Antiandrogenic effect of spirolactones: mechanism of action.

Spirolactones are aldosterone antagonists which inhibit the binding of aldosterone to the renal mineralocorticoid receptor. These molecules also possess an antiandrogenic effect which could be due, among other possibilities, to a  peripheral antagonism of androgens. This hypothesis has been tested in the present study. From in vivo experiments, spironolactone K+ canrenoate appear to inhibit the binding of [3H]5alpha-dihydrotestosterone [3H]DHT to the cytosolic and nuclear receptor of the rat ventral prostate. The doses used are in the same range as those used for demonstrating the antimineralocorticoid effect of these molecules. In vitro incubations and in vitro displacement studies show that spironolactone and K+ canrenoate are respectively about 20 and 100 times less effective than DHT in displacing 50 percent of 5 times 10- minus 10 M [3H]DHT from its receptor. Spirolactones are also able to compete with [3H]DHT for the specific 8 S cytosolic receptor. Neither spironolactone nor K+ canrenoate decreases prostatic 5alpha-reductase activity, even at a concentration as high as 10- minus 5 M. It seems likely that spirolactones, besides their action on testosterone biosynthesis, exert their antiandrogenic activity via a peripheral androgen antagonism.


Modulation by thyroid hormones of cytoplasmic estrogen receptor concentrations in reproductive tissues of the rat.

The influence of induced variations in circulating levels of thyroid hormones upon the estrogen receptor content of rat uterine, anterior pituitary and hypothalamic cytosol fractions has been investigated. Sucrose gradient centrifugation analyses showed no observable effects of thyroidectomy or acute induced hyperthyroidism upon receptor levels in uterus hypothalamus of ovariectomized rats, but an apparent positive thyroidal influence upon anterior pituitary receptor content. Qualitatively, the receptor complex sedimentation coefficients were unaltered. Binding assays showed that thyroid hormone administration to the ovariectomized rat resulted in a marked increase in pituitary receptor content, whereas the same treatment had no significant effect on the pituitary receptor level of the intact animal. Conversely, a marked fall in uterine receptor content was found in thyroxine-treated intact, but not castrate, animals. The changes in measurable receptor levels could not be attributed to competition for estrogen binding sites by thyroxine or triiodothyronine. Pituitary receptor content of ovariectomized rats was studied following administration of varying levels of thyroxine; 5 mug/day significantly depressed receptor levels, while enhancement of binding capacity was occasioned by levels of thyroxine ewual to or greather than 25 mug/day. The hypothalamus was completely unaffected by similar treatment and the uterus responded with decreased receptor level only at the highest dosage used. Thyroxine replacement reversed the decrease in anterior pituitary receptor concentration detectable following thyroidectomy. The effect of the thyroid hormones on the pituitary receptor content was not a result of changes in the capability of the tissue to respond to estrogen-induced synthesis of receptor.


Premature regression of corpora lutea in pseudopregnant rabbits following the removal of polydimethylsiloxane capsules containing 17 beta-estradiol.

17-beta-Estradiol, which is luteotropic in rabbits, was administered during pseudopregnancy via polydimethylsiloxane (Silastic) implants to determine the effects on serum progesterone concentrations. Implants which released estradiol at a rate of approximately 2 mug/day were place beneath the skin the day after sterile mating and ovulation (day 0). Blood (3 ml) was obtained from the marginal ear vein on days 3, 6, 9, 10, 11 and 12. Serum estradiol levels, determined by radioimmunoassay, were 2- to 3-fold higher in estradiol-treated rabbits (11.7 plus or minus 1.2 pg/ml) than in untreated pseudopregnant controls (5.9 plus or minus 1.4 pg/ml). Weights of corpora lutea in treated and control rabbits were not different at the conclusion of the experiment on day 12. Serum progesterone concentrations, also determined by radioimmunoassay, were not significantly different between treated and control animals. However, when estradiol implants were removed from other rabbits on day 10, a rapid decline in serum progesterone occurred, from 14.0 plus or minus 2.4 to 2.6 plus or minus 0.8 ng/ml 24 h later. By comparison, serum progesterone concentrations in rabbits with estradiol implants left in place and in untreated rabbits on day 12 were similar  (similar to 12 ng/ml). The premature decline in serum progesterone was accompanied by a decrease in the wet weight of corpora lutea. Other experiments revealed: 1) a precipitous fall in serum estradiol to basal values within 2 h after estradiol implants were removed, preceding the decline in serum progesterone by approximately 6 to 10 h; 2) reduced levels of estradiol in ovarian venous blood, but elevated levels of estradiol in peripheral arterial blood of rabbits with estradiol impants. The inability to elevated estradiol to increase serum progesterone or weights of corpora litea suggests that the luteotropic effect is maximal when estradiol is present at physiological concentrations. Following the continuous administration of estradiol, ovarian secretion of estradiol appears diminished and the corpora lutea become dependent upon the exogenous estradiol for luteotropic support. Although the ovaries continue to release measureable quantities of estradiol, this is inmeasurable quantities of estradiol, this is insufficient to prevent regression of corpora lutea when exogenous estradiol is rapidly withdrawn from the circulation.


Influence of ionic strength of incubation medium on the release of some enzymes from rat liver mitochondria.

Influence of ionic strength of incubation medium on the release of adenylate kinase (AK), isocitrate dehydrogenase (IDH) and glutamate dehydrogenase (GDH) from intact and injured rat liver mitochondria has been studied. It has been found that raised ionic strength (mu equal 0.16-0.32) has increased the release of GDH and AK activities - IDH to a lesser degree - for mitochondrial preparations only with damaged inner membrane. Treatment of mitochondria with potassium chloride solution of definite ionic strength has permitted to detect the alteration of these organelles occurring in vivo and in vitro.


The effect of dietary rape-seed oil on cholesterol-ester metabolism and cholesterol-ester-hydrolase activity in the rat adrenal.

The effects of stock diet and stock diet supplemented by olive oil and rape seed on rat adrenal cholesterol ester metabolism have been studied. Rats fed rape seed oil failed to gain weight at the same rate as rats fed olive oil. A prominent feature of the rats fed rape seed oil was an accumulation of high concentrations of cholesterol erucate in the adrenal lipid droplets. When these rats were subjected to an ether stress no percentage decrease in the amount of cholesterol erucate was observed. Adrenal cholesterol ester hydrolase activity was higher in rats fed the olive oil and rape seed oil diets than rats fed the stock diet. In rats fed stock or olive oil diets, a ten-minute ether anaesthesia stress resulted in a two-fold increase in activity of adrenal cholesterol ester hydrolase. Cofactor addition of ATP, cyclic AMP and MgCl-2 in vitro resulted in a stimulation of cholesterol ester hydrolase to a similar activity in both quiescent and ether-stressed rats. By contrast rats fed the rape seed oil diet gave no significant stimulation of cholesterol ester hydrolase activity when given an ether stress or when cofactors were added in vitro. Cholesterol erucate was hydrolysed at only 25% to 30% of the rate of cholesterol oleate in vitro in all groups of animals. Oleic acid added in vitro gave an inhibition of cholesterol ester hydrolase activity in rats fed stock diet while erucic acid activated the enzyme. The accumulation of cholesterol erucate in the adrenal when rats are fed rape seed oil could be due to the reduced ability of cholesterol ester hydrolase to hydrolyse this ester.


Control of pyrimidine biosynthesis in the perfused liver. Feedback inhibition of glutamine-dependent carbamoyl phosphate synthetase.

The site of feedback inhibition of the biosynthesis of pyrimidine nucleotides de novo was investigated in the isolated perfused rat liver. Hepatic uridine phosphate contents were specifically depleted by use of D-galactosamine. The effective activities of enzymes involved in the synthetic pathway were deduced from the rats of incorporation of labeled precursors into the acid-soluble uracil nucleotide pool and into some intermediates of the pathway. The labeling of hepatic urea was also monitored. When the uridine phosphate contents were less than 20% of controls, the incorporation of [14-C]-bicarbonate was stimulated about 20-fold. Label from [U-14C]oxaloacetate used as permeable precursor of intrace-lular aspartate was introduced into the uridylates to the same extent in normal and UTP-depleted livers. Similar results were obtained with labeled carbamoyl phosphate although the uptake of this compound by the liver was rather low. The lack of labeling of urea from exogenous carbamoyl phosphate does not indicate a free exchange of extra- and intramitochondrial carbamoyl phosphate. [ureido-14C]Ureidosuccinate produced in normal and D-galactosamine-treated livers almost identical labeling patterns of dihydroorotate, orotate and orotidine 5'-phosphate. The steady state concentrations of these intermediates were all below 15 nmol/g liver wet weight.


