhibits urinary cyclic AMP excretion in diabetic rats. Short term (30 min) infusion of cyclic somatostatin (50 microgram/rat), insulin (1 U/rat) or the two together significantly suppressed urinary cyclic AMP excretion in streptozotocin-diabetic rats. While somatostatin tended to increase cyclic GMP excretion, insulin had an opposite effect in diabetic but not in normal rats. It is suggested that somatostatin suppresses cyclic AMP excretion by inhibiting directly adenylate cyclase in liver and perhaps in other organs. The possibility that suppression of urinary cyclic AMP is due to inhibition of glucagon secretion is also considered.


Perfusion of the pancreas isolated from pertussis-sensitized rats: potentiation of insulin secretory responses due to beta-adrenergic stimulation. In order to study the mechanism by which pertussis-sensitized rats showed enhanced insulin secretory responses to various secretagogues (Sumi, T., and M. Ui, Endocrinology 97: 352, 1975), pancreases of rats receiving a single injection of Bordetella pertussis cells 3 days before were perfused with Krebs-Ringer solution, and release of insulin therefrom was compared with that from the pancreases of normal rats. Much more insulin was released from the pancreas of the pertussis-sensitized rat than from the pancreas of the normal rat in response to glucose, arginine, glibenclamide and 3-isobuty-l-methylxanthine. The inhibition of insulin secretion caused by epinephrine, norepinephrine or phenylephrine via alpha-adrenergic receptors in the pancreas of normal rats was no longer observable with the pancreas from pertussis-sensitized rats. Instead, the addition of epinephrine with or without phentolamine gave rise to a marked secretion of insulin from the pancreas of pertussis-sensitized rats which was prevented by propranolol. It is concluded that a single injection of B. pertussis into rats results in a sustained modification of insulin secretory processes in the pancreatic beta-cells in such a manner as to favor insulin secretory responses to beta-adrenergic stimulation and other secretagogues.


Reduction in hepatic triiodothyronine binding capacity induced by fasting. Triiodothyronine (T3) receptor kinetics were determined in liver nuclei isolated from fasting and fed rats. The results indicate that although affinity equilibrium constants (Ka) did not differ in the two groups, mean (+/- SE) maximal binding capacity (MBC) was reduced significantly to .30 +/- .05 nM/mg DNA in fasting compared to .46 +/- .07 nM/mg DNA (p less than .01) in the fed state. This observed decrease in MBC during fasting apparently could not be accounted for by a differential rates of loss of either DNA or of the receptor during the period of incubation.


Effects of concurrent administration of lead, cadmium, and arsenic in the rat. Humans are exposed to a number of toxic elements in the environment; however, most experiments with laboratory animals investigate only one toxic element. To determine if concomitant exposure to lead (Pb), cadmium (Cd), and/or arsenic (As) modified the changes produced by any one metal in various parameters of toxicity, 168 male, Sprague-Dawley, young adult rats were fed nutritionally adequate diets to which had been added 0 or 200 ppm Pb as Pb acetate, or 50 ppm Cd as Cd chloride, or 50 ppm As as sodium arsenate or arsanilic acid in a factorial design for a period of 10 weeks. At these concentrations, Cd and As reduced weight gain even when differences in food intake were taken into account; administration of both Cd and As depressed weight gain more than did either metal alone. Pb did not adversely affect food consumption or weight gain. Increased numbers of red blood cells (RBCs) were observed following administration of Pb, Cd, or As; usually more cells were observed when two or three metals were administered, compared to individual metals. Despite increasing numbers of circulating RBCs, hemoglobin and hematocrit were reduced, especially with the Pb-Cd combination and the Cd-arsanilic acid combination. Specific effects of Pb on heme synthesis were observed, including increased urinary excretion of delta-aminolevulinic acid; this increase was reduced by the presence of dietary cadmium. Analyses of blood showed values for the laboratory rat within normal ranges for blood urea nitrogen, creatinine, cholesterol, calcium, albumin, total protein, and bilirubin. Uric acid was increased by Pb, with little modification by dietary Cd or As content. Serum glutamate-oxalate transaminase activity was reduced by As. Serum alkaline phosphatase was greatly reduced by either As or Cd but not Pb. Combinations of As and Cd did not further reduce the activity of this enzyme. Kidney weight and kidney weight/body weight ratios were increased by Pb alone, with no effects of Cd or As alone or as interactions. Liver weight/body weight ratios were reduced in animals fed 50 ppm dietary Cd. Kidney histology shows predominantly Pb effects, namely, intranuclear inclusion bodies and cloudy swelling. Ultrastructural evaluation of kidneys from Pb-treated animals disclosed nuclear inclusion bodies of the usual morphology and mitochondrial swelling. Concurrent administration of Cd greatly minimized Pb effects on the kidney under conditions of this experiment. Liver histology suggests an increased rate of cell turnover with either As compound, but few specific changes.


Ultrastructural and biochemical effects of prolonged oral arsenic exposure on liver mitochondria of rats. This investigation was undertaken to further delineate the subcellular manifestations of arsenic toxicity following chronic exposure using combined ultrastructural and biochemical techniques. Male rats were given access to deionized drinking water solutions containing 0, 20, 40, or 85 arsenic as arsenate (As(+5)) for 6 weeks. In situ swelling of liver mitochondria was the most prominent ultrastructural change observed. Mitochondrial respiration studies indicated decreased state 3 respiration and respiratory control ratios (RCR) for pyruvate/malate but not succinate mediated respiration. Specific activity of monoamine oxidase which is localized on the outer mitochondrial membrane showed increases of up to 150% of control and cytochrome-C oxidase which is localized on the inner mitochondrial membrane showed increases in specific activity of 150-200%. Activity of malate dehydrogenase which is localized in the mitochondrial matrix was unchanged at any dose level. These studies indicate that decreased mitochondrial respiration is only one aspect of arsenic toxicity to this organelle. Marked arsenic-mediated perturbation of important enzyme systems localized in mitochondria which participate in the control of respiration and other normal mitochondrial functions are also important manifestations of cellular dysfunction.


