Solid-phase enzyme immunoassay for herpes simplex virus. An enzyme immunoassay was used for the rapid detection of herpes simplex virus type 1 (HSV-1). The test utilized specific antibodies to HSV-1 attached to a solid phase. A laboratory preparation of HSV-1 was detectable in amounts of greater than or equal to 50 50% tissue culture-infective doses by this method. Tests performed with clinical samples indicated a specificity of 95%. The sensitivity appeared to depend on the length of time the samples were stored. When samples were stored frozen for three to six months, the sensitivity was 46% of that of tissue culture isolations performed when the samples were first collected. The sensitivity was 89% in comparison with attempts at tissue culture reisolation with the same samples at the time of enzyme immunoassay. The immunoassay also detected HSV in seven of 10 positive samples that were stored frozen for no more than two weeks before testing.


Detection of soluble antigen of Epstein-Barr virus by the enzyme-linked immunosorbent assay. The enzym-linked immunosorbent assay (ELISA) was compared with the microcomplement-fixation test for sensitivity and reliability in detection of antibody to soluble antigen of Epstein-Barr virus. ELISA was found to be more sensitive (up to 160 times more antibody) in detecting levels of antibody than the complement-fixation assay. The ELISA test was shown also to be comparably reliable to the complement-fixation assay in detecting immunity to this antigen; only one false-positive response was found in tests of 21 sera. Because this assay is completely quantifiable, it provides an easy, rapid, and highly sensitive technique for the detection of antibody to antigen of Epstein-Barr virus.


Magnetic transfer devices for use in solid-phase radioimmunoassays and enzyme-linked immunosorbent assays. A method was developed for the simultaneous transfer of large numbers of solid-phase adsorbents in radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Specially coated ferromagnetic spheres (beads) were used as the solid phase. These beads were transferred from a reaction mixture through a rinse bath to another reaction mixture by magnetic probes. The quality of results obtained with this new methodology compared favorably with that obtained when solid-phase adsorbents were handled individually. The magnetic transfer system provided a high degree of mechanization, thus permitting many more tests to be performed at one time, under almost identical conditions. Certain mechanical features of the test and the micro scale of the reactions provided substantial economy in time and consumption of valuable reagents. The sensitivities of the RIA and ELISA for detection of viral antibody were about the same. An important advantage in using magnetic devices for transfer of beads is that the immune and enzyme-substrate reactions can be started and stopped instantly in either system.


Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination test for detection of antibody to cytomegalovirus. Thirty samples of serum were tested for antibody to cytomegalovirus by enzyme-linked immunosorbent assay (ELISA) and indirect hemagglutination (IHA). The two tests were in extremely close agreement. Of the 30 sera evaluated by ELISA, 16 were considered to be positive and 14 were considered negative. In all 16 positive samples, the titers by ELISA were at least twofold to 10-fold higher than the indirect hemagglutination titers. Two of the 14 sera evaluated as negative by the ELISA test had low indirect hemagglutination titers (1:8 and 1:4). They were not detectable at the initial dilution (1:50) of the ELISA test. These two sera might have been classified as positive if the initial dilution in ELISA had been lower than 1:50.


Uptake of very low density lipoprotein triglyceride by bovine aortic endothelial cells in culture. Primary monolayers of calf aortic endothelial cells were presented with isolated human very low density lipoproteins that had been labeled with radioactive triglyceride. The cells were observed to take up triglyceride over a 24 hr period; incorporation increased with exogenous lipoprotein concentrations, and up to 60% of the triglyceride taken up was converted to other cell lipids within 24 hr. When [2-3H]glyceryl tri[1-14C]oleate-labeled very low density lipoprotein was used, the 3H/14C ratio in the cell triglyceride was always similar to that of the exogenous lipoprotein triglyceride. Moreover, no significant hydrolysis of the exogenous very low density lipoprotein triglyceride was observed during the time of exposure to the cells. Similar experiments using doubly-labeled triglyceride exposed to endothelial cells in triglyceride-phospholipid liposome preparations also resulted in incorporation of the exogenous triglyceride without evidence of extracellular hydrolysis. The results indicate that primary monolayers of endothelial cells in culture are able to incorporate and metabolize very low density lipoprotein triglyceride. However, triglyceride does not appear to be significantly hydrolyzed during uptake, suggesting an absence of lipoprotein lipase activity in these cells.


Common characteristics of the cytochrome P-450 system involved in 18- and 11 beta-hydroxylation of deoxycorticosterone in rat adrenals. 18- and 11beta-Hydroxylation of deoxycorticosterone and side chain cleavage of cholesterol were studied in mitochondria and submitochondrial reconstituted systems prepared from rat and bovine adrenals. A mass fragmentographic technique was used that allows determination of hydroxylation of both exogenous and endogenous cholesterol. The following results were obtained. (1) Treatment of rats with excess potassium chloride in drinking fluid increased mitochondrial cytochrome P-450 as well as 18- and 11beta-hydroxylase activity in the adrenals. Cholesterol side chain cleavage was not affected. In the presence of excess adrenodoxin and adrenodoxin reductase, cytochrome P-450 isolated from potassium chloride-treated rats had higher 18- and 11beta-hydroxylase activity per nmol than cytochrome P-450 isolated from control rats. The stimulatory effects on 18- and 11beta-hydroxylation were of similar magnitude. (2) Long-term treatment with ACTH increased cholesterol side chain cleavage in the adrenals but had no effect on 18- and 11beta-hydroxylase activity. The amount of cytochrome P-450 in the adrenals was not affected by the treatment. It was shown with isolated mitochondrial cytochrome P-450 in the presence of excess adrenodoxin and adrenodoxin reductase that the effect of ACTH was due to increase of side chain cleavage activity per nmol cytochrome P-450. Side chain cleavage of exogenous cholesterol was affected more than that of endogenous cholesterol. (3) Gel chromatography of soluble cytochrome P-450 prepared from rat and bovine adrenal mitochondria yielded chromatographic fractions having either a high 18- and 11beta-hydroxylase activity and a low cholesterol side chain cleavage activity or the reverse. The ratio between 18- and 11beta-hydroxylase activity was approximately constant, provided the origin of cytochrome P-450 was the same. (4) Addition of progesterone to incubations of deoxycorticosterone with soluble or insoluble rat adrenal cytochrome P-450 competitively inhibited 18- and 11beta-hydroxylation of deoxycorticosterone to the same degree. Addition of deoxycorticosterone competitively inhibited 11beta-hydroxylation of progesterone with the same system. Progesterone was not 18-hydroxylated by the system. From the results obtained, it is concluded that 18- and 11beta-hydroxylation have similar properties and that the binding site for deoxycorticosterone is similar or identical in the two hydroxylations. The possibility that the same specific type of cytochrome P-450 is responsible for both 18- and 11beta-hydroxylation of deoxycorticosterone is discussed.


Characterization of human very low density lipoproteins containing two electrophoretic populations: double pre-beta lipoproteinemia and primary dysbetalipoproteinemia. Two discrete populations of very low density lipoproteins, with fast and slow pre-beta electrophoretic mobility, were found in 50% of normolipemic and 30% of hyperlipemic individuals selected at random. The two populations were isolated by preparative electrophoresis from five hyperlipemic subjects. The particles comprising the slow component were smaller than those of the fast component and the slow component contained a larger proportion of cholesteryl esters, free cholesterol, B-apoprotein, and arginine-rich apoprotein and a smaller proportion of triglycerides and the two most anionic apoproteins (R-glutamic acid and R-alanine). The properties of the slow component thus closely resemble those of "remnant" very low density lipoproteins that accumulate in blood plasma of functionally hepatectomized rats. The chemical composition of the slow component was also similar to that of the very low density lipoproteins with beta mobility found in primary dysbetalipoproteinemia. However, the proportion of cholesteryl esters and argininerich apoprotein was much higher in the latter. The argininerich apoprotein from very low density lipoproteins of most normolipemic and hyperlipemic subjects separates into three or four major bands upon isoelectric focusing electrophoresis in polyacrylamide gels, with pI varying from 5.57 to 6.03. In very low density lipoproteins from individuals with primary dysbetalipoproteinemia, this protein uniquely contains little or none of the two most cationic bands. The number of bands was constant in all subjects studied. The pattern was the same in very low density lipoproteins with fast and slow pre-beta mobility as well as in the beta and pre-beta components in primary dysbetalipoproteinemia. These results suggest that many individuals have "remnant" very low density lipoproteins in their plasma. However, the beta-migrating "remnant" that accumulated in large amounts in individuals with primary dysbetalipoproteinemia contains much more arginine-rich protein and this protein is structurally abnormal.


Apolipoprotein A-II content of human plasma high density lipoproteins measured by radioimmunoassay. A double antibody radioimmunoassay for human ApoA-II is reported. ApoA-II isolated from human plasma high density lipoprotein (HDL) by column chromatography migrated as a single band on polyacrylamide disc gel electrophoresis, had the appropriate amino acid composition, and provoked the production of monospecific antisera. (125)I-ApoA-II (iodinated by lactoperoxidase, purified by Sephadex G-75 chromatography) migrated with "cold" ApoA-II as a single band on disc gel electrophoresis in SDS. Its specific radioactivity was 5-12 mCi/ micro g. In assays, (0.05 M barbital buffer, 0.01% Triton X-100, pH 8.6) over 90% of (125)I-ApoA-II was bound by excess first antibody and over 95% was displaced by excess "cold" ApoA-II. Low density lipoprotein, very low density lipoprotein, ApoA-I, ApoC-II, and ApoC-III displaced no counts. Intraassay and interassay coefficients of variation for lipoprotein or plasma samples were 7 +/- 4 and 11 +/- 6%, respectively. As little as 1.0 ng of ApoA-II was detectable with a precision of 10%. ApoA-II made up 20-25% of the proteins of HDL (d 1.083-1.19), HDL(2) (d 1.083-1.124), and HDL(3) (d 1.124-1.19) on column chromatography. The ApoA-II contents of these HDL fractions were also 20-25% by radioimmunoassay. Similar results were obtained whether assays were carried out on intact or delipidated HDL samples. Thus, in contrast with ApoA-I (only 10% of which is detectable), all of the ApoA-II contents of intact HDL are detected with accuracy by this assay. Plasma levels of ApoA-II in young normolipemic subjects were approximately 40 mg/dl (n = 29). In these subjects, over 98% of ApoA-II was found in the d 1.063-1.21 density fractions.


