Effects of hypophysectomy, thyroidectomy, and thyroxine on specific prolactin receptor sites in kidneys and adrenals of male rats. The effect of T4 on specific PRL binding in the kidneys and adrenals was measured in intact, hypophysectomized, and thyroidectomized male rats. PRL binding in the kidneys was reduced from 12.1 +/- 1.1% in the intact group to 4.5 +/- 0.5% after hypophysectomy; 4-day replacement with T4 returned PRL binding to intact values. T4 administered to intact rats significantly increased PRL binding above intact values. Thyroidectomy reduced PRL binding in the kidneys from 14.9 +/- 1.2% to 7.0 +/- 0.6%, and T4 treatment restored PRL binding to intact values. PRL binding was measured at 2, 3, 5, 7, and 10 days after thyroidectomy in kidneys and found to decrease progressively from 8.2+/-0.5% in the intact rats to 2.3+/-0.3% on day 10. A single injection of T4 doubled PRL recetor binding in kidneys of thyroidectomized rats at 12 h and returned binding to intact levels at 24 h. In contrast to the effects of T4 on kidney PRL binding, adrenal PRL binding was only slightly altered by thyroidectomy and T4. These results suggest that T4 has a specific role in regulating PRL receptors in the kidneys but not in the adrenals. Since PRL receptors in the adrenals were only slightly altered by thyroid status, it is believed that the changes in kidney receptors represent a specific rather than a general metabolic effect of T4.


Dissociation of glucocorticoid inhibition of glucose transport and metabolism by increase in adipocyte age and/or size. Exposure of adipocytes from young rats (2--3 months old) to dexamethasone in vitro results in 40--50% inhibition of glucose transport and metabolism. Comparable experiments with older rats reveal that the hormonal effect on glucose metabolism is progressively reduced to about 20% in 12- to 13-month-old animals and about 5% in 24- to 26-month-old animals. However, no effect of dexamethasone on the glucose transport system can be detected in either of the older age groups when three different sugars are used to measure transport activity. The ability of dexamethasone to inhibit glucose phosphorylation seems to be closely related to inhibition of overall glucose oxidation in both young and old adipocytes. That the membrane may be generally more resistant to control of glucose transport is suggested by failure or reduced ability of insulin, vitamin K5, and hydrogen peroxide to stimulate this function in older cells.


Gonadotropin-releasing hormone action in cultured pituicytes: independence of luteinizing hormone release and adenosine 3',5'-monophosphate production. The secretory response of pituitary gonadotropes to stimulation by gonadotropin-releasing hormone (GnRH) has been extensively studied, but the mechanism by which GnRH evokes gonadotropin synthesis and release has not been clarified. In particular, there has been conflicting evidence about the role of cAMP in GnRH-induced release of LH. To examine this question in more detail, the actions of GnRH on LH release and cAMP production were analyzed in primary cultures of collagenase-dispersed rat pituitary cells. In this system, addition of 10(-10)--10(-6) M GnRh to cultured pituicytes caused rapid release of LH into the incubation medium. In contrast, GnRH caused no significant change in intracellular or extracellular cAMP or in occupancy by cAMP of the regulatory subunit of protein kinse. Neither dibutyryl cAMP nor methyl isobutylxanthine (MIC) stimulated LH production to the same level as GnRH, and neither agent potentiated the effect of the releasing hormone. Cholera toxin and prostaglandin E1 (PGE1), both of which stimulated cAMP production in cultured pituicytes, did not raise LH levels as markedly as GnRH. These results demonstrate the independence of LH release from cAMP accumulation in cultured pituicytes, suggesting that cAMP is not required for stimulation of LH release from these cells and that GnRH acts on LH secretion by a different mechanism.


Inhibitory effects of corticosterone on cell proliferation and steroidogenesis in the mouse adrenal tumor cell line Y-1. Monolayer cultures of mouse adrenal tumor cell line Y-1 have been used to investigate the effects of glucocorticoid on cell replication, [3H]thymidine incorporation into the trichloroacetic acid-precipitated cell fraction, steroidogenesis, and the ACTH receptors of adrenocortical cells. Corticosterone at a concentration of 5.0--50 micrograms/ml inhibited cell replication and [3H]thymidine incorporation into trichloroacetic acid-precipitated cell fraction in a dose-related manner. Corticosterone at a concentration of 0.5--50 micrograms/ml inhibited ACTH-induced steroidogenesis in a dose-related manner. Steroids which do not possess glucocorticoid action did not show such inhibitory effects on cell replication and steroidogenesis of Y-1 cells. The characteristics of the ACTH receptors of these cells remained unaffected by corticosterone. Our findings suggest that synthesized or secreted glucocorticoid may play an important role in the direct regulation of proliferation and function of adrenocortical cells under physiological conditions.


Metabolism of parathyroid hormone by fetal rat calvaria. These studies examine the metabolism of highly purified bovine parathyroid hormone [bPTH-(1--84)] by fetal rat calvaria. Enzymatically dispersed bone cells and intact (minced) calvaria were incubated with bPTH-(1--84) and the incubation medium was analyzed for degradation of PTH by polyacrylamide gel electrophoresis. Eluates of gel slices were assayed for immunoreactive PTH (iPTH) in carboxy- and amino-terminal RIAs. Both bone preparations metabolized bPTH-(1--84). The intact hormone progessively decreased with time and carboxy-terminal iPTH fragments were evident by 5 min of incubation. In the isolated cell preparations, intact hormone was completely degraded at submaximal doses of PTH (5 X 10(-9) M), as assessed by cAMP production. Degradation was incomplete in intact calvarial preparations at all doses studied. Intact calvaria were less sensitive to PTH with regard to cAMP production. No amino-terminal fragments were detected in the medium with either cell preparation. Oxidized (biologically inactive) bPTH-(1--84) was not metabolized in these systems. These findings contrast with studies in liver and kidney preparations, where oxidized bPTH has been shown to be degraded. These findings contrast with studies in liver and kidney preparations, where oxidized bPTH has been shown to be degraded. These data suggest that biological activity may be necessary for the metabolism of intact bPTH-(1--84) by bone cells and that skeletal tissue may contribute to the immunoheterogeneity of circulating PTH in the rat.


Intestinal calcium-binding protein and calcium absorption in cortisol-treated chicks: effects of vitamin D3 and 1,25-dihydroxyvitamin D3. Vitamin D3 in rachitic chicks stimulates calcium absorption and induces the synthesis of two pools of intestinal calcium-binding protein (CaBP), one soluble and the other membrane bound. Cortisol acetate caused a decrease in calcium absorption which was accompanied by a decrease in soluble CaBP. Cortisol was similarly effective in 1,25-dihydroxyvitamin D3-dosed chicks, suggesting that the glucocorticoid effect was not entirely due to the defective synthesis of this metabolite. Ca absorption was directly correlated with soluble CaBP and alkaline phosphatase and inversely related to the ratio of bound to soluble CaBP. It was further observed that the slope of the Ca absorption vs. soluble CaBP regression line was greater in chicks given 1,25-dihyroxyvitamin D3 compared to those given vitamin D3, and this is interpreted to mean that another factor or condition, in addition to assayed concentrations of soluble CaBP, determines the degree of calcium absorption.


Parathyroid hormone stimulation of collagenase secretion by isolated bone cells. Cells isolated from fetal rat calvaria were cultured in a [14C]glycine-labeled collagen gel. This method of culture places the cells in an environment similar to that in the intact tissue and provides a means for assaying the release of collagenase from the cells. Degradation of the collagen matrix begins within the first 3 h of cell culture, the earliest time point at which samples were taken, and continues for at least 12 h. When cells were prepared from calvaria incubated for 30 min with parathyroid hormone (PTH), there was a marked decrease in the collagenase content of the freshly isolated cells, indicating that secretion of collagenase had been enhanced by PTH. Upon subsequent culture of control and PTH-pretreated cells, there was an increase in lysis of collagen gels surrounding the hormone-treated cells within 8 h. The increased collagenolysis was prevented by exposure of the cells to puromycin before and during culture, suggesting that collagenase synthesis also was stimulated by PTH.


Oxidative metabolites of [2-14C]propylthiouracil in rat thyroid. The identities and relative amounts of the major metabolites in rat thyroids 6 h after the administration of [14C]propylthiouracil ([14C]PTU) have been investigated. Rat thyroid extracts were chromatographed on columns of Bio-Gel P-2 and DEAE-Sephadex and in various thin layer chromatography systems. The extracts contained protein-bound PTU metabolites; an unknown peak 3; peak 1, which was chromatographically similar to PTU--SO2H; peak 2, which was similar to PTU--SO3H; PTU; and small amounts of 6-n-propyluracil (PU). The major metabolites were isolated and purified by column chromatograpy. On the basis of chromatographic properties identical to cochromatographed standards in seven different systems and the products formed after treatment with various reagents, peak 1 was identified as PTU-SO2H and peak 2 as PTU--SO3H. Peak 3 was seen only on Bio-Gel P-2 columns, was very unstable, and was not similar to any known PTU standard. The properties of this compound suggest that it may be a thiolsulfonic ester (formula:see text), but the data are insufficient for positive identification. Approximately 85% of the radioactivity in the protein peak was bound to thyroglobulin. HCl converted 86.5% of the protein-bound radioactivity to PU, and H2S converted 91% to PTU, indicating that an oxidized S was involved in the linkage to protein. Dithiothreitol released 23.6% of the protein-bound radioactivity as PTU, and mercaptoethanol released 32.5%, indicating that 25-35% of the PTU is bound in disulfide linkage. Approximately 50% of the radioactivity released by mercaptoethanol was S-ethanol PTU, which suggests a PTU-protein bond similar to a thiolsulfonic ester. Quantitation of the metabolites revealed that protein-bound metabolites accounted for 21-29% of the total radioactivity, unknown peak 3 accounted for 7.1%, PTU--SO2H for 48-50%, PTU--SO3H for 8-10%, and PTU for 10.7-16.5%. Only traces of PU were observed. The data demonstrate that PTU--SO2H is the major PTU metabolite in rat thyroid and must be the compound X observed by other investigators and that all metabolites identified are oxidative products of PTU. These findings support the earlier conclusion of Taurog and Riesco that protein binding of PTU occurs as a consequence of oxidation.