Hydrolysis and isomerization of trans,trans-farnesyl diphosphate by Andrographis tissue-culture enzymes. Incubation of (3R,5S)-[5-3H1]mevalonate + (3RS)-[2-14C]mevalonate with Andrographis cell-free extract leads to trans,trans-farnesol and cis,trans-farnesol which both totally retain tritium. 2. This conflicts with our previous results which predict one third tritium loss in the cis,trans-farnesol. Inversion at C-1 during hydrolysis of trans,trans-farnesyl diphosphate to trans,trans-farnesol could explain this anomaly. 3. (1s)-trans,trans-[1-3H1]Farnesyl diphosphate and phosphate and (1R)-trans,trans-[1-3H1]-farnesyl diphosphate and phosphate, all prepared chemically, were hydrolysed with Andrographis phosphatase, and alkaline phosphatase and hydrogenolysed with lithium aluminium hydride and the product alcohols exchanged with liver alcohol hydrogenase. 4. Both Andrographis phosphatase and alkaline phosphatase hydrolyse trans,trans-farnesyl diphosphate and trans,trans-farnesyl phosphate with retention. 5. Hydrolysis of trans,trans-[1-18O]farnesyl diphosphate in H2(18O with both phosphatases supports P-O fission. 6. The C-1 configuration in (1S)-TRANS,TRANS-[1-3H1]farnesyl diphosphate and phosphate and (1R)-trans,trans-[1-3H1]farnesyl diphosphate and phosphate is progressively racemised in 0.01 M NH4OH/MeOH (1/9) AT - 20 degrees C.


Toxicological properties of lead.

Toxicological properties of lead. The pathological effects of lead on the renal, nervous, reproductive, endocrine, and immune systems have been reviewed. Emphasis is placed on reported subclinical effects due to chronic, low-level lead exposure. The crucial issue of whether subtle behavioral, intellectual, and developmental impairment occurs in young children, as a result of lead-induced CNS damage is discussed in detail. This issue remains unresolved. Further studies are needed in order to determine the long-term health effects of continuous, low-level lead exposure.


Dimerization of papain induced by mercuric chloride and a bifunctional organic mercurial. The bifunctional mercurial meso-1,4-bis(acetatomercuri)-2,3-diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium. The conformational change of papain necessary for this reaction is discussed.


Adenosine diphosphoribosylation of certain basic chromosomal proteins in isolated trout testis nuclei. Isolated trout testis nuclei rapidly incorporate [alpha-32P]NAD+ into chromosomal proteins. Three proteins, very-lysine-rich histone (H1), a specific trout chromosomal protein (H6) and the sperm-specific protamines, incorporate the label as adenosine diphosphoribosyl (ADP-Rib) residues. No significant labeling of the nucleosomal 'core' histones H2A, H2B, H3 and H4 was observed. The linkage of the [32P](ADP-Rib) residues to each protein was very labile at pH values greater than 7.0 but by working at acidic pH and low temperatures the ADP-Rib label could be shown to be covalently bound to protein by gel electrophoresis and ion-exchange chromatography. The [32P]ADP-Rib chains could be removed by digestion with snake venom phosphodiesterase with the formation of AMP and phosphoribosyl-AMP.


Reduction in beta-adrenergic response of cultured glioma cells following depletion of intracellular GTP. 1. When C6 glioma cells were incubated with mycophenolic acid, a potent and specific inhibitor of IMP:NAD oxidoreductase (EC 1.2.1.14) there was a marked depletion of the cellular content of GTP. The viability of the cells was unaffected. 2. The adenosine 3':5'-monophosphate (cyclic AMP) response of C6 glioma cells to the beta-adrenergic stimulant, (+/-)isoprenaline, was considerably reduced after treatment with mycophenolic acid. The diminished response to (+/-)isoprenaline was prevented by the inclusion of guanine in the culture medium along with mycophenolic acid. 3. The adenylate cyclase response to (+/-)isoprenaline of whole homogenates from C6 cells treated with mycophenolic acid was also depressed; the response was restored to normal by the addition of GTP. 4. The adenylate cyclase response to (+/-)isoprenaline of a membrane fraction prepared from homogenates of C6 cells was almost totally dependent on the presence of added GTP. Membrane fractions from control and mycophenolic-acid-treated C6 cells gave similar adenylate cyclase responses to (+/-)isoprenaline in the presence of GTP. 5. It is concluded that mycophenolic acid may depress the beta-adrenergic sensitivity of C6 cells by depleting the cellular content of GTP.


Purification of aldolase C from rat brain and hepatoma. An isolation procedure for rat brain aldolase C has been developed which also permits the isolation of aldolase C from experimental hepatomas. Certain enzymatic properties (specific activity and Michaelis constant towards the two specific substrates: fructose 1,6-biphosphate and fructose 1-phosphate) and physico-chemical properties (molecular weight, N-terminal amino-acid) of the two enzymes have been studied and compared. Moreover, an amino-acid analysis has been carried out for rat brain aldolase C. Within experimental errors, the two enzymes appear to be identical.


Nuclear-magnetic-resonance studies of Pseudomonas aeruginosa cytochrome c-551. Nuclear magnetic resonance (NMR) spectroscopy was used to study Pseudomonas aeruginosa cytochrome c-551. Assignments of resonances to specific residues have been made. A low-resolution X-ray structure was used to aid assignments. A structural comparison was made between P. aeruginosa cytochrome c-551 and mammalian cytochrome c, based on comparisons of NMR data.


Ethanolaminosis. A newly recognized, generalized storage disease with cardiomegaly, cerebral dysfunction and early death. A storage disease with cardiomegaly, generalized muscular hypotonia, cerebral dysfunction, failure to thrive and early death is described in two siblings. The first one died at the age of 10 months, the second at the age of 17 months. The symptoms were mainly due to lysosomal storage of a substance which had a positive reaction to PAS and Best's stain and which was resistant to diastase. This substance was stored in nearly all the organs, especially in the heart, liver, spleen and less in the brain and skeletal muscles. An increased renal excretion of ethanolamine, a greatly increased hepatic concentration of ethanolamine and diminished hepatic ethanolamine kinase activity could be demonstrated. Ethanolamine is essential for the synthesis of phospholipids. Both parents showed increased renal excretion of taurine. In several aspects, this syndrome is similar to the glycogenosis type II described by Pompe.


