Parathyroid hormone- and prostaglandin E1-response in a selected population of bone cells after repeated subculture and storage at -80C. Five different cell populations, designated I to V, were isolated from minced newborn rat calveria by 5-sequential 20 min incubations with an enzyme mixture containing collagenase, elastase and DNAse. In primary culture, all five populations responded to parathyroid hormone (PTH) to a different degree, population IV giving the highest increase in cyclic-3'5'-adenosine monophosphate (cAMP) level. None of the five populations gave any response to calcitonin. Upon subsequent subcultures, all populations, except population IV, either lost or considerably decreased their response to PTH. Population IV gave a two to three-fold increase in cAMP concentration in response to PTH up to the third subculture. No morphological differences could be observed among the five populations. The third subculture of population IV cells that had been stored in 10% glycerol at -80C for four months was subsequently thawed and subcultured to the sixth subculture. These cells still responded to PTH with an increase in cAMP level. In a second experiment, 5 different cell populations designated I to V were isolated in a similar way by incubation with collagenase and DNAse. The maximum response to PTH was found in population 3. The preservation of the PTH-responsiveness of this population, after subculturing, freezing, storing in 10% glycerol at -80 C and subsequent subculturing, was likewise demonstrated. The hormone-responsiveness of cells from the sixth subculture of previously frozen and thawed population IV cells was further analyzed. These cells responded to PTH at a concentration of 0.1 U/ml to 5U/ml and to prostaglandin E1 (PGE1) at a concentration of 0.1 microng/ml to 10 microng/ml. The time course of action on population IV of PTH was found to be different from that of PGE1, suggesting a possible difference in the regulation of intracellular cAMP levels by these hormones.


Opposite effects of ACTH and glucocorticoids on adrenal DNA synthesis in vivo. Administration of ACTH to rapidly growing weanling rats results in an increase of DNA synthesis in adrenal and a decrease in liver. Dexamethasone administration decreases both adrenal and liver DNA synthesis. When both hormones were administered to the same animals, the liver DNA synthesis was similar to that observed with dexamethasone alone, but the DNA synthesis in adrenal was lower than that obtained with ACTH alone, yet higher than that observed with dexamethasone. The plasma levels of corticosterone were similar in animals treated with ACTH or with ACTH plus dexamethasone. Aminoglutethimide stimulated adrenal DNA synthesis, but less than ACTH. This substance overcame partially the inhibitory effects of dexamethasone on liver DNA synthesis but did not in adrenal. When both ACTH and aminoglutethimide were given simultaneously, adrenal DNA synthesis was higher than that observed with each substance alone. In all experiments in which adrenal cytosol DNA polymerase was studied, the activity varied in the same direction as DNA synthesis. These results indicate opposing effects of ACTH and glucocorticoids on adrenal DNA synthesis. The finding of a glucocorticoid effect on the adrenal is supported by the demonstration of a glucocorticoid specific binding protein in adrenal cytosol. Cycloheximide blocks the stimulatory action of ACTH on both steroidogenesis and DNA synthesis. Actinomycin D, as well as dexamethasone, blocks only the DNA synthesis-promoting action of ACTH. This latter result suggests some differences in the metabolic pathways by which ACTH controls steroidogenesis and growth in the adrenal cell.


Adrenal steroidogenic actions of cyclic nucleotide derivatives in the rat. Fifteen 3',5'-cyclic nucleotides and related compounds were studied for ability to mimic the steroidogenic action of ACTH in rats in which secretion of ACTH and corticosterone were suppressed by treatment with betamethasone, or by hypophysectomy. Subcutaneous administration of 8-chloro-cAMP, at doses of 40 mg/kg or greater, elicited the secretion of corticosterone to normal plasma levels in both betamethasone-treated and hypophysectomized animals. Cyclic AMP, dbcAMP, 8-methylthio-cAMP, 8-hydroxy-cAMP and the 6-chloro-8-aminopurine cyclic ribotide analog of cAMP also displayed steroidogenic activity in the betamethasone-treated rat; cGMP, 8-bromo-cGMP and 8-benzylthio-cGMP were inactive. Each of the steroidogenic derivatives of cAMP also displayed ability to activate steroidogenesis in isolated rat adrenal cells. These experiments demonstrate that various derivatives of cAMP mimic the adrenal steroidogenic action of ACTH, in vivo. Structure-activity comparisons support a steroidogenic mechanism involving direct activation by the nucleotides of cAMP-dependent protein kinase of the adrenal cortex.


Interaction of cyclic AMP and alloxan on insulin secretion in isolated rat islets perifused in vitro. The interaction of alloxan and cyclic AMP on glucose-induced insulin secretion was studied with the use of isolated rat islet perifusion. Simultaneous perifusion with cyclic AMP and alloxan did not protect the islets against the effect of alloxan. However, addition of dibutyryl cyclic AMP to the alloxan solution produced 40% protection of glucose-induced insulin secretion. Partial protection was obtained with either theophylline (41%) or caffeine alone (54%), and the addition of 1 mg/ml glucose to the theophylline or caffeine solution provided greater than 68% protection. The levels of islet tissue cyclic AMP were more than 2.1 times that of islets not protected against the alloxan effect, when partial or nearly complete reversal of the inhibitory action of alloxan on glucose-induced insulin secretion was effected by theophylline or caffeine. These results suggest that cyclic AMP affords partial protection against the effect of alloxan on glucose-induced insulin secretion.


Evolution of prolactin receptors in rabbit mammary gland during pregnancy and lactation. The numbers and affinity of prolactin receptors in the rabbit mammary gland were determined during pregnancy and early lactation under conditions in which the endogenous lactogenic hormone was depleted by means of the compound CB 154. In untreated rabbits the number of prolactin binding sites per mg protein increased from 25 +/- 3 (SE) fmol at Day 14 of gestation to 54.8 +/- 5.8 fmol/mg at Day 22, after which binding declined to 14.2 +/- 8.5 fmol/mg, then increased in late pregnancy and during lactation to 110.5 +/- 11.5 fmol/mg at Day 28. In animals treated with CB 154, binding was always higher than in non-treated animals, with a peak during pregnancy of 149 +/- 24 fmol/mg at Day 22. After declining in late pregnancy, the number of receptors was highest at Day 6 of lactation (257.4 +/- 34.6 fmol/mg). There is an almost linear increase in the weight of the mammary gland from Day 14 of pregnancy until Day 6 of lactation and this increase is unaffected (except at Day 6 of lactation) by CB 154 treatment. It was observed that prolactin receptors remain at a relatively low and constant level while mammary development (mammogenesis) takes place. The onset of milk secretion parallels a striking increase (greater than 500%) in the number of prolactin receptors (expressed per total mammary gland or per cell) after parturition has occurred. These results are discussed with emphasis on the mechanisms through which hormonal balances during pregnancy and lactation may modulate the amount of receptors per cell, hence its sensitivity to lactogenic hormones.


Evidence for preferential effects of parathyroid hormone, calcitonin and adenosine on bone and periosteum. In order to explore the distribution of hormone-responsive cells in skeletal tissues, we have examined the effects of synthetic bovine parathyroid hormone N-terminal peptide (bPTH 1-34) and salmon calcitonin (sCT) on cyclic AMP levels in periosteum-free rat calvaria, segments of periosteum, and in isolated cells dispersed from each tissue by collagenase digestion. Synthetic bovine PTH increased cyclic AMP levels to a greater degree in calvaria and in isolated bone cells than in the periosteal segments and cells, whereas sCT was more effective in the periosteal than in the bone systems. Primary cultures prepared from bone and periosteal cell populations exhibited progressive increases in their responsiveness to bPTH (1-34) and progressive decreases in responsiveness to sCT. After six days in the culture, bone cells failed to respond to sCT, and sCT did not modify their response simultaneously added bPTH (1-34). Six-day periosteal cell cultures exhibited residual sCT responsivity and an additive response upon simultaneous exposure to high concentrations of bPTH (1-34) and sCT suggesting separate sites of hormone action. Adenosine, a known stimulator of bone cell adenylyl cyclase, caused a greater increase in periosteal cell than in bone cell cyclic AMP. bPTH (1-34)-responsive cells which enrich periosteum-free bone may be osteoblasts, in view of their histological prominence in this tissue and in the bone cell isolates. Periosteal cells which responded to sCT and to adenosine preferentially are unidentified. Although periosteal segments contained numerous fibroblast-like cells, skin fibroblasts cultured from the same fetuses were sCT-insensitive. Growth in primary culture appears to alter the number of hormone-responsive cells or responsiveness of existing cells to each hormone, or both.


Enhancement of [3H-methyl]thymidine incorporation and replication of rat chondrocytes grown in tissue culture by plasma, tissue extracts and vasopressin. A pituitary mitogenic peptide, which stimulates cellular replication of a variety of cells maintained in tissue culture, has been identified by other investigators. To study this mitogenic substance, we developed an assay to measure mitogenic substances utilizing fetal rat chondrocytes grown in monolayer culture. Mitogenic activity of added test substances was determined by [3H-methyl]thymidine incorporation into trichloroacetic acid insoluble cell products and increase in total cell number after 24 h exposure. Extracts of whole pituitary glands were more potent in stimulating these cellular indices than either those of liver or muscle, confirming that the chondrocytes are sensitive to the described mitogen. Identically prepared extracts of either anterior or posterior pituitary lobes were mitogenic indicating the presence of two or more mitogenic substances in crude pituitary extracts. Synthetic lysine vasopressin and a beef pitressin concentrate stimulated thymidine incorporation into chondrocytes in the absence of calf serum and this effect was additive to that of calf serum, suggesting that the mitogenic substance of posterior pituitary extracts was vasopressin. The maximum effective dose of vasopressin leading to an increase in either thymidine incorporation or total cell number was between 100 to 500 pg/ml, and as little as 50 pg/ml of hormone elicited an increase in total cell number. The mitogenic effect of both vasopressin and calf serum on chondrocytes was partially inhibited by 1 X 10(-4)M N, O'dibutryl cyclic adenosine 3',5' monophosphate suggesting that cell division of chrondrocytes may be under tonic control by the andenylyl cyclase system. We conclude that vasopressin is a potent mitogen for chondrocytes maintained in tissue culture and its presence must be rigorously excluded in evaluating mitogenic activity of pituitary or serum concentrates.