Effects of glucocorticoids on microsomal membrane synthesis in hepatocytes from adrenalectomized rats. Biochemical and morphological studies were performed on livers from normal, adrenalectomized (ADX), and ADX and dexamethasone (DEX)-treated rats to investigate the effects of glucocorticoids on microsomal membrane synthesis. Overnight fasted normal, ADX and ADX rats treated 2 or 4 h with DEX received [3H]leucine and [14C]glycerol. Livers were removed, and tissue specimens were prepared for electron microscopy and tissue fractionation. Liver microsomal subfractions were prepared and subsequently washed to produce rough and smooth microsomal membranes. Radioactivity and membrane composition were determined, and glucose-6-phosphatase activity was measured in washed microsomal membranes. Adrenalectomy caused decreased microsomal membrane synthesis. Two and 4 h of DEX administration restored microsomal membrane synthesis to normal levels. ADX also caused an alteration in composition of the microsomal membranes (reflected in decreased phospholipid-protein ratios), which was restored to normal levels by 4 h after DEX administration. The earliest effects of the hormone on membrane synthesis were observed in smooth microsomes as part of a smooth endoplasmic reticulum (SER) proliferation. These findings were supported by observations made with the electron microscope. The proliferating SER was enriched in at least one component: glucose-6-phosphatase. Although the specific relationship of SER to glucocorticoid action remains unclear, the interpretation is offered that SER proliferation and alteration in glucose-6-phosphatase distribution are component parts of the total response of the hepatocyte to glucocorticoids.


Adenosine 3',5'-monophosphate-dependent protein kinase activity in soluble thyrotropin receptor complex. cAMP-dependent protein kinase activity was present in a soluble TSH receptor fraction. The Km of this enzyme was 2.2 X 10(-6) M for casein substrate in the absence or presence of 10(-5) M cAMP. A [3H]cAMP-binding protein was also found in this fraction. The Ka for [3H]cAMP-binding was 0.11 X 10(6) M-1, with a total binding capacity of 3 nmol/mg protein. After fractionation using a continuous sucrose density gradient, one of the several [125I]iodobovine TSH-binding peaks corresponded to a [3H]cAMP-binding peak. After fractionation on a sucrose density gradient containing 0.4 M NaCl at pH 6.5, a major peak of protein kinase activity was shown. This protein kinase activity was stimulated by adding 10(-5) M cAMP. A peak of [3H]cAMP-binding activity corresponded to the same peak. Protein kinase activity in the receptor fraction was stimulated by adding 6 mg/ml bovine TSH. The soluble TSH receptor fraction also has an adenylate cyclase activity stimulated by TSH. These results suggest that some TSH receptors released from thyroid plasma membranes have associated adenylate cyclase activity and cAMP-dependent protein kinase activity. The receptor, cyclase, and kinase activities may exist in a functional primary receptor unit which is spontaneously released from plasma membranes.


Resensitization of the desensitized follicular adenylyl cyclase system to luteinizing hormone. LH-induced desensitization of the adenylyl cyclase system in a cell-free membrane preparation from preovulatory porcine follicles exhibits a critical dependence upon Mg and ATP (1). The membrane-rich preparation was found to contain endogenous cAMP-dependent and cAMP-independent protein kinases as well as phosphorprotein phosphatases. Endogenous phosphatase activity was enchanced by by Mn2+ and dithiothreitol. The addition of either Mn2+ or dithiothreitol to the porcine follicular membrane preparation incubated under desensitizing conditions promoted a specific concentration-dependent reversal of the LH-induced desensitization of the adenylyl cyclase system. The addition of exogenous phosphoprotein phosphatase, partially purified from procine follicular cytosol, also reversed LH-induced desensitization in a concentration-dependent manner. Boiling of the phophatase preparation prevented reversal of desensitization. The addition of either exogenous beef heart cAMP-dependent protein kinase or heat-stable protein kinase inhibitor did not modify LH-induced desensitization of the follicular adenylyl cyclase system. These results provide indirect evidence that while LH-induced desensitization is not mediated by a cAMP-dependent protein kinase, reversal of desensitization can be promoted by activation of endogenous phosphatase and the addition of a homologous phosphatase preparation.


Production of long term steroid-producing granulosa cell cultures by cell hybridization. Primary cultures of rat ovarian granulosa cells have been used extensively to study hormonal regulation of cellular function. To date, no long term cultures of ovarian cells which retain their differentiated functions have been developed. Hypoxanthine guanine phosphoribosyl transferase-deficient simian virus 40-transformed rat ovarian granulosa cells were fused with freshly prepared rat granulosa cells using inactivated Sendai virus. Putative hybrid cell strains obtained after selection in medium containing hypoxanthine, aminopterin, and thymidine were analyzed for progesterone synthesis. Neither the original simian virus 40-transformed granulosa cell nor its hypoxanthine guanine phosphoribosyl transferase-deficient derivative produced progesterone, but three of the hybrid strains produced progesterone at basal levels and in response to dibutyryl cAMP. One of these strains produced progesterone in a dose-responsive fashion when exposed to prostaglandin E2, cholera toxin, dibutyryl cAMP, and 2-chloroadenosine. Cell strains obtained by hybridization were remarkably similar to primary cultures of granulosa cells with respect to both the magnitude and temporal aspects of progesterone production in response to dibutyryl cAMP.


Estrogens increase the number of thyrotropin-releasing hormone receptors on mammotropic cells in culture. The number of plasma membrane receptors for TRH on tumor-derived mammotropic cells in culture, GH3 and GC cells, but not their affinity for TRH, was increased by estrogens. For GH3 cells, exposure to 10 nM 17 beta-estradiol for 48 h increased the receptor level from 54,000 to 90,000 sites/cell, while for GC cells, the number of receptors increased from 29,000 to 46,000 after 28 h. PRL accumulation in the medium was also increased by 17 beta-estradiol. 17 beta-Estradiol and diethylstilbestrol were equally potent in increasing the TRH receptor level, while estrone was only 1/10th as potent. Diethylstilbestrol bound to the cytoplasmic estrogen receptor with an apparent affinity approximately 2.5 times higher than 17 beta-estradiol in GH3 and GC cells, while the affinity for estrone was only 1/12th to 1/20th that of 17 beta-estradiol. Tamoxifen, an antiestrogenic compound, inhibited the increase in TRH receptor number induced by 0.3 nM 17 beta-estradiol and was capable of binding to the estrogen receptor. Modulation of the TRH receptor level on mammotropic cells by estrogens, which is likely mediated through cytoplasmic estrogen receptors, may be an important mechanism for regulation of TRH action.


Cyclic nucleotide-dependent protein kinase modulators in thyroid tissue. A heat-stable protein which inhibits cAMP dependent protein kinase was prepared from the bovine thyroid 100,000 X g supernatant. This protein inhibited the cAMP-dependent protein kinase both from bovine thyroid cytosol and bovine thyroid plasma membranes. The inhibitory effect was noncompetitive with histone as substrate of the cytosolic enzyme. The stimulatory modulator for cGMP-dependent protein kinase was separated from the 100,000 X g supernatant by Sephadex G-100 gel filtration. The stimulatory modulator had no stimulating effect on cAMP-dependent phosphorylation, and the inhibitory modulator of cAMP-dependent enzyme had no effect on cGMP-requiring phosphorylations. The inhibitory modulator may regulate cAMP-dependent protein kinase activity in cytosol and plasma membrane, and the stimulatory modulator for cGMP-dependent protein kinase may have a role in thyroid function independent of the cAMP-requiring system.


The effect of hypophysectomy on rat chorionic somatomammotropin as measured by prolactin and growth hormone radioreceptor assays: possible significance in maintenance of somatomedin generation. We have previously reported that serum somatomedin concentrations are maintained in pregnant rats after hypophysectomy. Because rat chorionic somatomammotropin (rCS) might replace pituitary GH during pregnancy, the concentration of rCS was measured at intervals after hypophysectomy on day of pregnancy. By day 16 of pregnancy, the serum rCS of hypophysectomized (hypox) rats was actually higher than that of normal pregnant rats when measured by a lactogenic radioreceptor assay. Increased levels of lactogenic radioreceptor activity (L-RRA) were maintained in the serum of hypox rats throughout the remainder of pregnancy. The GH radioreceptor activity (GH-RRA) of serum of hypox pregnant rats was also greater than that of normal rats during the last days of pregnancy, but the activity was only about 1/20th that of the L-RRA with the assays employed. There was no significant difference between placental L-RRA and GH-RRA of normal and hypox pregnant rats. The difference in concentration could not be attributed to differences in the number of fetuses. We conclude that the high levels of rCS were sufficient to maintain serum somatomedin concentration in hypox pregnant rats. This effect of rCS could have been due to binding by GH or PRL receptors of the maternal liver.


The effects of opiate agonist and antagonist on serum prolactin in primates: possible role for endorphins in prolactin regulation. Intravenous administration of the opiate receptor antagonist naloxone produced a significant reduction in basal serum PRL concentrations in four male Macaca arctoides. Significant decreases from basal levels were found 15, 30, 45, 60, 90, 120, 150, and 180 min after the iv injection of 0.05 and 0.25 mg/kg naloxone. The iv administration of 0.4 mg/kg morphine produced rapid and significant increases in PRL levels, while 0.04 mg/kg morphine or saline produced no change. Both the dopamine receptor-stimulating agent apomorphine (0.15 mg/kg) and naloxone (0.25 mg/kg) decreased basal serum PRL and blocked the morphine-induced increases in serum PRL. These data support the hypothesis that endorphins are involved in the stimulation of PRL secretion.


Studies on adrenocorticotropic hormone receptor using isolated rat adrenocortical cells. To clarify the function of ACTH receptors, the actions of ACTH on cyclic AMP formation, Ca2+-influx across cell membrane, and corticoidogenesis were examined using dispersed adrenocortical cells prepared from the rat adrenal gland. 1) There are two types of ACTH receptors from Scatchard analysis of 125I-ACTH1-24 binding to the cell, the one receptor is of high affinity and low capacity (dissociation constant (Kd1) = 2.6 x 10(-19) M and 7,350 sites per cell), and the other one is of low affinity and high capacity (dissociation constant (Kd2) = 7.1 x 10(-9)M and 57,400 sites per cell). 2) Both apparent dissociation constants derived from the effects of ACTH on corticoidogenesis and Ca2+ influx well correspond with Kd1 of the high affinity receptor, 3) Apparent dissociation constant obtained from the effect of ACTH on cyclic AMP formation is in good agreement with Kd2 of the low affinity receptor. Thus it could be deduced from these data that the high affinity receptor is concerned with an increased Ca2+-influx to regulate corticoidogenesis at physiological levels of ACTH, whereas the low affinity receptor is coupled to adenylate cyclase at supraphysiological concentrations of ACTH.