Central cardiovascular effects of SQ 14,225, an angiotensin-converting enzyme inhibitor in chloralose-anesthetized cats. Experiments were conducted in chloralose-anesthetized cats to investigate the central and peripheral cardiovascular effects of a new orally active angiotensin-converting inhibitor, SQ 14,225 (3-mercapto-2-D-methylpropanoyl-L-proline). I.v. administration of SQ 14,225 (0.31-3100 microgram/kg) antagonized the pressor responses to angiotensin I (AI) (310 ng/kg) i.v. in a dose-related manner but did not alter responses to angiotensin II (AII) (200 ng/kg) i.v. Similarly, intracerebroventricularly (i.c.v.) administered SQ 14,225 (0.31-310 microgram/kg perfused over 10 min) produced dose-related decreases in the centrally mediated pressor responses and heart increases elicited by AI (310 ng/kg) i.c.v. The central responses to AII (200 ng/kg) i.c.v. were not affected by SQ 14,225. Passage of SQ 14,225 out of the ventricular system into the systemic circulation was detected, but only at doses greater than that required to antagonize central AI responses. Doses of SQ 14,225 (310 microgram/kg and 3.1 mg/kg) i.v. which maximally inhibited i.v. AI responses, had no effect upon central AI activity. This finding suggests that passage of SQ 14,225 across the blood brain barrier into the central nervous system is restricted. Peripheral or central administration of SQ 14,225 produced only minimal transient decreases in blood pressure (less than 10 mm Hg). The results of these studies indicate that SQ 14,225 is a potent inhibitor of AI conversion in the brain as well as in the peripheral circulation.


Failure of alpha-adrenergic stimulation by phenylephrine to enhance renin secretion in the isolated rat kidney. The intrarenal effect of the alpha-receptor agonist phenylephrine on renin secretion was examined in the isolated rat kidney. Infusion of phenylephrine in non-vasoconstrictor doses resulted in secretion rates which were not significantly different from control values. Similarly no change in renin secretion was found when phenylephrine was infused at a dose that clearly caused renal vasoconstriction and increased vascular resistance. These results do not support the hypothesis of a role for the alpha-receptor in the stimulation of renin secretion by adrenergic activity.


Effect of phosphodiesterase inhibitors and dBcAMP on the inotropic and relaxing actions of histamine in cardiac muscle. The influence of phosphodiesterase inhibitors, imidazole and dBcAMP on the action of histamine on peak tension, on rate of tension development and rate of relaxation of ventricular muscle of guinea pig's heart was investigated. It was found that theophylline plus dBcAMP enhanced the peak tension, (dF/dt)max and the rate of relaxation and increased the relaxing action of histamine. Imidazole, an activator of phosphodiesterase, also potentiated the action of histamine on peak tension, (dF/dt)max and rate of relaxation. Caffeine, a well-known inhibitor of phosphodiesterase, suppressed the relaxing action of histamine and epinephrine. These discrepant results could be explained by assuming the possibility that these drugs have effects other than those involving inhibition and activation of phosphodiesterase.


Elevation of adenosine 3',5'-monophosphate in the perfusate of rat kidney after addition of dopamine. Dopamine (10-4 M) and vasopressin (1 mU/ml) were found to increase the level of cyclic AMP in the perfusate of rat kidney. There were some differences in the mode of action of these two drugs. Firstly, the effect of dopamine, but not of vasopressin, was completely antagonized by spiroperidol. Secondly, the maximal response was attained within 1 min after dopamine perfusion, but 8 min after vasopressin perfusion. These results suggest that a specific dopamine receptor which acts to increase the concentration of cyclic AMP is located in the vascular tissue of rat kidney.


Stress-induced ACTH release after removal of the hypothalamus in rats with atrophied neural lobe. Twenty-four hours after isolation of the pituitary by surgical removal of the medial hypothalamus, i.e. in rats with pituitary island, E. coli endotoxin significantly increased the plasma corticosterone level. Atrophy of the neural lobe, due to pituitary stalk section performed one month prior to removal of the medial hypothalamus, did not prevent the increase of ACTH release by E. coli endotoxin. E. coli endotoxin-induced ACTH release in MBH-deprived animals does not appear to be a function of mechanisms operating only in the innervated lobe.


Site of action of 5-hydroxytryptophan and 5-hydroxytryptamine on the hypothalamo-pituitary-adrenal system in vitro. 5-hydroxytryptamine at a concentration of 0.04 microgram/ml was able to block the ACTH release caused by hypothalamic tissue (CRF) while it was ineffective when a hypothalamic extract containing CRF  was used. 5-hydroxytryptophan added to rat adrenal tissue caused a dose-dependent increase in corticosterone production. In a dose of 0.04 microgram/ml, 0.4 microgram/ml and 4.0 microgram/ml, 5-hydroxytryptophan was able to inhibit the ACTH release caused by hypothalamic tissue in vitro. However 0.04 microgram/ml was ineffective on the increase in ACTH secretion elicited by hypothalamic extract CRF. The data suggest that the inhibitory action of 5-hydroxytryptophan and 5-hydroxytryptamine is exerted at the hypothalamic level by inhibiting the release and/or synthesis of the corticotrophic releasing factor.


Persistence of Sendai virus in a mouse breeder colony and possibility to re-establish the virus free colonies. An epizootic of Sendai virus infection occurred in a mouse breeder colony with respiratory signs, mortality, retarded growth in young and prolonged gestation period in adults. Thereafter, the infection persisted in this colony without any clinical signs and with 90 to 100% antibody positivity in adults. Weanlings had maternal antibodies and no pneumonic lesions. Antibodies were hardly detected in 6-week-old mice with high incidence of red hepatization or congestion in their lungs, but mice over 8 weeks of age had antibody without the lesions. After isolating weanlings and pregnant mice with antibodies from the infected colony, the isolated weanlings and offspring from the isolated dams became negative for Sendai virus antibodies.


Biologic effects of orally administered deuterium oxide on rat liver. Rats were made to drink D2O mixed water (30: 70) for 6 weeks in order to study the biological effects of orally administered D2O on the liver. Heavy water administration results in gradual decrease in the body weight whereas the liver showed marginal increase in weight throughout the experimental period. Phosphatases and dehydrogenases were analyzed biochemically. Acid phosphatase, glucose-6-phosphatase and adenosine triphosphatase registered fall in contrast to alkaline phosphatase, SDH and LDH, all of which showed a definite increase. Lipids, nucleic acids and proteins, estimated biochemically, gradually decreased throughout the experimental period in response to D2O feeding.


Non-synaptic chemical neurotransmission. Images with apparently gemmulofugal polarity in the EPL of the olfactory bulb are the result of sectioning, along misleading planes, gemmulopetal synapses containing postsynaptic vesicles. Unless one accepts a bidirectional conduction for chemical synapses, the internal granule cells lack actual gemmulofugal synapses and the neurotransmitter contained on their vesicles must act at non-synaptic membranes.


In vitro effects of eledoisin on amylase secretion and cyclic AMP concentration of rat's parotids. Amylase secretion from in vitro rat parotids was strongly stimulated by eledoisin. In the same experimental conditions the glandular cAMP concentration was not affected by the peptide. The results might indicate that the effect of eledoisin on rat parotids is not mediated by cAMP.