Modification of phenylalanyl-tRNA synthetase from baker's yeast by proteolytic cleavage and properties of the trypsin-modified enzyme.

Earlier studies have shown that native phenylalanyl-tRNA synthetase from baker's yeast contains two different kinds of subunits, alpha of molecular weight 73000 and beta of molecular weight 63000. The enzyme is an asymmetric tetramer alpha-2beta-2, which binds two moles of each ligand per mole. Incubation of the purified enzyme with trypsin results in an irreversible conversion: the alpha-subunit remains apparently unchanged but beta is rapidly degraded and yields a lighter species beta of molecular weight 41000. The trypsin-modified enzyme is an alpha-2beta-2 molecule which can still activate phenylalanine but cannot transfer it to tRNA-Phe; furthermore it does not bind tRNA-Phe but its kinetic parameters are identical to those of the native enzyme with respect to ATP and phenylalanine. Therefore the two beta subunits play a critical part in tRNA binding. Isolated alpha or beta subunits exhibit no significant activity and both types of subunit seem to be required for phenylalanine activation.


Characterization of the reaction between ferrocytochrome c and cytochrome c oxidase.

The reaction between cytochrome c oxidase and ferrocytochrome c has been investigated by the stopped-flow method. It has been found that only one electron acceptor, a heme group, in the oxidase is rapidly reduced by cytochrome c. The presence of N3- does not affect the reduction of the acceptor, which supports the hypothesis that this is identical with cytochrome a. The results are consistent with the existence of a simple equilibrium between cytochrome a and cytochrome c: c-2 + a-3+  in equilibrium c-3+ + a-2+ with an equilibrium constant corresponding to an oxidation-reduction potential of cytochrome a 30 mV higher than that for cytochrome c at pH 7.4. The oxidation-reduction potential of the a-3+ /a-2+ couple, 285 mV (based on a potential of 255 mV for cytochrome c), and the optical properties of the reduced form indicate that it is identical with neither of the reduced hemes seen in potentiometric titrations. The oxidase species resulting from the rapid reduction of cytochrome a by cytochrome c is proposed to represent a metastable intermediate state which, under anaerobic conditions, eventually is transformed into a more stable state characterized by a reduced high-potential heme.


Cyclic amp concentration and enzyme activities of cerebrospinal fluid in patients with epilepsy or central nervous system damage.

Cyclic adenosine-3',5'-monophosphate (cAMP) concentration in cerebrospinal fluid (CSF) was measured by the protein binding method of Gilman from 62 neurological patients, 46 of them were epileptics and 16 with a central nervous system (CNS) damage. In epileptic patients the CSF concentration of cAMP was significantly elevated (p smaller than 0.02) for 3 days after an attack when compared with those free from attacks for at least 2 weeks. The causality relationship between the rise of cAMP in CSF and an epileptic discharge is discussed. In patient with an active or rapidly progressing CNS damage the cAMP levels in the CSF were significantly higher (p smaller than 0.001) than in those with an old CNS damage. Also some enzyme activities in CSF were measured but no uniform alterations could be found. In damaging processes of the CNS, the cAMP values in the CSF seem to correlate to the activity of the disease.


Evidence for disordered cyclic amp metabolism in patients with cerebral infarction.

Systemic blood cyclic AMP levels were elevated but urinary excretion was unaltered in patients with recent cerebral infarction. Cerebral venous levels for cyclic AMP were significantly higher than arterial levels. CSF cyclic AMP levels were also elevated. After intravenous 10% glycerol, CSF cyclic AMP levels decreased and cerebral A-V differences were reduced. Elevation of cyclic AMP levels in plasma may be due to alteration of neuroendocrinal function after cerebral infraction and contributed to in part by release from infarcted brain. Cyclic AMP release into cerebral venous blood and CSF suggests (1) blood brain barrier damage and (2) extracellular cyclic AMP accumulation in infarcted brain secondary to abnormal neurotransmitter release and/or impaired cellular function which appears to be partially reversed by glycerol.


[Research on substances with antiviral activity. II. Derivatives of N-substituted 2-chloro-3-formylindoles].

Thiosemicarbazones and amidinohydrazones of N-substituted 2-chloroindole-3-aldehydes were synthesized and investigated for antiviral activity against RNA virus (parainfluenza type 3, HA-I/CR-8 stock) and DNA virus (vaccinia virus, HID stock). The thiosemicarbazones of 1-(o,m,p,-chlorobenzoyl)-2-chloroindole-3-aldehydes were more active than methisazone against vaccinia virus.


[The role of ACTH in the process of complex heparin compound formation in the blood during immobilization stress].

In rats during stress due to 30 min immobilization the total unenzymatic fibrinolytic activity becomes 4 times as high, and its share in the blood plasma total fibrinolytic activity 1.5 times as high. The same stress with ACTH added up increases these values still more: by 2.5 times. ACTH considerably increases formation of the heparin complexes also in absence of the stress. The ACTH effect on the heparin complexes formation is mediated through adrenal stimulation. In 96 hr after adrenalectomy the additional cortical tissue already responds to ACTH administration and, respectively, the stressor effect with ACTH added up entails still greater increase of the unenzymatic fibrinolytic activity than without ACTH.


Prevention of hypoglycemic attacks by propranolol in a patient suffering from insulinoma.

An insulinoma was diagnosed in a fifty-seven-year-old woman suffering from frequent hypoglycemic attacks. Propranolol--a beta-adrenergic blocker--in a dose of 80 mg. per day effectively prevented recurrent hypoglycemic attacks. It also corrected the basal hyperinsulinemia as well as the increased insulin secretion which results from stimulation with glucose or arginine.


Degradation of insulin by isolated rat liver cells.

The degradation of insulin by isolated rat liver cells has been studied. The phenomenon is time- and temperature-dependent. After sixty minutes' exposure to 1.5 times 10-6 cells/ml, about 50 per cent, 15 per cent, and less than 5 per cent of insulin at 1.5 muM. are degraded at 37 degrees C., 20 degrees, and 0 degrees C., respectively. The methods used to measure the hormone degradation effect the apparent Vmax. Higher values of Vmax are found when radioimmunoassay rather than precipitation by trichloracetic acid and absorption to talc is used. However, the apparent Km. (0.27 muM) is virtually the same with any of methods used. N-ethyl-maleimide and Trasylol are potent inhibitors, whereas GSH increases the hormone degradation. Proinsulin acts as competitive inhibitor (apparent Ki equals 0.35 muM.). Gel filtration patterns of incubation supernates suggest that several enzymatic systems may be operative in the degradation of insulin by the liver cells. Glutathione-insulin-transhydrogenase is suggested by the appearance of a component that has the same elution volume as the A chain, but the inhibitory effects of trasylol on insulin degradation, as well as qualitative and quantitative similarities with insulin proteases, suggest that a proteolytic similiarities with insulin proteases, suggest that a proteolytic mechanism is involved. The insulin-degrading system in isolated liver cells closely resembles that observed in purified liver plasma membranes and in the isolated perfused liver. Such similarities stress the possible significance of the degradation process in the regulation of insulin action. These studies are also important for the quantitative analysis of insulin interaction with its specific receptors in isolated liver cells.


Synthesis of proinsulin and large glucagon immunoreactivity in isolated Langerhans islets from EMC-virus infected mice.