Serum bile acid analysis: a rapid, direct enzymatic method using dual-beam spectrophotofluorimetry. The direct quantitative measurement of total bile acids in serum has been achieved using an enzymatic fluorescent method with a dual-beam spectrophotofluorimeter. By use of a 3alpha-hydroxysteroid dehydrogenase, oxidation of bile acids with NAD is completed in 200 seconds with the observed NADH fluorescence being proportional to the concentration of serum bile acids. This method is rapid (8 minutes per individual sample), has an intrinsic sensitivity of +/- micronM of total bile acids, requires no sample preparation and less than 0.8 ml of serum. Paired data analysis using enzymatic fluorescence and gas-liquid chromatographic methods gives a correlation coefficient (r) of 0.99 for 34 samples ranging from 2 to 530 micronM.


Innervation of the ventral diaphragm of the locust (Locusta migratoria). 1. Innervation and some electrical properties of the locust ventral diaphragm were investigated with electrophysiological and histological methods. 2. Muscle fibres are coupled electrically. Electrical stimulation evokes a graded active membrane response. 3. Each segment is innervated by four motor neurones as follows. Two motor neurones are situated in each abdominal ganglion. Branches of their axons supply the ventral diaphragm in the respective and the next posterior segment. 4. This pattern of innervation was confirmed by axonal Co and Ni staining of the motor nerve endings. 5. Neuromuscular junctions are excitatory. EPSPs show summation but no facilitation. 6. Spontaneous electrical activity of the diaphragm is to a certain degree coupled to activity of the main inspiratory muscles.


Adult mouse dorsal root ganglia neurons in cell culture. A method has been developed for the long-term culture of dissociated adult mouse dorsal root ganglia (DRG). Of critical importance to the success of this technique was a three-hour incubation in collagenase which softened the DRG and permitted gentle dissociation. The morphological and electrophysiological features of the dissociated adult DRG were similar to those observed in previous studies of immature (i.e., embryonic and newborn) DRG in culture and also to those of adult DRG in situ. With regard to electrophysiological work, the adult DRG neurons are superior to embryonic and newborn neurons because of their larger size and greatly increased survival in culture (no degeneration for first six days, and thereafter a relatively slow decrease). The adult neurons regenerated nerve fibers to an extent comparable to that of immature neurons. Therefore, the adult DRG cultures might be useful to study factors influencing regeneration in the adult mammalian nervous system. The adult cultures might also be useful to investigated factors influencing the aging process.


Fine structure of cutaneous nerves in ganglioside storage disease. Skin punch biopsies of six children suffering from infantile or late onset Tay-Sachs disease, juvenile Sandhoff disease, or GM gangliosidosis type I, contained axons which, when viewed with the electron microscope, were distended by large amorphous black deposits. These are nonspecific residual bodies. Their large numbers indicate severe disturbance of the nerve cell and may be part of the dying back process. The three cases with Tay-Sachs disease had also axonal zebra or complex membranous bodies which appeared to be specific. Cytoplasmic vacuolation of other cells was a feature in the patient with GM1 gangliosidosis. Biopsies of three parents were negative.


Mouse spinal cord in cell culture. II. Synaptic activity and circuit behavior. 1. Neurons in cell cultures of fetal mouse spinal cord (SC) and dorsal root ganglia (DRG) develop extensive synaptic interconnections. 2. No spontaneous synaptic activity was detectable in the presence of tetrodotoxin or an elevated magnsium ion concentration, but statistical analysis of evoked excitatory postsynaptic potentials (EPSPs) indicates that the quantal size was 200-250 muV, which was below the noise level of the recording system used. 3. In a sample of eight RDG-SC and seven SC-SC cell pairs linked by EPSPs, the quantal content of the SC-SC EPSPs was about 3.5-fold larger than for the DRG-SC EPSPs. 4. The extrapolated equilibrium potential for the SC-SC EPSP was about 20 mV positive. The IPSP reversed at a membrane potential of 60-80 mV negative. 5. Some examples of the types of synaptic circuits commonly encountered are given. Only one case of electrical coupling between neurons was found.


Concentration of 3',5' cyclic adenosine monophosphate in ventricular CSF of patients following severe head trauma. Previous studies have demonstrated that cerebrospinal fluid (CSF) from the lateral ventricle of patients without disturbance of sensorium or intracranial pressure, contains 15 to 30 nM 3',5' cyclic adenosine monophosphate (cAMP). The concentration of this cyclic nucleotide was measured by radioimmunoassay in 133 samples of CSF from the lateral ventricle of 26 patients who were comatose following acute head trauma for periods up to 40 days. Concentration of CSF cAMP in diminishing coma Grades V, IV, III, II, and I was 1.5 +/- 0.1 nM; 1.24 +/- 0.34 nM; 3.14 +/- 0.7 NM; 10.06 +/- 3.47 nM; and 13.36 +/-  1.38 nM, respectively. After the sensorium cleared (coma Grade 0), cAMP was 22.0 +/- 1.7 nM. The correlation between the grade of coma and cAMP concentration was -0.80 (p greater than 0.01). These results imply that alteration in the level of consciousness following head trauma is associated with a disturbance of cAMP metabolism within the central nervous system. Possible mechanisms explaining this observation as well as therapeutic implications are discussed.


Cerebrospinal fluid GABA reductions in seizure patients evoked by cerebellar surface stimulation. Lumbar cerebrospinal fluid (CSF) gamma-aminobutyric acid (GABA) levels determined by fluorometric assay in four seizure patients were found to be significantly lower during bilateral, continuous cerebellar stimulation than those determined after a 7-day period without stimulation. The CSF GABA concentrations during chronic unilateral, alternating cerebellar stimulation were reduced in three seizure patients but unchanged in a fourth patient. The percentage decrease in CSF GABA appeared to be independent of cerebellar stimulation frequency. These findings suggest that GABA-mediated neuronal transmission is depressed during cerebellar surface stimulation and this evoked reduction in GABA activity may compromise the efficacy of cerebellar stimulation in the treatment of epilepsy. Lumbar CSF cyclic guanosine monophosphate levels determined by radioimmunoassay were not significantly altered by either mode or frequency of cerebellar stimulation.


Influence of vitamin C restriction on guinea pig adrenal calcium and plasma corticosteroids. Some aspects of adrenocortical function were investigated in young male guinea pigs fed an ascorbic acid (AsA)-deficient diet for 7 days, followed by 0.1 mg AsA/100 g body weight/day for 4 days; pair-fed guinea pigs served as controls. Ninety minutes prior to killine, all guinea pigs received either an adrenocorticotropic hormone (ACTH) or saline injection, and 30 minutes prior to killing, all were injected with 20 muCi  45Ca/100 g body weight intraperitoneally. AsA restriction alone caused an 89% reduction in adrenal AsA concentration, but growth rate, adrenal weight and plasma ACTH were not different from those of pair-fed controls. Adrenal radiocalcium uptake, adrenal calcium content and plasma corticosteroids were similar in saline-treated guinea pigs restricted in AsA and the ACTH-treated controls, all of which were significantly higher than the values observed in saline-injected controls. Similar responses of the ACTH-treated controls and the saline-treated mildly deficient guinea pigs indicated that, at the adrenal AsA levels achieved (4.45 to 7.02 mg/100 g tissue), adrenal calcium and plasma corticosteroids increased significantly without the mediation of ACTH.


Current progress in the treatment of the child with cancer. Treatment for the child with cancer has increasingly been on a rational rather than an empiric basis. An understanding has developed of the importance for determining clinical and laboratory features present at diagnosis as an aid not only to establish a prognosis but also to design specific treatment regimens. A system has been developed for bringing new chemotherapeutic agents into clinical trials as effectively as possible. Through both clinical and laboratory studies, an increasing understanding of the biology of cancer is being developed. This understanding will provide the basis for more rational treatment programs in the future. Physicians of different specialty interests have learned to work together to develop coordinated programs of treatment so important to optimal care. By far the most important lesson learned, however, is that cancer in children is not of necessity a fatal disease, even when dissemination has occurred. For the furture, it will be necessary to develop even more effective methods of treatment, and research must provide a better understanding of this disease that may offer the opportunity for prevention, which, after all, is the number one interest of the pediatrician.


A viremic phase for herpes zoster in children with cancer. Eight childhood cancer patients with herpes zoster were serially tested for the presence of varicella-zoster virus in blood. Cell cultures of leukocyte-rich plasma from four patients were positive for the virus. In this study viremia was clearly related to dissemination of dermal lesions-the spread of zoster lesions outside an infected dermatome. The child with the longest viremic phase, five days, had the longest and most severe course of skin dissemination, as well as biochemical evidence of hepatitis. One patient with viremia had advanced embryonal carcinoma and died of disseminated tumor before her clinical course could be evaluated. These observations, the first to document a viremic phase for herpes zoster in immunosuppressed children, furnish an added criterion for evaluation of antiviral drugs and live-virus vaccines in the treatment and prevention of varicella-zoster infections.


Is calcium the final mediator of exocytosis in the rat parotid gland? Rat parotid slices were partially depleted of cellular Ca by long (80-100 minute) incubations in media containing no added Ca and 5mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). This treatment inhibited the secretory response (release of alpha-amylase) both to isoproterenol and to dibutyryl cyclic adenosine 3':5'-monophosphate. The isoproterenol-stimulated synthesis of cyclic adenosine 3':5'-monophosphate was inhibited by depletion of Ca but not to an extent sufficient to explain the effects of depletion of Ca on secretion. Isoproterenol did not affect influx of 45Ca but stimulated efflux of 45Ca suggesting release of Ca from intracellular stores. Isoproterenol caused vacuolation of the Golgi region and (in high concentration) enhanced the release of 86Rb, responses which are both believed to be mediated by an increase in cytoplasmic Ca concentration. The results of these experiments suggest that isoproterenol acts to increase the tissue level of cyclic adenosine 3':5'-monophosphate which in turn acts to release Ca from intracellular stores. The rise in intracellular Ca concentration is believed to mediate exocytosis.