Long-term prognosis of patients with infantile spasms following ACTH therapy. The influence of ACTH on the prognosis of patients with infantile spasms remains controversial. We have examined retrospectively the long-term benefits of initially successful ACTH therapy in patients treated at this institution between 1961 and 1974. Individuals with equivocal or minimal improvement during ACTH therapy were excluded from this study. Eighteen affected infants showed a favorable early response consisting of cessation of seizures for at least 3 weeks during ACTH therapy and concurrent disappearance of the hypsarhythmic EEG pattern. Modal age at last follow-up was 5 years (range, 15 months to 16 years). Infantile spasms recurred in 7 patients (39%), and 8 patients subsequently had other seizure types. All epileptiform (spike) activity disappeared from the EEGs of 8 patients during ACTH therapy, but in 4 of these cases epileptiform activity was present in later tracings. In the remaining 10 patients the hypsarhythmic pattern disappeared in association with ACTH therapy, but the EEG remained epileptiform (often only in sleep). Later EEGs were free of epileptiform activity in 5 of the 10 patients whose tracings contained spike discharges in the early follow-up period. Four patients (22%) were seizure free and without intellectual impairment when last evaluated.


Biological effects of raw and processed oil shale particles in the lungs of laboratory animals. Environmental and occupational health concerns will have an effect on the developing oil shale technologies. The mining and crushing of large volumes of rock will be a characteristic of at least some of these technologies, and above ground disposal of proceessed shale will require adequate control measures. Exposure by inhalation to the dusts that may arise from shale oil technologies may present a hazard both in the work force and in the local population. Animal studies dealing with the effects of oil shale-related materials in the lung are in progress. Experiments involving Syrian hamsters exposed by inhalation and by intratracheal instillation are described.


Morphological investigations of fibrogenic action of Estonian oil shale dust. A review of morphological investigations carried out to clarify the pathogenicity of industrial dust produced in the mining and processing of Estonian oil shale is given. Histological examination of lungs of workers in the oil shale industry taken at necropsies showed that the inhalation of oil shale dust over a long period (more than 20 years) may cause the development of occupational pneumoconiotic changes in oil shale miners. The pneumoconiotic process develops slowly and is characterized by changes typical of the interstitial form of pneumoconiotic fibrosis in the lungs. Emphysematous changes and chronic bronchitis also occur. The average chemical content of oil shale as well as of samples of oil shale dust generated during mining and sorting procedures is given. The results of experiments in white rats are presented; these studies also indicate a mild fibrogenic action of Estonian oil shale dust.


Experimental approach to the determination of pulmonary carcinogenic influences of shale oil effluents. Oil derived from oil shale deposits is known to contain many organic complexes. The formation of carcinogenic hydrocarbons is temperature-dependent and is associated with retorting of the oil. Furthermore, oil shale is a rich source of inorganic elements such as the metals. Biological studies have demonstrated that concentrated extract of tars from combustion of shale oil are carcingenic to the skin of mice. The purpose of the current project is to evaluate the potential carcinogenic hazard from inhalation of retort and combustion effluents for man. These studies will be carried out in pathogen-free rats by intratracheal instillation with and without added factors such supplemental particles and known carcinogens as interactants.


The 'enzyme-probe' method for characterizing metabolite pools. The use of NAD-glycohydrolase in human erythrocyte sonicate as a model system. 1. An approach for testing the homogeneity of metabolite pools is described. An alien enzyme that can attack the metabolite in question is introducted into the system studied. By analyzing the time-course of decomposition of the metabolite it can be decided whether the pool is homogeneous in respect of reactivity towards the probe-enzyme or can be divided into fractions of different reactivities. 2. The information obtainable from such experiments is illustrated by the case of human erythrocyte sonicate as model system with NAD-glycohydrolase as probe-enzyme. The nicotinamide adenine dinucleotide pool in the concentrated sonicate could be resolved into three fractions (I, II and III) with half-lives of about 1, 7 and 240 min, respectively. Fraction I is free NAD, fraction II is NAD bound to glyceraldehyde-3-phosphate dehydrogenase, and fraction III is coenzyme strongly bound to some, so far unidentified, protein. Sonicate glycolysis seems to require only fraction II and is unable to use fraction III under the experimental conditions applied. 3. The scope of application of the enzyme-probe method is discussed.


On the nature of biochemically generated hydroxyl radicals. Studies using the bleaching of p-nitrosodimethylaniline as a direct assay method. An efficient scavenger for radiolytically generated hydroxyl (OH) radicals, p-nitrosodimethylaniline, was used to try to substantiate the presence of this oxygen radical species in several biochemical systems. Most of these systems which were investigated had previously been assumed to generate OH radicals, e.g. the autoxidation of 6-hydroxydopamine, the hydroxylating system NADH/phenazine methosulfate, and the oxidation of xanthine or acetaldehyde by xanthine oxidase. We did not observe inhibition of the bleaching of p-nitrosodimethylaniline in oxygenated solutions by other scavengers of OH radicals nor, in the case of xanthine/xanthine oxidase, by catalase and superoxide dismutase. We therefore conclude that, under biochemical conditions as opposed to radiolysis or photolysis, no freely diffusable OH radicals are formed. Rather, a strongly oxidizing OH-analogous complex is considered to represent the p-nitrosodimethylaniline-detectable species formed under these conditions.


Structure of the disaccharide chain of galactosyl-N-acetylgalactosaminyl-protein synthesized in vitro. In order to obtain a [14C]galactosyl-N-acetylgalactosaminyl-protein which would be useful as an acceptor in studies on the specificity of glycosyltransferases, a porcine submaxillary gland microsomal galactosyltransferase preparation was used for the galactosylation in vitro of N-acetylgalactosaminyl-protein (desialylated ovine submaxillary mucin). The newly formed oligosaccharide unit was obtained as a reduced disaccharide after alkaline borohydride treatment of the [14C]galactosyl-N-acetylgalactosaminyl-protein product and as glycopeptides by proteolytic digestion of the glycoprotein. The reduced disaccharide consisted of equimolar amounts of galactose and N-acetylgalactosaminitol and was characterized by thin-layer chromatography, high-voltage electrophoresis and gas-liquid chromatography. Periodate oxidation studies on the reduced disaccharide revealed that [14C]galactose was linked to position C-3 on the N-acetylgalactosaminyl residue. Digestion of the reduced disaccharide and the glycopeptides with galactosidases gave equivocal results as to the anomeric configuration of the [14C]galactose residue. Nuclear magnetic resonance of the reduced disaccharide, however, definitely indicated that the configuration was beta. The specificity of the porcine submaxillary gland galactosyltransferase thus can be defined as a uridine diphosphogalactose: alpha-D-N-acetylgalactosaminyl-protein beta 1 leads to 3 transferase activity.


Cell-mediated immunity to antigens associated with murine sarcoma virus-induced tumors: augmentation of cytolytic T lymphocyte activity by successive specific and nonspecific stimulation in vitro. It has been shown previously that lymphoid cells from mice which have rejected a tumor induced by the Moloney sarcoma virus-leukemia virus (MSV-MLV) complex develop high levels of specific H-2-restricted cytolytic T lymphocyte (CTL) activity after in vitro stimulation with syngeneic, irradiated MLV-induced lymphoma cells in mixed leukocyte-tumor cell cultures (MLTC). Attempts to further increase lytic activity by restimulating long-term MLTC cells with syngeneic, irradiated lymphoma cells have met but with limited success. This report shows that, in contrast to the lack of increased activity observed after specific stimulation with lymphoma cells, nonspecific stimulation of long-term MLTC cells either with supernatants from secondary mixed leukocyte cultures (2 degrees MLC SN) or with supernatants from concanavalin A-stimulated spleen cells leads, on a per cell basis, to a further 5 to 10-fold increase in CTL activity. The stimulatory activity of 2 degrees MLC SN is due to a factor(s) of apparent mol. wt. of 25,000 to 40,000. The activity of the CTL populations formed under these conditions is at least 100-fold higher against syngeneic as compared to allogeneic MLV-induced or unrelated tumor cells.


Changes in the fibroblastoid colony forming unit population from mouse bone marrow in early stages of Soule virus induced murine leukemia. Fibroblastoid cells from mouse bone marrow belong to the hemopoietic inductive microenvironment involved in the regulation of hemopoiesis (1). We observed a decline in the incidence of precursors of these cells (fibroblastoid colony forming units) in the early stages of viral leukemogenesis. This was not accompanied by similar changes in bone marrow derived hemopoietic colony forming cell populations (CFU-s and CFU-c). Cultured fibroblastoid colonies from the bone marrow of young AKR mice or Soule murine leukemia virus inoculated BALB/c mice were found to produce type-C ecotropic virus. No such production was observed when similarly cultured granulocyte macrophage colonies (CFU-c) from the bone marrow of these mice were examined. The possibility that the fibroblastoid cell population is a major source of ecotropic leukemia viruses in the early stages of viral leukemogenesis is discussed.


Seasonal variation of androgen interconversion in testicular tissue of Rana temporaria in vitro. Testicular steroid metabolism of winter and spring frogs, Rana temporaria, were studied by in vitro incubation with radioactively labelled dehydroepiandrosterone and androst-4-ene-3,17-dione. Marked seasonal differences were observed which are in line with the findings of others.


Increase of interferon antiviral activity by exogenous cyclic adenosine-3':5'-monophosphate (cAMP). Some effects of cAMP on replication of Semliki Forest Virus in chick embryo fibroblast cell cultures are described. Depending on concentration, the incorporation of [3H]-uridine into viral RNA or the formation of plaque-forming units is inhibited; the highest concentration tested was 8 mM. Cyclic AMP has an effect of its own and increases the Interferon action in the lower concentration ranges of Interferon (up to 1 unit/ml). The effect of cyclic AMP is fast, needs no induction and is also visible in late phases of viral replication. However, these experiments do not establish a causal relation between cAMP and Interferon.


Functional consequences of treating turkey liver fructose-1,6-bisphosphatase with penicillin G. Treatment of turkey liver fructose-1,6--bisphosphatase with penicillin G progressively inactivated the enzyme and desensitized the enzyme toward high substrate inhibition. The treatment also led to reduced sensitivity to AMP inhibition and the loss of cooperative interaction among AMP-binding sites. These altered properties were not reversed by dialysis, but were prevented when treatment with penicillin G was perfomed in the presence of substrate.


Effects of insulin on plasma fibrinogen levels in rats submitted to tissue injury or ACTH administration. Insulin is necessary to produce an increase of plasma fibrinogen in rats submitted to tissue injury or ACTH administration. This increase is more significant when endogenous or exogenous excess of insulin is present, while in uninjured rats the absence or excess insulin does not modify plasma fibrinogen.


Cytotoxicity of ethyl methanesulfonate in mice spermatogonia. Enumeration of different types of spermatogonia, following a single i.p. administration of different doses of ethyl methanesulfonate in mice, showed a survival of A1-A4 and in spermatogonia is markedly reduced due to cell killing while the remaining types of spermatogonia were affected marginally. The cell killing effect was dose-dependent, and replenishment of these cells was observed by the end of one cycle of the seminiferous epithelium comprising of 8.5 days.