The protein synthesis in normal and in EMC-virus infected mouse islets of Langerhans was investigated. Mouse large glucagon immunoreactivity was determined by an immunoassay after chromatographic separation. It was characterized as a peptide of 16 000 MW with in intracellular half-life of 35-45 min. The proportional reduction of void volume proteins, large glucagon immunoreactivity and proinsulin synthesis after infection shows, that both alpha- and beta-cells are damaged by the virus. A reduction in the synthesis of the three protein fractions was already found 6 hrs after inoculation of the virus and remained nearly constant for 48 hrs. An almost complete breakdown of protein synthesis occurred 60 to 70 hrs after infection and was paralleled by the first light microscopic changes in the islets. The stimulation of proinsulin synthesis by glucose was preserved for 48 hrs after EMC-virus infection.


The mechanism of alloxan toxicity: an indication for alloxan complexes in tissues and alloxan inhibition of 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid (SITS) binding for the liver cell membrane.

It is shown that alloxan inhibits binding of SITS to liver cells. This indicates the cell membrane as a site of alloxan action. Alloxan is found to react with tissues to form complexes that are detectable up to 3 hrs after alloxan treatment. On the basis of the present findings, an assumption is made that alloxan inhibits a cell membrane processes by blockade of functionally importnat groups.


Electroencephalographic changes with age in male mice.

Electroencephalographic (EEG) changes, as measured by the awake state, slow-wave sleep (SWS), rapid-eye movement (REM) patterns and ratio of REM/total sleep, were recorded in aging male mice of DBA/2J and C57BL/6J strains. Results indicate that there is a significant increase in the awake state accompanied by significant decrease in SWS with advancing age for both strains, although these changes appear more pronounced in DBA/2J mice than C57BL/6J mice. Of considerable significance is the finding that REM sleep is absent in mice of DBA/2J strain at 23.5 months of age. Based on these findings, the conclusion was reached that strain DBA/2J ages significantly faster than C57BL/6J. The difference in aging between the two strains emphasizes the need for additional studies dealing with genetic aspects of aging.


Biochemical and histochemical changes in energy supply enzyme pattern of muscles of the rat during old age.

Senile muscles of the rat (28-36 months) show loss of overall activity of glycolytic and aerobic enzymes. However, there is a differential loss and shift of enzyme activity pattern in the three types of muscles. The extensor digitorum longus (EDL) and diaphragm show a decrease of ratios of glycolytic to aerobic-oxidative enzymes. This shift to a more oxidative type of metabolism is not observed in the soleus muscle. Decrease of enzyme activities is least marked in the diaphragm muscle. Biochemical analysis shows a trend to levelling out of metabolic differences between the different muscle types. This trend of 'dedifferentiation' is most marked when comparing EDL and soleus, least marked when comparing EDL and diaphragm muscle. The histochemical analysis shows a shift from the original mixed to a more uniform pattern of muscle fibres in the EDL and soleus muscle; this levelling-out of differences between enzymatic activities of different muscle fibres is not observed in the diaphragm muscle. Preferential atrophy and loss of ATPase activity in II muscle fibres in the soleus muscle and the occurrence of 'type grouping' are further characteristic features of senile muscle change. The findings show general (i.e. loss of enzyme activities) and differential trends of biochemical and histochemical enzyme changes in different types of muscles.


A case of syndactyly in the white-lipped tamarin Saguinus nigricollis.

A case of syndactyly in a captive bred white-lipped tamarin (Saguinus nigricollis) is reported which involved the fusion of metacarpals 3 and 4 of both hands. Variations in the vertebral formula among the parents and offspring were also found.


[Carotid angiography with subtraction for glomus tumours of the middle ear (author's transl)].

Four patients with glomus tumours of the middle ear are described, in whom carotid angiography with subtraction was carried out. In three patients the tumour could be clearly demonstrated by using the subtraction method. In a fourth case, in whom the diagnosis was made at operation, angiography was unsuccessful.


The value of fine needle aspiration biopsy and intravenous pyelography in the diagnosis of renal masses.

On the basis of an analysis in 118 patients with space occupying lesions in the kidney demonstrated on the pyelogram it is conluded, that the combination of intravenous pyelography and fine needle aspiration biopsy is a safe diagnostic procedure, and it could be a valuable supplement to angiography and explorative incision.


Using volunteer therapists to reduce hospital readmissions.

Two aftercare programs using volunteers as therapists for former inpatients have been effective in reducing hospital readmissions. The volunteer therapists ensure that patients take medications, evaluate them for decompensation, help them find housing and jobs, and give them supportive counseling. In the first program the recidivism rate after one year for 36 chronic schizophrenic women was 11 per cent for the group treated by volunteers and 34 per cent for the control group receiving traditional aftercare. In the second program the recidivism rate after one year for 11 men and women with various types of schizophrenia was 9 per cent for the treatment group and 37 per cent for the control group.


The placement of long-term "criminally insane" patients in open settings.

Twenty long-term psychiatric patients with criminal charges against them were transferred from the Iowa Security Medical Facility to open hospitals and aftercare settings between 1969 and 1971. Staff prepared the patients for transfer and persuaded authorities to permit the placements. Follow-up shows 19 have adapted to the new settings. The authors speculate that age is a factor in predicting aftercare adjustment; 18 of the 19 patients were at least 44 years old. The authors also feel that clinical staff must take the initiative to reduce the needless confinement of such patients.


Serum and tissue proteins in tuberous sclerosis. I. Serum and red-cell polymorphic systems.

5 serum protein polymorphic systems (haptoglobin, alkaline phosphatase, group-specific (Gc) proteins, beta2-glycoprotein 1 and leucine aminopeptidase) and 6 red-cell polymorphisms (adenosine deaminase, adenylate kinase, phosphoglucomutase, glutamic-pyruvic transaminase, phosphogluconate dehydrogenase and acid phosphatase) have been investigated in 54 subjects with tuberous sclerosis. The frequencies of all systems were compared with those of a control sample drawn from a similar mentally retarded population and abnormal distributions were detected in the haptoglobin and Gc system. Quantitative estimation of the serum levels of the Gc protein failed to detect any inter-group differences. Data on the deviations from the Hardy-Weinberg equlibrium, Haldane's Log ratio test between groups, and gene frequencies of both test and control groups are given. It is suggested that selection by mortality is the possible causation for the abnormal distribution of the Gc phenotypes, but the haptoglobin phenotype distribution requires further investigation with care being taken in the selection of control subjects.


Red cell enzyme polymorphisms in the greek populations.

The frequency of variant forms of 6 red cell enzymes, adenylate kinase, adenosine deaminase, phosphoglucomutase, acid phosphatase, 6-phosphogluconate dehydrogenase and glutathione reductase, were determined in 9 Greek populations. The frequencies of the variants in these populations were similar to those previously reported in most other European populations. However, several differences, particularly in the 6-phosphogluconate dehydrogenase, phosphoglucomutase and acid phosphatase alleles, were found in a comparison of Greeks and Bulgarians, in accordance with their separate ethnic origins. The Macedonians resembled the other Greeks and differed from the Bulgarians.


Cytogenetic studies in 12 patients with itai-itai disease.

Among 12 Itai-Itai disease patients examined, 8 patients showed a remarkably high frequency of chromatid aberrations, whereas the other 4 patients showed a much lower frequency of such aberrations, although a significant number of stable type aberrations was observed also in the latter patients. The frequencies of aneuploid cells of all 12 patients were significantly higher than those of the controls. The abnormalities were found in 50-hour and 72-hour cultures, from which it can be concluded that the aberrations occurred in the blood stem cell of the patients. In addition to these structural and numerical aberrations, satellite associations of the D and G group chromosomes were often observed.


Immunochemical studies on the human pathogen Sporothrix schenckii: effects of chemical and enzymatic modification of the antigenic compounds upon immediate and delayed reactions.

The rhamnose-containing polysaccharide-peptide compound derived from the cells of the pathogenic fungus Sporothrix schenckii has been shown to contain 87.1% carbohydrate and 12.5% peptide and to give rise to both immediate- and delayed-type reactions in sensitized guinea pigs. The capacity to induce immediate-type reaction, passive cutaneous anaphylaxis, was completely lost by degradation of the carbohydrate moiety by periodate, whereas the ability to induce the delayed-type reactions of migration inhibition (in vitro) and the sporotrichin reaction (in vivo) were only slightly affected by periodate treatment. On the other hand, delayed "reactivities" to the compound were considerably reduced by treatment with papain, whereas the immediate-type reaction remained positive. These results lead to a conclusion that the rhamnose-containing polysaccharide of the polysaccharide-peptide antigenic compound plays an important role in the immediate-type reaction, whereas the peptide is largely responsible for the delayed-type reaction.