Growth rate and secretion of pituitary hormones in relation to age and chronic treatment with thyrotropin-releasing hormone in prepubertal dairy heifers. Holstein heifers were treated with synthetic thyrotropin-releasing hormone (TRH) or saline twice daily from one week through 6 mo of age. Plasma concentrations of prolactin (PRL) and thyrotropin (TSH) were elevated (P less than .01) within 30 min after the first TRH injection (1 week of age). At 1 and 3 mo of treatment, PRL and TSH increased in response to TRH, although the TSH response was reduced (P less than .01) as compared to the first day of treatment. Although plasma growth hormone (GH) appeared to be elevated following the first TRH injection, this effect was not statistically significant (P less than .05), nor was it significantly influenced by treatment following subsequent TRH injections. None of the 3 hormones, PRL, TSH or GH, was elevated following the final TRH injection at 6 mo of age. In contrast, plasma concentrations of PRL and TSH were increased in a control heifer injected with TRH at 6 mo. These data indicate that hormonal responsiveness to TRH stimulation decreases with continued twice daily treatment at doses of TRH used in the present studies. Examination of weight gains indicated that chronic treatment with TRH was associated with increased growth rate through 6 mo of age (10.6% increased average daily gains P less than .10), which was exhibited in a steeper slope (P less than .05) of the growth curve in the TRH group. Feed intake was slightly greater in TRH heifers, although feed efficiency (kg feed/kg gain) was not different between the two groups. Plasma concentrations of PRL increased (P less than .01) with age (r = +0.938) in control heifers while plasma TSH and GH were not significantly related to age. This observation establishes a positive correlative relationship between PRL secretion and the approach of puberty in the dairly heirfer. It was also noted that elevation of PRL secretion by TRH treatment was associated with significant advancement of age at first observed estrus (9.4 vs. - 10.5 mo) suggesting that a functional relationship between PRL secretion and puberty may exist in dairy heifers.


Identification of "angiotensin immunoreactive material" in rat kidney. The reported presence of large quantities of a high molecular weight form of angiotensin I and II in renin granules from rat kidney cortex was investigated. Subcellular fractionation by differential centrifugation and isopycnic gradient centrifugation confirmed the presence of 'angiotensin I immunoreactive material,' but the distribution resembled that of lysosomes rather than renin granules, mitochondria or protein. The material did not possess pressor properties, was stable to incubation at 37 C and was precipitated by ethanol. Incubation of subcellular fractions with peptidase inhibitors known to inhibit the activity of renal angiotensinases (EDTA, diisopropyl phosphorofluoridate and 2,3-dimercaptopropanol) decreased the level of 'angiotensin I immunoreactive material' in kidney fractions treated by radioimmunoassay. By paper chromatography it was apparent that subcellular fractions were capable of degrading 125I-labelled angiotensin I used as tracer in the radioimmunoassay. The degree of degradation followed the distribution of lysosomes among the fractions and was decreased by the angiotensinase inhibitors. The apparent molecular weight of the major portion of angiotensinase activity persisting despite the presence of angiotensinase inhibitors was 75,000, a value similar to that of 'angiotensin immunoreactive material'inkidney fractions treated with non-ionic detergent. On the basis of the present findings it is suggested that 'angiotensin immunoreactive material' may be largely artifactual, beingcreated by the hydrolysis of tracer by angiotensinase enzymes during radioimmunoassay to form fragments that are no longer capable of binding to the specific antibody. This would produced results that would appear the same as those produced had genuine angiotensin immunoreactivity indeed been present in the samples. Such an effect could, in principle, occur in any competitive protein binding assay and should be tested.


Effects of biogenic amines on the formation of adenosine 3', 5'-monophosphate in human thyroid slices. The effects of various concentrations of biogenic amines on the formation of adenosine-3', 5'-monophosphate (cyclic AMP) and their interactions with other thyroid stimulators were investigated in human thyroid slices from normal and Graves' disease. Most of biogenic amines were found to have the stimulatory effects to some extent. Among the biogenic amines tested, histamine was the most potent thyroid stimulator, norepinephrine and serotonin, the intermediate in terms of cyclic AMP formation. The effect of histamine was almost as potent as TSH in thyroid slices from Graves' disease. This stimulatory effect of histamine was blocked by metiamide, a histamine H2-receptor antagonist, but not by chlorpheniramine, a histamine H1-receptor antagonist. The effect of norepinephrine was completely inhibitied by propranolol, but not by phentolamine. Polyphloretin phosphate did not inhibit norepinephrine- or histamine-induced cyclic AMP formation, while it significantly depressed cyclic AMP formation induced by prostaglandin E2. The maximal effect of histamine was additive to that of TSH. It is suggested that biogenic amines, histamine and norepinephrine, in particular, have the thyroid receptors different from that of TSH or prostaglandin E2 and could play an important role in thyroid physiology.


Effects of concanavalin A and neuraminidase on cyclic AMP levels and 14C-1-glucose oxidation in dog thyroid slices. Treatment with concanavalin A at 100 micron/ml or higher concentrations significantly increased 14C-1-glucose oxidation in dog thyroid slices as reported in other tissues. This treatment exerted no effect on tissue cyclic AMP levels. Neuraminidase at the same concentrations also had similar effects on these parameters. Neither concanavalin A nor neuraminidase at the concentrations up to 100 microng/ml had the TSH effect on both tissue cyclic AMP and 14C-1-glucose oxidation. These results indicate that modification of carbohydrate moieties of glycoproteins on the cell surface may cause an increase in glucose metabolism without any critical effect on cyclic AMP system and in the process of TSH response.


Hypothalamic pituitary adrenal function in patients with anorexia nervosa. Hypothalamic pituitary adrenal function was studied in 14 patients with anorexia nervosa. Although basal plasma cortisol levels in the morning were elevated in most cases, basal plasma ACTH levels were not suppressed. Oral administration of 1 mg dexamethasone 10 hr before blood sampling failed to suppress plasma ACTH and cortisol levels in most patients with anorexia nervosa. Apparent biological half-life of exogenous cortisol was prolonged in all 4 patients with anorexia nervosa tested. The cortisol response to insulin-induced hypoglycemia and exogenous ACTH appeared to be blunted in these patients. It is concluded that anorexia nervosa has dysfunctions of hypothalamic pituitary adrenal axis, especially an abnormal feedback mechanism on ACTH secretion.


Binding of mouse choriomammotropin to prolactin receptors in the mouse mammary gland. The interaction between mouse choriomammotropin and mouse mammary glands was examined by radioreceptor assays using ovine prolactin (NIH-P-S9) iodinated by lactoperoxidase as a tracer. Mouse pituitary extracts and placental extracts were subjected to 10% acrylamide gel electrophoresis. Gels were cut into 2-mm segments after electrophoresis, and stored in 1 ml 0.05 M phosphate buffer (pH 7.4) containing 0.05 M NaCl overnight for elution. Lactating mammary tissues from D strain mice were incubated for 120 min in 1 ml Medium 199 containing 6 ng of 125I-prolactin and 0.1 ml of each eluate. Pituitary extracts displaced 125I-prolactin only at the position which coincides with the prolactin band. Displacement was observed at two positions of the gel when placental extracts were used. Relative mobilities (Rm) were 0.21 and 0.71, respectively. The slowly migrating component of choriomammotropin inhibited the binding of 125-I-prolactin more strongly that the rapidly migrating one. Neither of them was identified as a distinct band in stained gels. The molecular weight of ovine prolactin, mouse pituitary prolactin and the slowly migrating component of mouse choriomammotropin was estimated to be 23000 using disc electrophoresis but the ion charges of these hormones were considerably different.


Plasma human calcitonin (hCT) levels in normal and pathologic conditions, and their responses to short calcium or tetragastrin infusion. Plasma hCT levels were less than 50 pg/ml in 50 normal subjects. In 16 patients with medullary carcinoma of the thyroid (MCT), plasma hCT levels were distinctively elevated and they fell significantly after total thyroidectomy, but in 11 of them plasma levels were still high, indicating the presence of metastases. In 74 patients with the other types of malignancy, plasma hCT levels were found to be high in 9 cases (3 oat cell carcinoma of the lung, 4 malignant carcinoids, one malignant pheochromocytoma and one acute myelocytic leukemia). Except for the leukemic case, all these tumors were derived from neural crest. In 12 patients with primary hyperparathyroidism, plasma hCT levels were less than 20 pg/ml. In 13 hypoparathyroid patients, two with pseudohypoparathyroidism and one with pseudoidiopathic hypoparathyroidism, plasma hCT levels were slightly elevated. Some patients with uremia had elevated plasma hCT levels, but there was no relation between plasma levels of hCT and those of PTH, urea nitrogen or creatinine. In response to Ca (4.5 mg/kg/10 min) or tetragastrin (4 mug/kg/5 min) infusion, a marked increase in plasma hCT was observed in all patients with MCT, but not in normal subjects. In 5 hypoparathyroid patients, a significant increase to both stimuli was also observed in all cases. Two patients with pseudopseudohypoparathyroidism responded to the Ca load. These results indicate that the determination of plasma hCT levels especially after a short Ca or tetragastrin infusion is important to study various pathological conditions.


Use of affinity elution chromatography for the microassay of adenosine 3':5'-monophosphate. Using the principle of affinity elution chromatography, a microassay has been developed for the determination of picomolar and even smaller quantities of adenosine 3'.5' -monophosphate (cyclic AMP) in biological samples. In principle, cyclic AMP along with the other anions is adsorbed onto an anion exchange cellulose and subsequently, the cyclic nucleotide is specifically eluted with cyclic-AMP -dependent protein kinase. In the actual assay cyclic AMP is determined by competitive displacement of radiolabelled cyclic AMP by non-labelled cyclic nucleotide. The described procedure results in a linear dependence for the displacement of the radioactive nucleotide on cyclic AMP concentration and obviates the usual procedure of sample enrichment by lyophilization. The degree of interference of different nucleotides including cyclic GMP was found to be minimal and in this respect the use affinity elution chromatography was comparable to radioimmunoassay.


Calf-ovary protein kinases dependent on adenosine 3':5' -monophosphate. Analysis by electrophoresis and electro-focusing on polyacrylamide get. High resolving power and quantitative application polyacrylamide-gel electrophopresis at various pore sizes and electrofocusing provide resolution of a calf-ovarian protein-kinase system at an increased level of magnification, as well as optimal preparative routes. Three protein kinases dependent on adenosine 3':5' -monophosphate are distinguished by polyacrylamide gel electrophoresis in calf ovarian cytosol. These enzymes which are observed in the pH range 7.5--10.2, appear to be aggregates of a commonsubmit or monomer. The three kinases are, by the criteria of polyacylamide gel electrophoresis, distinct from three adenosine-3':5' -monophosphate-binding proteins found in the calf ovarian system. Analysis by electrofocusing on polyacrylamide gel shows that conventionally purified preparations of the major kinase of cytosol contain an overwhelming majority of contaminant proteins.