Histamine mediation in muscular vasodilatation induced by beta adrenoceptor stimulation in dogs. This study was designed to investigate the possibility of a histamine mediation in muscular vasodilation induced by beta adrenoceptor stimulation. Accordingly, in seven dogs the effects of isoproterenol administration on the release of 14C-histamine from the perfused gracilis muscle were studied. Beta adrenoceptors stimulation induced a vasodilatation, as shown by a decrease in perfusion pressure(-43 +/- 12 mm Hg); simultaneously, a significant increase of the radioactivity measured in the venous blood effluent from the gracilis muscle was observed. Both these events were blocked by propranolol. In the other five dogs, chlorpheniramine was able to reduce the vasodilatation induced by the injection in the gracilis muscle of isoproterenol. Under control conditions, isoproterenol induced a fall in perfusion pressure of 44 +/- 5 mm Hg while, after chlorpheniramine, perfusion pressure decreased by only 24 +/- 4 mm Hg. The results of this study seem to confirm the possibility of a histamine mediation in isoproterenol-induced vasodilatation. However, further investigation is needed in order to identify the exact role of histamine in the geneis of this phenomenon.


The hepatic microsomal mixed-function oxidase system in man: cofactor effects and the influence of cholestasis. Microsomal preparations from 22 surgical specimens of normal, cholestatic or severely diseased human liver were analyzed with respect to the amounts of cytochromes P-450 and b5 and NADPH-cytochrome c reductase present. Normal human liver contained slightly less NADPH-cytochrome c reductase and cytochrome P-450 (mean 102.6 +/- 14.6 nmol/mg of protein per min. and 0.60 +/- 0.10 nmol/mg of protein, respectively) than is found in the adult rat, but wide variations were observed. Cytochrome b5 was present in comparable amounts to that in rat liver, and cytochrome b5/P --450 ratios were slightly increased in human liver as compared with rat liver. Cholestatic liver did not show significant alterations in the specific contents of any of these three components of the microsomal mixed-function oxidase system, but the two livers with severe parenchymal disease showed substantial reductions in all three. In vitro investigations of cofacter interrelationships in the N-demethylation of ethylmorphine by human liver microsomes suggested that NADPH was the favored electron donor, but NADH had a substantial (ca. 20%) synergistic effect (which was not enhanced by cyanide addition). These observations are in keeping with cytochrome b5 having a possible role in cytochrome P-450-mediated drug hydroxylation in man in both the normal state and after onset of cholestasis.


The contribution of cholinergic postganglionic neurotransmission to contractions of rabbit detrusor. Field stimulation was used to elicit a contractile response in muscle strips from rabbit detrusor. The blockade of this response by tetrodotoxin (1 X 10(-7) M) ranged from 100% at 1 Hz to 86% at 40 Hz. At concentrations which produced strictly muscarinic antagonism (up to 4 X 10(-7) M) atropine depressed the frequency-response curve by about 42% at maximum but was much less effective at frequencies below 10 Hz. Similarly, treatment of the strips with hemicholinium-3 (5.2 X 10(-4) M) for 90 minutes in the presence of field stimulation at 60 Hz, depressed the frequency-response curve by 52% at maximum but produced less depression below 10 Hz. The hemicholinium-3-resistant response was neither depressed further by atropine (4 X 10(-7) M) nor potentiated by physostigmine (2 X 10(-6) M). Although hemicholinium-3 has antimuscarinic and anticholinesterase properties, these were found not to interfere with the tests for residual cholinergic transmission. Therefore, it was concluded that only part of the motor neurotransmission in rabbit detrusor is cholinergic. The remaining portion is predominant at frequencies below 10 Hz and is postulated to involve a chemical mediator other than acetylcholine.


The effect of catecholamines on Na-K transport and membrane potential in rat soleus muscle. 1. The action of catecholamines on the transport and the distribution of Na and K and the resting membrane potential (E(M)) has been investigated in soleus muscles isolated from fed rats.2. In a substrate-free Krebs-Ringer bicarbonate buffer adrenaline (ADR) (6 x 10(-6)M) increased (22)Na efflux by 83%, (42)K influx by 34%, and E(M) by 10%. Similar effects were exerted by noradrenaline (NA), phenylephrine, salbutamol and isoprenaline. The effects of ADR on Na-K transport and E(M) were suppressed by ouabain (10(-3)M) and propranolol (10(-5)M), but not by thymoxamine (10(-5)M) or tetracaine (10(-4)M).3. Following 90 min of incubation in the presence of ADR (6 x 10(-6)M), the intracellular K/Na-ratio was increased threefold. NA produced almost the same change, and both catecholamines seem to induce a new steady-state distribution of Na and K which can be maintained for several hours in vitro.4. The effect of ADR on (22)Na efflux and E(M) could be detected at concentrations down to 6 x 10(-9) and 6 x 10(-10)M, respectively, and half-maximum increase was obtained at around 2 x 10(-8)M. NA was at least one order of magnitude less potent.5. The effect of low concentrations of ADR on (22)Na efflux was potentiated by theophylline (2 mM). When added together, dibutyryl-cyclic AMP and theophylline mimicked the action of ADR on (22)Na efflux, (42)K influx, Na/K content and E(M). Ouabain (10(-3)M) also suppressed the effect of dibutyryl-cyclic AMP and theophylline on Na-K transport.6. Following the addition of ouabain (10(-3)M), E(M) rapidly dropped from a mean of -71 to -63 mV, and then showed a slow linear fall for up to 4hr.7. The hyperpolarization induced by ADR was associated with a decrease in membrane conductance, (22)Na influx and (42)K efflux. The time course and the response to ouabain suggests that all of these effects are secondary to stimulation of the active coupled transport of Na and K.8. It is concluded that in rat soleus muscle, the active Na-K transport is electrogenic and susceptible to stimulation by catecholamines via beta-adrenoceptors. This effect is mediated by adenyl cyclase activation and may account for the increase in E(M) and the intracellular K/Na ratio.


Regulation of amylase release from dispersed pancreatic acinar cells. 1. A study has been made of factors influencing release of amylase from dispersed pancreatic acinar cells. 2. In the basal, unstimulated, condition cells released 2-3% of the total amylase present in 30 min. 3. The rate of amylase release was stimulated 50-70% by C-terminal octapeptide of cholecystokinin (CCK-OP, maximally effective concentration, 3 X 10(-10) M); carbamylcholine (maximally effective concentration, 10(-5 M); secretin (maximally effective concentration greater than 10(-6) M); vasoactive intestinal peptide (VIP, maximally effective concentration, 10(-8) M); and adenosine 3':5' monophosphate (cyclic AMP) and guanosine 3':5' monophosphate (cyclic GMP) as well as their dibutyryl derivatives (maximally effective concentrations, 10(-3) M). 4. The responses to CCK-OP or carbamylcholine were potentiated by secretin, VIP or dibutyryl cyclic AMP. 5. The responses to secretin or VIP were potentiated by CCK-OP, carbamylcholine, or dibutyryl cyclic GMP. 6. There appear to be two pathways for the regulation of amylase release from pancreatic acinar cells: one pathway can be stimulated by cholecystokinin or cholinergic agonists, and the response to these stimuli is mediated by cyclic GMP; the other pathway can be stimulated by secretin or VIP, and the response to these stimuli is mediated by cyclic AMP.


Capillary supply of the quadriceps femoris muscle of man: adaptive response to exercise. 1. Five subjects trained for 8 weeks on a bicycle ergometer for an average of 40 min/day, four times a week at a work load requiring 80% of the maximal oxygen uptake (V(O2 max.)). V(O2 max.) determinations were performed, and muscle biopsies from the quadriceps femoris muscle (vastus lateralis) were taken before, as well as repeatedly during, the training period. The muscle biopsies were histochemically stained for fibre-types (myofibrillar ATPase) and capillaries (amylase-PAS method), and analysed biochemically for succinate dehydrogenase and cytochrome oxidase activities.2. The training programme resulted in a 16% increase in V(O2 max.), a 20% increase in capillary density, a 20% increase in mean fibre area, and an approximately 40% increase in the activities of succinate dehydrogenase and cytochrome oxidase.3. The capillary supply to type I, IIA and IIB fibres, expressed as the mean number of capillaries in contact with each fibre-type, relative to fibre-type area, increased equally.4. The present study shows that endurance training constitutes a powerful stimulus for capillary proliferation in human skeletal muscle.


A study of excitatory neuromuscular transmission in the bovine trachea. 1. The excitatory innervation of bovine tracheal smooth muscle has been studied with the sucrose-gap apparatus.2. Single 2 ms electrical stimuli applied to the whole tissue excited intrinsic nerves, and produced a small transient depolarization of the smooth muscle, the excitatory junction potential (e.j.p.). The e.j.p. caused a twitch-type contraction; twitches and e.j.p.s summated during repetitive stimulation but facilitation was not observed, and action potentials were never elicited.3. The effects of electrical stimulation could be abolished by atropine (5 x 10(-7) mol/l) and augmented by neostigmine (4 x 10(-6) mol/l), and were mimicked by exogenous acetylcholine (1.0 mug/ml).4. With the electron microscope, the density of innervation was found to be low (one axon per ninety smooth muscle cells). Axons were found in small groups in the clefts between bundles of cells, but no axons penetrated within the muscle bundles. Naked axon varicosities containing agranular vesicles were seen, but no axon approached within 200 nm of a smooth muscle cell.5. It is difficult to reconcile the sparsity of innervation with the dependence of the tissue on nerve excitation to initiate activity.