Production, isolation, and partial characterization of three foot-and-mouth disease virus temperature-sensitive mutants.

Three high temperature-sensitive (ts) mutants of foot-and-mouth disease virus were characterized by their relative abilities to grow at 33 or 38.5 C, to kill infant mice, to infect guinea pigs, and to produce foot-and-mouth disease in steers. Mutants ts-24 and ts-42 did not grow at 38.5 C; both may have produced considerable quantities of noninfectious virus particles at 33 C. A third mutant, ts-22, appeared "leaky" because it multiplied to a limited extent during prolonged incubation at the nonpermissive temperature. Mutant ts-42 was pathogenic for infant mice, whereas ts-22 and ts-24 were essentially apathogenic. Mice were protected against the lethal effects of the wild-type (wt) virus if injected 1 week earlier with ts-22, apparently as a result of specific antibody development. One-half of the guinea pigs injected with the mutant viruses showed lesion development, but only at the site of injection. Antibody development was also much less than in those animals injected with the wt virus. The onset of FMD in steers was delayed and the severity of the disease was diminished after intradermalingual injection of the mutant viruses.


Immunological sequelae of Trichinella spiralis infection in mice: effect on the antibody responses to sheep erythrocytes and Japanse B encephalitis virus.

Immunosuppression of the antibody response to Japanese B encephalitis (JBE) virus and sheep erythrocytes (SRBC) was observed in mice infected with Trichinella spiralis. This suppression was paralleled by the presence of fewer antibody-forming cells to SRBC in the spleens of parasitized mice. Both primary and secondary complement-fixing antibody responses to JBE virus were suppressed, but the development of immunological memory was not affected. Parasitized mice demonstrated a normal blastogenic response to phytohemagglutinin and normal serum clearance rate of injected (125)I-labeled immunoglobulin G(2b), although the size of the extravascular fluid compartment was significantly increased. The data presented here and in previous reports suggest that sequential antigenic competition is a possible explanation for the humoral immunosuppression to heterologous antigens caused by T. spiralis infection.


Interactions between aerolysin, erythrocytes, and erythrocyte membranes.

Aerolysin, a hemolytic and lethal exotoxin of Aeromonas hydrophila, was analyzed for amino acids. Assuming 8 histidine residues/mol, the purified toxic protein has, by summation, a molecular weight of 49,000, a value in agreement with earlier estimates by other methods. Erythrocytes from different animal species differ greatly in sensitivity to aerolysin's lytic action. There is some correlation between sensitivity and phosphatidyl choline content. Erythrocyte membranes of different species bind the toxin, and the efficiency of binding is a function of sensitivity to lysis. Binding is temperature independent, is not dependent upon membrane sialic acid, and is decreased by prior treatment with phospholipase C and proteases. Preparations of aerolysin convert substantial amounts of membrane phosphorus to water-soluble form; the conversion is concentration and temperature dependent. Most of the conversion is attributable to contaminating phospholipase(s) that is separable from the toxin. Aerolysin purified by electrophoresis in polyacrylamide gel retains some phospholipase activity, and this activity may or may not be a contaminant.


Adeno-associated virus in adenovirus type 3 conjunctivitis.

Although human infection with adenovirus-associated virus (AAV) has been demonstrated, there is no evidence that disease results from such infections. The proportion of adenovirus infections which are dual infections with AAV is virtually unknown, since special methods are required to demonstrate infection with AAV. To search for AAV, we re-examined a collection of specimens from 40 persons involved in an epidemic of pharyngoconjunctival fever associated with a swimming pool. Virological and serological studies indicated that the etiological agent was adenovirus type 3. When the 91 original eye, throat, and fecal specimens were re-examined, using methods suitable for detection of adenovirus and AAV, 37 strains of adenovirus type 3 and 35 strains of AAV type 3 (AAV3) were isolated. Surprisingly, 19 AAV3 but only 11 adenovirus isolates were found in eye specimens, whereas adenovirus isolates were equally distributed in all types of specimens. Four AAV3 strains were isolated from adults. Significant (fourfold or greater) rises in AAV3 complement-fixing antibody titers were seen in six of 14 persons shedding AAV3, whereas nine of 10 persons shedding adenovirus type 3 showed significant rises in adenovirus complement-fixing antibody. These results raise the question whether AAV persists better in eyes than adenovirus or that a possible association with conjunctivitis might be present. In contrast to the results in the specimens from the swimming pool epidemic, only one of 36 adenovirus strains isolated in other Seattle-based studies yielded AAV. Complement fixation tests on serial sets of sera collected from 60 children not involved in the swimming pool episode revealed nine AAV2 and 12 AAV3 infections during a 4-year period.


L-arginine elution of measles virus adsorbed on  monkey erythrocytes.

l-Arginine (1 M) eluted measles virus adsorbed on monkey erythrocytes without affecting the infectious and hemagglutinating activities of the virus. This phenomenon was also observed for encephalomyocarditis virus and Newcastle disease virus.


Occurrence of BK virus and BK virus-specific antibodies in the urine of patients receiving chemotherapy for malignancy.

Urine specimens from 23 children and 9 adults who were undergoing treatment for malignancy as well as urines from 40 normal individuals were concentrated and examined for evidence of papovavirus infection. Papovavirus particles were detected in 6 of 64 urines examined by electron microscopy. Three of the particle-positive urines induced BK virus-specific immunofluorescence after inoculation of WI38 cells, and three isolations of BK virus were made by inoculation of urines from virus-excreting patients into Vero cells. BK virus-specific hemagglutination-inhibiting and immunofluorescence neutralizing antibodies were found in a majority of urines from adult patients, in about a fifth of pediatric patients, and less often in normal urines. Urines of virus-excreting patients generally had antibodies. In indirect fluorescent antibody tests, BK virus-specific antibodies of the immunoglobulin G class were found in five urine specimens from patients; immunoglobulin A antibodies were not detected in any urine. These data suggest that activation of BK virus is related to immunosuppression and not to transplantation itself and that the occurrence of virus-specific antibodies in urine may be indicative of virus multiplication in the urinary tract.


Interferon production in mice by the capsular polysaccharide of Klebsiella pneumoniae.

The capsular polysaccharide of Klebsiella pneumoniae (CPS-K) type 1, Kasuya strain, induces interferon production in the blood of mice when injected intravenously. CPS-K resembles bacterial endotoxin (lipopolysaccharide) in the time pattern of interferon production, with peak levels 2h after injection. CPS-K on a weight basis exhibits a more potent interferon-inducing effect than lipopolysaccharide. The active substance responsible for the interferon-inducing activity of CPS-K is the neutral CPS-K antigen which is antigenically distinct from the O antigen and from acidic CPS-K (the type-specific capsular antigen). Neutral CPS-K from the Kasuya strain has been already found to exhibit a strong adjuvant effect on antibody responses to various antigens in mice. Preparations of neutral CPS-K from other strains of K. pneumoniae, of which adjuvant action is only very weak, exhibit interferon-inducing activity similar to the preparation from the Kasuya strain. Heterologous and homologous tolerance to re-induction of interferon is produced by a prior injection (one each) of LPS, neutral CPS-K, and acidic CPS-K. No simple correlation exists between the inducing and tolerogenic capabilities of these substances.


Inhibition of Herpesvirus hominis replication by human interferon.

The effect of human interferon on single-cycle replication of Herpesvirus hominis type 1 (HV-1) and vesicular stomatitis virus (VSV) in human foreskin fibroblast cultures (HFF) was studied. After treatment of HFF cultures with low concentrations (58 U) of human interferon, a variable but statistically significant inhibition of HV-1 was observed. At higher concentrations (greater than 95 U) yield reduction of HV in interferon-treated cultures approached that of VSV. Preliminary data indicate that antiviral activity decays more rapidly for HV-1 than for VSV after removal of interferon from cultures.


Establishment of a nonproductive herpes simplex virus infection in rabbit kidney cells.