Nucleotide sequence of a region in 23-S RNA adjacent to peptidyl transferase catalytic center of Escherichia coli ribosomes. N-Iodoacetylphenylalanyl-tRNAPhe was used as an affinity label to localize the RNA components intimately involved in the catalytic center of Escherichia coli ribosomes. This analogue could alkylate the specific region of 23-S RNA that waslocated within 2000 nucleotides from the 3' terminus of the molecule. Sequence analysis revealed that the alkylation by the active substrate (N-iodoacetylphenylalanyl-tRNAPhe) was directed to 5'-terminal adenosine residue of a heptanucleotide, A-U-U-U-U-A-Gp, which seemed to be derived from a heptadecanucleotide, U-U-A-A-A-A-A-C-A-C-A-U-U-U-U-A-Gp, in the original 23-S RNA. The significance of the unique sequence in the ribosomal functions is discussed.


Prolyl 3-hydroxylase: partial characterization of the enzyme from rat kidney cortex. The formation of 3-hydroxyproline was studied with crude rat kidney cortex extract as a source of enzyme and chick embryo tendon protocollagen and procollagen or cartilage protocollagen as a substrate. Synthesis of 3-hydroxyproline was observed with all these substrates and the formation of 3-hydroxyproline ranged up to seven residues per pro-alpha-chain. The highest rate of 3-hydroxylation took place at 20 degrees C and the reaction required Fe2+, O2,2-oxoglutarate and ascorbate. The formation of 3-hydroxyproline was affected by chain length and the conformation of the substrate, in that longer polypeptide chains proved better substrates, while the native triple-helical conformation of protocollagen or procollagen completely prevented the reaction. Formation of 3-hydroxyproline with tendon procollagen as a substrate was not inhibited by antiserum to prolyl 4-hydroxylase or by poly(L-proline) when these substances were used in concentrations which clearly inhibited 4-hydroxyproline formation with tendon protocollagen as a substrate. Furthermore, pure prolyl 4-hydroxylase did not synthesize any 3-hydroxyproline under conditions in which the crude rat kidney cortex enzyme would readily do so. The data thus strongly suggest that prolyl 3-hydroxylase and prolyl 4-hydroxylase are separate enzymes.


Physicochemical properties of T4 polynucleotide kinase. Some physicochemical properties of T4 polynucleotide kinase (EC2.7.1.78) have been studied. The enzyme is an oligomer of one polypeptide chain. The molecular weight of the monomer is 33000, as determined from the amino acid analysis. Phenylalanine is the N-terminal amino acid. Each monomer contains two --SH groups, one exposed and one more buried. Circular dichroic spectra suggest a high content of alpha-helical structure, 45--55%. Excitation at 280 nm gave a strong emission fluorescence spectrum with a maximum centering at 340 nm. Sedimentation studies suggested the enzymically active form to be a tetramer. High ionic strength (0.1 M KC1), spermine, and the substrates ATP and thymidine 3'-monophosphate were found to be essential factors in order to stabilize the protein in an oligomeric structure. The association constants for ATP, thymidine 3'-monphosphate, and P1 were determined fluorimetrically to be 7.9 x 105, 4.8 x 105, and 7.2 x 10(2) M-1 respectively.


Thyrotropin stimulation of the phosphorylation of serine in the N-terminal of thyroid H1 histones. The analysis of thyroid nuclear proteins by polyacrylamide gel electrophoresis has demonstrated that thyrotropin and dibutyryl adenosine 3':5'-monophosphate stimulate specifically the phosphorylation of H1 histones in an intact cell system. This effect does not require new protein synthesis and implicates the phosphorylation of secrine residue(s) situated in the N-terminal part of H1 histones.


Effect of neuraminidase on the chromatographic behaviour of eleven acid hydrolases from human liver and plasma. 1. The elution profiles of eleven acid hydrolases from human liver and plasma were directly compared using a system whereby a single salt gradient was simultaneously applied to two DEAE-cellulose chromatographic columns. 2. Plasma alpha-L-fucosidase, alpha-mannosidase, alpha-galactosidase and alpha-glucosidase isoenzymes were eluted at higher salt concentrations than the corresponding liver isoenzymes whereasbeta-N-acetylglucosaminidase, beta-galactosidase, beta-glucosidase, exo-1,4-beta-xylosidase and alpha-L-arabinofuranosidase isoenzymes were eluted at lower salt concentrations. The elution profiles of beta-glucuronidase and acid phosphatase weremore complex. 3. After incubation with neuraminidase most plasma hydrolases were eluted at lower salt concentrations, however the elution patterns of beta-glucosidase, beta-xylosidase and acid phosphatase were not altered. 4. Preincubation with neuraminidase had no effect on the elution profiles of six liver hydrolases whereas the major isoenzymes of alpha-mannosidase, beta-galactosidase and alpha-L-arabinofuranosidase were eluted at markedly lower salt concentrations. Liver alpha-fucosidase and alpha-galactosidase were eluted at slightly lower salt concentrations afterincubation with neuraminidase. 5. The results are discussed in relation to thepathogenesis of Mucolipidosis II (I-cell disease), and the synthesis and packaging of lysosomal enzymes.


The role of leukocyte subpopulations in the indirect leukocyte adherence inhibition assay in the mammary tumor virus system. A modification of the leukocyte adherence inhibition test for the detection of cellular immunologic reactivity of mice to the mammary tumor virus (MTV) has been described. It involves the transfer of the leukocyte adherence inhibition factor (LAIF) produced by spleen cells from immunized animals when cultured with antigen to indicator cells, for which peritoneal exudate cells from normal mice are used. The method proves to be sensitive and highly reproducible. By crude separation of leukocyte subpopulations it became established that for the production of LAIF the following sequence is needed: 1. incubation of adherent cells with MTV;2. transfer of a soluble factor SF1 produced by the adherent cells to T cells; 3. transfer of another soluble factor SF2 released by T cells to the adherent cells.


An involvement of alpha-adrenergic stimulation in exercise-induced hypoglycemia. Hypoglycemia developed in fasted rats during forced swimming. This hypoglycemia was mostly abolished by phentolamine, an alpha-adrenolytic agent, or by hexamethonium; was potentiated by propranolol, a beta-adrenolytic agent, of by 5-methoxyindole-2-carboxylic acid, a gluconeogenic inhibitor; and was not affected by anti-insulin serum. The turnover rate of blood glucose estimated from the decay curve of blood [14C]glucose increased significantly during exercise. There was a slight but significantly increase during exercise in the transfer of 3-O-methyl-[14C]glucose into muscle and adipose tissues, when it was corrected for by [3H]mannitol transfer to the same tissues. It is concluded that the alpha-receptor-mediated action of endogenous catecholamine stimulates peripheral glucose utilization leading to hypoglycemia during exercise. The action of alpha- and beta-adrenergic mechanisms, directly on peripheral tissues or via insulin secretion, in fine regulation of blood glucose level is discussed.


Positive dromotropic effect of dibutyryl cyclic adenosine 3',5'-monophosphate on the atrioventricular node. The effect of atrioventricular (A-V) conduction of N6-2'-0-dibutyryl cyclic 3',5'-adenosine monophosphate (dibutyryl cyclic AMP) was investigated in comparison with those of norepinephrine, cyclic 3',5'-adenosine monophosphate (cyclic AMP), adenosine-5'-monophosphate (5'-AMP), and adenosine by the use of the isolated, blood-perfused A-V node preparation of the dog. Single injections of dibutyryl cyclic AMP (3-300 micronmol) and norepinephrine (0.1-1 nmol) into the posterior septal artery of the preparation (the upper part of the A-V node is mainly perfused through this artery) produced a dose-dependent decrease in the A-V conduction time. The time to the peak effect and the duration of the effect of dibutyryl cyclic AMP were much longer than with norepinephrine. The positive dromotropic effect of dibutyryl cyclic AMP was resistant to the beta-adrenoceptor blocking action of propranolol. Unlike dibutyryl cyclic AMP, cyclic AMP (above 30 nmol), 5'-AMP and adenosine (above 1 nmol) injected into the posterior septal artery prolonged the A-V conduction time in a dose-dependent manner. The results indicate that dibutyryl cyclic AMP has a mode of action on A-V nodal cells which differs distinctly from that of either norepinephrine or cyclic AMP, 5'-AMP, and adenosine.


Ethanol-induced changes in gastric mucosal content of cyclic AMP and ATP in the rat. The stomach of urethane-anaesthetised rats was perfused with 10% (v/v) ethanol. At 40 min, the secretion of acid was strongly inhibited and the contents of cyclic AMP and ATP were lowered in the superficial mucosa, but not in whole gastric mucosa. After discontinuation of the ethanol perfusion, the rate of gastric acid output as well as the cyclic AMP and ATP levels recovered. Most of the acid-secreting parietal cells were found in the superficial mucosa where the changes of cyclic AMP and ATP took place. The results suggest that lowering of the mucosal contents of cyclic AMP and ATP may be involved in the ethanol-induced inhibition of the gastric acid output.


Involvement of central alpha-adrenoceptors in the hypotensive and bradycardic effects of (--)-delta 9-trans-tetrahydrocannabinol. I.v. administration of (--)-delta9-trans-tetrahydrocannabinol (delta 9-THC) to chloralose-anesthetized cats produced a decrease in blood pressure and heart rate. Studies conducted to investigate the mechanism of these changes revealed that the hypotensive and bradycardic effects of delta 9-THC (0.1 mg/kg, i.v.) could be significantly antagonized following intraventricular (i.v.t.) perfusion with alpha-adrenergic receptor blocking agent phentolamine, while a larger dose of delta 9-THC (0.25 mg/kg, i.v.) still produced significant hypotension and bradycardia. I.v.t. perfusion of phentolamine (100 mug/min for 30 min) caused a fall in blood pressure and heart rate which were reversible and produced blockade of central alpha-adrenergic receptors. These results provide evidence for the involvement of central alpha-adrenergic receptors in the hypotensive and bradycardic actions of delta 9-THC and indicate that other receptor mechanisms may also be involved in mediating these responses observed following administration of delta 9-THC.