[Role of alpha- and beta-adrenoreceptors in catecholamine regulation of the natriuretic function of the kidneys in rats]. In acute tests set up on rats subject to investigations was the significance of alpha- and beta-adrenoreceptors in the mechanism of the catecholamines influence on the natriuretic function of the rats' kidneys. Phentolamine did not obstruct the stimulating action of epinephrine on the tubular reabsorption of sodium, but perverted its inhibitory effect on the glomerular filtration. Inderal did not affect the glomerular action of epinephrine, but fully prevented its effect on reabsorption of sodium in the tubules of the kidney. When used in a dose failing to produce any significant changes in the systemic arterial pressure isopropylnorepinephrine increased glomerular filtration, but inhibited excretion of sodium, augmenting its tubular reabsorption. The resulting implication is that alpha-adrenoreceptors participate in the mechanism of the catecholamines regulation of the intrarenal hemodynamics, whereas beta-adrenoreceptors play the part of mediating their activating influence on the tubular transport of sodium.


[Effect of non-steroid anti-inflammatory agents on components of the kinin system in animals with experimental inflammation]. The influence exerted by a number of non-steroid antiphlogistic agents on the fermentative mechanism responsible for the formation and decomposition of kininines in an inflammation focus and on the permeability of vessels in a skin changed under the effect of exogenous bradykininine was studied. Bradykininine, chigamin, sodium salicylate and mephenaminate are shown to inhibit "in vitro" the kininogenic and intensify the kininase activity of the exudate in rats with turpentine-induced pleuritis, while indometacin affects only the activity of kininogenases. Butadion, chingamin and sodium mephenaminate merely reduce the reactivity of the skin with respect to exogenous bradykininine.


Virus-specific RNA in selected cell lines derived from RSV-induced tumour. The presence of virus-specific RNA in mammalian tumour cell lines of RSV aetiology was studied using molecular hybridization between the PR-RSV 3H-DNA probe synthetized by the endogenous RNA-directed DNA polymerase reaction and cellular RNA. It was found that both virogenic XC cells harbouring the full PR-RSV genome and cryptovirogenic RVP3 cells harbouring an incomplete PR-RSV genome contain virus-specific RNA. The degree of homology between such RNA and PR-RSV 3H-DNA probe reached 10--12% in XC cells and 28--30% in RVP3 cells. It was found that RVP3 cells contain larger amounts of virus-specific RNA than XC cells and that it is localized in the gradient zone corresponding to 20--25S.


Histopathology of carcinomas of the liver in mice ingesting heptachlor or heptachlor epoxide. C3H male and female mice, ingesting 10 ppm of the pesticides heptachlor or heptachlor epoxide in the diet, developed highly significant incidences of carcinomas of the liver. The carcinomas varied from well-differentiated to poorly differentiated and undifferentiated and were capable of invasion and metastasis.


Differential cytology of breast cancer.

Differential cytology of breast cancer. The aim of the study was to investigate the possibility of predicting the histologic type of different breast cancers by semi-quantitative screening of cytologic criteria. Cytologic material was obtained by the aspiration technique. The frequency of ten cytologic criteria was correlated to five histologic types of breast cancer. Hitherto, it has not been possible to define histologic types of breast carcinomas on the basis of cytologic criteria alone. While the occurrence of cellularity, intercellular cohesion, foam cells and necroses often varies from slide to slide within one particular carcinoma, other features such as nuclear diameter, polymorphism, anisonucleosis, apocrine carcinoma cells, and enlarged nucleoli are more or less uniformly distributed. The application of these cytologic criteria thus uniformly represented in a tumour may prove to be a more reliable method for classifying breast carcinomas than the use of histologic growth patterns which are often inhomogenous within each individual neoplasm.


A review of the current status of oil adjuvants in foot--and--mouth disease vaccines. Review of lipovaccines since 1916, including oil adjuvants (1935-1943) and the two Freund's adjuvants. The first oil-adjuvanted vaccines appeared in 1961. Criticism of the water-in-oil and oil-in-water vaccines and description of the mineral oil adjuvants which are available today. The authors set forth the advantages of oil adjuvants and secondary reactions which might occur; they are particularly interested in the foot-and-mouth disease vaccine intended for use in swine and express their regret that no method of standardization has yet been adopted for oil-adjuvanted vaccines which are promising but which necessitate still further study.


Efficiency testing of foot--and--mouth disease vaccines prepared from strain "C" with different adjuvants. Various oil adjuvants were tested for immunogenicity enhancing action in "C" type FMD monovaccine preparations. The following composition proved to be optimal in mouse tests: Bayl F 85%, Arlacel A 14% Tween 80 1%. As to the quantitative relations of antigen adjuvant, 40% oil to 60% antigen was the best among all proportions tested. Oil-adjuvanted vaccines failed to evoke an immune response in calves, but proved to be the preparation of choice for adult cattle. Oil containing and DEAE-dextran-containing vaccines had an equal efficiency in pigs; both conferred full protection on one in three pigs treated with 2.5-fold bovine doses. The number of vaccinated pigs was too small to permit definitive conclusions, but it seems clear that only oil dextran are efficient adjuvants of FMD vaccines for pigs, and that the antigen component of such vaccines should be increased considerably in order to obtain a greater potency.


[Use of anti-foot-and-mouth disease vaccine in oil adjuvant in young cattle]. A comparative study of the activity of foot-and-mouth disease vaccines, both aqueous- and oil-adjuvanted, was carried out on young bovines from vaccinated mothers. The immunity of these animals against foot-and-mouth disease was controlled after the first vaccination and booster by serology (neutralizing antibody count and immunology virulence testing). After having followed the kinetics of decrease in seroneutralizing antibodies of maternal origin the animals were vaccinated at the age of 11 weeks either with aqueous-adjuvanted or oil-adjuvanted vaccine. A booster dose was given two months later. Blood samples were taken at regular intervals and two virulence tests were made, one 21 days after the first vaccination and the other six months after the booster dose.


[Production, control and use in swine of anti-foot-and-mouth disease vaccine in oil adjuvant]. A certain number of parameters involved in the manufacture, control and use of an efficacious vaccine against foot-and-mouth disease have been studied. This report considers the problems raised by the standardization of the raw material constituent to the vaccine as well as the different types of innocuity and activity testing carried out on sows, young pigs and bacon pigs. Special attention is given to virulence testing methods, to the criteria allowing evaluation of the quality of the vaccines and to the transmission of maternal immunity of the sows to their piglets and its incidence on the vaccination schedule.