A nonproductive infection of rabbit kidney cells was established with a type 2 strain of herpes simplex virus after incubation of the virus-infected cells at 41 C. Although infectious virus could not be recovered from cells disrupted by freeze-thawing or sonication after incubation at 41 C, spontaneous reactivation of virus growth occurred in 84% of the cultures after lag periods of variable length when cultures of viable cells were transferred to 37 C and incubated. Forty-one percent of the cultures had lag periods of 4 or more days, and 24% had lag periods of 7 or more days. The longest lag period was 45 days. A similar infection was established in WI-38 cells, but attempts to establish the infection in human kidney cells was not successful. This nonproductive infection is being used to study the effects of various physical and chemical agents on herpes simplex virus in a latent state.


Effect of human leukocyte interferon on vaccinia-and herpes virus-infected cell cultures and monkey corneas.

Pretreatment of human fibroblast cultures with human leukocyte interferon (HIF, 1,000 IU/ml) resulted in a 24-h delay of virus replication after infection with vaccinia virus and herpes simplex virus type 1 and type 2. Additional HIF treatment 24 h after infection effectively lowered the maximum yield of viral infectivity. Equal results were obtained in simian cells with 3,000 IU of HIF per ml. The spread of two cell-bound herpesviruses, varicella zoster virus and Medical Lake macaque herpesvirus, was inhibited by 2,000 IU of HIF per ml in human fibroblasts and Vero cells, respectively. Varicella zoster virus infectivity was notably reduced by HIF, whereas the latter system showed a low sensitivity. To study the effect of HIF in the infected cornea, keratitis was induced experimentally in both eyes of 12 rhesus monkeys and 12 African green monkeys by inoculation with vaccinia virus and herpes simplex virus, respectively. In each monkey one eye served as a control for the full cycle of disease. In the other eye HIF treatment was initiated prophylactically 15 h before or simultaneously with the challenge virus infection or 6 to 20 h postinfectionally or therapeutically after onset of the disease, and the treatment was continued for 2 to 7 days. Prophylactic and simultaneous administration equally resulted in inhibition of both vaccinia and herpes keratitis. Postinfectional and therapeutic administration of interferon moderated the course of keratitis slightly and shortened the period of virus shedding.


Herpes simplex virus infection in guinea pigs: an animal model for studying latent and recurrent herpes simplex virus infection.

Footpad infection of guinea pigs with herpes simplex virus led to an acute local inflammatory reaction, followed by a persistent latent infection of lumbosacral dorsal root ganglia. Spontaneous reactivation of the latent virus occurred, leading to recurrent lesions at the site of the initial infection. Clinical observations and virological studies during the acute latent and recurrent infection are reported.


Role for cell-mediated immunity in the resistance of mice to subcutaneous herpes simplex virus infection.

The role of cell-mediated immunity in the resistance of young adult mice to subcutaneous herpes simplex virus (HSV) type I infection was studied in mice receiving immunosuppressive doses of antilymphocyte sera (ALS) or antithymocyte sera (ATS). The effectiveness of these treatments to reduce cell-mediated responses was measured by their ability to prolong the life of allografts transplanted to ALS- or ATS-treated mice. It was found that subcutaneous infection of these mice with HSV resulted in spread of virus from the site of inoculation to the central nervous system. Neutralizing antibody could not be detected in the sera of ALS- or ATS-treated mice after HSV inoculation. Passive transfer of neutralizing antibody to ATS-treated mice did not restore resistance to subcutaneous HSV infection. However, adoptive transfer of HSV-sensitized spleen cells did provide significant protection against infection unless the spleen cells were treated with ATS prior to transfer. These experiments suggest that lymphocytes are involved in a cell-mediated response to subcutaneous HSV infection and demonstrate the importance of a noncompromised immune response in controlling spread of HSV from localized areas of infection.


Growth and cytopathology of Mycoplasma synoviae in chicken embryo cell cultures.

Mycoplasma synoviae was tested for its ability to grow and induce cytopathogenic changes in chicken embryo cell cultures. M. synoviae grew to high titers by day 5 in the presence of chick cells, but showed no growth in the tissue culture medium alone even though it was enriched with nicotinamide adenine dinucleotide and swine serum. Infected chick cell cultures showed a progressive cytoplasmic degeneration on successive days of examination. Early changes involved cytoplasmic granularity and mild vacuolation. On the last day of examination the cytoplasm of most cells was completely degenerated and some showed nuclear degeneration. M. synoviae was shown to be cytophilic for the chick cell membranes where the mycoplasmas reproduced and formed microcolonies which, on successive days, increased in size. The attachment site on the chick cell membrane was shown to be neuraminidase sensitive.


Factors in the inactivation of Encephalomyocarditis virus in aerosols.

Encephalomyocarditis virus in aerosols is inactivated rapidly at relative humidities below 50%. In glycerol-water mixtures a similar decrease of infectivity occurs when the glycerol concentration exceeds 78% (wt/wt), corresponding to a relative humidity of 50%. The decay in aerosols does not involve oxygen or surface-dependent factors. Variation of temperature shows the inactivation to be a low-energy process with an activation enthalpy of 15 kcal per mol. The damage could be ascribed to dehydration of the virion, presumably proceeding to removal of structurally essential water molecules. This might trigger irreversible changes in the protein coat, resulting in disintegration of the virion.


Failure of attenuated temperature-sensitive influenza A (H3N2) virus to induce heterologous interference in humans to parainfluenza type 1 virus.

The present investigation was undertaken to determine if a candidate live vaccine virus, influenza A/Hong Kong/68-ts-1 [E] (H3N2), induced heterologous interference against an interferon-sensitive, wild-type, parainfluenza type 1 challenge virus. The parainfluenza virus was administered 7 days after Hong Kong/68-ts-1 [E] virus infection. The clinical response, daily quantitative virus shedding, interferon production, and serum and nasal wash antibody responses were determined in an experimental group (influenza A virus followed by parainfluenza virus) and 10 volunteers in a control group (parainfluenza virus only). The volunteers were selected on the basis of susceptibility to the two viruses, i.e. serum hemagglutination-inhibition antibody titer of is less than or greater to 1:8 for influenza virus and low nasal wash antibody titer (is less than or greater to 1:8) for parainfluenza virus. Despite a 100% infection rate in the Hong Kong/68-ts-1 [E] vaccinees, no heterologous interference was induced against the parainfluenza type 1 virus challenge.


Depressed specific cell-mediated immunity to Herpes simplex virus type 1 in patients with recurrent herpes labialis.

Cell-mediated immunity to herpes simplex virus type 1 (HSV-1) was assessed by a lymphocytotoxicity 51-Cr-release microassay procedure, using the MA-160 human prostatic adenoma cell line chronically infected with HSV-1 and its parent cell line as control. The specific immune release mean plus or minus standard deviation for nine asymptomatic patients with recurrent HSV-1 infections was 13.7 plus or minus 8.1%, compared to 28.9 plus or minus 8.4% in seven normal seropositive controls (P is less than 0.01). In four additional patients studied serially, the cell-mediated immunity was significantly increased during the recrudescence of herpetic infection, with a mean specific immune release value of 51.7 plus or minus 27.8%, compared to 8.7 plus or minus 1.5% during the convalescent period 2 to 10 weeks later (P is less than 0.05). These findings suggest that patients with recurrent HSV-1 infections have vigorous cellular immune responses during the acute phase but a specific impairment of cell-mediated immunity during the quiescent period, which may in part account for their susceptibility to recurrent herpetic infections.


The occurrence of tumours in F1, F2 and F3 descendants of bd rats exposed to N-nitrosomethylurea during pregnancy.

N-nitrosomethylurea (NMU) was administered once IP at a dose of 20 mg/kg to BD female rats on the 18th day of pregnancy. The first generation descendants (F1) were mated to produce a second generation (F2), which were then mated to produce a third generation (F3). While F1 rats were exposed directly to NMU during intrauterine life, it can be reasonably excluded that F2 and F3 descendants were exposed to NMU or its metabolites. F2 and F3 rats received no other treatment and untreated rats served as controls. Kidney and nervous-tissue tumours were observed in F1 descendants and, with a lower incidence, in F2 descendants. In F3 rats, tumours of nervous tissue, but not of kidneys, were observed. These results confirm previous observations that exposure to a carcinogen during prenatal life results in an increased cancer risk which may persist for more than one generation.