[Virus-induced autoimmunity phenomena in experimental animals (author's transl)]. It may happen that the virus infection causes changes of the immunogenicity of the host cells that may theoretically result in disturbances of the "self-recognition" of the organism. In this paper we report on the immunization of rabbits by autologous testes cells infected with herpes-virus hominis (HVH) types 1 and 2. In 20 rabbits the excision of one testis was carried out. The testes cells were cultivated in L 15-medium (Leibowitz) and lactalbumine hydrolysate supplemented with 12% autologous serum. The virus strains herpesvirus hominis type 1 (Kupka) and type 2 (US) were passaged three times on autologous testes cells, in order to remove host-foreign proteins out of the envelope. Thereafter, the monolayer cultures grown in Demeter flasks were infected with 15 ml virus suspension containing 10(4) to 10(5) TCID50. For the immunization three antigen preparations were applied: a) Supernatant of virus-infected cell cultures, b) Pellets of the virus-infected cell cultures, c) Pellets of the non-infected cell cultures. Two times at an interval of 3 weeks the animals were immunized by intramuscular and intraperitoneal injections with prepared antigens. Altogether, 20 animals were immunized: 15 rabbits with virus antigen, 5 control animals with non-infected autologous cells. After 28 days, 13 rabbits were bled, the second testis, liver, spleen, and kidneys were taken out. From 7 rabbits, only the second testis was taken out. These animals got a further injection on the 40th day post infectionem, and on the 50th day, they were bled. From the tissue samples, frozen sections were cut, fixed with acetone, and treated with fluorescein isothiocyanate-labelled antirabbit globulin, in order to elicite the tissue-bound antibodies. For histological studies, paraffin sections were stained with hematoxylin and eosin, and with azan, besides the PAS reaction was carried out. The determination of humoral antibodies was done by means of indirect-immunofluorescence antibody technique (IFAT). As antigen, autologous cells grown on slides were used; a part of them was infected with HVH types 1 or 2, while another part was not infected. After immunization with autologous testes cells which were infected with HVH types 1 or 2, histologically homogeneous eosinophilic PAS-positive substances were detected in 14 out of 15 rabbits, mainly in the interstitium of the testes (fig. 2). These substances corresponded to globulin deposits that could be demonstrated by the reaction of labelled antirabbit globulin (fig. 1). The testes sections of thoses 5 rabbits that were injected with noninfected autologous cells, did not react. On the 28th day post infectionem, the antibody levels against virus antigen were found at the degree of 1:32 to 1:1024. Only after the removal of the second testis, there occurred antibodies against autologous testes cells. The immunization conditions exclude the formation of isoantibodies...


Changes of the phosphatidylcholine content and the number of synaptic vesicles in relation to the neurohumoral transmission in sympathetic ganglia. In sympathetic ganglia stimulated in the presence of HC-3, the reduction in number of synaptic vesicles was observed to be accompanied by a significant decrease of the ganglionic phosphatidylcholine content.


Effect of prostaglandin E1 on rat gastric motility and cyclic nucleotide content. Administration of exogenous prostaglandin E1 resulted in an increase in contractility of rat fundic muscle measured in vivo; a significant decrease in fundic tissue levels of cyclic- adenosine monophosphate and a significant increase in cyclic-guanosine monophosphate.


Effects of penfluridol of dopamine-sensitive adenylate cyclase in corpus striatum and substantia nigra of rats. Penfluridol, a neuroleptic with diphenylbutyl piperidine structure, blocked the dopamine-sensitive adenylate cyclase in homogenates of corpus striatum and substantia nigra of rats, probably by a competitive antagonism versus dopamine.


Arabinose nucleoside triphosphates are no inhibitors for DNA-dependent RNA polymerases. 1-Beta-D-arabinofuranosylcytosine-5' -triphosphate and 9-beta-D-arabinofuranosyladenosine-5' -triphosphate were found to have no inhibitory potency for both mammalian DNA-dependent RNA polymerase II and E. coli DNA-dependent RNA polymerase.


Rosette formation by human T and B lymphocytes in the presence of adrenergic and cholinergic drugs. It was shown that adrenergic drugs, which increase the intracellular levels of cAMP, inhibit the rosette formation by T-lymphocytes, but stimulate the rosettes produced by B-lymphocytes. Cholinergic drugs, which increase the levels of cGMP, on the contrary, stimulate the formation of rosettes by T-lymphocytes but inhibit those produced by B-lymphocytes.


Fluorometric study of interaction between ACTH fragments and bovine adrenocortical membranes. Corticotropin 1-24 and [Gly1]corticotropin1-18 amide increased the fluorescence of 1-anilinonaphthalene-8-sulfonate which bound to the bovine adrenocortical membranes. The two ACTH fragments interacted with the protein of the membranes and membranes and increased the net positive charge of the membranes.


Investigations on the turnover of adrenocortical mitochnodria. VII. Effects of ACTH on the half-life of mitochondria from the zona reticularis of the rat adrenal cortex. The half-life of mitochondria from the zona reticularis of the rat adrenal was calculated by determining the radioactivity decay curves of the mitochondrial compartment of 3H-thymidine-injected animals, using autoradiographic methods. ACTH was found to enchance significantly the half-life of the organelles.


Snake venom action: are enzymes involved in it? Enzymes were the first clearly recognized components of snake venoms. When several more were discovered, attempts were made to correlate venom action with enzymic functions. The last few years have seen most successful efforts in the identification, isolation and structrual elucidation of highly toxic polypeptides present in snake venoms, in particular of 'neurotoxins' and membrane-active toxins. Following this development the polypeptides were called the true toxic components and the enzymes lost their previous central position in venom pharmacology. The time, therefore, has come re-evaluate the role of enzymes in the complex interaction between snake and prey. While highly active polypeptides indeed dominate the actionof hydrophiid venoms, they appear to play a lesser role in crotalid venom action as compared with enzyme components. Enzymes are involved in many levels of venom action, e.g. by serving as spreading factors, of by producing very active agents, such as bradykinin and lysolecithins in tissues of preys or predators. Some toxins, e.g. the membrane-active polypeptides appear to participate in the interaction between membrane phospholipids and venom phospholipases. The classical neurotoxin, beta-bungarotoxin, has been recognized as a powerful phospholipase. Several instances are known which indicate that some enzymes potentiate the toxic action of others; the analysis of a single enzyme may, therefore, not fully reveal its biofunction. For 3 enzymes,ophidian L-amino acid oxicase, ATPpyrophosphatase, and acetylcholinesterase, some of the problems pertaining to venom toxicity are discussed.


Cyclic nucleotide levels in the perfused rat heart subjected to hypoxia. Isolated rat hearts were subjected to hypoxic perfusion on a recirculating Langendorff apparatus. Following a 30-min-period of aerobic stabilization the hearts were perfused for 30 min with media equilibrated with 84% N2, 12%O2 and 4% CO2. At the end of the hypoxic period myocardial concentrations of cyclic AMP and cyclic GMP were determined by radioimmunoassay. Exposure to hypoxia resulted in a significant increase in cyclic AMP (p less than 0.01) and a decrease in cyclic GMP (p less than 0.05) as compared to hearts perfused for 60 min with media gassed with 96% O2. 4% CO2.


Stimulation of glucagon and inhibition of insulin secretion evoked from carotid baroreceptors. The influence from carotid baroreceptors on portal immuno-reactive glucagon and insulin levels and on arterial plasma glucose concentration was studied in vagotomized cats by sectioning of the sinus nerves. Such a complete elimination of the afferent baroreceptor discharge caused a prompt and pronounced increase in the glucose and glucagon levels, whereas the insulin concentration significantly decreased. The role of vascular barorecptors in the hyperglycemic response to hemorrhage is discussed.


Effects of TSH and cyclic AMP on the human thyroid cells cultured in a chemically defined medium. In a serum-free, chemically defined medium human thyroid cells elongated remarkably and resembled fibroblastic cells. They retained the cyclic AMP response to TSH and the supplement of medium with TSH or dibutyryl cyclic AMP permitted the preservation of epithelial nature by the cells. Cyclic AMP of the cells of epithelial nature was higher than those of fibroblastic appearance.


Hepatic alcohol oxidation and its metabolic liability. The pathways responsible for ethanol oxidation and the toxic results of its metabolism are reviewed. The predominant pathway for ethanol oxidation at low ethanol concentrations involves alcohol dehydrogenase. However, at high alcohol concentrations, up to 50% of ethanol uptake is 4-methylpyrazole-intensitive. Oxidation of ethanol under these conditions is associated with a change in the steady-stage concentration of catalase-H2O2. Based on recent evidence, we conclude that it is unnecessary to postulate that ethanol is oxidized directly via cytochrome P-450. Acetaldehyde production from ethanol via the microsomal subfraction can be accounted for by the combined activities of catalase-H2O2 and alcohol dehydrogenase. The metabolism of ehtanol via alcohol dehydrogenase produces a marked reduction in the hepatocellular NAD-NADH sytems. This reduction is indirectly responsible for the inhibition of glycolysis, gluconeogenesis, citric acid cycle activity, and fatty acid oxidation and may be related to some of the pathological effects observed following chronic consumption of alcohol. Attempts in inhibit alcohol dehydrogenase with alkylpyrazoles and activate catalase with substrates for peroxisomal H2O2-generating flavoproteins, while successful, may have limited applicability because of the native toxicity of the substrates themselves...


The opposing physiological effects of high pressures and inert gases. The physiological effects on mammals of elevated pressures (approximately 100 atmospheres) must be considered in the context of the inert gases breathed. The most striking effect of pressure per se is a central hyperexcitability manifest at first by trembling of the entremities and finally by convulsions. Paralysis and death occur at higher pressures. The primary effects of the inert gases breathed are inert gas narcosis and general anesthesia. The exciting effects of pressure per se and the depressive effects of the inert gases tend to oppose each other. Thus consciousness may be restored to anesthetized mice by raising the pressure, and conversely the threshold pressure that causes convulsions is elevated in the presence of anesthetics. These mutually antagonistic effects can be rationalized in terms of model which proposes that both anesthetics and pressure non-specifically perturb thelipid bilayer regions of neutral membranes. This model is termed the critical volume hypothesis. Anthesthetics dissolve in and expand these lipid bilayer regions, while pressure causes mechanical compression. Expansion leads to anesthesia and compression to convulsions if a critical degree of change is achieved. At elevated partial pressures of inert gas the gas-induced expansion is opposed by the compression of pressure per se. With very insoluble gases, such as helium, this expansion is so small that net compression results and the effects of helium differ little from those of pressure per se. With more soluble gases, such as nitrogen, net expansion results in inert gas narcosis and anesthesia. The critical volume hypothesis enables "safe" mixtures of "expanding" and "compressing" gases to be defined. These enable higher pressures to be better tolerated by mammals.


Nutritional factors in relation to heavy metal toxicants. An increased environmental exposure to various toxic heavy metals such as lead, cadmium, or mercury seems to be a fact of 20th-century life. But relatively little attention has been paid to the possible implications of sucy exposure for the nutritional status of humans and animals. This review summarizes the information available concerning the effect of various nutritional factors in resistance to metal toxicants and the effect of heavy metal toxicity on nutritional status. In particular, the following questions are considered: 1) Are there any examples of heavy metal toxicity that are potentiated by a nutritional deficiency? 2) Is there any evidence that nutritional deficiency can be caused by heavy metal toxicity? 3) Is there any proof that heavy metal toxicity can be decreased by an excess intake of nutrients: 4) Is there any proof that heavy metal toxicity can be increased by an excess intake of nutrients? The discussion is focused primarily on studies with animal models but, wherever possible, implications for human health are pointed out.