[Comparison of a bivalent anti-foot-and-mouth disease vaccine with oil adjuvant to a vaccine with DEAE-dextran adjuvant in swine]. The report compares the compatibility, efficacity and serology response of two bivalent anti-foot-and-mouth disease vaccines (OC). One is oil-adjuvanted and the other based on diethyl-amino-ethyl-dextran (DEAE-Dextran). After vaccination no general clinical or local reactions are normally observed. Tissular reactions at the inoculation site are more severe with the oil-adjuvanted vaccine than with the DEAE-Dextran vaccine. Meat inspection, about three months after injection of the vaccine, showed the lesions to be in regression and negligible. The control of activity has been performed by the virulence test. For the oil-adjuvanted vaccine, protection against O1-Lausanne, 5, 21 and 90 days after vaccination is 90%, 80-100% and 55% respectively. For the DEAE-Dextran vaccine, these levels of protection are 80% after 5 days and 20-70% after 21 days. The percentage of swine protected against C-Noville 35 days after vaccination is 100% for the oil-adjuvanted vaccine and 70% for the DEAE-Dextran vaccine. The evolution of the amount of serum antibodies was followed for three months by the method of seroneutralization on cell cultures.


Data on a FMD virus of type "A" isolated in Italy in 1975. A strain of FMD virus type A was isolated in a disease outbreak in Alessandria, Italy, in December 1975. Affected animals were young unvaccinated bovines and pigs of a very restricted region. No further spread has been observed. The strain was compared with some subtype strains of type A, including the Italian vaccinal strain. This Italy 1975 strain differs from all the other viruses included in the performed tests.


[Serological study of several strains of foot-and-mouth disease virus type "O" isolated in Europe between 1971 and 1975: application of the biomathematical system of classification]. Nine strains of foot-and-mouth disease virus type "O" received in our laboratories since 1971 have been studied serologically by Osler's quantitative method of complement fixation (50% hemolysis). The results, submitted to the biomathematical system of bidimensional classification, allow to conclude that at present in Europe there are two groups of foot-and-mouth disease strains of type "O"; one has reference to our vaccinal strain "O Lausanne 1965" and the other to "O Romania 1972" strain, which has certain points of similarity with subtype "O2". In addition, strain "O Hungary 1975" has been studied on bovines; the immunological tests confirm the serological test.


Examination of differences between foot-and-mouth disease virus strains using a radioimmunoassay techinque. A radioimmunoassay (RIA) technique was used to compare different samples of type SAT 2 foot-and-mouth disease (FMD) viruses. The reaction involved the measurement of the competition of heterologous virus with homologous virus for previously titrated homologous antiserum. The results showed that differences occurred between the viruses examined. Viruses could be grouped according to their ability to compete with the homologous virus, and statistically significant differences between virus 'groups' were observed. A comparison of the relationships between the viruses using RIA and complement fixation tests was made. The results did not always correlate. Certain homologous antigen/antibody combinations were more discriminating. The method might prove useful in the examination of a large number of field strain viruses using a well-characterized vaccine virus homologous system, particularly if the method is adapted to use antisera specific for the neutralizing antigen of the homologous test virus.


[Classification of strains of foot-and-mouth disease virus according to the relationship-dominance model for a better understanding of the concept of serologic and immunologic subtype]. The homologous and heterologous serological titers of a specific serum can be integrated in a bidimensional system which characterizes the criteria of relationship and dominace. This bidimensional classification specifies more precisely the serological properties of each strain compared with the others (taxonomic purpose) and allows eventual guidance in the choice of vaccinal strains (epizootiologic and prophylactic purposes).


A survey of FMD type 'O' strains from the Far East. A survey of 'O' strains submitted from Far Eastern countries has been conducted, comparing the strains with standard reference strains and with each other. The results indicate differences between these strains but they cannot be arranged in clearly distinct groups. There is, rather, a continuous variation with cross-relation between strains occurring more or less haphazardly. This makes the subgrouping of a large number of strains extremely difficult and supports the view expressed in other papers in this symposium that current concepts of subtyping should be revised to take into account multiple antigenic relationships.


Serological and immunological relationships among type A foot-and-mouth disease strains in South America. Field strains of foot-and-mouth disease (FMD) virus which differ immunogenically from vaccine strains appear from time to time and may present serious problems for FMD campaigns. In order that appropriate measures can be taken it is important to determine the degree of differences between the vaccine strain and the variant strain soon after its appearance. With the A strains studied it was observed that a serological relationship by complement fixation test of greater than or equal to 0.40 would indicate sufficient protection of the vaccine strain for the field strain. With a lower relationship, however, the vaccine strains usually do not fully protect against the field strain. The mouse protection test can be used to determine how serious the lack of protection may be. With this test routine it is possible to issue alerts for intensified epidemiological surveillance within 2-3 weeks after the field strain is received at the Center.


New media and their advantages in the production of suspended cells and foot--and--mouth disease virus. The problems related to the use of serum in cell culture are reviewed. The possibility of substituting the serum with peptones has already been shown. Different peptones have been tested: one of the best is a peptone obtained from meat and casein pepsin pancreatically digested. BHK 21/13 cells were cultivated in serum-free media for 35 passages. The 0.81 cultures without automatic pH control had a cycle length of 3 days; starting with concentrations of 0.4 X 10(6) cells/ml, concentrations higher than 3 X 10(6) cells/ml were often obtained. We did not obtain satisfactory results when the volume of cultivation was increased to more than 6 1, perhaps because of different requirements for agitation, pH, O2. It is also necessary to check whether the results obtained up to now are consistent with reference to the source of reagents and cell strain used. The absence of serum in the medium did not influence virus replication when infectivity and complement fixation titers were tested.


[Methods of testing of safety of the anti-foot-and-mouth disease vaccine]. Innocuity testing in bovines remains the reference method but other methods using mice and especially cell cultures may be employed. Different techniques (dialysis, neutralization, elution, etc.) allow the elimination of toxic constituents of the vaccine while respecting the eventual presence of small quantities of active virus. The sensitivity of the cell cultures is certainly equal if not superior to that of the tongue of bovines. Interference phenomena do not intervene and, furthermore, there is not reason to fear them solely on cell cultures. From the purely statistical standpoint, it is hardly possible to prove the innocuity of a vaccine but the credit which sould be accorded to the innocuity test can be reinforced by a good control of the inactivation process and, a posteriori, by the repetition of negative results.