Effect of cordycepin on the replication of type-c RNA tumor viruses.

Cordycepin (3'-deoxyadenosine) was previously shown to inhibit virus production induced by iododeoxyuridine from murine fibroblasts (Wu et al., 1972). We now report that the inhibitory effect of cordycepin results in a reduction of the number of cells producing virus as measured by the infectious center assay and fluorescent antibody technique. Cordycepin has a much greater inhibitory effect on viral replication than on transformation of normal rat kidney cells by murine sarcoma virus since viral production was greatly reduced (seven- to 35-fold) with 5-10 mug/ml of cordycepin while viral transformation was only slightly inhibited (two-fold reduction in focus-forming units) with the same concentration of cordycepin. Inhibition of viral production is most effective if the compound is present during the first 24 h after injection.


Activation of C-type viruses during skin graft rejection in the mouse. Interrelationships between immunostimulation and immunosuppression.

Both mouse-tropic and zenotropic C-type RNA viruses were activated in BALB/c mice during rejection reactions against skin grafts from mice differing at the major histocompatibility locus (A/J) or sharing this locus (DBA/2). Animals that maintained their skin grafts following immunosuppression with antilymphocyte serum (ALS) or cyclophosphamide had a much higher incidence of virus-positivity than animals that did not receive ALS or cyclophosphamide, or that rejected their skin grafts despite immunosuppressive therapy. ALS itself activated type-C viruses from a small proportion of BALB/c mice whereas cyclophoshamide alone did not. Type C viruses were detected in spleens and regional nodes draining skin-graft sites between 1 and 2 weeks following transplantation; thereafter they reached maximum titers in spleens. Viruses were not detected at skin-graft sites, in tails or in thymuses of grafted animals. Virus activation was associated with splenic histological changes consisting of germinal center formation, diffuse hyperplasia of reticulum cells, depletion of periarteriolar lymphocytes and hyperplasia of red pulp hematopoietic elements.


EB Virus-associated nuclear antigen production and cell proliferation in adult peripheral blood leukocytes inoculated with the QIMR-WIL strain of EB virus.

A nuclear antigen, apparently the Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA), was detected by anticomplement immunofluorescence (ACIF) tests in adult peripheral blood leukocytes infected with the QIMR-WIL strain of EBV. EBNA was not detectable at 24 h but appeared in about 11% of the cells by 3 days, and by 5 days up to 64% of the cells were positive. Proliferation of EBNA-positive cells at this stage was confirmed by autoradiography. There was a good correlation between the concentration of virus and the number of EBNA-positive cells in the first 5-7 days. The subsequent course of events was found to be influenced by the initial cell concentration and the time of subculture. EBNA production was delayed in cells infected with higher dilutions of virus but subsequently appeared in a high proportion of cells. Indirect immunofluorescence failed to detect viral capsid antigen (VCA) or early antigen (EA) by 10 days. The results show that EBV infection was abortive and that the critical events of viral transformation occurred within the first few days.


Tumor-specific and forssman antigens of guinea-pig hepatoma cells: comparison of tumor cells grown in vivo and in vitro.

Cloned tumor-cell lines were derived from two antigenically distinct ascites variants of diethylnitrosamine-induced guinea-pig hepatomas (designated line 1 and line 10). Cell-surface antigens on the ascites and in vitro-grown tumor cells were analyzed with immunofluorescence, C1 fixation and transfer, and antibody-complement-mediated cytotoxity tests. Tumor-specific and Forssman antigens continued to be expressed during 3-6 months in vitro cultivation. Differences between ascites and cultured cells were noted in the degree of antigen expression and sensitivity to antibody-complement-mediated cytotoxicity. Cells grown in vitro exhibited a greater number of Forssman and tumor-specific antigen sites than in vivo grown cells as determined by the quantitative C1 fixation and transfer test. Immunofluorescent staining indicated that some cloned lines were considerably more homogeneous in terms of antigen expression than were the cultured, non-cloned parent cells or ascites-grown cells. Cloned lines were frequently more sensitive to the cytotoxic action of antibody and complement than were the in vivo grown and cultured non-cloned parent tumor cells. Sensitivity to cytotoxicity did not necessarily correlate, however, with the degree of antigen expression. These results suggest that; (1) the expression of Forssman and tumor-specific antigens does not diminish on cells cultivated in vitro and (2) ascites hepatoma cells in vivo are a heterogeneous population of cells differing in their degree of antigen expression and sensitivity to antibody-complement-mediated cytotoxicity.


Helper specificity for retrieval of defective friend virus.

A Friend virus-induced reticulum-cell sarcoma from BALB/c mice has been cultivated in vitro in our laboratory for more than 11 years and contains no detectable evidence of virus. However, Friend virus could be retrieved readily from the tissue cultures by any of several lymphatic leukemia viruses belonging to the Friend-Moloney-Rauscher (FMR) group, as long as the helper viruses were actively replicating in a culture. Helper activity appeared to be highly specific and limited to the FMR group including the B-tropic Tennant leukemia virus which produced a B-tropic Friend virus pseudotype. The naturally occurring type AKR murine leukemia viruses and the related Gross passage A virus failed both in vivo and in vitro to retrieve the Friend virus genome from the virus-free tumor cells.


The detection of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) by anticomplement immunofluorescence. Immunoglobulin class of antibodies and role of complement.

The Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was detected by anti-complement immunofluorescence (ACIF) in Raji lymphoblastoid cell line, and the mechanism of the reaction explored, using Ig fractions of anti-EBNA sera and purified components of complement. Following fractionation of serum from 13 donors, anti-EBNA antibodies were always found to be associated with IgG, but were also detectable in the IgM fraction of four sera. Sequential addition of functionally pure complement components in the ACIF reaction showed that the classical sequence of complement activation in involved. Anti-EBNA antibody reactions in Raji cell nuclei can also be detected by anti-Ig immunofluorescence with a low level of sensitivity. The same pattern of granular fluorescence was observed when C3 (beta1A/beta1C), or C4 or IgG anti-EBNA antibodies were revealed with the corresponding flurescein-conjugated reagents. Blocking of the ACIF reaction was achieved with Fab 2 anti-EBNA antibody fragments, which therefore provided an appropriate specificity control for the detection of EBNA.


Expression of SV40 T antigen during the cell cycle of sv40-transformed cells.

The expression of the nuclear SV40-induced T antigen was measured by microfluorimetry on individual, asynchronously growing SV40-transformed cells which had been stained with hamster T-antiserum by the indirect immunofluorescence method. The same individual cells were first measured for T antigen and then for DNA by Feulgen microspectrophotometry. A linear correlation was observed between the two parameters. T antigen expression was also measured in cell populations arrested at different phases of the cell cycle. Results of both types of experiments show that the expression of the gene (s) for T antigen in transformed cells increases during DNA replication and reaches its highest level in G2 nuclei. During mitosis T antigen is found in the cytoplasm.


Immunofluorescence and anti-complement immunofluorescence absorption tests for quantitation of Epstein-Barr virus-associated antigens.

Immunofluorescence absorption methods are described which permit quantitative estimation and differentiation of Epstein-Barr virus (EBV)-associated antigens (virus capsid antigen, VCA, early antigen, EA and EBV-determined nuclear antigen, EBNA) in cell extracts. EBNA was present in all cell lines (producer and non-producer) which carried the EBV-genome, while VCA and EA were present in producer lines only. All the antigens were absent from a lymphoid cell line (MOLT-4) which lacked the EBV-genome, as well as from leukemia cells from peripheral blood. The techniques demonstrated antigenic identity of the various antigens when prepared from different cell lines.


Studies of Epstein-Barr virus (ebv)-associated nuclear antigen. I. assay in human lymphoblastoid cell lines by direct and indirect determination of 125I-IgG binding.