The angiotensin I converting enzyme.

The angiotensin I converting enzyme. The angiotensin I converting enzyme (kininase II; peptidyl dipeptidase; EC3.4.15.1) has a dual function: it converts angiotensin I to angiotensin II and it inactivates bradykinin. Lung, kidney, guinea pig plasma and testicles are among the richest sources of the enzyme. Vascular endothelial cells and bursh borders of renal proximal tubular cells contain high concentrations of the enzyme. The availability of synthetic peptide inhibitors was a great help in establishing the function of converting enzyme in normal and pathological conditions.


The renin-angiotensin system and the central nervous system. One of several factors affecting the secretion of renin by the kidneys is the sympathetic nervous system. The sympathetic input is excitatory and is mediated by beta-adrenergic receptors, which are probably located on the membranes of the juxtaglomerular cells. Stimulation of sympathetic areas in the medulla, midbrain and hypothalamus raises blood pressure and increases renin secretion, whereas stimulation of other parts of the hypothalamus decreases blood pressure and renin output. The centrally active alpha-adrenergic agonist clonidine decreases renin secretion, lowers blood pressure, inhibits ACTH and vasopressin secretion, and increases growth hormone secretion in dogs. The effects on ACTH and growth hormone are abolished by administration of phenoxybenzamine into the third ventricle, whereas the effect on blood pressure is abolished by administration of phenoxybenzamine in the fourth ventricle without any effect on the ACTH and growth hormone responses. Fourth ventricular phenoxybenzamine decreases but does not abolish the inhibitory effect of clonidine on renin secretion. Circulating angiotensin II acts on the brain via the area postrema to raise blood pressure and via the subfornical organ to increase water intake. Its effect on vasopressin secretion is debated. The brain contains a renin-like enzyme, converting enzyme, renin substrate, and angiotensin. There is debate about the nature and physiological significance of the angiotensin II-generating enzyme in the brain, and about the nature of the angiotensin I and angiotensin II that have been reported to be present in the central nervous system. However, injection of angiotensin II into the cerebral ventricles produces drinking, increased secretion of vasopressin and ACTH, and increased blood pressure. The same responses are produced by intraventricular renin. Angiotensin II also facilitates sympathetic discharge in the periphery, and the possibility that it exerts a similar action on the adrenergic neurons in the brain merits investigation.


Blockade of renin or angiotensin for understanding human hypertension: a comparison of propranolol, saralasin and converting enzyme blockade. To understand the role of the renin-angiotensin-aldosterone system in the pathogenesis of human hypertension, in serial studies we have blocked the system using three different pharmacologic probes: 1) reduction of renin secretion by administration of the beta receptor blocker, propranolol; 2) blockade of the action of angiotensin II by infusion of saralasin, a competitive antagonist of angiotensin II; and 3) blockade of the enzymatic conversion of angiotensin I to angiotensin II by infusing a nonapeptide competitive inhibitor. The depressor responses induced by either propranolol or the nonapeptide expose a significant to major involvement of excess renin--angiotensin in maintaining the hypertension of some 50 to 70% of common forms of hypertension including "essential" hypertension. This subgroup includes nearly all patients with high or "normal" renin--sodium profiles. The considerably lower estimates for a renin factor in essential hypertension suggested by saralasin testing now appear due to the partial agonism of this drug. Further studies are required to determine whether this relative or absolute excess of renin secretion is primarily involved in the hypertension and if not why it fails to shut itself off. Similar studies of normal subjects are also needed to determine whether renin support of blood pressure is proportionately greater or less than in hypertensive subjects. Meanwhile the validation provided by these three different pharmacologic probes portends a burgeoning clinical role for renin--sodium profiling not only in screening for renal and adrenal cortical hypertensions but also for characterizing the vasoconstrictor and volume elements involved in various individual patients and thus enabling more specific treatments of the various subtypes of essential hypertension.


[Studies on the thyrotropin receptor in the thyroid: positive cooperativity of Concanavalin A binding and its biphasic effects on thyroid activation induced by thyrotropin (author's transl)]. Concanavalin A (Con A) was tested for its ability to affect thyroid activation induced by the thyroid stimulators in mouse thyroid tissues. Con A was found to have the biphasic stimulatory and inhibitory effects of thyrotropin (TSH)-induced cyclic AMP formation and endocytosis, a step in thyroid hormone secretion, in mouse thyroid tissues. Low concentrations of Con A potentiated TSH-stimulated cyclic AMP formation and endocytosis. In contrast, high concentrations of Con A markedly inhibited TSH stimulations. These effects were reversed by the addition of methyl-alpha-D-glucoside to the second preincubation medium (without Con A) prior to TSH. A high concentration of Con A alone did not depress the basal levels of cyclic AMP or basal glucose oxidation in thyroid tissues. A high concentration of Con A also inhibited cyclic AMP formation induced by prostaglandin E2 and the long-acting thyroid stimulator (LATS). Binding of 125I-labeled Con A to thyroid tissues increased with time up to 75 min and was very slowly reversible after attainment of equilibrium. Binding was directly proportional to tissue weight. Scatchard plot analysis on the binding of 125I-labeled Con A to thyroid tissues indicated a positive cooperativity which seemed to be well correlated to the biphasic effects.


Hepatocarcinogenesis by thioacetamide: correlations of histological and biochemical changes, and possible role of cell injury. Hepatocarcinogenesis in rats produced by prolonged feeding of thioacetamide appears as a progressive phenomenon in which morphological changes are associated with important biochemical modifications. It seems most likely that changes in the permeability of cell membrane induced by the carcinogen are responsible for increased intracellular accumulation of Ca2+, and for the ensuring of cell injury produced by Ca2+ overloading of the mitochondria. This calcification of the mitochondria may play a role in the neoplastic transformation of the cell, especially as far as it concerns metabolic behavior and the genetic specification of the permeability characteristics of the transformed cell membrane. The increased synthesis of acid glycosaminoglycans suggests their involvement in calcium-mediated control of tumor development and growth.


Carcinogenicity of N-nitrosamines related to N-butyl-N-(4-hydroxybutyl)nitrosamine and N,N,-dibutylnitrosamine in ACI/N rats. Carcinogenic effect of 14 N-nitrosamines related to N-butyl-N-(4-hydroxybutyl(nitrosamine (BBN) and N,N-dibutylnitrosamine (DBN) was studied in ACI/N male rats by administration in the drinking water. BBN homologs having methyl, ethyl, or pentyl group selectively induced urinary bladder tumors, but a homolog with tert-butyl group did not have any carcinogenic effect. N-Ethyl-N-(3-carboxypropyl)nitrosamine, the principal urinary metabolite of the ethyl homolog of BBN, did also induce bladder tumors selectively, thus providing an additional evidence that N-alkyl-N-(3-carboxypropyl)nitrosamines are responsible for the selective induction of bladder tumors by BBN homologs. N-Butyl-N-(carboxymethyl)nitrosamine and BBN analogs having 3-hydroxypropyl chain together with ethyl or butyl group were found to be noncarcinogenic. N-Propyl-N-butylnitrosamine and DBN induced hepatomas, but simultaneous development of esophageal tumors were observed only with the former. N-Butyl-N-(3-hydroxybutyl)nitrosamine, one of the principal metabolities of DNB, did not induce any  tumors, but its further transformation product, N-butyl-N-(3-oxobutyl)nitrosamine as well as N-butyl-N-(2-oxobutyl)nitrosamine, another metabolic intermediate of DBN, induced hepatomas. Possible correlation of structure and metabolism with organotropic carcinogenesis by N-N-dialkylnitrosamines is discussed, with special reference to selective induction of urinary bladder tumors.


Effect of sulfated polysaccharides on blood-borne pulmonary metastasis in rats. The inhibitory effect of sulfated polysaccharides on blood-borne metastasis was examined. As a model of blood-borne metastasis, the ascitic form of hepatoma AH-109A tumor was injected intravenously into Donryu strain rats. Examination of the pulmonary metastatic nodules developed 2 weeks later showed inhibitory effect of the five sulfated polysaccharides tested. Xylan sulfate was the most inhibitory, and exerted its inhibitory effect when the tumor cells were in the pulmonary capillary beds. However, fromthe rapid disappearance of radioactivity from the lungs after injection of 125IUDR-labeled AH-109A cells, tumor cells seemed to be retained in the lungs for only a very short time. Measurement of the anticoagulative and fibrinolytic activities of three sulfated polysaccharides showed that the inhibitory effect of these compounds on blood-borne metastasis was proportional to their anticoagulative and fibrinolytic activities, xylan sulfate showing the highest activities. These results suggest that sulfated polyaccharides may inhibit blood-borne pulmonary metastasis by inhibiting the lodging of tumor cells in the pulmonary capillary beds.


Induction of papillary ependymomas and insulinomas in the Syrian golden hamster by BK virus, a human papovavirus. Newborn hamsters were inoculated intracerebrally with BK virus. Between 3 and 6 months after inoculation, they developed papillary ependymoma (8 hamsters, 42%) or functional malignant islet cell tumors of the pancreas (insulinoma, 8 hamsters), or both (1 hamster). Both tumors contained an antigen reactive to SV40 T-antibody, suggesting that at least a part of BK virus genomen has been integrated into the tumor cells. No infectious virus was detected in the extract of these tumors.


Electron microscopic evidence of surface immunoglobulins in Yoshida ascites sarcomas and ascites hepatomas. Yoshida ascties sarcoma and its variants and several azo dye-induced ascites hepatomas of rats were examined by an immuno-peroxidase technique to demonstrate surface immunoglobulins. More than 60% of the tumor cells in 8 ascites tumors showed positive surface staining (Group A), whereas the other 9 tumors were less than 15% positive (Group B). One tumor showed 34% positive reaction. The staining did not show capping. Six of the 8 Group-A tumors were single cell type, while 5 of the 9 Group-B tumors were island type. Intravenous transplantation rate was very low (less than 10%) in 2 of the 6 Group-B tumors, but only 1 of the 7 Group-A tumors showed moderately low (40%) intravenous transplantation rate. All of the 8 Group-A tumors showed survival period of less than 35 days after intraperitoneal transplantation, while the survival period was more than 45 days in 3 out of the 9 Group-B tumors.


Iron incorporation into liver cells and ferritin of tumor-bearing rats. Measurement of the incorporation of iron into liver cells and liver ferritin of the rat revealed that both incorporated less iron under tumor-bearing condition than in the normal state.