Investigations on the suitability of mice for potency testing of FMD vaccines. Potency testing of FMD vaccines by a three-point method was elaborated. Mice myotropic FMD virus was used as challenge virus. With this test the immunogenicity of FMD vaccines based on BHK, calf kidney or swine kidney was examined. The results are reproducible and allow quantification of the antigenic value of FMD vaccines within statistical limits.


Studies on the correct quantitative relations of antigen components in mono-, bi- and trivalent foot-and-mouth disease vaccine preparations. Waldmann and frenkel type mono-, bi- and trivalent FMD vaccines, with different quantitative relations of the antigen components, were prepared and tested by the E, K and S index methods. Potency tests in adult mice showed that the antigen components of the bivalent vaccines are usually synergistic, but a 47-57% excess of antigen "O1" over the other two types is always required for a similar immunogenic action (E index value). For the same reason, the "O1" content of the trivalent vaccine should be as high as the total quantity of its other two components. Trivalent FMD vaccines in which the quantitative relations of the antigen components correspond with the proportions assessed as optimal in the mouse test prove also to be satisfactorily immunogenic in cattle in respect of all three components.


Determination of antigen content and effectiveness of foor-and-mouth disease vaccines. The following paragraphs are in reality the conclusion of a communication which was not presented at the symposium owing to Dr Czelleng's absence. Taking the place of his colleague, Dr Sólyom gave a brief account of the complement fixation test in the estimation of immunity against foot-and-mouth disease. The test conditions are improved by using a standardized complement; this method allows to meausre very precisely the amount of antigen used for the production of vaccine.


Attempts at potency testing of foot-and-mouth disease vaccines by evaluation of the complement fixing capacity of eluates. A method has been developed to evaluate the amount of antigen in foot-and-mouth disease vaccines by complement fixation after elution from aluminum hydroxide. The 12 S antigen is eliminated by treatment with Arcton 119 so that only the 140 S antigen may be determined. The concentration of 140 S antigen eluted from the vaccine is compared with the degree of protection conferred to cattle. 333 heads of cattle were vaccinated with 111 vaccines containing detectable amounts of 140 S antigen. Only one animal was not protected. In a repeated test of the questionable vaccine with 6 animals all were found to be protected. From 32 vaccines the amount of eluted 140 S antigen was compared with the PD50 in guinea pigs. Ten vaccines from 11 without detectable 140 S antigen had a PD50 value in guinea pigs lower than the limit indicating a satisfying potency for cattle.


[Routine physico-chemical testing of concentrated virulent and inactivated foot-and-mouth disease virus antigens]. The ponderal quantity of 140 S antigens and their peptide distribution are controlled in concentrated virulent and inactivated preparations proir to their being transformed into vaccines. A certain variability in the amount of 140 S particles and in the titer of peptide sensitive to trypsin (VP1) has been demonstrated in the virulent preparations. After treatment by inactivating agents (glycidaldehyde, binary ethyleneimine and formaldehyde) two types of alteration of the viral capsid are shown : one leads to the partial cleavage of VP1 (glycidaldehyde and binary ethyleneimine), the other leads to the detaching of an important quantity of VP1 from the capsid (formaldehyde). The degradation by formadehyde of the 140 S particles of type O and type C into 12 S particles is underlined, as well as the relatuve stability of type A and the relatuve lability of type C. The results are discussed in terms of immunogenicity.


[Immunogenic quality of foot-and-mouth disease virus]. Routine analysis of suspensions of foot-and-mouth disease virus and eluates of vaccine by the isopycnic method in analytical ultracentrifuge demonstrates the important heterogeneity of the viral population. This heterogeneity increases during inactivation of the virus by formol. In view of this dispersion of the physical characteristics of viral particles it may be asked (a) whether the immunogenic value is linked to the total quantity of particles which, we know, are very different from each other and of which we can determine only the partial or total parameters or (b) whether, on the contrary, the immunogenic value is linked to the activity of marginal particles, very small in number, and perhaps even quantitatively indistinguishable by the physical methods available. However, the hypothesis of particles possessing different properties but converging in the final result cannot, a priori, be excluded. Consequently it cannot be hoped that a simple relation between the concentration in weight of a viral suspension and its immunogenic property may be determined.


An evalutation of some methods of assay of foot-and-mouth disease antigen for vaccines. The relative merits of various in vitro assay systems for the measurement of the quality and quantity of foot-and-mouth disease virus vaccine antigen will be discussed. The assay systems include : viral infectivity, complement fixing activity, particle counts, radial diffusion titre and single radial haemolysis titre. The predictive value of the tests for the immunogenicity of the final vaccine product will be evaluated against the results of 50% Protective Dose titres determined in guinea pigs and cattle by antibody assay and by challenge.


[Testing the activity of anti-foot-and-mouth disease vaccine in cattle: quantitative methods of antigenic extinction]. Two methods of evaluating the activity of the foot-and-mouth disease vaccine are compared, leading to the evaluation of the number of protective doses 50% or potency. These methods differ in sensitivity, precision and equivalence potency-percentage of protection. For the same vaccine the value of the PD50 is always larger than that of Pb. We chose the Pb method using a diluent containing no adjuvant. Standards based on the evaluation of average potency have not been taken into consideration; only those standards based on the evaluation of minimum potency ensure respect of OIE's recommendation. This recommendation: 70% minimum potency with 0.95 probability is not an easier alternative, since the average protection observed corresponds in fact to at least 90% taking into account the biological variability of bovines.


[Combined vaccines including 1 or several anti-foot-and-mouth valences]. Combined vaccines are used  for many human and animal diseases. However, we have very few examples of combinations containing anti-foot-and-mouth valence which would allow easy immunization against differnet diseases without additional handling. Until now foot-and-mouth vaccination has been applied simultaneously with anti-brucellic vaccination (Baldoni, Renoux), andi-anthrax (Nobili) and vaccination against swine fever and Aujeszky's disease (Lysenko). During the last ten years we have particularly studied the association of anti-foot-and-mouth disease vaccines with anti-brucella and anti-rabies vaccines. We have shown the possiblity of combining these different antigens, either in classical aqueous vaccines in aluminum hydroxide, or in emulsified vaccines containing oily adjuvants. The anti-brucella valences may be agglutinogenic (H 38) or non-agglutinogenic (MacEwen 45/20). The combined vaccines "rabies + foot-and-mouth disease" and "brucellosis + foot-and-mouth disease" can be produced on an industrial scale. For many years millions of animals have been successfully vaccinated in France by means of a combined "rabies + foot-and-mouth disease" vaccine.