A quantitative assay for EBNA in human lymphoblastiod cell lines has been developed. The assay employs EBNA-positive and -negative 125-I-IgG preparations as reagents and can be used in a direct or indirect manner. EBNA specificity has been demonstrated in a number of ways. The antigenic relationship between EBNA  present in Raji cells and in a number of other human lymphoblastiod cell lines of diverse origins and between the cell-associated antigen present in FT Raji cells and that present in isolated Raji nuclei has been studied. The possibility of carrying out blocking titrations for anti-EBNA determination has been demonstrated and the effect of glutaraldehyde and formaldehyde upon antigenic activity in FT Raji cells has been studied.


Serum factors modifying cell mediated immunity to rat hepatoma d23 correlated with tumour growth.

Sera from rats bearing a progressively growing transplanted aminoazo-dye-induced hepatoma (hepatoma D23) have been examined for the presence of hepatoma D23-specfic antigen, antibody and immune complexes throughout the course of tumour growth. The levels of these factors have been correlated with the in vitro blocking and inhibition of cytotoxic lymph-node cells from immunized animals for cultured tumour cells. Sera from animals bearing small tumours (7-14 days after tumour implantation contain free tumour-specific antigen whilst immune complexes could not be detected. Although these sera were neither blocking nor inhibitory under the normal conditions of the test, when concentrated two and fourfold, inhibition but not blocking of lymph-node cell cytotoxicity could be detected. In comparison sera from animals bearing large tumours (24-28 days) blocked but did no inhibit in vitro lymph-node cell cytotoxicity and this correlates with the presence of tumour-specific immune complexes in antibody excess. Animals with intermediate-sized tumours had high levels of both blocking and inbibitory activity in the serum, these effects becoming apparent when neither free antibody nor free antigen could be detected. The relevance of these findings to the mechanism by which a growing tumour may escape specific cellular immune destruction is discussed.


Rat cell line 3y1 and its virogenic polyoma- and sv40- transformed derivatives.

Cell lines were established from cultures derived from Fischer rat embryos according to the transfer schedule described by Todaro and Green (1963) for mouse 3T3 cells where cell crowding and serum exhaustion were kept to a minimum. Cell growth rate did not decline greatly during the course of successive 3-day transfers. Like 3T3 cells the rat cell lines possess very low saturation desities under standard culture conditions. A clonal cell line with a relatively high plating efficiency as obtained from one of the cell lines, 3YL. In these cloned cultures, virus growth was not detectable upon infection with SV40, while a small amount of virus was produced upon infection with polyoma virus. Morphological transformation of the cloned 3Y1 cells by SV40 and polyoma virus could be assayed with single-hit kinetics and with effieiencies comparable to those of the previously available transformation systems for each virus. Independent cell lines transformed by SV40 were consistently virus-free and all the lines tested produced SV40 upon fusion with permissive monkey cells. Most of the independent transfromed cell lines isolated after polyoma infection appeared to be virus-free, although the cultures of some lines produced a small amount of polyoma virus spontaneously after a prolonged cultivation. Most of the virus-free polyoma-transformed lines produced virus upon fusion with permissive mouse cells.


Control of nuclear division in sv40 and adenovirus type 12 transformed mouse 3t3 cells.

Untransformed, non-tumorigenic mouse cells respond to cytochalasin B (CB) with limited nuclear division. BALB/c mouse embryo fibroblasts (MEF) and both BALB/c 3T3 and Swiss 3T3 cells become binucleated in the presence of CB and cells with three or more nuclei are very rare or undetectable. MEF are diploid (40 chromosomes) and 3T3 cells are subtetraploid (74-76 chromosomes). Transformation of MEF by SV40 produces a dramatic change in response to CB. These cells, which contain SV40 T-antigen, become highly multinucleated in the presence of CB. More than 20% of the cells have at least seven nuclei. Also premature chromosome condensation (PCC), an abnormality rare in CB-treated normal cells but one which is common to highly multinucleated cells, is frequent and occurs in at least 10% of mitoses. SV40-transformed MEF have 40 or 80 chromosomes, e.g. are diploid or tetraploid. Transformation of 3T3 by SV40 or adenovirus type 12 does not result in a marked change in the response to CB. Although some trinucleate and tetranucleate cells are formed, cells with more nuclei are undetectable or rare. PCC is also rare. These cells show chromosome numbers somewhat lower than their untransformed parents and in one line the chromosome number appears to decrease with passage of the cells. This failure to undergo a marked change in responsiveness to CB following transformation is not a characteristic of all transformed 3T3 cells. SVT2, a line of 3T3 which was transformed by SV40 prior to its establishment as a continuous line, responds to CB with a high degree of multinucleation. These cells are diploid. These results suggests that 3T3 cells are constitutive for controlled nuclear division and that this feature may be related to chromosome constitution.


Subpopulations of multiparous rat lymph-node cells cytotoxic for rat tumour cells and capable of suppressing cytotoxicity in vitro.

Lymph-node cells (LNC) from multiparous pregnant rats were separated on columns prepared from nylon wool, and tested for cytotoxicity against target tumour cells. Reactivity of LNC towards hepatoma D23 and mammary carcinoma AAF57 was demonstrated in cell populations retained on the nylon wool, and not with cells eluted from the column. Although only 25% of the samples of unfractionated LNC were cytotoxic for tumour cells, retained cell fractions were cytotoxic in 11 out of 12 tests (p = less than 0.05). Similarly retained LNC were also cytotoxic for 15-day-old embryo cells but not for normal adult rat fibroblasts. Using multiparous rat serum it was shown that the reactivity of the retained LNC population could be abrogated in eight out of 11 tests (p = less than 0.05). The LNC population recovered from the nylon wool constituted 28 to 35% of the original LNC preparation, and consisted of 60-70% Ig-bearing cells together with a subpopulation of cells responding to soluble PHA. Separation of multiparous LNC on glass beads coated with rat Ig and then rabbit anti-rat Ig (in excess) also demonstrated the retained cell population to be cytotoxic against tumour cells. Approximately 17-20% of the original cell population was recovered from cells retained on the column, and consisted of an enriched Ig-bearing cell population (65-80% Ig-bearing cells) and LNC responsive to PHA. Carbonyl iron treatment of multiparous rat LNC was found to remove detectable cytotoxicity from multiparous rat LNC preparations. The cytotoxicity of multiparous rat LNC retained on nylon wool was also abolished following incubation with carbonyl iron. Definite conclusions as to the nature of the effector cell cannot be drawn from this test, since carbonyl iron treatment was found to remove not only phagocytic cells from LNC preparations but also a proportion of other cell populations including Ig-bearing lymphocytes... In addition to detecting a cytotoxic LNC population reactive towards tumour-associated embryonic antigens (retained fractions from nylon-wool column separation), a subpopulation of multiparous rat LNC was demonstrated in cell fractions eluted from the nylon wool which was shown to suppress the cytotoxicity of the retained multiparous LNC population. The exact nature of this subpopulation of LNC and the mechanism of action is at present not known.


Epidemiological studies on feline leukaemia virus infection. I. A serological survey in urban cats.

A survey of the incidence of feline leukaemia virus (FeLV) infection in cats in a large urban area was made by studying the prevalence of antibodies to feline leukaemia virus-associated cell membrane antigens. Two serological tests were used, immunofluorescence and a mixed immunoglobulin rosette technique. The overall incidence of cats with antibodies was 40%, contrasting with 6% in the surrounding rural area. Only 6% of urban kittens were positive while 50% of adults had antibodies. The incidence in adults rose from 29% at 5-6 months to 74% in cats over 3 years. Stray cats had an incidence twice as high as that of domestic pets. These results support and extend earlier findings that FeLV infection is common and is horizontally transmitted.


Oncogenic transformation of primary hamster cells by herpes simplex virus type 2 (hsv-2) and an hsv-2 temperature-sensitive mutant.

Six oncogenically transformed cell lines were obtained following infection of hamster embryo fibroblasts with UV-irradiated herpes simplex virus type 2 (HSV-2) or with an HSV-2 temperature-sensitive mutant. All lines produced undifferentiated fibrosarcomas in newborn hamsters, four of the six lines produced metastatic tumors in the lung, and sera from tumor-bearing hamsters contained neutralizing antibody to HSV-2. HSV-specific cell-surface antigen (s) was detected by immunogluorescence tests in all six of the lines established from primary tumors. Antibody to an early, HSV-specific, non-structural polypeptide (VP134) reacted by immunofluorescence with antigen(s) in fixed preparation of three of the six tumor-cell lines.