On the effects of thiamphenicol and chloramphenicol on nucleic acid and protein synthesis in rabbit bone marrow cells in vivo and in vitro. Besides the in vivo effects of thiamphenicol (TAP), this study shows the in vitro effects of TAP and D- and L-threo-chloramphenicol (CAP) on the synthesis of DNA, RNA, protein and hemoglobin in marrow cells and reticulocytes. The experiments make it likely that TAP exerts its action on a stem cell in a proliferating phase.


[Evaluation of the pharmacological properties of a new steroid curarelike agent, RGH 1106]. Tests on cats showed the compound RGH 1106 to possess an antidepolarizing mechanism of action. The sequence of myorelaxation, arising under the effect of this agent, is characterized by relaxation in the first place of the musculus masseter, muscles of limbs, then of abdominal muscles and those of the diaphragm and, finally, of the intercostal muscles. RGH 1106 doses not possess cardiotropic m-cholinolytic action, does not exert a ganglion blocking effect, nor affects the central nervous system. It neither changes the blood supply of the myocardium and the uptake of oxygen by the latter, nor inhibits the acetylcholinesterase.


[Effect of aggregation inducers and inhibitors on the pentosephosphate pathway enzymes of glucose conversion in the thrombocytes]. Experiments with rabbit platelets in vitro revealed differences in the effect of the aggregation inducers (ADP and thrombin) and the aggregation inhibitor cyclic AMP on the activity of the enzymes of pentosephosphate pathway of glucose oxidation. ADP and thrombin decrease significantly the activity of glucoso-6-phosphate dehydrogenase (G-6-PDH) and riboso-5-phosphate-metabolizing enzymes (R-5-PME) in platelets during aggregation, whereas cyclic AMP produces no appreciable effect on the G-6-PDH activity, but increases significantly the R-5-PME activity. ADP decreases and cyclic AMP raises substantially the activity of R-5-PME. The differences were also revealed in the effects produced by cyclic AMP and cyclic GMP on the enzymes of pentosephosphate pathway of glucose oxidation. Unlike cyclic AMP, cyclic GMP decreased significantly the activity of G-6-PDH. The activity of R-5-PME and cyclic GMP transketolase was more pronounced than that of cyclic AMP. Like cyclic AMP, cyclic GMP differs from ADP and thrombin in the action produced on the enzymes of pentosephosphate pathway of glucose oxidation.


Long-lasting synaptic potentials and the modulation of synaptic transmission. Long-lasting postsynaptic potentials (PSPs) generated by decreases in membrane conductance (permeability) have been reported in many types of neurons. We investigated the possible role of such long-lasting decreases in membrane conductance in the modulation of synaptic transmission in the sympathetic ganglion of the bullfrog. The molecular basis by which such conductance-decrease PSPs are generated was also investigated. Synaptic activation of muscarinic cholinergic receptors on these sympathetic neurons results in the generation of a slow EPSP (excitatory postsynaptic potential), which is accompanied by a decrease in membrane conductance. We found that the conventional "fast" EPSPs were increased in amplitude and duration during the iontophoretic application of methacholine, which activates the muscarinic postsynaptic receptors. A similar result was obtained when a noncholinergic conductance-decrease PSP--the late-slow EPSP--was elicited by stimulation of a separate synaptic pathway. The enhancement of fast EPSP amplitude increased the probability of postsynaptic action potential generation, thus increasing the efficacy of impulse transmission across the synapse. Stimulation of one synaptic pathway is therefore capable of increasing the efficacy of synaptic transmission in a second synaptic pathway by a postsynaptic mechanism. Furthermore, this enhancement of synaptic efficacy is long-lasting by virtue of the long duration of the slow PSP. Biochemical and electrophysiological techniques were used to investigate whether cyclic nucleotides are intracellular second messengers mediating the membrane permeability changes underlying slow-PSP generation. Stimulation of the synaptic inputs, which lead to the generation of the slow-PSPs, increased the ganglionic content of both cyclic AMP and cyclic GMP. However, electrophysiological analysis of the actions of these cyclic nucleotides and the actions of agents that affect their metabolism does not provide support for such a second messenger role for either cyclic nucleotide.


Functional role of serotonergic neuromodulation in Aplysia. The serotonergic metacerebral cell (MCC) of the mollusk Aplysia produces slow synaptic potentials in motor neurons of the buccal muscle, and increases the rate of ongoing rhythmic burst output of the buccal ganglion. In addition, the MCC acts peripherally to enhance the strength of buccal muscle contractions that are produced by firing of motor neurons. The potentiation of contraction is not associated with any detectable changes of resting membrane potential of muscle cells. Although MCC activity produces a small enhancement of excitatory junctional potentials, several experiments clearly indicate that the MCC has a direct potentiating effect on excitation-contraction coupling. The data suggest that potentiation of contraction might be mediated by cAMP. For example, activity of the MCC enchances the rate of accumulation of cAMP in buccal muscle, application of phosphodiesterase resistant analogs of cAMP potentiates muscle contraction, and a phosphodiesterase inhibitor enhances the effect of MCC stimulation. Recordings from free-moving animals indicate that the MCC becomes activated by exposure of the animal to food stimuli, and that the activation parallels the presence of a food-arousal state. Food-arousal is characterized by enhanced strength and increased frequency of biting responses. Both these effects can result from activity of the MCC. Thus, in this system, modulatory synaptic actions function to provide the substrate for a type behavioral modulation.


The role of sodium transport in thyroid thermogenesis. The hypothesis that augmentation of Na+ transport activity mediates a significant fraction of thyroid thermogenesis is supported by studies on a wide range of target tissues. Recent criticisms based on theoretical and experimental considerations do not appear to negate the validity of this concept. A basic difficulty that remains to be resolved, however, is the development of techniques for assessing the contribution of the Na+ transport pathway to energy turnover in the intact animal.


Cellular events underlying catecholamine-induced thermogenesis: cation transport in brown adipocytes. Evidence supporting the hypothesis that the active transport of Na+ and K+ contributes significantly to catecholamine-mediated brown fat thermogenesis is reviewed. Such evidence indicates that the Na+/K+ membrane pump is activated following norepinephrine interaction with alpha- and beta-adrenergic membrane receptors. That the involvement of the pump in the thermogenic response is considerable is indicated by the finding that pump inhibition is accompanied by a 50--70% reduction of the catecholamine-evoked respiratory stimulation of isolated hamster brown adipocytes. This inhibitory effect of pump blockade suggests that in the norepinephrine-activated brown adipocyte, the pump generates a stimulatory signal for pathways of substrate oxidation, the operation of these being associated with conversion of chemical energy to heat.


Cyclic nucleotides and neuromuscular transmission. A review of the research on cyclic nucleotides and neuromuscular transmission suggests that cAMP is involved in the release of transmitter from motor nerve endings. Lipid-soluble derivations of cAMP cause depolarization of unstimulated nerve endings and prolong the after potentials of stimulated nerve endings. They also increase the frequency of miniature end plate potentials and increase the quantal content of stimulus evoked end plate potentials. Similar effects are produced by compounds that activate adenylate cyclase or inhibit phosphodiesterase. The responses to the derivatives of cAMP and activators of cyclase are enhanced by inhibitors of phosphodiesterase and prevented by compounds that block the flux of calcium into nerve endings. There is no evidence that suggests that cyclic nucleotides are involved in the postjunctional response to transmitter. Thus, it seems likely that cAMP is involved in the regulation of calcium in motor nerve endings and the exocytosis of transmitter. Additional study should expand our knowledge of neuromuscular transmission and contribute to an understanding of the functions of cyclic nucleotides in other synapses.


Cyclic nucleotides in central synaptic function. A wide array of interdisciplinary experiments have served to strengthen the general premise that some central synaptic effects of neurotransmitters may be mediated by cyclic nucleotides. Specific instances of such second messenger mediation are most strongly supported for certain noradrenergic connections of the locus coeruleus (LC) and for dopaminergic connections within the caudate nucleus. In these sites catecholamines selectively activate intraneuronal cyclic AMP synthesis, and exogenously applied cyclic AMP closely mimics the biophysical actions of the catecholamine on target neurons. In cerebellar cortex, iontophoresis of norepinephrine, stimulation of LC, or iontophoresis of cyclic AMP lead to hyperpolarization and increased membrane resistance. Although overtly inhibitory when examined in isolation, the effects of the LC system coupled to adenylate cyclase can initiate a holistic set of target cell responses which can enhance or "enable" the actions of other synaptic inputs to the target cells. Electrophysiologic and immunocytochemical evidence suggests that this heterosynaptic interaction may arise from phosphorylation of the synaptic membrane substrate of cyclic AMP-dependent protein kinase, or Protein 1.


Toxic substances and cell membrane function. The exposed location and functional importance of cell membranes make them particularly susceptible to the toxic effects of many chemicals. The likelihood of such effects has been appreciated for many years. However, the recent advent of new techniques has greatly increased our understanding of the complexities of membrane structure and function. These data make it quite clear that the interaction of toxic compounds with either the protein or the lipid component of cell membranes may substantially alter membrane function. This paper summarizes the current concepts of membrane structure and function and discusses the techniques currently in use to study cell membranes. Several examples are presented in which xenobiotics significantly alter membrane function. These include effects of heavy metals on passive ion permeability, impairment of osmoregulation and calcium transport by organochlorine pesticides, inhibition of the transport of neurotransmitter metabolites by phenoxyacetic acid herbicides in choroid plexus, and reduction in intestinal nutrient transport by heavy metals. Hence the study of the interactions of foreign compounds with membrane function may enhance our understanding of mechanisms both of toxicity and of basic membrane function.


Effect of prolactin on the calcium binding and/or transport of ejaculated and epididymal human spermatozoa. The prolactin concentration in human seminal plasma and in human epididymal cauda fluid was assessed by radioimmunoassay. The prolactin concentration in cauda plasma was found to be similar to that found in male blood serum (5.5 to 9.1 ng/ml) and significantly lower than the concentration of the hormone found in seminal plasma obtained either from euspermic (48 +/- 12 ng/ml) or from vasectomized volunteers (50 +/- 10.2 ng/ml) (P less than 0.001). Calcium binding and/or transport in ejaculated spermatozoa were found to be little (0.29 +/- 0.08 nmoles/10(8) cells) and dependent on a quickly saturable process. The addition of 200 ng of human prolactin/ml induced a 60% increase in this parameter, while 50 ng of prolactin/ml were ineffective. Epididymal human spermatozoa differ from ejaculated sperm cells in showing greater, time-dependent, calcium binding and/or transport under basal conditions (2.00 +/- 0.35 nmoles/10(8) cells/hour), and in being more susceptible to the stimulating action of prolactin (4.4 +/- 0.68 nmoles/10(8) cells/hour in the presence of 50 ng of prolactin/ml).