An analysis of the actions of prostaglandin E1 on membrane currents and contraction in uterine smooth muscle. 1. The effects of prostaglandin E(1) have been studied on the transmembrane potentials, ionic currents, and contractions in isolated myometrial strips from pregnant rats by means of a double sucrose gap apparatus.2. At low concentrations (10(-8) g/ml.), prostaglandin E(1) reduced the duration (though not the amplitude) of the action potential, but significantly increased the contraction. The inward current was unchanged, as well as the phasic component of the contraction. However, the tonic contraction, recorded when the transmembrane calcium influx was blocked selectively with D 600, was stimulated significantly, and the outward current secondarily increased.3. At maximally effective doses (10(-6) g/ml.), the electrical response to prostaglandin E(1) consisted of a slight depolarization, while a large contracture developed. The depolarization and contracture were unaffected by the removal of external calcium. The inward current was reduced by prostaglandin E(1) and the reversal potential was shifted towards less positive values of voltage, indicating a decrease in the driving force and consequently, an increase in the internal calcium concentration.4. Dibutyryl-c-AMP (5 x 10(-4)M) produced a marked relaxation of the resting tension and a slight hyperpolarization of the uterine membrane. Under these conditions, a triggered action potential was able to evoke a larger contractile response. However, prostaglandin E(1) is known to increase tissue c-AMP, so that its contractile effect cannot be mediated by c-AMP.5. It is suggested that prostaglandin E(1) acts essentially by increasing the intracellular calcium concentration. This might result from the translocation of membrane-bound calcium from surface microvesicles or sarcoplasmic reticulum. The increase in internal calcium concentration could, in turn, lead to an increase in the outward current intensity.


Improving patient care through measurement: goal importance and achievement scaling. Sophisticated treatment record keeping requires something more than a focus on problems. An emphasis on positive aspects of treatment, i.e., on goals may be more appropriate for certain patients or for certain types of treatment or at specific stages of an illness. Unfortunately, currently available methods of quantifying goal importance and achievement are not entirely satisfactory for general everday use. GIA is proposed as a simple procedure for quantifying judgments of the importance of a treatment goal, of the extent to which that goal is achieved and of determining the social value of what treatment has accomplished. GIA ratings have considerable potential for improving patient care through measurement.


3-N-Substituted aminomethyl derivatives of rifamycin SV. A convenient method of synthesis, cyclization of certain derivatives, and anticellular and antiviral activities of several derivatives. A new synthesis of Mannich bases of rifamycin SV using the Borch2 procedure with rifaldehyde is described. This new synthesis offers two advantages over the previously published method. It provides a route to monoalkyl-aminomethylrifamycins (le-h) and to unsubstituted aminomethylrifamycins that were not accessible by the old procedure. The new method also offers a preparative route to Mannich bases 1a and 1b were needed in multigram quantities for biological testing. In addition, the cyclization of certain of the monoalkylaminomethylrifamycins to the novel N,15-didehydro-15-epi[methano(alkylimino)]rifamycin SV derivatives (2) is described. The anticellular and antiviral effects of representatives of both series of compounds against cultured mouse cells and murine oncornavirus are are discussed.


Prostaglandin isosteres. 1. (8-Aza-,  8,10-diaza-, and 8-aza-11-thia)-9-oxoprostanoic acids and their derivatives. A series of novel (8-aza-, 8,10-diaza-, and 8-aza-11-thia)-9-oxoprostanoic acids has been synthesized and evaluated for their ability to mimic the E series prostaglandins in stimulating cAMP formation in the mouse ovary and in binding to the rat kidney plasma prostaglandin receptor. 7-[2-(3-Hydroxyoctyl)-1,1,4-trioxo-3-thiazolidinyl]heptanoic acid markedly stimulates cAMP formation at reasonable pharmacological concentrations and avidly binds to the rat kidney prostaglandin receptor.


11,12-secoprostaglandins. 4. 7-(N-alkylmethanesulfonamido) heptanoic acids. A series of 7-(N-alkylmethanesulfonamido) heptanoic acids has been prepared which represents an extension of our 8-aza-11,12-secoprostaglandin studies. The studies. The compounds mimic the natural prostaglandins in that they markedly stimulate cAMP formation in the mouse ovary assay.


Synthesis of spin-labeled nitroxyl esters of steroids. The preparations of spin-labeled nitroxyl esters of prednisone, cortisone, deoxycorticosterone, and cholesterol are described. The ESR spectra indicate that the esters of the three steroids with an oxygen atom at the 11 position have narrower band widths than those with only protons at that position. Mass spectral, chemical, and ESR studies confirmed the structures and purity of the compounds prepared. Reaction of four esters with prednisone antibodies showed reversible binding and large crossover binding.


Cell proliferation and histologic classification of bronchogenic carcinoma. The rate of cell proliferation of 99 bronchogenic carcinomas (94 primary tumors and 5 metastases) was evaluated from the labeling index after in vitro incorporation of [3H]thymidine; the rate was then correlated with the histologic tumor type according to the classification of the World Health Organization (WHO). Cell proliferation was significantly slower in adenocarcinoma (WHO type III) than in squamous cell carcinoma (WHO type I), small cell anaplastic carcinoma (WHO type II), and large cell carcinoma (WHO type IV). Cells proliferated at a significantly higher rate in large cell carcinoma than in the squamous cell type, whereas no significant difference was observed between the other cell types. Dedifferentiated forms of squamous cell carcinomas had a higher rate of cell proliferation than did differentiated forms of the same cell type. Metastases of small cell anaplastic carcinoma did not differ in cell proliferation from primary tumors of the same cell type.


B-lymphocytes and T-lymphocytes in three types of bovine lymphosarcoma. Lymphoid cells of peripheral blood, lymph nodes, and thymus from clinically normal cattle, cattle infected with bovine leukemia virus (BLV), and cattle with lymphosarcoma were characterized for T- and B-cell surface markers. B-cells were detected by the erythrocyte-antibody-complement (EAC) rosette test and the surface immunoglobulin (sig) immunofluorescence assay. Peripheral blood from BLV-infected cattle had a higher than normal percentage of B-cells by both EAC rosette and sig immunofluorescence assays. Lymphoid cells from tumorous lymph nodes of cattle with the adult type of lymphosarcoma had a higher than normal percentage of sig-bearing cells, but in the same cell preparation the EAC rosette-positive cells were fewer than sig-positive cells. T-cells were detected by the erythrocyte rosette test. The percentage of T-cells by this test in lymph nodes of adult type lymphosarcoma was lower than that in normal cattle. A distinctly lower than normal percentage of lymphocytes could be characterized as either B- or T-cells in lymph nodes thymus, and peripheral blood from the calf type and thymic type of lymphosarcoma.


Pathology of human-mouse somatic cell hybrid tumors. Somatic cell hybrids between mouse peritoneal macrophages and simian virus 40-transformed human cells injected sc into nude mice resulted in a formation of tumors with two distinct growth rates and growth patterns: 1) rapid growing, irregularly circumscribed, with necrotic foci and prominent vascular channels lined by tumor cells and 2) slow growing, small, well-circumscribed, and nonvascular. Individual tumor cells were ultrastructurally classified as undifferentiated mesenchymal cells indistinguishable within these two tumor types. Characteristics of each tumor type were retained during subsequent passages in nude mice.


Detection of bovine leukemia virus in B-lymphocytes by the syncytia induction assay. Bovine peripheral blood lymphocytes (PBL's) from 3 cows and 1 steer infected with bovine leukemia virus (BLV) were separated by fractionation through nylon wool columns into nylon-adherent and nonadherent cell populations. Nylon-adherent cells were enriched in B-lymphocytes, as determined by the presence of surface membrane immunoglobulins (slg), whereas nylon-nonadherent cells or "non-B-lymphocytes" contained few slg-bearing cells. PBL's and separated B- and non-B-lymphocyte populations were assayed for the presence of BLV by the induction of syncytia in bovine embryonic spleen cells. PBL's and B-lymphocyte populations both produced many syncytia, whereas non-B-lymphocytes yielded few or no syncytia. The specificity of syncytia formation by anti-BLV serum. PBL's from 2 control animals were negative for syncytia induction. This study presents further evidence that B-lymphocytes are the target cells for BLV infection.


Subcellular distribution of the tumor-specific transplantation antigen of simian virus 40-transformed cells. TU-5, a simian virus 40 (SV40)-transformed cell line of BALB/c origin, expressed the SV40-specific T-antigen and a transplantation  antigen (TSTA). Nuclei and plasma membranes were prepared from these cells and shown on the basis of the distribution of T-antigen and histocompatibility (H-2) antigens to be relatively free of cross contamination. Most of the TSTA, estimated by tumor rejection, was associated with the nuclear fraction.


Biologic and biochemical properties of detergent-solubilized tumor-specific transplantation antigen from a simian virus 40-induced neoplasm: brief communication. The detergent Nonidet P40 was used to solubilize tumor-specific transplantation antigens (TSTA) from crude membranes obtained from dissociated cells of simian virus 40-induced sarcoma of BALB/c mice. A good recovery of specific tumor rejection activity was observed. One fraction, fraction V, was obtained following polyacrylamide-agarose filtration of the solubilized material, and this fraction contained most of the activity. An increased specific activity followed gel filtration. Preliminary data from lectin column chromatography of the active fraction V indicated a separation of TSTA activity from H-2 activity.


Some factors affecting the expression of Epstein-Barr virus-associated membrane antigens. The effect of two factors, L-arginine concentration and phytohemagglutinin (PHA) stimulation, on the expression of Epstein-Barr virus (EBV)-associated membrane antigens (MA) was evaluated. An EBV-producer cell line, AV-1, was cultivated in Eagle's basal medium with Earle's salts, supplemented with various L-arginine concentrations ranging from 0.0 to 20.0 mM. In most of the L-arginine concentrations, the number of cells expressing MA decreased within the first 24 hours. This decrease was followed by a marked increase in MA-positive cells at 48 hours and a slight decrease between 48 and 72 hours. Statistical evaluation, however, revealed no significant differences in the level of MA expression among cells exposed to the various L-arginine concentrations. These findings were discussed in relation to the cell cycle dependence of L-arginine-deficient media as a virus-activating agent. AV-1 cell cultures were treated with PHA and observed at 24-hour intervals for 72 hours. Within the first 24 hours, the percentage of cells showing MA was markedly increased. This was followed by a rapid decline in percentage of MA-positive cells within the next 24 hours. Treatment of an EBV-nonproducer cell line, NC37, with PHA resulted in the production of MA in approximately 11% of the cells within the first 24 hours. The number of MA-positive cells gradually declined over the next 48 hours. No viral capsid antigens were detected in these cells. The data suggested either complete or partial activation of the latent EBV genome by PHA.


Epstein-Barr virus and nasopharyngeal carcinoma: is there an etiologic relationship? Antibody titers to all the Epstein-Barr virus (EBV)-related antigens were significant elevated (P less than 0.001) in patients with nasopharyngeal carcinoma (NPC) compared with those of controls. EBV, early antigen, and anti-diffuse titers of Chinese-American and black patients with NPC were higher than those of white patients with NPC. Epstein-Barr viral capsid antigen antibody titers were elevated in white patients with squamous cell carcinoma of the pharynx, regardless of subsite. These results raise doubts about the specificity of the association between EBV and NPC.