Cytochrome P-450 in hyperplastic liver nodules during hepatocarcinogenesis with N-2-fluorenylacetamide in rats. Amount of cytochrome P-450 in the hyperplastic liver nodules was measured during hepatocarcinogenesis with N-2-fluorenylacetamide in the rat. Amount of cytochrome P-450 in the liver microsome decreased in early stage of hepatocarcinogenesis. Concentration of cytochrome P-450 in the hyperplastic nodules and their surrounding tissues was examined, using homogenate from each of them. Its amount was significantly less in the hyperplastic nodules in the 13th week than in their surrounding tissues, and became more in hyperplastic nodules in the 19th week. The hyperplastic nodules of the animals treated with phenobarbital showed almost the same amount of cytochrome P-450 as that in the controls. Amount of cytochrome P-450 in hepatoma tissues also showed similar values as that in hyperplastic nodules.


Further observation of Kashara isoenzyme in patients with malignant diseases. The Kasahara isoenzyme of alkaline phosphatase was found in cancer tissues from patients with gastric carcinoma, maxillary carcinoma, pulmonary carcinoma, and carcinoma of the urinary bladder, in addition to hepatoma. This fact suggests that the Kasahara isoenzyme may not be a specific marker protein of liver cancer but could occur in a variety of neoplasms.


Squamous carcinoma of the stomach after luetic linitis plastica. This report illustrates many of the long term complicatio of syphilis involving the stomach. A 49-year-old woman with diffuse squamous metaplasia of the gastric mucosa developed invasive pure squamous cell carcinoma. The literature is reviewed and the probable pathophysiology is discussed.


Cytochemical studies of rat liver glycogen and alkaline phosphatase under the effect of hepatocarcinogens. Cytochemical changes of glycogen and alkaline phosphatase were studied under sequential effect of p-dimethylaminoazobenzene (DAB) feeding and CCl4 injections. It has been found that in liver cells of experimental animals, the concentration of glycogen and the activity of alkaline phosphatase were decreased.


Streptococcal extracellular NAD-glycohydrolase. Optimal temperature and activation by cysteine. Streptococcal extracellular NAD-glycohydrolase (EC 3.2.2.5) exists in two distinct states with respect to the optimal reaction temperature. Bacteria produce the enzyme form with optimum activity at about 40 degress C. Probably due to oxygen action, the enzyme is converted to a form with optimum activity at 30 degrees C. Compounds of the type of cysteine restore the initial state. The conversions are accompanied by enzyme activity fluctuations.


[Burkitt's lymphoma].

[Burkitt's lymphoma]. The Burkitt's tumor is a neoplastic disease which occurs mostly in the tropics. Sporadic cases however are distributed all over the world. This undifferentiated lymphosarcoma predominates in children. Extralymphnodal in origin, quick growth and lack of pain are very characteristic signs. Unattended most of the patients die within a few months. Cytostatic treatment in connection with x-ray therapy and surgical proceedings show some results. The Epstein-Barr-virus is incriminated to play a causative role in the pathogenesis of Burkitt's lymphoma. But the experimental proof of its oncogenic activity in human beings has not yet been demonstrated. The problem of an active immunization is to be controlled.


[Wilms tumor in hemihypertrophy].

[Wilms tumor in hemihypertrophy]. The case of a 4-year-old boy with Wilms' tumor and hemihypertrophy is described. Wilms' tumors are frequently associated with congenital malformations of the urinary tract, with aniridia and hemihypertrophy. Hemihypertrophy is a relatively rare malformation (1:14000) in the common population, but in patients with Wilms' tumors its frequency is about 1:49. Besides Wilms' tumors tumors of the adrenal cortex and hepatoblastomas are frequently observed together with hemihypertrophy.


The contribution of hepatic triglyceride lipase to plasma post-heparin lipolytic activity in the rat. Plasma post-heparin lipolytic activity (PHLA) was reduced by 50% in functionally hepatectomised compared with shamoperated rats. Inhibition of PHLA by protamine sulphate was significantly increased in functionally hepatectomised compared with shamoperated rats. Hepatic triglyceride lipase contributes significantly to total plasma PHLA. Its function is not yet clear.


A comparison of the pathogenicity of three strains of bovid herpesvirus 2 in calves. The isolates of Bovid herpesvirus 2 share common morphological, physical and antigenic features, but differences in their pathogenicity for cattle have been found among the strains. In order to investigate this aspect in more detail a comparative study was made of the skin lesions produced in calves by three strains of the virus: Allerton BA, bovine mammilitis and 69/1L0. The results show that Allerton BA was less pathogenic for calves than the other two strains used in the experiment. This conclusion is supported by the observations that very few inclusion bodies were found in the lesions produced by Alerton BA, and the maximum titre of virus in the lesions was lower than the titres obtained with bovine mammillitis and 69/1L0. Moreover the thickness and diameter of the lesions with Allerton BA were less than those with the other strains. It is speculated that Allerton BA strain, which originated from a buffalo, might behave similarly to bovine mammillitis and 69/1LO following serial passages in cattle.


Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary. Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.


The gross and histological findings in the liver of pigs with Aujeszky's disease. The gross and histological changes were studied in the liver of young pigs naturally infected with Aujeszky's disease. In the majority of the cases examined it was possible to detect small necrotic foci, scattered throughout the liver parenchyma, which corresponded histologically with intralobular or plurilobular areas of coagulative necrosis. At the periphery of the larger necrotic foci, inclusion bodies were also found in the hepatocytes of 9 of the 17 pigs examined. These intranuclear inclusion bodies showed various developmental features, similar to those reported quite frequently in herpesvirus infections of animals.


Fine structural studies of a human thyroid adenoma, with special reference to psammoma bodies. The fine structural morphologic features of a microfollicular thyroid adenoma from a 28 year old female were examined. Although the patient had been laking exogenous thyroxine therapy for 14 months, the morphology of the adenoma was characterized by numerous small to medium sized follicles composed of metabolically active, well differentiated columnar cells with numerous colloid droplets, dilated granular endoplasmic reticulum, large numbers of coated vesicles and lysosomes, large colloid containing "lakes," microtubules, microfilaments, and prominent apical microvillous projections. Of special inetrest were small spherical psammomatous calcospherites ecountered in histiocytes and the interstitium. Also noteworthy were ropelike configurations observed in most of the follicular lumina. Structural-functional correlations and potential origins of psammoma bodies and calcospherites are discussed.


A new variety of hereditary sensory neuropathy. A pedigree with a new form of hereditary sensory neuropathy is described. Ataxia and scoliosis rather than loss of pain and ulcerating acropathy are the principal clinical feature. Analysis of the pedigree suggests a dominant mode of transmission with variable age of onset and perhaps reduced penetrance.


Evidence for X-linkage of phosphoribosylpyrophosphate synthetase in man. Studies with cultured fibroblasts from a gouty family with mutant feedback-resistant enzyme. Skin fibroblast cultures were utilized to study the mode of inheritance of a mutant feedback-resistant phosphoribosylpyrophosphate synthetase in a gouty family with purine overproduction. Selective conditions were applied to allow the survival in culture of mutant cells only. Whereas in the male gouty propositus the cell culture was homogenous for the mutant enzyme, in the cell culture from his nongouty mother two cell populations were demonstrated, one normal and the other mutant. The mosaicism in the mother is compatible with X-linkage of the enzyme. From this finding, together with the clinical and biochemical data available, it is concluded that in this family the enzyme mutation is transmitted in a X-linked recessive pattern.


Ultrastructural and immunofluorescent studies of the replication of herpesvirus saimiri in cultured lymphocytes of infected owl monkeys. The lymphocytes of five owl monkeys infected with herpesvirus saimiri (HVS) and of three control owl monkeys were studied by electron microscopic (EM) and immunofluorescent (IF) techniques. Buffy coats of whole blood immediately after bleeding were also prepared for study. At the time of the study, two of the five infected monkeys were leukaemic; within 50 days after the study, four of the five had died with malignant lymphoma and lymphocytic leukaemia. HVS virions were demonstrated by EM and HVS antigens by IF in 1-20% of the lymphocytes from infected monkeys in 2/5 cultures at 24 hours AC, 4/5 at 48 hours AC, and 5/5 at 72 hours AC. There was quite good agreement between the EM and IF data. None of the control monkey cultures and none of the buffy coat preparations contained HVS virions or antigens. By EM, the great majority of the virus particles were nucleocapsids within the nuclei of lymphocytes. Enveloped virions were rare. There was some evidence to suggest that the higher the percentage of lymphocytes containing HVS, the poorer the prognosis for the monkey. HVS was isolated from all five infected monkeys by co-cultivation of lymphocytes with Vero cells. Control lymphocyte co-cultivations were negative.


Response of marmosets to experimental infection with Epstein-Barr virus. The response of cotton-topped (CT) or white-lipped (WL) marmosets, inoculated with material containing Epstein-Barr virus (EBV), was studied. Twenty-five marmosets inoculated with 10(6)-10(8) cells from human or simian EBV-carrying lymphoblastoid cell cultures developed no detectable clinical disease. Transitory antibody titres to VCA were detected in sera from several of these marmosets. Thirty-eight marmosets inoculated with P3 virus (10(9) particles) and seven marmosets inoculated with 10(5) infectious units of HR-1 virus likewise developed no recognizable clinical or haematological disease over several years of observation. Fourteen marmosets were inoculated with 10(2)-10(4) transforming units (TFU) of EBV strain B95-8; five of 11 animals inoculated with 10(3)-10(4) TFU of virus developed malignant lymphoma and died 31-110 days PI. Antibodies to one or more EBV-specified antigens were detected in sera from nine of the inoculated marmosets. EBV-carrying lymphoblastoid cell cultures were established from the spleen and lymph-nodes of one animal that died with malignant lymphoma and from another animal that died 19 days PI from undetermined causes.


Humoral and cellular immunity to EBV and lymphoid cell line antigens in human lymphoma. Although elevated antibody levels to the Epstein-Barr virus (EBV) have been reported in a number of lympho-proliferative neoplasms, it has not been possible to determine whether these antibodies were the result of a specific response to an oncogenic agent (EBV), whether they were a non-specific humoral compensation for depressed cell-mediated immunity (CMI), or whether a different mechanism was responsible. We have previously shown in a group of lymphoma patients that depressed cellular immunity to a number of standard antigens (Candida, SKSD, etc.) is not associated with an increase in antibody to EBV. In this study, we tried to compare CMI to possible EBV and lymphoid cell line antigens with humoral antibody to EBV. The two basis CMI assays utilized were lymphocyte cytotoxicity (LC) and skin testing (ST) for delayed hypersensitivity. In the LC assay, an EBV-containing cell line (F265) was used as the target. Reactivity against F265 was stronger in normal individuals than in cancer patients, suggesting a relationship to general cellular immune competence. ST studies showed that membrane extracts from lymphoid cell lines derived from patients with Burkitt's lymphoma and nasopharyngeal carcinoma (NPC) were more likely to elicit a delayed hypersensitivity in lymphoma and NPC patients than cell lines derived from normal individuals. Patients with ST reactivity against the membrane preparations from the tumour-derived cell lines were as likely to have elevated EBV antibodies as patients without such reactivity. The data strongly indicated that the elevated EBV titres in lymphoma patients are not related to a specific or non-specific depression of CMI.