Somatostatin: lack of effect of cyclic AMP release and amino acid transport in isolated rat hepatocytes. The aim of the present study was to determine whether or not somatostatin can directly affect amino acid transport and cyclic AMP (cAMP) release in isolated rat hepatocytes. Somatostatin at 1.5 microgram/ml (1mumol/l) had no effect on basal uptake of alpha-aminoisobutyric acid (AIB). Similarly, the peptide was without effect on basal cAMP release. Somatostatin exerted a slight but statistically not significant inhibitory effect on glucagon-stimulated AIB uptake and cAMP release. These observations do not support the possibility that somatostatin might directly interfere with hepatic glucose metabolism by altering the entry of amino acids into the liver and--or--by affecting the level of endogenous cAMP.


Effect of somatostatin on the potentiating action of glucagon, cyclic adenosine monophosphate and theophylline on glucose-induced insulin release. The effect of glucagon on the inhibition of the insulin response to glucose induced by somatostatin was investigated in humans and in the isolated perfused rat pancreas. Both in vivo and in vitro somatostatin suppressed glucose-induced insulin release. This inhibitory effect of somatostatin was overcome by glucagon. Similar results have been observed in vitro by the infusion of theophylline or cyclic adenosine monophosphate.


Analysis of the progress of hormone-receptor amplification during differentiation in regenerating planarians. Planarians developing from isolated regenerates, when pretreated with epinephrine at different periods of their second regeneration, show a higher rate of sugar uptake than the controls, which were not treated with epinephrine. The hormone receptor development can be influenced most effectively by pretreatments performed during the 1--3 and 6--9 day periods, though some effect can be evoked at any time in the course of the second regeneration. The head regenerates developing from the anterior end of the original midpiece (and regenerating tails the second time, B1) were found to be most sensitive, while the similar regenerates developing from the hindpiece of the original planarian (C) showed the weakest reactivity. The experiments provide further evidence that the hormone receptors' activity can be amplified in the course of regenerative development, leading thus--at a later stage--to a more intense sugar uptake response of the cells.


Titration of total binding sites for growth hormone in rabbit liver. Quantitative modifications of these sites during pregnancy. The present work outlines various kinetic parameters of the interaction between bGH (bovine growth hormone) and a receptor located on the membranes of rabbit liver. A dissociation procedure, which strips the hormone off its receptor has been worked out, by varying MgCl2 concentrations and times of contact; exposure to 4 M MgCl2 for 15--30 min was found optimal for dissociation, without denaturing the receptor, as shown by the possibility of rebinding the hormone to the desaturated membranes. This method has been applied to titrate growth hormone receptors in rabbit liver, during pregnancy and lactation. The first half of pregnancy is characterized by a gradual increase of receptor levels and low saturation by endogenous hormone; conversely at parturition, there occurs a striking increase in receptors, which, however, may be demonstrated only after desaturation. During the lactational period, the receptors reamin in part saturated. The results suggest that growth hormone levels increase at parturition and during lactation in the rabbit and hence may play an important role during lactation. The factors which modulate receptor levels at the same period remain unknown.


Specific binding of prostaglandin F2 alpha to membranes of rat corpora lutea. Prostaglandin F2 alpha (PGF2 alpha) binds specifically to a partially purified membrane preparation from rat corpora lutea. The high affinity, low capacity binding component asa a Kd = 4.7 nM and has a capacity of 0.38 pmol/mg protein. Binding kinetics were temperature-dependent with an association rate constant of 2.5 x 10(5) 1/mol-sec and a dissociation rate constant of 4.3 x 10(-4) sec-1 at 22 degrees C. Little competition for binding was shown by other prostaglandins and prostaglandin metabolites; the PGF2 alpha analogue ICI 81008 (16-m-trifluoromethylphenyl-prostaglandin F2 alpha) showed a binding affinity similar to that of PGF2 alpha. The specific binding of PGF2 alpha to luteal cell membranes was confirmed by electron microscopy using a ferritin--PGF2 alpha conjugate. Ferritin--PGF2 alpha was found predominantly on luteal cell surfaces; little binding occurred on other types of cells present. These data demonstrate specific binding of PGF2 alha to rat luteal membranes. It is suggested that the luteolytic action of PGF2 alpha in the rat may be receptor-mediated.


Down-regulation of testicular androgen biosynthesis and LH receptor levels by an LHRH agonist: role of prolactin. Inhibition of plasma prolactin levels by 2-bromo-alpha-ergocryptine (CB-154) caused a 60% decrease and potentiated the inhibitory effects of [D-Ala6,des-Gly-NH2(10)]LHRH ethylamide on testicular LH receptor levels. Animals treated with the LHRH agonist showed reduced plasma and testicular testosterone levels and elevated progesterone concentration. This progesterone rise was further increased in animals having high circulating prolactin levels but was  prevented by CB-154. These data demonstrate that: (1) treatment with the LHRH agonist induces a blockage in the steroidogenic pathway at a step between progesterone and testosterone and (2) prolactin levels to an apparent accentuation of this blockage reflected by higher progesterone levels.


Suppression of follicular cAMP and steroid response to gonadotrophins by pretreatment with gonadotrophin. This study examines the effect of preincubation of sheep ovarian follicles with hCG or FSH on the ability of isolated theca and granulosa to increase cAMP levels in response to a second incubation with hCG or FSH. Secondly, the effect of preincubation of small follicles with hCG on androstenedione and testosterone production is examined in intact follicles. Preincubation with hCG resulted in a decrease in the ability of theca from large (4--6 mm in diameter) and small (1--3 mm) follicles and granulosa from large follicles to increase cAMP levels in response to a second exposure to hCG. Similarly, preincubation with FSH suppressed the response of isolated granulosa from small and large follicles to a second exposure to FSH. A partial, but significant inhibition of the response to the other hormone was observed after preincubation with both FSH and hCG. Intact small follicles pretreated with hCG were unable to increase testosterone secretion in response to a second exposure to hCG.


Oestrogen-induced increase in uterine cGMP content: a true hormonal action? The dependency of the oestrogen-induced increase in uterine cGMP content towards the cytosol-nuclear receptor system was investigated. The following observations were made: (1) With oestradiol-17 beta (E2-17 beta), U11-100A (UA) or CI-628 (CI) the cGMP response elicited in the uterus of immature rats followed a course that was parallel to (yet delayed by about 1 h from it) the known time-course evolution of nuclear occupancy by the complex formed by each compound with the oestrogen-receptor. (2) While a marked (about 2-fold) increase in uterine cGMP content was obtained with 0.1 microgram E2-17 beta, oestradiol-17 alpha (E2-17 alpha) given at the same dose had no effect on uterine cGMP. (3) The 2--3 h response to E2-17 beta (or to UA) could not be obtained in animals that had received a first injection of E2-17 beta, 2 h, or of one of the anti-oestrogens UA or tamoxifen, 20--22 h prior to the test injection of E2-17 beta. Those 3 treatments have in common that, at the time indicated, they create a state of depletion in the uterine cytosolic receptor population. The cGMP response to E2-17 beta was restored 20--22 h following a first injection of E2-17 beta. This time is known, in this case, to correspond to full replenishment of the cytosol-receptor population. In all those tests, the wet weight increase, measured in the same organs, behaves exactly as did the cGMP response. These results support the conclusion that the increase in uterine cGMP after oestrogen administration to the immature rat, represents a true hormonal action which, like other uterotrophic actions of oestrogens, involves binding of the hormone by the cytosol receptor.


Use of reflected green light for specific identification of islets in vitro after collagenase isolation. A simple technique is described that permits specific identification in vitro of isolated islets as contrasted with samll lymph nodes.


High concentration of gamma-aminobutyric acid in pancreatic beta cells. The gamma-aminobutyric acid (GABA) concentration of pancreatic islets in rats treated with streptozotocin (STZ) and of human insulinoma tissue was studied. Seven hours after the administration of 65 mg/kg body weight of STZ, a distinct increase in serum insulin concentration and at the same time a decrease in blood glucose level were seen. Twenty-four hours after the injection of STZ, however, the level of serum insulin decreased much, whereas that of blood glucose increased considerably. On the other hand, the GABA concentration of the islet was reduced dramatically to about one-tenth the control level after both 7 and 24 h. The histologic investigations of the islets revealed the destruction of B cells but no changes in A and D cells 7 and 24 h after the treatment of STZ. Nerve fibers and nerve endings in the islets were preserved intact all through the study. The GABA and insulin contents of the two cases of human insulinoma were determined. One insulinoma, which was compactly occupied with B cells according to its histologic features, contained a high concentration of GABA. The other tumor, having a rather sparse distribution of B cells in it as compared with the former case, possessed a lower concentration of GABA, but it was still high compared with that of its surrounding tissues. The present observations indicate that a large amount of GABA is available in the B cells of the pancreatic islets.


Hepatic effects of chlorpropamide: inhibition of glucagon-stimulated gluconeogenesis in perfused livers of fasted rats. In perfused livers of rats fasted for 24 h, glucagon (5 x 10(-10) M) significantly elevated tissue and perfusate levels of cyclic AMP and caused a twofold increase in glucose formation from lactate. Chlorpropamide (0.8 x 10(-3) M) consistently blocked these effects. Measurements of metabolic intermediates suggest that chlorpropamide may inhibit gluconeogenesis by antagonizing the action of glucagon on the phosphoenolpyruvate cycle. In the experiments described, chlorpropamide did not lower hepatic ATP concentration or energy charge, and exerted its effects at perfusate concentrations comparable to serum concentrations reported in patients on maintenance doses of the drug.


Hepatic glucose-6-phosphatase activities and correlated ultrastructural alterations in hepatocytes of diabetic rats. Cell fractionation, enzyme analysis, and electron microscopy were used to study the effects of streptozotocin-induced diabetes and insulin replacement on liver structure and function. In liver homogenates from diabetic rats, glucose-6-phosphatase (G-6-Pase) activity was stimulated about 2 1/2-fold over that found in normal animals. Analyses of isolated rough and smooth microsomes from diabetic rats for G-6-Pase activity showed a fourfold increase in the smooth microsomes and a small increase in enzyme activity in rough microsomes when compared with these fractions from control animals. Associated with the increased enzyme activity was a reduction in liver glycogen. Insulin treatment of the diabetic rats caused a fall in homogenate G-6-Pase levels to approximately normal values and stimulated the accumulation of hepatic glycogen. Administration of insulin to these animals also caused a decrease in G-6-Pase activity, which was most pronounced in the smooth microsomes. Studies with the electron microscope revealed ultrastructural alterations in livers of the diabetic rats, which were most striking in the periportal region of the lobule. Periportal hepatocytes from diabetic rats displayed dispersed particles of glycogen separated by cytoplasm rich in SER rather than dense masses of glycogen with little SER, as is characteristic of these cells in normal animals. Centrilobular cells from the diabetic animals displayed some disorganization of the RER and a dispersed pattern of glycogen with abundant SER, similar to the pattern found in these cells from normal animals. After insulin treatment the periportal cells appeared normal morphologically, whereas the centrilobular hepatocytes displayed regions of both dense masses and dispersed glycogen. In the glycogen masses, little SER was found; however, in the areas of dispersed glycogen particles, an abundance of this organelle was evident. We conclude from these studies that diabetes causes an increase in amount of hepatic smooth endoplasmic reticulum (SER), especially within periportal hepatocytes. The results of cell fractionation indicate that membranes of the smooth endoplasmic reticulum are enriched in G-6-pase. We interpret these results to indicate that diabetes causes hepatocytes to form additional smooth endoplasmic reticulum with specialized membranes, at least with respect to G-6-Pase. It is suggested that this cellular specialization is a response of the hepatocyte to the diabetic state, namely, a demand for increased hepatic glucose production and release into the blood stream, thus contributing to the hyperglycemia characteristic of this disease. Insulin administration to the diabetic animals reverses the above alterations.