[Anti-foot-and-mouth vaccines combined with other bacterial or viral vaccines]. Medical and sanitary prevention of a growing number of major infectious diseases of bovines has led IFFA-Mérieux to investigate the association of vaccines and to define methods of utilization. Care has been taken to limit the number of interventions and at the same time to retain in the combined valences the efficacy they had when in a state of single constituent. The work which has been carried out for the last ten years, including the study of adjuvants as well as of incompatibility of associations, has permitted the improvement of formulas, reduced in volume (5 ml for a plurivalent dose), in aqueous excipient (aluminum hydroxide and saponin) or oily excipient (oil in water type). These formulas combine 3 foot-and-mouth disease valences with a rabies valence (fixed strain grown on cell culture) or with a brucellosis valence (strain B. abortus MacEwen 45/20 and B. melitensis H 38). The innocuity of these associations allows them to be used in sanitary compaigns; their efficacy is equal to that of monovalent vaccines. A logical vaccination calendar is proposed.


Simultaneous vaccination of cattle with foot-and-mouth disease and vesicular stomatitis live virus vaccines. Four- to six- month-old cattle were simultaneously vaccinated with foot-and-mouth disease (FMD) and vesicular stomatitis (VS) live virus vaccines by injection of FMD vaccine in one side of the body and VS vaccine in the other side. These cattle were again vaccinated with FMD and VS vaccines given simultaneously three and nine months after the first vaccination. The antibody levels of the vaccinated animals were studied by serum protection test in suckling mice and results were compared with those obtained when cattle are vaccinated either with FMD alone or with VS vaccine alone. It appears that an interference mechanism is responsible for the lack or the low levels of antibody formation observed in the animals simultaneoulsy vaccinated with the two vaccines.


[Preliminary study of the combination of anti-foot-and-mouth disease and anti-brucellosis vaccines]. The results obtained with Brucella B19 strain, used as adjuvant and stimulant in the preparation of hyperimmune anti-foot-and-mouth disease serums on guinea pigs, have led the authors to carry out a series of experiments with a combined anti-foot-and-mouth disease and anti-brucellosis vaccine (strain B 19 Buck and Cotton). The search for neutralizing antibodies for foot-and-mouth disease has been undertaken by different methods. In the search for antibrucellic antibodies reactions such as seroagglutination, fixation of the complement, etc. have been used. The results obtained with combined vaccine are identical with those obtained with each of the monovalent vaccines.


One year experience on the Lindholm B medium used in large--scale FMD virus production on BHK cells in suspension. The propagation of FMD virus on BHK 21 clone 13 suspension cells was based on the Lindholm B medium during the production period August 1975 to June 1976. The medium was used in connection with a production cycle including decreasing amounts of serum in the cell growth medium and inoculation of the cells while they were still in growth. This method of virus production, compared with the traditional method, is cheaper due to the lowered medium cost. The improved utilization of the fermenter capacity and the decreased consumption of serum have practical as well as economic aspects. The results obtained during the period indicate that virus production by this method is reliable and at least on the same level as by traditional methods.


Certain aspects of foot--and--mouth disease. Virus production in growing BHK suspended cell cultures. Production of FMDV in growing BHK suspended cell cultures offers several technical advantages: no medium change and no sedimentation of cells prior to infection are needed, thus saving time and medium. However, the final product should be as free of serum proteins as possible. For this purpose polyethylene-glycol-(PEG)-treated serum can be used for the stimulation of cell growth in combination with precipitation of the virus with PEG. With this method virus preparations are obtained in which practically no serum proteins can be estimated. Recently the use of serum-free medium has been reported for a BHK line which had been adapted to this medium. This also offers possibilities of virus cultivation in growing cells. Two of our BHK lines also grow in a cheap serum-free medium in which amino acids are replaced by a combination of lactalbumin hydrolysate and peptic peptone. With BHK cells adapted to this medium high cell yields are obtained. The use of PEG-treated serum will be compared with the use of the low cost serum-free medium with respect to FMDV production.


Studies on the susceptibility to foot--and--mouth disease virus of BHK cell cultures derived from various sources. Cultures of BHK monolayer and suspension cells obtained from a number of laboratories and a group of cloned sub-lines derived from suspension cells were examined for their susceptibility to three FMD virus strains. It was found that the various cultures were sensitive to the test virus strains to differing degrees. It was shown possible to obtain a clone with high susceptibility to Asia 1 Iran 1/73 virus from a parent culture which had a low susceptibility to that virus. The implication of these findings is discussed.


Reproducibility of yields of foot--and--mouth disease virus from BHK monolayer and suspension cells. The production of FMD virus from BHK 21 C13 monolayer and suspension cells was examined under standardized conditions and in different systems. The yields of virus from suspension cells did not significantly exceed the yield from monolayer cells, whereas the monolayer cell was capable of producing virus from some strains of FMD virus which would not grow in suspension cells. When different production systems were examined, the scale of operation did not significantly influence the yield of virus from suspension cells, while the plastic spiral film propagator produced significantly less virus than the other monolayer systems examined. The use of unit monolayer systems on the large scale is discussed.


[Characteristics of viruses multiplying in cell lines in suspension and their correspondent vaccines]. The characteristics of the viruses produced from cell lines developed in suspension and the vaccines for which they are used should be judged by methods which allow to ensure in all cases their innocuity and efficacity. Innocuity is essentially dependent upon the purity of the viral preparation which should contain a minimum of foreign proteins (due either to the cell or the culture medium) and certain antibiotics. Furthermore, the absence of oncogenic properties of the inactivated viral suspensions should be shown, in addition to their specific innocuity. Efficacity is linked with the concentration of viral particles and with their immunogenic properties after inactivation and incorporation of adjuvants of immunity. Methods of appreciation of the different parameters are proposed.


The use of suspension cultures for FMD vaccine production. Criteria for the evaluation of cells, virus and vaccine. A survey of problems connected with the cell suspension culture for the production of FMD vaccine is presented, in order to stimulate a discussion. Introductory remarks are made on the following topics: 1) suspension cell lines used for FMD vaccine production, 2) terminology, 3) criteria for the characterization of a cell culture, 4) evaluation of FMD virus produced according to the cell suspension technique and correlation existing between in vitro tests and antigenicity in domestic animals, 5) criteria which must be met by FMD vaccine produced from virus cultivated on cell suspension.