Experimental carcinoma of liver in macaque monkeys exposed to diethylnitrosamine and hepatitis B virus. The effects of treatments with diethylnitrosamine (DENA) and hepatitis B virus (HBV) on macaque monkeys were investigated by virus serology and by light and electron microscopy. The experimental groups comprised 43 newborn or juvenile cynomolgus and rhesus monkeys of both sexes. HBV neither had a carcinogenic effect nor increased the oncogenic effect of DENA. However, HBV given to juvenile primates before treatment with DENA resulted in subsequent gross and microscopic alterations consistent with mild hepatitis and postnecrotic cirrhosis; multifocal liver carcinoma apparently developed within these cirrhotic nodules. The pathologic findings in the experimental animals were strikingly similar to those observed in liver cancer patients.


Susceptibility of common marmosets (Callithrix jacchus) to oncogenic and attenuated strains of Herpesvirus saimiri. Adult common marmosets, inoculated with either of 2 oncogenic Herpesvirus saimiri (HVS) strains, developed fatal lymphoproliferative disease within 23-25 days post inoculation (PI). The disease was identical to HVS-induced lymphoma in cotton-topped and white-lipped marmosets. Common marmosets inoculated with an attenuated HVS strain developed persistent infection; virus has been recovered from cocultivated lymphocytes of these animals for more than 384 days PI.


Elution of tumor-directed antibody from kidneys of mammary tumor-bearing mice. Immunoglobulin-containing eluates, prepared from kidneys of Paris RIII mammary tumor-bearing mice approximately 10 months old, were incubated with frozen sections of mouse mammary tumor and normal lactating mammary gland and examined by immunofluorescence. The eluates diffusely strained the tumor but did not stain the nonneoplastic lactating mammary gland. Mammary tumor and normal lactating mammary gland were both stained by an antiserum to mammary tumor virus (MuMTV). The euglobulin portion of the eluates, when fractionated on a Sephadex G-200 column, yielded an IgG fraction which was shown by immunodiffusion to react with Paris RIII mammary tumor extract and with anti-mouse globulin but not with nonneoplastic mouse tissue extracts. Since the lactating mammary glands of these animals contain MuMTV, the antibody-containing fraction eluted from the kidneys appeared to be tumor directed rather than virus directed.


Transformation of rat liver epithelial cells by Kirsten murine sarcoma virus. We studied the transformation of epithelial, diploid cell lines (RL-33 and RL-34) derived from W rat liver by the Kirsten murine sarcoma virus. On days 4-5 after virus infection, the epithelial cells began to pile up focally, forming small projections and releasing round cells from the foci. The epithelial cells grew in  chains or as islets and grew in suspension above the cells attached to the bottom of the flasks when the cultures reached the confluent stage. The virus titration pattern was "one-hit." Three classes of transformed cells were isolated with respect to virus release and antigen expression: 1) virus producer, 2) non-producer, and 3) sarcoma-positive, leukemia-negative cells. When transplanted sc into newborn rats, the transformed cells produced sarcomas. The transformed cells formed within 1-3 days larger aggregates than those of their normal counterpart cells when suspended in liquid growth medium above an agar base. Aggregate  properties (size, viability, and proliferation) of transformed cells correlated with growth in soft agar and tumorigenicity. RNA-dependent DNA polymerase and type C virus particles were readily induced in the normal rat liver epithelial cells after exposure to 5-iodo-2'-deoxyuridine.


Induction of simian virus 40 (SV40) transplantation immunity in mice by SV40-transformed cells of various species. Specific tumor rejection was obtained with the use of simian virus 40 (SV40)-transformed cells from several species including man, rat, ape, sheep, and hamster. Growth of the syngeneic sarcoma mKSA in BALB/c mice was strikingly inhibited following a single immunization with as few as 10(3) intact, viable cells. Non-SV40-transformed cells did not induce tumor rejection activity nor did SV40-transformed lines induce immunity against  the 3-methylcholanthrene-induced sarcoma Meth A, syngeneic with BALB/c mice. A close relationship existed between the tumor rejection antigen, the tumor-specific transplantation antigen (TSTA) located on the plasma membrane, and the intranuclear tumor antigen (T-ag). Both were associated with the DNA sequence of the early region of the SV40 genome, and TSTA activity was found in the nucleus. However, we did not observe a close parallelism between T-ag activity and TSTA. Neverthesless, the results strongly suggested that TSTA, like T-ag, was encoded by the virus.


Inhibitory effect of a polychlorinated biphenyl (Aroclor 1254) on aflatoxin B1 carcinogenesis in rainbow trout (Salmo gairdneri). Duplicate lots of 120 rainbow trout (Salmo gairdneri) fingerlings were fed for 1 year semipurified diets containing 6 ppb aflatoxin B1 (AFB1), 100 ppm Aroclor 1254 (a polychlorinated biphenyl), and 6 ppb AFB1 plus 100 ppm Aroclor 1254. Appropriate controls were also maintained. Samples were taken at 1, 2, 4, 6, 9, and 12 months to monitor tumor incidence, Aroclor 1254 accumulation, and histopathology of liver, spleen, and kidney tissues. At the end of the year, 26 of 37 (70.3%) trout fed 6 ppb AFB1 had hepatocellular carcinomas, compared to 14 of 46 (30.4%) trout fed 6 ppb AFB1 plus 100 ppm Aroclor 1254, a highly significant reduction in tumor incidence in the trout on the Aroclor 1254-containing diet. None of the control or Aroclor 1254-fed fish had liver tumors. Levels of Aroclor 1254 increased rapidly during the first 6 months, then plateaued at approximately 80 ppm on a whole-fish basis. AFB1 inhibited growth but Aroclor 1254 did not. Glycogen depletion of hepatocytes and hyperemia, and white pulp depletion of the spleen were the only changes induced by Aroclor 1254.


Transmission of mammary tumor virus in mouse strain DD: further support for the uniqueness of strain GR. Outcrosses between high tumor mouse strain DD and low-incidence strains BALB/c and C57BL to produce reciprocal F1 susceptible offspring of both types resulted in high incidences of mammary tumors in (DD female X BALB/c male)F1 and (DD female X C57BL male)F1 females but not in the reciprocal hybrids with DD as the male parent. The finding that transmission of murine mammary tumor virus (MuMTV) in DD was through the milk, as most often observed in mouse strains, further supported the uniqueness of strain GR in which MuMTV is transmitted by either parent.


High incidence of ependymomas induced by BK virus, a human papovavirus: brief communication. Ependymomas were produced in 44 of 50 Syrian golden hamsters and in 9 of 31 outbred Swiss mice inoculated intracerebrally with high-titer, purified BK virus (BKV). Tumors contained a T-antigen that reacted with BKV-specific T-antibody in immunofluorescence and complement-fixation tests. A proportion of tumor-bearing animals had antibodies to BKV T-antigen in their sera. BKV could be rescued from two tumor cell lines by Sendal virus-mediated fusion with Vero cells. A low, or lack of, oncogenic activity was displayed by BKV inoculated sc, ip, or iv.


Characterization of a new type of human papillomavirus that causes skin warts. A human papillomavirus (HPV) was isolated from the lesions of a patient (ML) bearing numerous hand common warts. This virus was compared with the well-characterized HPV found in typical plantar warts (plantar HPV). ML and plantar HPV DNAs have similar molecular weights (5.26 x 10(6) and 5.23 x 10(6), respectively) but were shown to be different by restriction enzyme analysis. When the cleavage products of both DNAs by endonuclease EcoRI, BamI, HpaI, or Hind were analyzed by electron microscopy, one, two, one, and four fragments were detected for ML HPV DNA instead of the two, one, two, and six fragments, respectively, detected for plantar HPV DNA. In contrast to plantar HPV DNA, a high proportion of ML HPV DNA molecules were resistant to these restriction enzymes. Most, if not all, of the molecules were either resistant to BamI and sensitive to EcoRI or sensitive to BamI and resistant to EcoRI. After denaturation and renaturation of the cleavage products of ML HPV DNA by a mixture of the two enzymes, the circular "heteroduplexes" formed showed one to three heterology loops corresponding to about 4 to 8% of the genome length. No sequence homology was detected between ML and plantar HPV DNAs by cRNA-DNA filter hybridization, by measuring the reassociation kinetics of an iodinated plantar HPV DNA in the presence of a 25-fold excess of ML HPV DNA, or by the heteroduplex technique. The two viruses had distinct electrophoretic polypeptide patterns and showed no antigenic cross-reaction by immunodiffusion or immunofluorescence techniques. Preliminary cRNA-DNA hybridization experiments, using viral DNAs from single or pooled plantar or hand warts, suggest that hand common warts are associated with viruses similar or related to ML HPV. The existence of at least two distinct types of HPVs that cause skin warts was demonstrated; they were provisionally called HPV type 1 and HPV type 2, with plantar HPV and ML HPV as prototypical viruses, respectively.


State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations. F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication.


Evidence for simian virus 40 (SV40) coding of SV40 T-antigen and the SV40-specific proteins in HeLa cells infected with nondefective adenovirus type 2-SV40 hybrid viruses. HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND1, Ad2(+)ND2, Ad2(+)ND4, and Ad2(+)ND5) synthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methionine-containing tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific protein (100,000 daltons) from Ad2(+)ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2(+)ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virus-infected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and -transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.