Heterotransplantation of human stomach carcinoma in nude mice. Thirty-five primary tumors and 14 metastatic lymph nodes from stomach cancer patients were transplanted in nude mice. Successful transplantation was obtained in 16 cases of primary tumors and 5 cases of metastatic lymph nodes. The tumors growing in the nude mouse displayed histological features similar to those of the original human tumors. Eight cases of the successful takes have been maintained by consecutive transplantation. Nine cases have been stored in liquid nitrogen. The transplanted tumors were observed to spread to the distant lymph nodes in 3 cases out of the 17 successive transplants.


Immunological cross reaction between sera from patients with breast cancer and mouse mammary tumor virus. Sera of Japanese women with breast cancer and without disorders of the mammary gland gave positive immunofluorescence reaction with mouse mammary tumor cells (MMT cells) producing type-A and type-B virus particles. Among these, 55.1% (49/89) of the sera from patients with breast cancer reacted with MMT cells, whereas 60% (3/5) of the sera from patients with benign mammary hyperplasia (2 of 3 mastopathies, 1 of 2 cystosarcoma phyllodes), 30% (12/40) of the patients with other than breast cancer (4 of 14 stomach cancers, 6 of 20 uterine cancers, 1 of 3 lung cancers, 1 of 3 rectum cancers), and 26.5% (18/68) of the sera from apparently healthy women showed positive reaction with MMT cells. Distribution pattern of the specific fluorescence obtained with positive human sera was similar to that resulting from the reaction with rabbit antiserum of type-A particles. Results of absorption studies and blocking tests also suggested that the reaction was due to the antigenic components common to type-A and -B particles. Furthermore, the results of membrane immunofluorescence tests suggested that some human sera also react with envelope antigen(s) of B particles. Antinuclear antibody was found in sera from 15.7% (14/89) of breast cancer patients by the indirect immunofluorescence tests, and antibody cross-reacting with mouse mammary tumor virus was not found in most of the sera from breast cancer patients who have antinuclear antibody.


Clinical studies on carcinoembryonic antigen in pancreatic cancer. We studied serum carcinoembryonic antigen (CEA) levels in 82 patients. Thirty-four of these had benign diseases while 48 had malignant diseases. Highest incidence and levels of CEA occurred in the sera of patients with pancreatic cancer and stomach cancer. In this paper we focused our particular attention on the serum CEA of 25 pancreatic cancer patients, and examined differences in serum CEA levels in relation to histologic differentiation and sites of pancreatric cancer. No statistical difference in serum CEA level was found among various histologic types and sites of the pancreatic cancer. Clinical courses of two patients with pancreatic cancer were also studied. Serial determinations of CEA levels are most useful in assessing the effect of operation or chemotherapies and are a useful indicator for differentiating pancreatic cancer from chronic pancreatitis but cannot be a conclusive factor for the diagnosis. Finally, we correlated serum CEA levels with those of RNase and confirmed a positive correlation.


Well-differentiated peripheral cholangiocarcinoma with an unusual clinical course. A patient with an unresectable well-differentiated bile duct tumor who survived for 15 yr after biopsy diagnosis is presented. Histologic examination of the tumor revealed bland features of bile duct adenoma despite extensive spread within the liver. Over its subsequent course, the tumor progressively replaced the liver, achieving huge size, although there was no evidence of metastases until shortly before the patient's death. This clinical course was very unusual for either bile duct adenoma or cholangiocarcinoma, but would be more characteristic of another tumor of intrahepatic bile duct origin, the biliary cystadenoma. However, this latter diagnosis was excluded with both gross and microscopic pathologic criteria. Evidence is presented to support classification of this tumor as an unusual varient of peripheral cholangiocarcinoma which requires correlation of the clinical and pathologic findings for correst diagnosis.


RNA synthesis of vesicular stomatitis virus. VIII. Oligonucleotides of the structural genes and mRNA. The single-stranded RNA genome of vesicular stomatitis virus (VSV, Indiana serotype, San Juan strain) yields approx. 75 RNase T1-resistant oligonucleotides ranging in size from 10 to 50 bases. Each of the five structural genes, isolated as duplex RNA molecules hybridized to complementary mRNA, contains two or more of these large oligonucleotides. One of the oligonucleotides is identified as part of the non-coding region near the 3' end of the genome. Comparison of these results with others indicate that the RNA sequence of VSV is apparently stable in the laboratory but not in the wild. RNase T1-resistant oligonucleotides are also shown for all five VSV mRN species. Whether the mRNA for these digestions are are isolated from duplex RNA molecules or as single-stranded RNA species, the oligonucleotide patterns for each mRNA are virtually identical, indicating that each mRNA is transcribed from contiguous sequences on the genome. Comparison with published oligonucleotide patterns obtained from other isolates of VSV or from VSV deletion mutants indicate that identity and changes in their genome structure can be correlated with specific structural genes.


Decrease in serum receptor-reactive somatomedin in diabetes. Somatomedin in rat serum has been measured by a sensitive radioreceptor assay using 125I-labelled human somatomedin and human placental membrane. In rats made diabetic with strepotzotocin, receptor-reactive somatomedin levels were decrease by up to 75%. The decrease followed the time course of increasing serum glucose and occurred to the same extent in rats aged between 4 and 40 weeks. Endogenous serum receptor-reactive somatomedin appeared exclusively in high molecular weight fractions on gel chromatography. In diabetes the decreased somatomedin was due to a fall in this high molecular weight activity, but was not accompanied by a fall in somatomedin binding protein. These results suggest a role for insulin in maintaining serum somatomedin levels.


Normal fluctuations in the concentration of corticosteroid and adrenocorticotrophin in the plasma of foetal and pregnant sheep. Changes in the concentration of adrenocorticotrophin and corticosteroid in the plasma of pregnant and foetal sheep have been followed at different times of day. Wide fluctuations were seen in the concentrations of both in the foetus and ewe, although no evidence for a diurnal rhythm in the ewe was obtained. The foetal plasma adrenocorticotrophin was higher at 07.00-11.00 h than at 21.00-01.00 h, but no corticosteroid rhythm was observed. A consistently close relationship between maternal and foetal hormone concentrations was not observed. The diurnal rhythm in adrenocorticotrophin concentration in the foetus is discussed in relation to rhythms in indicators of central nervous activity.


Direct effects of trypan blue on thyroid secretion. Trypan blue directly inhibited in vitro thyroid secretion (butanol soluble 125I release to the media) induced by both thyroid stimulating hormone (TSH) and dibutyryl cAMP. Intracellular colloid droplet counts were also decreased. Inhibition was directly proportional to dye concentration and could be overcome by supramaximal TSH and dibutyryl cAMP. Inhibition could be observed as early as 20 min of incubation, was not increased by preincubation, and could even be demonstrated after TSH in vivo. Trypan blue, in vivo, produced similar inhibition of thyroid secretion. Incubation of 125I-thyroglobulin with lysosomal enzymes revealed inhibition with much lower concentrations of dye. Inhibition of lysosomal enzyme(s) would not appear to explain the marked decreases in colloid droplets, and this may represent two separate effects of trypan blue on thyroid secretion.


Predicting patient outcome: the contribution of prehospital, inhospital, and posthospital factors. Data on variables often used in recidivism studies were gathered from state hospital records of 129 patients released within a two-year period to a three-county area in West Virginia. The variables were categorized as prehospital, inhospital, or posthospital; the relationship between each variable set and recidivism was determined. The three criteria of recidivism were readmission within one year following discharge, number of days in the community, and number of days to first readmission. Inhospital variables were found to be the best predictors for all three outcome measures. When the number of variables in the set was controlled for, the posthospital set and the inhospital set had the same predictive power for readmission within one year of discharge and number of days to readmission. Posthospital variables were poorest at predicitng, among recidivism criteria, the number of days in the community.


Viral vaccines: new problems and prospects. The problems are many: how best to apply the swine flu lessons to the Russian flu "invasion;" whether enough is known about the cytomegalovirus to attempt vaccine development; how to deliver established vaccines to the millions who need them.


The viral hepatitides.

The viral hepatitides. Vaccines and chemotherapy may soon strengthen the armamentarium for control of type B hepatitis. The incidence of type A is still declining in the U.S., and now that type non-A, non-B has been transmitted to animals, a marker for it is being widely sought.


Cushing's syndrome: a tripartite entity. The three distinct etiologies of Cushing's syndrome usually reflect the presence of a neoplasm, most often but not necessarily of the pituitary, and all ultimately result in hypercortisolism. Recent advances in diagnosis and treatment have vastly improved the outlook for many patients. Several therapeutic options are outlined, the success of which depends on accurate identification of the cause of hypercortisolism.


Metabolic requirements for renal function. Renal mass accounts for only 0.5% of body weight, yet the kidney consumes 7% of the body's total oxygen uptake. Most is used for tubular transport, but a significant portion contributes to less well-recognized aspects of renal function. Cortical gluconeogenesis capacity, for example, exceeds that of the liver per gram of tissue. Basal and transport metabolic processes are examined in terms of clinical relevance.


Structural organization of human cytomegalovirus DNA. HCMV DNA preparations in which purity was characterized by sedimentation, buoyant density, and restriction enzyme cleavage contain two molecular weight size classes of HCMV DNA molecules (150--155 x 10(6) and 100 x 10(6) daltons) in addition to some shear products. Partial denaturation mapping reveals that both size classes contain the same sequences and that molecules of 150--155 x 10(6) daltons contain the apparent full complement of sequences which are variously contained in the smaller molecules. The complete genome (150--155 x 10(6) daltons) seems to be divisible into regions of about 78--82% and 18--22%, which undergo complete inversions with respect to one another. Enriched or purified preparations of molecules of 150--155 x 10(6) daltons generate cleavage products identical to those from total HCMV DNA preparations.