[Allergic reactions in cattle vaccinated against foot-and-mouth disease with vaccines prepared from viruses cultivated in BHK cells in roller bottles]. Tests on bovines vaccinated 3 or 4 times against foot-and-mouth disease with vaccines based on viruses cultivated on BKH cells, in roller bottles, have allowed to define the nature of some of the principal allergens: on the one hand residuary horse serum used to saturate the filters employed in preparing the virus intended for the manufacture of vaccines and, on the other hand, the substances liberated by the cytopathogenic action of the virus on the BHK cells. The horse serum is the most active and causes general and local reactions which are sometimes severe. It is therefore replaced by calf serum intended to reduce the importance of the problem for bovines. BHK antigens (BHK extracts and virus) produce only local reactions which are generally less severe.


Critical factors of the vacuum-oven technique which influence the estimation of moisture in veterinary biologics. When striving toward standardization of lyophilized products, it is often difficult to correlate biological characteristics and residual-moisture content because of the great variety of assay procedures being used to estimate residual moisture. Even when moisture assays are performed according to the same general assay procedure, there are often subtle differences that cause large variations in results. Critical factors of the vacuum-oven technique such as: homogeneity and number of samples, size of samples, moisture adsorption of dry weighing dishes, efficacy of disiccator jars, influence of ambient conditions, controlling time-sequence of the test, tolerances of the test, etc. are discussed and data presented to demonstrate how some of these factors can strongly influence the results of the assay. The validity of the assay and the subsequent judgement about the quality of the final product are greatly dependent upon how well these factors are controlled.


Plasma cyclic nucleotide levels in juvenile-onset diabetes. In patients with juvenile-onset diabetes, plasma concentrations of 3',5'-adenosine cyclic monophosphate (cAMP) were significantly lower than those of norman subjects [16 +/- 4 and 24 +/- 7 pmol per milliliter (p less than 0.025), respectively] as determined in this laboratory; whereas there were essentially no differences in plasma levels of 3',5'-guanosine cyclic monophosphate (cGMP). Because cAMP inhibits cell growth and cGMP stimulates it, these findings may represent an important factor in the atherosclerotic and obliterative angiopathies of diabetic individuals. We observed that cyclic nucleotide values were the same whether or not the subjects were receiving insulin. Those given insulin plus enough glucose to maintain hyperglycemia revealed modest elevations in cyclic nucleotide levels. Thus, the ratio of cAMP to cGMP, abnormally low in juvenile-onset diabetes, is relatively independent of short-term variations in plasma levels of either glucose of insulin.


Cyclic nucleotide phosphodiesterase. Insulin activation detected in adipose tissue by gel electrophoresis. In rat adipose tissue, insulin (100 muU./ml.) increases the activity of cyclic AMP (but not cyclic GMP) phosphodiesterase (PDE). Radioisotopic assay, autoradiography, and histochemical stains demonstrated that cyclic nucleotide PDE activity is associated with multiple bands of protein separable by polyacrylamide gel electrophoresis. The insulin activation of cyclic AMP PDE, however, was limited to a single band corresponding to the "low" Km enzyme specific for cyclic AMP; insulin had no effect on the "high" Km enzyme or on PDE bands with activity directed toward cyclic GMP. These data support the concept that insulin may modulate the activity of at least one of the cyclic AMP phosphodiesterases.


Virus induced diabetes and the immune system. I. Suggestion that appearance of diabetes depends on immune reactions. The participation of immune reactions in the EMC virus induced diabetes of the mouse was studied by immunosuppression with 500 R sublethal X-irradiation or 120 mg/kg Asta 5122, a cyclophosphamide derivative. Average glucose levels after X-irradiation and infection remained normal, while virus, infected, otherwise untreated mice, had significantly higher mean glucose levels, indicating that immune reactions are necessary for the development of virus induced diabetes. Immune suppression by the cyclophosphamide derivative led, in contrast, to a significantly increased mean glucose level and increased insulitis in comparison with the controls only infected. This indicates an important role of the cellular immune reaction, insulitis in the destruction of the islets.


Metabolic and membrane aspects of gastric H+ transport. Metabolic properties of dog gastric mucosa, investigated by substrate level measurements, implicate the Krebs cycle as the major energy-yielding metabolic pathway but are equivocal in terms of an ATP-based H+ secretion. Purification of gastric membranes by centrifugation and free flow electrophoresis results in a class of membrane vesicles enriched in K+-ATPase and capable of ATP-energized H+ uptake. Immunohistochemistry shows these to be derived from the parietal cell. H+ uptake by the vesicles is accompanied by K+ efflux, and movement of either ion is not potential-coupled. The simplest interpretation of these transport studies is uptake of KCl by the vesicles by passive diffusion followed by active H+:K+ exchange. In some respects, however, this model fails to conform to the expectations from in vitro studies. It may be, therefore, that another pump (i.e., redox) or another membrane component (i.e., Cl- conductance) is lost during purification. The properties of the vesicles are such, however, as to establish their role in H+ secretion by the stomach.


Effect of cholecystokinin variant (CCK39) on dispersed acinar cells from guinea pig pancreas. In dispersed acinar cells from guinea pig pancreas, cholecystokinin variants (CCK39 and CCK33) or carboxyl-terminal octapeptide of cholecystokinin (CCK-OP) caused significant increases in outflux of 45Ca, cyclic GMP, and release of amylase. In homogenates of acinar cells each peptide caused a significant increase in adenylate cyclase activity. For each function tested (1) CCK39 was equipotent with with CCK33, (2) CCK39 and CCK33 were 10 to 30 times less potent than CCK-OP, (3) the efficacies of CCK39, CCK33, and CCK-OP were the same, and (4) none of these effects were altered by concentrations of atropine sufficient to abolish the action of muscarinic cholinergic agents.


Distribution of intrinsic factor-vitamin B12 receptors in human intestine. A new rapid and accurate method for detecting vitamin B12-intrinsic factor (IF-B12) receptors was used to study the distribution of mucosal IF-B12 receptors in three human small intestines. IF-B12 receptors in human intestine are present in significant amounts in the entire distal three-fifths of intestine. Triokinase activity is absent in the distal two-fifths of intestine, and tryptophan oxygenase is present only in the distal one-sixth, or most terminal part of the ileum. These data suggest (1) biochemical functions in the ileum have different anatomical distributions, (2) IF-B12 receptors correspond most closely with the accepted anatomical definitions of the ileum, and (3) vitamin B12 malabsorption seen in resection or disease of terminal ileum may not be attributable solely to a deficiency of ileal receptors.