Epstein-Barr virus (EBV)-associated antibody patterns in relation to the deficiency of cell-mediated immunity in patients with Hodgkin's disease. Sera from unselected and untreated patients with Hodgkin's disease (HD) were examined for antibodies to Epstein-Barr viral (EBV) capsid antigens (VCA). Delayed cutaneous hypersensitivity reactions were carried out with purified tuberculoprotein (PPD). Highly purified blood lymphocytes of the same patients were studied morphologically and classified for cell surface markers. Incorporation of 14C-thymidine was used as a measure of spontaneous DNA synthesis and DNA synthesis after exposure to different concentrations of three mitogens (PHA, Conconavalin A and pokeweed mitogen) and PPD. The distribution of EBV titres was in good agreement with previous reports. Most patients were lymphopenic, due to subnormal levels of T lymphocytes. The lymphocyte stimulation and skin tests showed different degrees of impairment in a considerable number of the patients. The results in 43 patients indicated that a relation exists between the immune defect and the anti-VCA titres. High serological anti-VCA reactivity was related to a poor cutaneous response to PPD, a decreased level of T lymphocytes in the blood and a depression of mitogen-induced DNA synthesis.


Immunogenetic aspects of nasopharyngeal carcinoma (NPC) III. HL-a type as a genetic marker of NPC predisposition to test the hypothesis that Epstein-Barr virus is an etiological factor in NPC. HL-A typing of 144 NPC patients and 236 controls revealed an increased frequency of 1st locus HL-42 (relative risk = 2.24) and an increased frequency of unidentified antigens at the 2nd locus (relative risk = 2.60) in the NPC patients. HL-A2 and the 2nd locus "blank" appeared to act together (HL-A2 blank haplotype) in determining NPC risk in highest-risk Cantonese, whereas in relatively lower-risk non-Cantonese Chinese (Hokkiens, Teochews) they appeared to act independently. Only the "blank" had an increased frequency in Malay NPC patients. Thus there was an indication that the strength of the HL-A association with NPC reflected the 30-50-fold difference in incidence between highest-risk Cantonese and lowest-risk Indians. In Singapore, HL-A segregation patterns in families of nine Chinese NPC patients confirmed that the "blank" was a genetic phenomenon. A new 2nd locus antigen (Singapore-2) has recently been detected. Singapore-2 occurs more frequently in NPC patients and appears to be associated with a high risk for NPC. Since HL-A2 and Singapore-2 are not the risk factors, it is likely that the HL-A association with NPC reflects the existence of disease-susceptibility (DS) genes in linkage disequilibrium with alleles of the HL-A loci. It is proposed that NPC-DS genes may determine differences in immune responsiveness to environmental agents, and thereby determine differences in NPC incidence. If the known altered immune responsiveness of NPC patients to EBV reflects the function of DS genes linked to the high NPC risk HL-A type, then the hypothesis that Epstein-Barr virus has an etiological role in NPC can be tested by several types of prospective studies.


Alteration in Epstein-Barr virus-human lymphoid cell interactions caused by the presence of type-C viral genome. We have recently established a cell line, designated FVNC, by infection of non-EBV-productive human lymphoid NC-37 cells with the type-C virus FLV. The FVNC cell line has been maintained free of detectable EBV- and FLV-related immunofluorescent antigens for three years but both viral genomes exist in the cells in a repressed form. The FVNC cells were morphologically similar to the uninfected NC-37 cells but a D-group chromosome change was consistently seen. The colony-forming capacity of FVNC cells in semi-solid agar medium was about 1/10 of that of NC-37, although the former grew rather faster than the latter in fluid culture. FVNC cells were three times more sensitive to EBV superinfection than NC-37, as shown by immunofluorescence. In addition, an obvious induction of FLV antigenic markers occurred in FVNC cells on exposure to EBV. Nucleic acid hybridization experiments showed a striking decrease in the number of EBV genomes in FVNC cells. The number of genomes was too small to be measured, but EBNA was clearly present in all FVNC cells as well as in NC-37 cells. The implications of these findings were discussed from the point of view of a possible co-carcinogenesis in man by EBV and the type-C virus.


Analysis and description of procedures used in the study of the relationship of herpes simplex virus "non-virion" antigens to certain cancers. After a brief review of the relationship of herpes simplex viruses to certain human cancers, a description is given of procedures used in the study of herpesvirus "non-virion" antigens, and various points on which disagreement currently exists are discussed. Complement-fixation tests on these markers with both cancer and control sera as well as with "non-virion" antisera, which did not react with non-anti-complementary stored virion antigens, are described, and it is suggested that another parameter is now available for studies of squamous-cell carcinomas. The finding in certain tumours that expression of a repressed viral genome takes place proves that there is a continuing release of virus-specific messages and would indicate an important role for the virus in the development of the tumour.


Sero-epidemiology of the Epstein-Barr virus: preliminary analysis of an international study - a review. Samples of Chinese, Indian, African and Caucasian populations, randomly selected in Hong Kong, Singapore, the West Nile District of Uganda, and Nancy, France, were titrated for antibodies to EBV, viral capsid (VCA) and complement-fixing soluble (CF/S) antigens. The age-specific prevalence of infection (as reflected by the proportion of VCA-positive individuals) varied greatly up to the age of 10 years in the four populations studied, the West Nile District of Uganda being outstanding in having an early and massive infection rate. Differences were also observed between ethnic groups in Singapore, where the Chinese appeared to have a delayed infection rate compared to the Indians. Immune response, as measured by the prevalence of CF/S antibodies and by the geometric mean titres (GMT) of VCA and CF/S antibodies, differed significantly between ethnic groups, Ugandan infants (1-3-year age-groups) having a humoral response to VCA and CF/S antigens as high as or higher than that of Burkitt's lymphoma patients, decreasing thereafter to levels lower than those of any other ethnic group observed. Indians in Singapore, known to be at no risk for NPC and BL, exhibited a higher and steadier immune response to VCA in going from young to old age-groups than did the Chinese.


Studies of the nature of herpesvirus-induced tumour-associated antigens induced by herpes simplex virus type 1 and further analysis of their relationship with squamous-cell carcinomas of the head and neck region. The differences seen in levels of antibody response to HSV-TAA between non-laryngeal and laryngeal cancers in cured and untreated patient populations were due to the use of filtered sera. In previous and present studies using unfiltered sera, these differences were not noted. Of unfiltered sera from patients with squamous-cell carcinomas of the head and neck, 89% (49 out of 55) were positive for CF reactivity to HSV-TAA, and 44% of these unfiltered sera had titres of 1:4 or greater to 44 microng of antigen. The upper band of the antigen appears to be present in early provirus formation and is highly unstable whereas the lower band does not seem to be part of the assembly process.


Antibodies to herpesviruses in patients with cervical cancer and controls. The indirect immunofluorescent test was used to detect antibodies to herpesviruses types 1 and 2, cytomegalovirus and EBV in sera from patients with cervical carcinoma, condyloma acuminatum and controls from Cali, Colombia, and a control group from the USA. No significant differences were found in the prevalence of antibodies to viral capsid antigens of HSV-1 and CMV among the groups studied. However, titres for HSV-2 were higher in the three groups from Cali (cervical carcinoma, condyloma and controls) than in the control groups from USA. High EBV antibody titres (VCA and early antigens) were found in the three groups from Cali. Antibodies to early antigens of CMV were detected in a sub-sample of the subjects and with higher frequency in patients with cervical cancer. The significance of these findings is discussed.


Genetic analysis by chromosome-mediated gene transfer. A general method is presented for stable transfer of genetic information to eukaryotic cells, utilizing metaphase chromosomes as the vehicle. Recent progress, current problems and large areas of uncertainty in this field are reviewed; particular consideration is given to frequency of transfer, size of the transgenome, evidence of cotransfer of linked genes and serial chromosome transfer. A reasonable model for chromosome transfer is considered with respect to the available information, and various descrepancies are noted. The utility of this method for fine structural mapping, cloning small regions of the eukaryotic genome and other potential applications are discussed.


Effects of cholera exotoxin on Fc receptor activity of lymphoid cells and mononuclear phagocytes. The effect of cholera exotoxin and aminophylline on Fc receptors in a murine lymphoid-cell line and in rabbit pulmonary alveolar macrophages has been investigated. Although both agents elevated intracellular cyclic AMP levels in macrophages and lymphoid cells, the effects on Fc receptor expression were distinct. Cholera toxin at 10 microng/ml reversibly inhibited Fc-receptor activity in the murine lymphoid cell line. In contrast, cholera toxin at 10 microng/ml or 0-01 microng/ml was ineffective in altering pulmonary alveolar macrophage receptor expression. Fc receptor activity on the macrophage was reduced by 20-30 per cent following incubation with aminophylline, (10(-3)M) from 0-6 h. There was no direct correlation between Fc-receptor activity and cyclic AMP levels in the cells studied. The differential susceptibility of these lymphoid and phagocytic cell populations to cholera toxin and also toward aminophylline suggests that there may be fundamental differences in topography on the membrane surface, or in the intracellular regulation of Fc receptors between lymphoid and phagocytic cells.


In vivo interference in vesicular stomatitis virus infection. Inactivated defective interfering and complete particles of vesicular stomatitis virus given intracerebrally to adult mice protect them against challenge with homologous virus whether this is given at the same time or several days later. Two separate protective processes appear to be involved. The first, which comes into operation immediately after inoculation, is also effective against heterologous strains of vesicular stomatitis virus, rabies (another rhabdovirus), and a neurotropic strain of foot-and-mouth disease virus. The second, later effect, which is strain specific, appears to be correlated with the appearance of circulating neutralizing antibody. Our results suggest that the protective effect that Holland and his colleagues described using defective interfering particles of vesicular stomatitis virus may also be accounted for by an immunological mechanism rather than one involving interference.