Characterization of the autoregulation of simian virus 40 gene A. Cells infected by tsA mutants of simian virus 40 (SV40) overproduce early RNA. Overproduction results from failure of the temperature-sensitive A protein (T antigen) to inhibit early transcription. The amount of early RNA in the cytoplasm, determined quantitatively from the kinetics of hybridization to labeled complementary SV40 DNA, was elevated at both permissive (32 degrees C) and nonpermissive (41 degrees C) temperatures in all the early mutants tested (tsA7, -30, -58, and -209), but not in the late mutant tsB4. The amount of early RNA in a culture maintained at 32 degrees C for 72 h and then shifted to 41 degrees C was maximum when each cell was infected initially with at least one plaque-forming unit of tsA58. Azidocytidine (2'-deoxy-2'-azidocytidine), which inhibits initiation of DNA synthesis, did not cause overproduction of early RNA in cells infected with wild-type SV40, showing that the effect seen with tsA mutants is not due to interference with initiation of DNA synthesis per se. In parallel infections at 41 degrees C, the amount of early RNA per copy of viral DNA was as much as 2,000 times greater with tsA58 than with wild-type SV40, even though there was no replication of the tsA58 DNA. Synthesis of late RNA could not be detected during the first 20 h of an infection by either virus at 32 degrees C, indicating that late and early transcription are under different control. In three cell lines transformed by tsA mutants, the amount of early RNA increased moderately after a shift from 32 to 41 degrees C, whereas with homologous cells transformed by wild-type virus, the amount of early RNA decreased, indicating that the A protein may be able to repress transcription of integrated SV40 DNA. All the observations are consistent with a simple model in which the binding of A protein at the origin of replication blocks either binding of RNA polymerase to the early promoter or its progress through the early gene(s).


Electron microscopic characterization of the defectiveness of a temperature-sensitive mutant of Moloney murine leukemia virus restricted in assembly. The effect of temperature shiftdown on the assembly of ts3 virions was investigated by both scanning (SEM) and transmission (TEM) electron microscopy. Ts3 is a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus (Mo-MuLV) which previous studies indicated to be defective in assembly or release of the virions. In the present study, both SEM and TEM revealed the following: (i) there were more cell-associated virions in ts3-infected cells grown at the nonpermissive temperature (39 degrees C) than either in cells grown at the permissive temperature (34 degrees C) or in wild-type MuLV-infected cells grown at 39 degrees C; (ii) there were more normal single particles than multiploids (virions with two or more pieces of genomic RNA) in ts3-infected cells grown at the nonpermissive temperature; (iii) there were more multiploids in ts3-infected cells grown at the nonpermissive temperature than either in cells grown at the permissive temperature or in wild-type MuLV-infected cells grown at the nonpermissive temperature; (iv) upon temperature shift from 39 to 34 degrees C, about 90% of the cell-associated virions dissociated from the cell surface. TEM studies also indicated that upon temperature shiftdown, virion assembly rapidly occurred. The above observations suggest that faulty assembly, which results in the production of multiploids, may not be the reason why ts3 virions accumulate on the cell surface at the nonpermissive temperature. The relatively higher proportion of multiploids found in ts3-infected cells grown at 39 degrees C compared with those grown at 34 degrees C may be due to the higher density of budding virions at the cell surface at the nonpermissive temperature, which increases the possibility of two or more particles assembling close to one another. The accumulation of ts3 virions in all stages of assembly at the nonpermissive temperature, together with the fact that rapid assembly and release of ts3 virions occurred on temperature shiftdown, indicates that virion assembly is restricted after it has been initiated. The probable role of altered glycoprotein(s) in restricting virion assembly is discussed.


Anatomy of herpes simplex virus DNA. IX. Apparent exclusion of some parental DNA arrangements in the generation of intertypic (HSV-1 X HSV-2) recombinants. We are reporting the physical location of parental DNA sequences in 28 recombinants produced by crossing herpes simplex viruses (HSV) 1 and 2. The parental crosses were of two kinds. In the first, temperature-sensitive mutants of HSV-1 and HSV-2 were crossed to produce wild-type recombinants. In the second, temperature-sensitive mutants of HSV-1 rendered resistant to phosphonoacetic acid were crossed with wild-type HSV-2, and recombinants that multiplied at nonpermissive temperature and were resistant to the drug were selected. The DNAs of the recombinants were mapped with XbaI, EcoRI, HpaI, HsuI, BglII, and, in some instances, KpnI restriction endonucleases. The results were as follows. (i) We established the colinear arrangements of HSV-1 and HSV-2 DNAs. (ii) There was extensive interchange of genomic regions, ranging from the exchange or the entire L of S component of HSV DNA to substitutions of regions within the same component. In some recombinants, the reiterated sequences ab and ac bracketing the L and S components of HSV DNA were heterotypic. Most recombinants grew well and showed no obvious defects. (iii) The number of crossover events ranged from one to as many as six. Although crossover events occurred throughout the DNA, some clustering of crossover events was observed. (iv) Analysis of recombinants permitted localization of several markers used in this study and appears to be a useful technique for marker mapping. (v) As previously reported, HSV DNA consists of four populations, differing in relative orientation of the L and S components. All recombinants could be displayed in one arrangement of L and S such that the number of crossover events was minimized. The data are consistent with the hypothesis that only one arrangement of the parental DNA participates in the generation of recombinants.


Human cytomegalovirus genome: partial denaturation map and organization of genome sequences. Contour-length measurements of both nondenatured and partially denatured DNA from purified extracellular human cytomegalovirus indicate that more than one size class of viral DNA is encapsidated. In addition to a size class averaging about 100 x 10(6) daltons, a much less abundant class of larger viral DNA molecules, 150 x 10(6) to 155 x 10(6) daltons, was extracted from purified extracellular virus. As predicted by melting-curve analysis, partial denaturation of human cytomegalovirus DNA generates denaturation maps showing distinctive adenine plus thymidine (A+T)-rich and guanine plus cytosine (G+C)-rich localizations. Alignment of partial denaturation maps of both 100 x 10(6)- and 150 x 10(6)- to 155 x 10(6)-dalton molecules from maximum overlap of common A+T- and G+C-rich zones clearly shows six unique zones contained in a length equal to the longest class, 150 x 10(6) to 155 x 10(6) daltons. However, various alignments of the smaller class of the molecules within the confines of the approximately 100 x 10(6)-dalton-length equivalent are nondistinctive. Of the six unique A+T- and G+C-rich zones, five are linked in a specific sequence and maintain the same relative orientation; these features indicate the absence of major inversions within these zones. The sixth unique zone may occur at either end of this five-zone series, but it was never found at both ends of the same molecule. Additionally, this terminal zone appears to undergo complete inversions at least at one end of the alignment, and perhaps at both. These data indicate that 150 x 10(6)- to 155 x 10(6)-dalton molecules comprise human cytomegalovirus-specific genetic information.


Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro. Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.


Structure and formation of circular dimers of simian virus 40 DNA. Most of the viral DNA extracted from simian virus 40 (SV40)-infected African green monkey kidney cells consists of circular molecules about 5.3 kilobases in contour length. However, about 1% of the viral DNA was found to occur as closed circular dimers that appeared to be formed, preferentially, late in infection. The monomeric units of dimers were organized in a head-to-tail, tandem arrangement; moreover, the monomeric units were not defective; i.e., they lacked deletions or other rearrangements. After infections with dimer DNA, nondefective monomers were formed. These findings suggest that dimers are not intermediates in the production of defective SV40 genomes. The majority of the dimers formed in mixed infections with two mutants were homodimers, but about 5% of the circular dimers were heterodimers and must have arisen by intermolecular recombination.


[125I]deoxycytidine used in a rapid, sensitive, and specific assay for herpes simplex virus type 1 thymidine kinase. [125I]deoxycytidine was a good substrate for herpes simplex virus type 1 thymidine kinase (TK), whereas [125I]deoxycytidine was a very poor substrate for cellular TK. Simple, sensitive, and specific assays for viral TK could be carried out in vivo and in vitro even in the presence of cell TK. Autoradiographic detection of incorporated [125I]deoxycytidine provided a rapid and simple method for detection of and screening for viral TK in infected as well as viral TK-transformed cells.


Relationship between the methionine tryptic peptides of simian virus 40 and BK virus tumor antigens. The monomer form of BK virus (BKV) tumor antigen (T Ag) was immunoprecipitated from extracts of BKV-transformed cells and had a molecular weight of approximately 113,000. This compared with 97,000 for the molecular weight of either BKV or simian virus 40 (SV40) T Ag from lytically infected cells. The SV40 and BKV T Ag's from productively infected cells were compared by examining their methionine-labeled tryptic peptides. Out of a total of 20 SV40-and 21 BKV-specific peptides, there were seven pairs of similar peptides on the basis of ion-exchange chromatography, These coeluting peptides contained approximately 25 to 30% of the total methionine radioactivity. Similar results were obtained when the tryptic peptides of SV40 T Ag from lytically infected cells were compared with those of BKV T Ag from virally transformed cells.


Prolongation of herpes simplex virus latency in cultured human cells by temperature elevation. Treatment of herpes simplex virus type 2 (HSV-2)-infected human fibroblast cells with cytosine arabinoside (ara-C) at 25 microgram/ml resulted in complete inhibition of virus replication. Removal of ara-C after 7 days of treatment ultimately resulted in renewed virus replication, but after a delay of at least 5 days. If however, the temperature was elevated from 37 degrees C to 39.5 to 40 degrees C at the time of ara-C reversal, infectious HSV-2 did not reappear. As long as the cultures were maintained at 39.5 to 40 degrees C (up to at least 128 days), HSV-2 was latent and infectious virus was undetectable. If the temperature was reduced to 37 degrees C at any time during the latent period, infectious virus was always reactivated, but only after a period of incubation at 37 degrees C of a least 11 days. Infectious-center assays performed with latent cultures indicated that only a very small fraction of cells could reactivate virus. The infectious-center titer did not show significant changes during much of the period of latency. This seemed to argue against the possibility that the latent cultures were synthesizing very small amounts of infectious virus. Additional studies were aimed at determining the minimum incubation period at 37 degrees C required to reactivate infectious HSV-2. Latent cultures reduced from 39.5 to 40 degrees C to 37 degrees C for less than 96 h did not yield infectious HSV-2, but those incubated at 37 degrees C for 96 h or more did.


Temperature-sensitive mutants of human cytomegalovirus. Eight temperature-sensitive mutants of human cytomegalovirus have been isolated after mutagenesis with nitrosoguanidine. Three of these mutants have been classified into three separate complementation groups and are capable of synthesizing virus DNA at the nonpermissive temperature (39.5 degrees C). Two others appear unable to synthesize virus DNA at the elevated temperature.