Physical mapping of herpes simplex virus-coded functions and polypeptides by marker rescue and analysis of HSV-1/HSV-2 intertypic recombinants. A number of temperature-sensitive (ts) mutants and one pyrimidine deoxyribonucleoside kinase-deficient mutant of herpes simplex virus (HSV), have been located on the physical map of the genome by means of marker rescue experiments and by the analysis of the crossover points in intertypic recombinants between HSV types 1 and 2. The physical map is compared to the genetic map and certain anomalies identified. Analysis of infected-cell polypeptides specified by intertypic recombinants has allowed tentative map co-ordinates to be assigned to the structural genes (or genes which cause post-translational modification) for many of the polypeptides. Immediate-early, phosphorylated, glycosylated and structural as well as non-structural polypeptides have been analysed in this way and it can be concluded that there is no restriction of any of these groups of polypeptides to either the long or the short regions of the genome. One of the recombinants, 2853, is at least partially "frozen" in one orientation of the long region. This orientation is also the one which exhibits a minimum number of crossovers in three other recombinants.


Integration of Epstein-Barr virus DNA.

Integration of Epstein-Barr virus DNA. The application to herpesviruses of different standard methods fo measuring tumour-virus integration is discussed. The evidence for the presence of integrated virus DNA in EBV-transformed cells is summarized.


Episomal viral DNA in herpesvirus saimiri-transformed lymphoid cell lines. Structural analysis of episomal viral genomes from two herpesvirus saimiri (HVS)-transformed tumour cell lines (No. 1670 and 70N2) showed that both types of episomes have a higher molecular weight than linear virion DNA. The arrangement of unique (L) and repetitive (H) DNA in No. 1670 episomes was studied by partial denaturation mapping. Part of the L-sequences present in linear virion DNA was found to be missing, part was found to be duplicated in the episomes. The episomal L-DNA regions were correlated with the known physical gene maps of linear HVS DNA.


Herpesvirus papio: state of viral DNA in baboon lymphoblastoid cell lines. Baboon lymphoblastoid cell lines (LCL) transformed with herpesvirus papio were evaluated for the state of HVP DNA in the cells by caesium chloride density-gradient centrifugation and DNA-cRNA filter hybridization. Five HVP producer cultures and one non-producer culture were studied and non-integrated and integrated HVP DNA were detected in all producer cultures whereas only integrated HVP DNA could be demonstrated in the non-producer LCL. Non-integrated HVP dna had a density of 1.715--1.716 g/cm3 in caesium chloride corresponding to a guanine + cytosine content of 56--57%.


Annealing of alkali-resistant HSV DNA strands and isolation of S and L components. DNA isolated from highly purified virions of herpes simplex virus type-1 (HF strain) was denatured by centrifugation in alkaline sucrose gradients. DNA molecules corresponding to intact single-stranded virion DNA (50 x 10(6) daltons) were isolated and adjusted to neutral pH. The DNA was annealed under conditions permitting reassociation of intact single-stranded molecules and studied by electron microscopy. Three classes of DNA molecules showing double-stranded sequences were observed: (a) fully double-stranded DNA molecules the size of the intact HSV DNA genome, namely 52 micron; (b) DNA hybrids with a region of partial double-strandedness ranging from 5 to 12 micron, plus long single strands; and (c) DNA hybrids with a double-stranded region of 32--40 micron, plus short single strands. (These results suggest that the alkali-resistant single-stranded HSV DNA molecules are composed of several subclasses that permit annealing of either the total genome or the S or L components.) The 5 micron double-stranded region probably constitutes the S component of HSV DNA and the sequences longer than 5 micron and shorter than 12 micron represent annealing of the repeat sequences on either or both sides of the S component. The double-stranded sequences with a length of 32--40 micron may represent the L component. Treatment of the annealed, partially double-stranded hybrid DNA molecules with S1 endonuclease to remove the single-stranded termini and centrifugation in neutral sucrose gradients yielded two distinct peaks. Centrifugation of fractions from the two peaks in caesium chloride density gradients showed that the small DNA component (possibly the S and the repeat sequences) had a higher buoyant density and the longer (possibly the L) DNA component had a lower density than the HSV DNA marker. Annealing of alkali-resistant viral DNA strands therefore provides a means of isolating the L, S and repeat sequence regions of HSV DNA.


Analysis of herpes simplex virus low molecular-weight native proteins by polyacrylamide gel electrofocusing. This communication has demonstrated the potential application of gel electrofocusing for the isolation and further characterization of HSV-2 native proteins. At least eight major HSV-2 specific proteins with characteristic isoelectric points could be reproducibly resolved by gel isoelectric focusing. Their isoelectric points ranged from 5.0 to 7.5 and analysis by two-dimensional electrophoresis on SDS gels indicated that their molecular weights range from 11,000 to 49,000.


Crossed immunoelectrophoretic analysis and viral neutralizing activity of five monospecific antisera against five different herpes simplex virus glycoproteins. Crossed immunoelectrophoresis of Triton X-100 solubilized HSV-infected cells has identified several major HSV glycoprotein antigens in the precipitating profile. Immunization of rabbits with the corresponding precipitates resulted in the production of monospecific antisera against the antigen part of the precipitate. The immunological importance of five different HSV glycoproteins in respect of their representation on the outer virion envelope as targets for neutralizing antibodies was evaluated by comparison of immunoprecipitating activity and neutralizing potency of the corresponding monospecific antisera.


Altered biological and biochemical properties of a phosphonoacetate-resistant mutant of herpesvirus of turkeys. A phosphonoacetate (PA)-resistant mutant of the herpesvirus of turkeys (HVT) was isolated and characterized. The mutant (HVTpa) replicates in growth medium containing 300 microgram/ml of PA and shows in vitro temperature sensitivity at 41 degrees C (its 37 degrees C/41 degrees C efficiency of replication is about 5). HVTpa replicates poorly in chickens and fails to provide complete protection against MDV challenge. The HVTpa-induced DNA polymerase has an apparent inhibition constant for PA 10 times as great, an apparent inhibition constant for pyrophosphate, twice as great, and an apparent Michaelis constant for dCTP 2.5 times as great as the respective figures for the HVTwt-induced enzyme. The HVTpa-induced enzyme is also more thermolabile.


Regulation of herpes simplex virus type 1 DNA synthesis: temperature-shift studies with DNA-negative temperature-sensitive mutants. The regulation of expression of viral genes involved in the synthesis of herpes simplex virus type 1 DNA was studied using three DNA- temperature-sensitive (ts) mutants (B, C, and D). These mutants were examined for their ability to synthesized viral DNA and polypeptides following temperature shift-down in the presence or absence of the transcription inhibitor actinomycin D. The results demonstrated that the B gene product is required transiently early in infection and apparently controls a transciptional step required for HSV DNA synthesis. The C gene product is required continuously during infection and also controls a transcriptional step needed for viral DNA synthesis. In contrast, the product of the D gene does not directly control a transcriptional step, is required continuously, appears to be directly involved in HSV DNA synthesis, and is probably the gene for viral DNA polymerase. The results further showed that recovery of viral DNA and polypeptide synthesis following temperature shift-down in the absence of inhibitor was greater for the D mutant than for the mutants blocked in viral DNA synthesis at the level of transcription.


Virus transcript mapping studies in cells infected with temperature-sensitive mutants of herpes simples virus type 1. Nuclear and cytoplastic transcripts, synthesized in cells infected with six DNA-negative temperature-sensitive (ts) mutants of HSV-1 (ts B, ts D, ts E, ts K, ts S and ts T) under non-permissive conditions, were isolated and hybridized to unlabelled fragments of HSV-1 DNA, generated by restriction endonuclease digestion and immobilized on to nitrocellulose membranes by the method of Southern (1975). In this way it has been possible to map those regions of the HSV-1 genome represented by stable transcrips in cells infected with these mutants and compare them with those regions transcribed in cells infected with the wild-type virus at early and late times post infection (before and after viral DNA replication) and in the presence of DNA- and protein-synthesis inhibitors. Viral transcription in ts D, ts T and ts K-infected cells is restricted, the patterns of hybridization being similar, but not identical to that observed with immediate early RNA. Since these three mutants fall into two complementation groups, these experiments suggest that at least two viral products are required for the switch-on of early transcripts. In contrast, transcript mapping with the other early mutants (ts B, ts E and ts S) has shown a much less restricted transcriptional pattern, the pattern obtained resembling that with early, rather than late RNA.


The regulations of gamma (structural) polypeptide synthesis in herpes simplex virus types 1 and 2 infected cells. The polypeptides specified by herpes simplex viruses types 1 and 2 form at least three groups designated as alpha, beta and gamma. The structural polypeptides are largely contained in the gamma polypeptide group. In this study, we investigated the transition from beta to gamma polypeptide synthesis. Our data show the following: (i) gamma polypeptide synthesis in the presence of inhibitors of DNA synthesis in the presence of inhibitors of DNA synthesis is multiplicity-dependent. Some gamma polypeptides are not detectable in cells infected with 1 PFU per cell form a significant fraction of viral polypeptides in cells infected with 25--500 PFU per cell. (ii) The mRNAs specifying these polypeptides have a relatively short half-life as measured by the relative rate of decay of gamma polypeptide synthesis in infected cells following exposure to actinomycin D. Our data thus suggest that: (i) the transition from beta to gamma polypeptide synthesis does not require the synthesis of viral DNA; and (ii) the rate of synthesis of viral structural (gamma) polypeptides is linked to the size of the viral DNA pool as a consequence of a relatively short-lived mRNA.


Location of non-temperature-sensitive genes on the genetic map of herpes simplex virus type 1. By constructing crosses involving the genes coding for the enzyme deoxypyrimidine kinase and for resistance to the drug phosphonoacetic acid, it has been possible both to extend the genetic analysis of HSV-1 such that the eight possible progeny genotypes which arise from a three-factor cross can be identified and also to precisely locate the gene for the plaque-morphology marker syn, the gene for deoxypyrimidine kinase dPYK and the gene for resistance to phosphonoacetic acid PAAr.


The thymidine kinase gene of herpes simplex virus type 1: cell-free protein synthesis and substrate specificity studies. The HSV thymidine kinase genetic system has a number of advantages for the study of virus and cell genetics. Both forward and reverse mutants are readily selected. The enzyme can be assayed directly in the presence of the cell TK by the choice of the appropriate substrate, for example, 125IdC. The gene product, HSV TK, can be identified by SDS-polyacrylamide gel electrophoresis and mutants can be found with alterations in the electrophoretogram. The in vitro synthesis of HSV TK in response to HSV mRNA has been achieved and can be used to analyse specific mutant virus.