C-type virus protein p30 in blood from inbred mice correlates with their later incidence of leukemia. The major core protein, p30, of mouse C-type viruses was quantitated radioimmunologically in lymphoid organs and blood from inbred strains of mice. The concentration of p30 in thymus and spleen had a weak and moderate correlation, respectively, to leukemia frequency. In contrast, the concentration of p30 in blood from mice with a high incidence of leukemia (strains AKR and C58) was 100-fold increased at 2 months of age compared with 10 strains with a low incidence of the disease. The SJL mice, which have a high incidence of reticulum cell neoplasms, showed generally elevated, but variable, values. The high concentration in AKR blood developed during the first weeks of life. Approximately one-third of the DBA/2 mice had elevated levels after 4 to 5 months, whereas the values from mice of the 129 strain were low irrespective in their age. The major part of p30 appeared to be associated with the erythrocytes. The concentration of p30 in the blood seems to reflect the presence of replicating virus in mice. It identifies among the inbred strains a high leukemia group one-half year prior to disease.


Persistence of cytomegalovirus in human lymphoblasts and peripheral leukocyte cultures. The in vitro susceptibility of human peripheral lymhpocytes and lymphoblastoid (F265) cells to infection by human cytomegalovirus was examined. Infection of these cell types with cytomegalovirus resulted in a persistent type of infection rather than the typical growth curve observed with permissive fibroblastic cells. When infection of peripheral lymphocytes was associated with a blastogenic response, the virus persisted for a longer time and at a higher titer than in cells in which a blastogenic response did not occur. Autoradiographic studies and infectious-center assays indicated that only a small number of cells, resembling lymphocytes, were involved in virus persistence. Whether or not the persistence of the virus indicates release of input virus or synthesis or new virus was not determined.


Oncogenicity of avian leukosis viruses of different subgroups and of mutants of sarcoma viruses. Leukosis viruses of seven subgroups were tested for oncogenicity in chickens susceptible to virus infection and to development of lymphoid leukosis (LL) tumors. All subgroup A viruses and the subgroup B virus tested produced a high incidence of LL and other related neoplasms. Viruses of subgroup C and RAV-61 of subgroup F produced a low level of LL. The RAV-50 of subgroup D produced osteopetrosis. In these tests, the viruses of subgroup E and G and one virus of subgroup F were not pathogenic, possibly because infection was not established in the chickens, the chickens were not susceptible to tumor development by these viruses, or the viruses lacked oncogenicity. All temperature-sensitive mutants of Rous sarcoma virus produced sarcomas, but the level varied. One nontransforming mutant produced sarcomas, and the other three tested produced LL. All three mutants that cause cells to grow as colonies in agar produced a high incidence of sarcomas. Thus, sarcoma viruses, by back-mutation, may lose the ability to transform cells in vitro, to make cells grow in agar colonies, or to induce sarcomas in vivo, yet they retain the ability to produce LL. Conversely, it was previously shown that leukosis viruses may be changed into viruses that transform cells in vitro and produce sarcomas in vivo by suitable passage in chicks.


Effect of rheumatoid factor and anti-immunoglobulin G antibodies on complement-mediated lysis of herpes simplex virus-infected human fibroblasts. The effect of various anti-immunoglobulin G (IgG) antibodies on the complement-mediated lysis of herpes simplex virus-infected human fibroblasts was determined. IgM rheumatoid factor, a naturally occurring anti-human Fc, inhibited lysis, whereas rabbit anti-human IgG serum potentiated immune cytolysis. We attempted to explain this disparity by determining the effect various classess of anti-IgG's with differing specificities had on complement-mediated lysis. Inhibition of cytolysis occurred with IgM anti-Fc and all of the IgG antiglobulins (anti-IgG, Fab, and Fc). In contrast, IgM anti-Fab enhanced lysis. IgM anti-IgG suppressed immune cytolysis when high concentrations of antiviral serum were incubated with the virus-infected cell, but augmented lysis when low concentrations of anti-herpes simplex virus antibody were exposed to the fibroblasts. The experiments indicated that whether a particular antiglobulin potentiates or inhibits lysis depends on the concentration of antibody bound to the target cells as well as the class and specificity of the antiglobulin exposed to the antibody-coated cell.


Detection of latent cytomegalovirus in murine salivary and prostate explant cultures and cells. After infection of adult mice, cytomegalovirus was detectable in salivary gland suspensions by tissue culture inoculation for up to 3 months. After these cultures had become negative, virus apparently latent in these tissues could be detected in explants of salivary and prostate glands and in cell lines derived from these explants. In some cases cycles of virus-induced cell injury and regrowth were observed. Murine cytomegalovirus plaque efficiency and morphology were evaluated in prostate and salivary gland cell cultures derived from previously infected and uninfected mice. No evidence of interference was detected, although plaques size was altered (larger) in prostate cells from previously infected mice. These studies indicate the presence of a range of suppression, latency, or effects of murine cytomegalovirus detectable after the resolution of active infection and provide methods for additional study of the establishment and activation of virus latency.


Interactions between calf alveolar macrophages and parainfluenza-3 virus. Cells washed from the lungs of freshly killed calves (lung wash cells; LWC) were cytotoxic for calf kidney (CK) target cells infected with parainfluenzavirus type 3 (Pi-3) when assayed by chromium release. LWC collected from 25 calves, including two gnotobiotic animals that had not previously been infected with Pi-3, were all cytotoxic, giving a specific chromium release between 11 and 50%. Cytotoxicity was detected at ratios of LWC to target cell as low as 5:1. The cytotoxic reaction required viable LWC, was inhibited by Pi-3 antiserum, and was not the result of virus-induced damage to the target cells. The cytotoxic cells in the LWC population were identified as alveolar macrophages from observations on glass adherence, phagocytic activity, killing by silica and fine-structural appearance. When LWC were added to CK cells or organ cultures of bovine trachea infected with Pi-3, the yield of virus was reduced for the first 2 to 3 days. However, subsequently, Pi-3 virus replicated in the LWC. Infection of LWC with Pi-3 virus reduced their cytotoxic activity. The significance of these interactions between alveolar macrophages and Pi-3 virus is discussed.


JC virus, a human polyomavirus associated with progressive multifocal leukoencephalopathy: additional biological characteristics and antigenic relationships. JC virus, a human polyomavirus, failed to grow or produce cytopathic effects in any of a variety of cells tested other than primary human fetal glial (PHFG) cells. Cells tested included other primary human cells and glial cells from other animals. Only a rare cell in inoculated insusceptible human cell cultures produced T or virion antigen. In PHFG cell cultures JC virus produced subtle cytopathic effects, and the majority of progeny remained cell associated. Only a few cells in the heterogenous PHFG cell cultures contained T antigen at 24 h postinoculation, and virion antigen was not detected until 48 h postinoculation. The infectivity of JC virus was resistant to inactivation by ether and by heating at 50 degrees C for 1 h. A three-way minor antigenic relationship was demonstrated among the virion antigens of JC virus, BK virus, and simian virus 40 by neurtralization and/or hemagglutination inhibition tests. Serological evidence is presented for the existence of JC virus as a distinct entity before the use of simian virus 40-contaminated poliovirus vaccines and for the nonexistence of an animal reservoir for JC virus infection.


Adjuvant effect of amorphous silica on the immune response to various antigens in guinea pigs. The adjuvant effect of a subcutaneously injected amorphous silica (Aerosil) to various antigens was studied in guinea pigs. It was demonstrated that silica is able to enhance the humoral immune response to particulate and soluble antigens widely differing in molecular weight and that, in the system employed, adsorption of antigen on silica particles is not absolutely required for its adjuvanticity.


Heterogeneity of Epstein-Barr virus originating from P3HR-1 cells. I. Studies on EBNA induction. Infection of EBV-negative human B-lymphoma cells of the lines BJAB and Ramos with EBV from P3HR-1 or B95-8 cells resulted in gradual conversion of these cells to EBNA synthesis. Whereas B 95-8 virus-infected cells exhibited a uniform brilliant EBNA fluorescence, two distinct fluorescence patterns were observed in P3HR-1 virus-converted BJAB and Ramos cells, a faint granular and a brilliant fluorescence, with predominance of the faint granular pattern. Cloning of P3HR-1 virus-converted BJAB cells resulted in 20 clones, 11 of them showing the heterogenous parental pattern, six revealing exclusively faint granular EBNA staining, and three with brilliantly stained nuclei, containing also a varying percentage of EBNA-negative cells. Further subcloning of one of the latter clones resulted in 26 subclones with brilliant EBNA expression, always segregating a significant percentage of EBNA-negative cells and one entirely EBNA-negative subclone. Reassociation kinetics did not reveal striking differences in the genome content of clones showing exclusively the faint granular or the brilliant type of EBNA expression. The EBNA-negative clone did not contain detectable amounts of EBV-DNA. Upon superinfection of the converted clones by the parental P3HR-1 virus, a significant increase in EA induction was noted when compared to non-converted BJAB and Ramos cells. This accounted in particular for cells with faint granular EBNA expression. These data support previous interpretations (Fresen and zur Hausen, 1976), suggesting the existence of at least two populations of EBV molecules within P3HR-1 cells. The reason for the apparently labile association of P3HR-1 EBV genomes inducing the brilliant EBNA flourescence in BJAB cells still remains obscure. The possible existence of a "helper" effect, exerted by the faint granular EBNA-inducing virus in stabilizing the persistence of the former, is discussed.


Tumour-specific antibodies reactive with cell-surface antigens in children with Wilms' tumour. Tumour-specific antibodies directed against membrane antigens were demonstrated by immunofluorescence in three of 45 patients with Wilms' tomour. Antibody capable of collaborating with K cells to kill Wilms' tumour was present in two additional patients. No patient exhibited both membrane immunofluorescence and K-cell collaborating antibody. Of 27 patients with non-renal solid tumours and 52 age-matched controls, none possessed tumour-specific antibody.


Lymphocyte responses to EBV-associated antigens in infectious mononucleosis, and Hodgkin's and non-Hodgkin's lymphoma patients, with the leukocyte adherence inhibition assay. The leukocyte adherence inhibition (LAI) assay was utilized as a test for cellular immunity to Epstein-Barr virus (EBV) antigens in 22 patients with infectious mononucleosis (IM), 47 patients with lymphoma, 101 carcinoma patients, and 84 subjects without cancer. Response to EB virion ("v") antigen was generally present at the time of diagnosis in the IM patients but the response to EB soluble ("S") antigen was delayed. An increased CMI response to "v" antigen was found in patients with IM, Hodgkin's disease and non-Hodgkin's lymphoma as compared to controls with and without cancer. Patients with Hodgkin's disease had depressed responses to the EBV-associated "S" antigen. The finding of increased LAI responses to "v" antigen in Hodgkin's disease patients with high EBV antibody titers conflicts with previous reports attributing high antibody responses against EBV to a generalized depressed cell-mediated immunity.