Size and genetic content of viral RNAs in avian oncovirus-infected cells. Viral complementary DNA (cDNA) sequences corresponding to the gag, pol, env, src, and c regions of the Rous sarcoma virus genome were selected by hybridizing viral cDNA to RNA from viruses that lack the env or src gene or to polyadenylic acid [poly(A)]-containing RNA fragments of different lengths and isolating either hybridized or unhybridized DNA. The specificities, genetic complexities, and map locations of the selected cDNA's were shown to be in good agreement with the size and map locations of the corresponding viral genes. Analyses of virus-specific RNA, using the specific cDNA's as molecular probes, demonstrated that oncovirus-infected cells contained genome-length (30-40S) RNA plus either one or two species of subgenome-length viral RNA. The size and genetic content of these RNAs varied, depending on the genetic makeup of the infecting virus, but in each case the smaller RNAs contained only sequences located near the 3' end of the viral genome. Three RNA species were detected in Schmidt-Ruppin Rous sarcoma virus-infected cells: 39S (genome-length) RNA; 28S RNA, with an apparent sequence of env-src-c-poly(A); and 21S RNA, with an apparent sequence of src-c-poly(A). Cells infected with the Bryan high-titer strain of Rous sarcoma virus, which lacks the env gene, contained genome-length (35S) RNA and 21S src-specific RNA, but not the 28S RNA species. Leukosis virus-infected cells contained two detectable RNA species: 35S (genome-length) RNA and 21S RNA, with apparent sequence env-c-poly(A). Since gag and pol sequences were detected only in genome-length RNAs, it seems likely that the full-length transcripts function as mRNA for these two genes. The 28S and 21S RNAs could be the active messengers for the env and src genes. Analyses of sequence homologies among nucleic acids of different avian oncoviruses demonstrated substantial similarities within most of the genetic regions of these viruses. However, the "common" region of Rous-associated virus-0, an endogenous virus, was found to differ significantly from that of the other viruses tested.


Genetic variation in the RNA transcripts of endogenous virus genes in uninfected chicken cells. Uninfected cells from two different phenotypes of chicken embryos express significant amounts of endogenous viral information, though they do not produce virus particles. Cells of the phenotype gs(+)chf(+) are positive for both group-specific (gs) antigens and chicken helper factor (chf) activity, whereas cells of a second phenotype, gs(L)chf(+)(h(E)), demonstrate noncoordinate expression of these two viral activities (very low amounts of gs antigens, but extremely high helper activity). RNA from these cells was analyzed to determine the size, genetic content, and relative abundance of virus-specific RNAs in cells of each phenotype. Two major size classes of polyadenylic acid-containing RNA, homologous to the avian leukosis virus genome, were detectable in cells of both types. The larger RNA, which contained most of the sequences of the leukosis virus genome, was of different sizes in the two phenotypes, 31S in gs(+)chf(+) cells but 35S in the noncoordinate cell type. Analysis of the viral RNA with gene-specific complementary DNA probes revealed the following characteristics. (i) The 31S RNA appeared to lack portions of the gag and pol genes. (ii) A smaller RNA species, which sedimented at 21S in both cell types, was a transcript of the 3'-proximal portion of the viral genome, consisting of the env gene and the "common" sequences. (iii) The amount of env-specific RNA in the 21S region was more than six times higher in the noncoordinate cell type than in the gs(+)chf(+) cells; this difference was concordant with the 5- to 10-fold higher chf activity in the noncoordinate cells. (iv) The endogenous viral RNA in uninfected cells and the RNA from Rous-associated virus-0 virions hybridized only partially with DNA complementary to the common region of the Rous-associated virus-2 genome, whereas the RNA of all exogenous viruses tested hybridized almost completely to this complementary DNA. Small amounts of src-specific polyadenylated RNA were also present in uninfected chicken cells. This RNA sedimented as a single peak at 26S and was not covalently linked to any other identifiable virus-specific RNA sequences. The amount of src RNA was the same in the above two types of expression-positive cells and also in cells that were gs(-)chf(-), indicating that the transcription of the cellular sequences homologous to the src gene is independent of the transcription of the other endogenous viral genes.


Visna virus RNA synthesis.

Visna virus RNA synthesis. Visna is a classical slow infection in which virus characteristically persists in the face of the host immune response. The agent of this disease belongs to the retravirus group. The persistence of infection and the slow spread of virus are at least in part a consequence of restriction of the expression of virus genetic information in tissues of an infected animal (A. T. Haase et al., Science 195:175-177, 1977), but the point at which the virus life cycle is interrupted in vivo and the mechanism of restriction are unknown. We have embarked on a molecular analysis of restriction, focusing first on transcription. In this paper we have established the levels of viral RNA synthesis under permissive conditions, as a base line for subsequent studies in vivo. We show that (i) uninfected cells do not contain RNA sequences related to the visna virus genome, (ii) parental RNA is rapidly transported to the nucleus of the infected cell, (iii) virus RNA is synthesized in the nucleus and then transported to the cytoplasm (iv) synthesis of RNA proceeds mostly exponentially to reach levels of about 4,000 copies per cell at the end of the growth cycle, (v) nuclear and cytoplasmic RNA sediment in two size classes, 35S and 10-20S, (vi) viral mRNA  has the same polarity as genome RNA and also sediments in two size classes of 35S and 10-20S.


Crossed immunoelectrophoretic studies of the solubility immunogenicity of herpes simplex virus antigens. The nonionic detergent Triton X-100 was capable of solubilizing 90% of the protein content in herpes simplex virus (HSV)-infected rabbit cornea cells. The solubilized HSV antigens formed well-characterized precipitates by crossed immunoelectrophoresis in Triton X-100-containing agarose gel, allowing both identification and relative quantitation. Water-soluble and detergent-requiring HSV antigens were identified by different solubilization procedures in buffer with and without detergent. Five glycoprotein antigens were solubilized only in the presence of detergent, indicating their membrane-bound state. One non-glycosylated antigen was present in both a water-soluble and a membrane-bound form. Based upon the crossed immunoelectrophoretic precipitating patterns of Triton X-100-solubilized HSV antigens, it has been estimated that infected cells yield an amount of virus-specific protein equivalent to 2,000 enveloped virions per cell. Rabbits inoculated intracutaneously with Triton X-100-solubilized HSV antigens developed neutralizing antibodies against HSV almost as effectively as rabbits with an active HSV infection. Precipitins against individual HSV antigens in sera from rabbits infected with HSV and immunized with the Triton X-100-solubilized HSV antigens were assayed by the crossed immunoelectrophoretic technique. Sera from infected rabbits reacted more strongly and with a higher number of HSV antigens than sera from immunized rabbits.


Further biochemical characterization, including the detection of surface glycoproteins, of human, calf, and simian rotaviruses. Polyacrylamide gel electrophoretic analysis of purified preparation of the simian rotavirus SA-11 indicated eight polypeptide components that migrated in a manner remarkably similar to those of the previously characterized human and calf rotaviruses. Analyses of preparations of single-shelled and double-shelled particles of human, calf, and simian an rotaviruses have also permitted assignment of the polypeptides to the inner or outer shells. The major components of the outer shells of each virus have been identified as glycoproteins, and the importance of this in terms of host cell specificity is discussed. Sensitivities of the various rotaviruses to acid, proteases, and glycosidases were also investigated.


Genome of infectious bronchitis virus.

Genome of infectious bronchitis virus. Techniques are described for the growth and rapid purification of the avian coronavirus infectious bronchitis virus (IBV). Purified IBV has a sedimentation coefficient of 320S and a buoyant density of 1.22 g/ml in sucrose-deuterium oxide equilibrium gradients. IBV RNA extracted by proteinase K in the presence of sodium dodecyl sulfate and further purified by phenol extraction and gradient centrifugation is single stranded and has a sedimentation coefficient of 64S, as determined by isokinetic gradient centrifugation. Analysis on sucrose gradients under both aqueous and denaturing conditions together with agarose gel electrophoresis in the presence of the chaotropic agent methylmercuric hydroxide gave a value of 8 X 10(6) for the moleclar weight of IBV RNA. This value was confirmed by RNase T1 fingerprinting, which also indicated that IBV RNA is haploid. No evidence was found of subunit structure in IBV RNA. From these results together with the recently reported observation that IBV RNA is infectious and contains a tract of polyadenylic acid (Lomniczi, J. Gen. Virol., in press), we conclude that the genome of the coronaviruses is a single continuous chain of about 23,000 mononucleotides that is of messenger polarity.


Inflammatory fibrous histiocytoma presenting as a renal pelvic and bladder mass. The first case of simultaneous presentation of a bladder and renal pelvic malignant fibrous histiocytoma is reported. Although only a small percentage of fibrous histiocytomas are malignant the lesion is extremely aggressive. Radiotherapy and chemotherapy have been used but only sporadic remissions have been reported. If the tumor is localized wide surgical excision is recommended.


Wilms' tumor metastatic to the orbit.

Wilms' tumor metastatic to the orbit. In a 2 1/2-year-old boy, a proptotic right lower lid developed one year after a primary abdominal mass proved to be Wilms' tumor. An orbital abscess or fungal infection was considered because the child was receiving chemotherapy. However, echography demonstrated a firm orbital mass, delineated its dimensions, and showed destruction of the orbital floor. The biopsy specimen showed metastatic tumor cells. Lile neuroblastoma and certain hematologic and reticuloendothelial malignant neoplasms, Wilms' tumor may secondarily invade the ocular adnexa.


Effects of angiotensin III (DES-1-asp-angiotensin II) and angiotensin III analogue (DES-1-asp-8-ile-angiotensin II) upon adrenal steroidogenesis and blood pressure. Effects of angiotensin III and angiotensin III analogue upon adrenal steroidogenesis and blood pressure were studied in rats, rabbits and a man. Pressor effect of angiotensin III was about one fifth of that of angiotensin II in all the species. Degradation rate  of pressor effect of angiotensin III in plasma was more rapid than that of angiotensin II. Different from the effects of angiotensin III upon blood pressure, its effect upon aldosterone was similar to that of angiotensin II. The effect of angiotensin III upon other adrenal steroids, such as DOC and cortisol, however, seemed to be slightly less than that of angiotensin II. Angiotensin III producted an additive effect to that of ACTH, but it didn't produce an additive effect to that of angiotensin II. Angiotensin III analogue, itself, stimulated adrenal steroidogenesis, but it inhibited the effects of angiotensin III and angiotensin II upon aldosterone. Effects of ACTH upon plasma DOC and cortisol were not inhibited by angiotenesin III analogue, but the effect of ACTH upon aldosterone was blunted slightly.