Cell-free systems for in vitro translation of herpes simplex virus messenger RNA. A cell-free system active in translation of HSV mRNA was obtained by fractionation of reticulocyte lysates. Endogenous protein synthesis was further reduced by preincubation with micrococcal nuclease, although this treatment did not affect the translation of infected (or uninfected) BHK cell RNA. Polypeptides synthesized by the fractionated reticulocyte cell-free system were very similar to those produced by unfractionated reticulocyte lysates.


Analysis of viral gene functions by means of early temperature-sensitive mutants of human cytomegalovirus. Fifteen ts mutants of CMV, which are unable to form normal plaques at 39 degrees C on HEL cells but do form plaques at 34 degrees C, have been isolated following mutagenesis by ultraviolet light. Five mutants defective in viral DNA synthesis at 39 degrees C were divided into two different complementation groups and examined for various phenotypic characteristics.


Cell DNA induction by human cytomegalovirus. The studies presented indicate that in cells pretreated with IUDR: (1) a temporal relationship exists between CMV and cell DNA synthesis; (2) in cells pretreated with IUDR only a fraction of the cell genome is induced by the virus to replicate; and (3) cell DNA induced by the virus to replicate represents unique rather than repetitive DNA sequences.


Effect of cyclic nucleotides on the response of cells to infection by various herpesviruses. The effect of varying the intracellular cyclic nucleotide concentration of mouse cells infected with HSV-1 or of duck embryo fibroblasts infected with Marek's disease virus has been studied. It has been shown in each case that agents that elevate the intracellular cAMP levels increase the yield of infectious virus or viral DNA, but inhibit cell division. cGMP enhances synthesis of viral DNA but allows continued synthesis of cell DNA.


Identification and partial purification of two EBV-associated DNA polymerases. Virally induced DNA polymerases have been demonstrated in cells infected with a variety of herpesviruses. These include herpes simplex virus (Weissbach et al., 1973), Marek's disease virus (Boezi et al., 1974), equine herpesvirus (Allen et al., 1977), and cytomegalovirus (Huang, 1975a). Recently a new iododeoxyuridine (IUDR)-induced intracellular DNA polymerase in an Epstein-Barr virus (EBV)-producing hybrid cell line has also been reported (Miller et al., 1977). We present evidence that there are two different DNA polymerase activities associated with EBV, one intracellular and the other virion-associated. Both of these enzymes have certain biochemical characteristics which distinguish them from each other, and from the host-cell DNA polymerases found in lymphocytes.


The use of intertypic recombinants for analysis of gene organization in herpes simplex virus. Analysis of the DNA sequence arrangement and polypeptides specified by 28 HSV-1 x HSV-2 recombinants show the following: (i) Recombinants with heterogeneous L and S components or with heterogenous inverted repeats are viable. (ii) HSV-1 and HSV-2 genes appear to be functionally equivalent and with few exceptions co-linearly arranged. Co-linear DNA maps have been established. (iii) At most two arrangements of HSV DNA are capable of replication. This is consistant with current studies suggesting that sequence arrangements are the consequence of obligatory post-synthesis repair. (iv) alpha Polypeptides map at the termini of the L and S components of HSV DNA. Although alpha ICP 27 maps entirely within the reiterated region of the L component, the template for alpha ICP 4 may lie only in part within the reiterated sequences of the S component. Of note is the finding that cells infected with a recombinant that contains both HSV-1 and HSV-2 DNA sequences in the S component, produced alpha ICP 4 of both HSV-1 and HSV-2. (v) Templates specifying beta and gamma polypeptides may in the L component and appear to be randomly distributed. (vi) The genes specifying thymidine kinase, resistance to phosphonoacetic acid and syncytial plaque morphology mapped in the L component. In addition, we have taken advantage of the rapid inhibition of host protein synthesis to map the gene(s) specifying this inhibition in the L component.


Mapping of the herpes simplex virus DNA sequences in three herpes simplex virus thymidine kinase-transformed cell lines. We have made use of a novel filter hybridization approach in order to map the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase (TK)-transformed cell lines. The cell line 33A+ which was produced by infection of 3T3 TK- cells with UV-irradiated HSV-2 (333) was found to contain one contiguous stretch of viral DNA sequences which maps between 0.15 and 0.57 on the HSV-2 genome. The sequences mapping from 0.31 to 0.37 were present in 3--4-fold higher abundance than the rest of the viral DNA sequences in this cell line. Cell lines 5A and 8N were produced by transfection of mouse CL1D cells with sheared HSV-1 (1023) DNA. The cell line 5A was found to contain a contiguous set of viral DNA sequences mapping between 0.26 and 0.41 on the HSV-1 genome. The cell line 8N was found to contain three non-contiguous sets of viral DNA sequences, mapping between 0.09 and 0.41, 0.53 and 0.58, and 0.94 and 1.0 on the HSV-1 genome. These results seem to indicate that many different sets of viral DNA sequences can be incorporated into the cell during HSV-mediated biochemical transformation.


DNA-mediated transfer of herpes simplex virus TK gene to human TK- cells: properties of the transformed lines. Human TK- cells carrying the HSV-2 TK gene express a TK activity of viral origin and maintain the TK+ phenotype when grown in HAT medium. Under non-selective or counterselective conditions, however, reversion to a TK- phenotype occurs with a significant frequency characteristic of each transformed line. The TK- phenotype appears to be stable since no instances of TK- to TK+ reversion have been observed.


Oncogenic transformation of non-permissive murine cells by viable equine herpesvirus type 1 (EHV-1) and EHV-1 DNA. Primary cultures of BALB/c mouse embryo fibroblasts infected with as much as 100 PFU per cell of EHV-1 do not exhibit cytopathology or synthesize detectable amounts of EHV-1 specific RNA, DNA, or infectious virus. Addition of 1--2 microgram of non-fragmented EHV-1 DNA as a co-precipitate with calcium phosphate to monolayers of such non-permissive mouse cells resulted in the appearance, after 4--6 weeks, of foci of piled-up, morphologically altered cells. Cell lines established from such transformed foci exhibited a greatly increased growth rate, unlimited growth potential, aneuploid karyotype, and grew with colony formation in soft agar. Inoculation of 10(6) transformed cells into newborn syngeneic mice resulted in the formation of serially transplantable tumours (undifferentiated fibrosarcomas) with a 100% incidence within eight weeks. Infectious virus could not be rescued from the EHV-1 transformed or tumour-derived cell lines by growth in the presence of IUDR, by cocultivation with permissive horse cells, or by attempts to transfect permissive cells with transformed or tumour cell DNA. However, EHV-1 specific membrane antigens were detected in the transformed cells by immunofluorescence with hyperimmune anti-EHV-1 mouse serum, and the presence of a fragment of the EHV-1 genome was demonstrated in both the transformed and tumour cells. These results indicate that cells nonpermissive for replication of EHV-1 remain susceptible to neoplastic transformation by the EHV-1 genome.


Enhanced efficiency of transfection of HSV DNA in HSV-1 DNA-transformed hamster cells. The efficiency of transfection, i.e., the number of plaques per microgram of DNA, was compared for herpes simplex virus (HSV) type 1 and type 2, and herpesvirus eidolon (antigenically unrelated to HSV) DNAs in HSV DNA-transformed cells (EH/A44) and control hamster cells (EHT). The efficiency of transfection of HSV-1 and HSV-2 DNAs was significantly higher in EH/A44 cells than in EHT cells, showing that an early step in HSV infection was involved in the partial resistance of HSV DNA-transformed cells to superinfection by various intact herpesviruses.


Mapping of putative transforming sequences of EBV DNA. The linkage of restriction enzyme fragments of DNA of the B95-8 strain of Epstein-Barr virus has been determined. Two approaches are being employed to define which EBV DNA sequences are needed to initiate and maintain the transformation of lymphocytes to lymphoblasts capable of long-term growth in culture. The first approach is to determine the differences between the DNA of strains of EBV which possess transforming capacity and the DNA of the HR-1 strain which cannot transform. The data indicate that EBV (HR-1) DNA lacks approximately 2--3 x 10(6) daltons of DNA contained largely in the HsuI B and EcoRI (J-K) and A fragments of EBV (B95-8) DNA and in the EcoRI A and HsuI B fragment of the W91 strain. The DNA common to HsuI B and EcoRI A fragments lies between 27 and 42 x 10(6) daltons from the HsuI A end of the molecule. This finding is compatible with the hypothesis that the inability of the HR-1 strain to transform is due to the absence of DNA needed for transformation. The second approach is to identify and map the DNA encoding polyadenylated viral RNA in cultures of restringently infected cells which contain the EBNA antigen and show no evidence of abortive or productive infection. Previous data indicated that viral RNA species encoded by 5% of the viral DNA are adenylated and identified in the polyribosomes of restringently infected cells. The data indicate that these RNAs are encoded primarily by the HsuI A (and to a lesser extent, B) fragment of EBV (B95-8) DNA. This would place the DNA encoding the viral RNA processed in restrigently infected cells adjacent to and possibly overlapping the small DNA segment deleted from the DNA of the non-transforming HR-1 strain.


In vitro lymphocyte transformation by Epstein-Barr virus (EBV)-like viruses isolated from Old-World non-human primates. EBV-like viruses and lymphoid cell lines have been isolated from baboons and an orangutan. The cell lines have properties of B- or undifferentiated lymphocytes and have antigens and DNA related to those of EBV. The baboon virus has a broad in vitro transformation host range among lymphocytes of Old-World simian species whereas the orangutan isolate has a narrower host range. Baboon and orangutan viruses as well as EBV have shown transforming activity for gibbon lymphocytes. Baboon virus is infectious for rhesus monkeys and baboons but has not induced neoplastic disease in these species.


Transcription of Epstein-Barr virus genomes in human lymphoblastoid cells and in somatic-cell hybrids of Burkitt's lymphoma. Expression of latent Epstein-Barr virus genomes in lymphoblastoid cells and somatic-cell hybrid of Burkitt's lymphoblastoid cells has been studied. IUDR treatment induced the formation of early antigen (EA), virus capsid antigen (VCA) and virus DNA replication in D98/Raji and D98/HR-1 cells whereas only EA was induced in Raji cells. HR-1 clone No. 9 did not respond to IUDR treatment. The pattern of transcription of virus genomes in these cell lines without IUDR treatment was uniform with 20--25% of virus DNA transcribed. IUDR treatment enhanced the transcription of virus DNA to 50% in D98/Raji, D98/HR-1 and Raji cells but no enhancement of virus genome transcription was observed in HR-1 clone No. 9 The amount of virus RNA in the cells calculated from DNA-RNA hybridization kinetics was found to be proportional to the number of virus genomes per cell indicating that every copy of virus DNA in these cells is actively transcribed.