[The effect of natural and artificial light via the eye on the hormonal and metabolic balance of man (author's transl)]. Examinations of hormone levels in blind persons compared with almost blind and people with normal vision and hormone evaluation with increased artificial light exposure show a definite stimulating effect of light on the human hormonal balance. The effect of light is mediated by an intact perception of light by the eye over an "energetic part" of the visual pathway (Hollwich 1948) - hypothalamus - hypophysis - peripheral endocrine gland. Increasing the intensity of artificial light with "neon-tubes" (fluorescent tubes) leads to "light stress" proved by increased hormone production - especially the stress hormone Cortisol. The belief that artificial light is the same as natural light and that it can fully replace it, is inappropriate in the medical view and needs correction.


[Effect of clofibrate on lipoprotein-lipids in patients with type IV hyperlipoproteinaemia (author's transl)]. The effect of clofibrate treatment (1.5 g/day for four weeks) on triglycerides, cholesterol and phospholipids in very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) was investigated in 12 patients with type IV hyperlipoproteinaemia. The decrease of serum triglycerides was due to the reduction of VLDL-triglycerides. Furthermore, LDL-hypertriglyceridemia was normalised by clofibrate. -- There existed a significant decrease of VLDL- und HDL-cholesterol as well as of VLDL-phospholipids. The changes in LDL-cholesterol and LDL-phospholipids by clofibrate depended on their initial concentrations which were negatively correlated to the serum triglycerides before treatment. -- The normalisation of lipid concentrations in VLDL and LDI could be explained by a stimulated delipidation of the apo B-lipoproteins.


[Model studies on virus-induced tumors and their immunological treatment (author's transl)]. After a review of the general biological properties of C-type oncorna viruses, results are presented on the structure of an exogenous murine leukemia virus (FLV) and on the serobiological properties of its structural proteins. Our findings suggested a major role of the viral surface glycoprotein gp71 in immunological defense mechanisms. This was confirmed by vaccination experiments with isolated gp71 in mice. The induced immunity was highly specific and not operative against endogenous murine C-viruses belonging to other serotypes. Surprisingly the latter were found to be activated by the vaccination with gp71 of FLV. In heterologous animal species isolated FLV-gp71 induced the formation of broadly reacting antibodies. They were found to be effective in the therapy of infections with FLV in mice as well as with feline leukaemia virus in cats. Most impressive results were obtained with an antiserum prepared against feline leukaemia virus in a goat. This serum completely suppressed sarcomas induced by infection with feline sarcoma virus.


[Factors involved in the development of human tumors using the Epstein-Barr virus as an example (author's transl)]. Several viruses induce tumors in animals under experimental or natural conditions. It is likely therefore that some human malignancies are also caused by viruses. Proof of this hypothesis can be provided only by indirect evidence based on the following criteria: (1) detection of viral antigens or viral genetic information in a given tumor; (2) transformation of normal human cells by the virus in tissue culture; (3) induction of tumors in animals by the virus; and (4) demonstration of enhanced titers of antibodies to the virus in patients bearing the tumor. These criteria have been fulfilled to support a causal relationship of the Epstein-Barr virus (EBV) in Burkitt's lymphoma and nasopharyngeal carcinoma. It is clear, however, that factors of a genetic, immunologic or environmental nature must play an additional role because EBV, the cause of infectious mononucleosis, is widely disseminated yet development of the tumors is a rare event.


The gastrin-producing cells in tissue cultures of the rat pyloric antrum. Tissue culture methods were used to study the gastrin-producing cells isolated from the pyloric antral mucosa of rats. Cultures were maintained for up to 7 days. Gastrin cells were identified by means of immunofluorescent and unlabeled antibody-enzyme techniques. The tissue culture medium was monitored for basal gastrin secretion. In addition, stimulation of gastrin release was achieved by the use of dibutyryl cyclic-AMP and theophylline. This is the first report of the successful application of tissue culture methods to the functional study of antral gastrin cells. The procedure has proven to be useful in assessing the mechanisms of gastrin secretion.


The place of electrical stimulation in the physiotherapy of leprosy. The production of nerve and muscle impulses by faradic and interrupted direct current, and the "Strength-duration curves" plotted for normal, denervated, and partially denervated muscles, are described. The advantages and disadvantages of such electrical stimulation in the testing of recent paralysis, the treatment of recent paralysis, and following tendon transfer surgery, in leprosy patients, are discussed. In the light of these, electrical stimulation is concluded to have only a minor role in the Physiotherapy of leprosy.


Pattern of reactions in leprosy: a clinical appraisal. The term 'reactions' is applied in the present text, to describe the acute episodes, recognized with the different types of leprosy. The incidence of reactions was found to be 9.7% of 1053 cases examined. They were seen in all types of leprosy, but their frequency and severity was marked in lepromatous and borderline cases, and in majority of them, the age ranged from 20-40 years. The precipitating factor(s) could not be established in many; in some dapsone was incriminated, followed by bacterial infection and malnutrition. The exacerbations of the existing lesion(s) either alone or in combination with new lesions (E.N.L.) and E.N.L. lesions alone, were the presenting clinical features. The constitutional symptoms were observed largely in the types other than the tuberculoid. Foot drop and contractures of the fingers were the common sequels of the reactions. An attempt has been made to explain their mechanisms in the light of the recent literature.


Hormonal regulation of hepatic amino acid transport. The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na+ dependent and the present finding that the uptake of 2-aminobicyclo [2,2]heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na+ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.


Perspectives and limitations of resolutions-reconstitution experiments. Reconstitutions of membranous activities can tell us how many components are required and what their functions are. The mitochondrial proton pump is used as an example. Moreover, the biological activity, such as Pi transport, can be used in reconstituted vesicles as an assay during the isolation of the transporter. Reconstitution experiments reveal the importance of membrane asymmetry and allow us to study conditions of vectorial assembly. The mechanism of action of ion pumps has been successfully analyzed in reconstituted liposomes. We can study the movement of ions and the electrogenicity of the system without interference by other unrelated processes. Based on studies with the resolved Ca2+-ATPase of sarcoplasmic reticulum, we propose a novel formulation of the mechanism of ATP-driven ion pumps in which cyclic binding of Mg2+ plays a key role.


[Granular cell tumour of the parotid gland. The differential diagnostic criteria (author's transl)]. Granular cell tumours (synonyms: myoblastmyomas, granular neuromas, granular cell myoblastomas) are localized predominantly in the head and neck area, especially in the region of tongue and larynx. Only 2 observations of an occurrence in the parotid gland exist till now in the world literature. This paper reports a further case of an occurrence in the parotid gland in a 6-years-old girl. The about cherrygreat tumour was distinctly limited and showed histologically a typic lobular arrangement of acidophilic granular cells. No recidive was observed in the prevailing postoperative phase of 3 years. The differential diagnosis of the granular cell tumorus is demonstrated to other tumour types with similar histological arrangement, expecially to the alveolar soft-part sarcoma, the acinic cell tumour and the non-chrom-affine paraganglioma (chemodectoma).


Antigenic variation of poliovirus caused by antibody components with different specificities. The possible role of antibody as a selective pressure on antigenic mutants of poliovirus in nature was investigated in vitro. A mutant resistant to a monospecific antibody with a definite specificity was readily obtained by several cycles of neutralization of Mahoney strain with a monospecific antibody and multiplication in monkey kidney (MS) cells. Mutants resistant to more than two different monospecific antibodies were also readily obtained in a similar manner. Studies on the antigenicity of these mutants by kinetic neutralization tests revealed that the Mahoney strain underwent a progressive serological variation as it became successively resistant to one to five different monospecific antibodies isolated from anti-Mahoney serum.


Restitution of hemagglutinating activity to spikeless particles of HVJ (Sendai virus) by glycoprotein components of Newcastle disease virus. Spikeless particles of HVJ (Sendai virus) lacking in hemagglutinating (HA) activity were obtained by enzymatic digestion of virions with trypsin followed by centrifugation through a sucrose gradient. When they were mixed with glycoprotein components of Newcastle disease virus (NDV) obtained by treatment of purified virions with deoxycholate (DOC), the mixture showed hemagglutination reaction, which was inhibited by anti-NDV serum, but not by anti-HVJ serum. Sedimentation profile of the HA active agents was then examined by centrifugation of the mixture of spikeless particles of HVJ (labeled with 3H-uridine) and glycoproteins of NDV (labeled with 14C-amino acid mixture). The results showed that the peak of HA activity had both of the radioactivities, and that the sedimentation rate of the HA was faster than that of spikeless HVJ but slower than that of intact HVJ. Electron micrographs of such HA active structures showed that they were morphologically closely similar to intact virion of HVJ, although they had neither hemolytic activity nor infectivity. The mixture of spikeless HVJ and glycoproteins of HVJ or NDV which were removed from virions by proteolytic enzymes, on the other hand, did not show any detectable hemagglutinating activity.


[Respiratory chain of Candida mycoderma]. The respiration chain of the yeast Candida mycoderma was studied during its growth on glucose. Electrons can be transported from the pyridine nucleotide (PN) pool to oxygen by two pathways, as was shown in experiments with intact cells on the basis of inhibitory analysis of respiration and the extent of reduction of electron carriers. The first pathway is inhibited by cyanide and antimycin A; it includes PN, flavoprotein, (FP) and cytochromes b, c and a + a3. The second pathway of electron transport is resistant to cyanide (CrPET), includes PN and FP, lacks cytochromes, and is blocked by salicyl- or benzhydroxamic acid. Two flavoprotein NADH-dehydrogenases are present in the cells of C. mycoderma: one is sensitive to rotenone, the other is resistant to it. The terminal electron acceptor can be not only O2, but also H2O2; in the latter case, electron transport involves PN, FP, and cytochromes b and c. At the exponential growth phase, only the first pathway of electron transport, including rotenone-resistant NADH-dehydrogenase, functions. At the phase when the rate of growth decreased, both pathways of electron transport and the two dehydrogenases are involved. At the stationary phase, mainly CrPET functions, but both NADH-dehydrogenases participate.