Antibody patterns to herpesviruses in kaposi's sarcoma: serological association of european kaposi's sarcoma with cytomegalovirus.

Sera from patients with Kaposi's sarcoma (KS) were examined for antibody titres to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV) types 1 and 2 by four techniques: indirect haemagglutination (IHA), complement fixation (CF), virus neutralization (NT) and indirect immunofluorescence (IF). The patients were classified, according to the stage of disease, as progressive and regressive. Control sera were obtained from healthy adults, matched for age, sex, race, socioeconomic status and geographic location, as well as from patients with melanoma, some of whom were receiving chemotherapy similar to that given to the KS patients. All KS sera contained CMV-neutralizing antibodies. Seventy-five percent of the European KS patients, mainly regressors, showed elevated anti-CMV titres by IHA with a significant increase in the geometric mean over the corresponding healthy adult group and the melanoma group. An overrepresentation of high anti-CMV titres, although less marked, was found by CF. There was no significant association with antibodies to EBV, HSV-1 and HSV-2 related antigens. By contrast, the African KS patients, mainly progressors, did not show a serologic association with CMV or with EBV and HSV-1 and 2. The implication of these results is discussed.


Ion-selective electrodes and enzyme electrodes in environmental and clinical studies.

Electrodes have been developed for the assay of glucose, urea, amino acids, uric acid, phosphate, nitrate and perchlorate. The electrodes for the organic compounds are enzyme electrodes which are prepared by chemically immobilizing an enzyme over the outside of a conventional ion-selective electrode. These electrodes will be discussed in depth. The progress and the development of the electrodes that show sensitivity and selectivity for phosphate, nitrate and perchlorate will be outlined. The basis of these sensors is a complex of a transition metal of either an analog of thiourea or an organic chelator, such as 1,10-phenanthraline. Such electrodes respond linearly to phosphate, nitrate or perchlorate, and show selectivity over sulphate, halides and acetate. The linear range of all these electrodes is approx. 10(-1)-10(-5) M with a near Nernstian slope and a reproducibility of 1%. The electrodes are stable and can be used continuously.


Synergistic effects of fad and acth on the 32-p turnover in the adrenal of rats treated with a one shot administration of dexamethasone phosphate.

The effects of Flavin Adenine Dinucleotide (FAD) and of vitamin A for two days to 25 days old rats has been studied on liver of rats treated with a one shot administration of dexamethasone phosphate were investigated. Efforts were made to determine whether or not ACTH participates in the action of FAD which affects adrenal atrophy induced by steroid hormone administration. The large decrease in adrenals 32-P turnover in rat treated with a one shot administration of dexamethasone phosphate was reduced by intraperitoneal administration of FAD or ACTH alone. This tendency of reduced loss was further promoted by an administration of ACTH simultaneously with the FAD. These results suggest the presence of a new physiological role for FAD i.e. FAD is able to potentiate the action of ACTH. The role was discussed of a possible synergism between FAD and ACTH with regard to their action on the induction of adrenal atrophy by steroid administration.


Interference of pregnancy steroid hormones with FPN test for estimation of phenothiazine derivatives in urine.

Interference of pregnancy steroid hormones with FPN test for estimation of some phenothiazine derivatives in urine has been investigated. The FPN test was positive in 97.9% of urine samples obtained from pregnant women. It is supposed that metabolites of estrogens and progestagens present in higher amounts in urine samples of these women are responsible for this positivity.


Ongoing occipital rhythms and the VER. I. Stimulation at peaks of the alpha-rhythm.

As a first step toward determining the source of variability in the visually evoked response (VER) between subjects, we have examined the relationships between the VER and the alpha-rhythm. This was done by comparing the VER's to photic stimuli delivered at positive and negative peaks of the alpha-rhythm (alpha-synchronized stimuli) with VER's to flashes delivered at a fixed interval, unsynchronized with the alpha-rhythm. Average VER's for the three classes of stimuli were calcuated for each of ten subjects. In addition, a grand average VER (GVVER) WAS CALCULATED FOR EACH STIMULUS CLASS BY AVERAGING THE INDIVIDUAL AVERAGE VER's. Quantitative comparisons between GVER's were made using cross- correlograms. Cross-correlograms were calculated between each of the GVER's, and between the GVER's to alpha-synchronized stimulation and their controls. These data show that the first 120 msec. poststimulus interval of the Gver's to alpha-synchronized stimulation are highly correlated with the prestimulus alpha- rhythm. The data also show a component occurring in al GVER's which is independent of the alpha-rhythm. This component begins 120 msec. After stimulation, reaches peak amplitude at 200 msec, and decays to baseline at 240 msec. Thses findings suggest that much of the variability in VER recordings may be due to alpha-activity which has been insufficiently attenuated by averaging.


Growth of calcium oxalate crystals. II. Inhibition by natural urinary crystal growth inhibitors.

A method is described for quantitatively determining the inhibitory activity of pure components, isolates, or mixtures of components (such as urine) on the growth of calcium oxalate crystals. Results with known calcium phosphate crystal growth inhibitors--magnesium, citrate, and pyrophosphate--suggest that these components contribute little to the ability to normal urine to inhibit the growth of calcium oxalate crystals. As yet unidentified urinary components seem to be responsible for most of this activity. The urinary crystal growth inhibitors appear to function by adsorbing on  the surface of the growing crystals, thereby preventing the further incorporation of lattice ions. The fit of experimental data to the Langmuir absorption isotherm supports this conclusion.


Pathogenesis of pyelonephritis. Escherichia coli-induced renal ultrastructural changes.

Escherichia coli-induced renal ultrastructural changes were observed 4 hr after the intravenous injection of 2 times 10-minus 6 bacteria. The initial changes consisted of mitochondrial swelling followed by intracellular vacuolation, nuclear membrane dilation, and increased lysosomal phagocytic activity. Alterations in the appearance of proximal tubular lysosomes included a loss of osmiophilic staining properties. Many of the involved cells showed necrosis and can presumably act as a nidus for bacterial proliferation.


A new fluorescent method for the demonstration of macromolecular aldehydes.

The fluorochrome, p-hydrazinoacridine (HA), is used to stain selectively natural macromolecular aldehydes as occur in elastic tissue as well as periodic acid (oxidized) or hydrochloric acid (hydrolyzed) engendered polyaldehydes (e.g., respectively of glycogen, mucosubstances and deoxyribonucleic acid). The reaction is independent of SO2 required in Schiff-type reactions. The mechanism of the staining reaction is presumably via the formation of fluorescent hydrazones from aldehyde condensation with HA. Aldehyde blocking agents, i.e., thiosemicarbazide and sodium borohydride, block the staining reactions with HA. In view of the controversy surrounding the mechanism of Schiff-type reactions and the lack of an adequate explanation for the affinity of miscellaneous fluorochromes for elastic tissue, p-hydrazinoacridine appears to be an ideal reagent, combining a simple staining procedure, excellent histological results and a straightforward mechanism for its selectivity.


Ultrastructural demonstration of NAD-pyrophosphorylase activity in mouse liver nuclei.

Ultrastructural demonstration of NAD-pyrophosphorylase activity (E.C.2.7.7.1) in isolated mouse liver nuclei was investigated with the use of an electronhistochemical procedure based on the precipitation of pyrophosphate ions with lead ions under conditions permitting simultaneous ATPase inhibition by formaldehyde/ethanol prefixation. In isolated mouse liver nuclei activity of NAD-pyrophosphorylase was found in nucleoli, in interchromatin granules, coiled bodies and strand-like structures in nucleoplasm.


Ultrastructural demonstration of phosphatases by perfusion fixation followed by perfusion incubation of rat liver.

An alternative to previous methods (tissue chopper, frozen sections) for the ultrastructural demonstration of phosphatases is described. The present approach is based on a short vascular perfusion of rat liver with glutaraldehyde through the inferior caval vein, followed by vascular perfusion incubation with a medium containing the enzyme substrates. The effect of glutaraldehyde on three different types of phosphatases was investigated, namely a lysosomal enzyme (acid phosphatase) a tightly bound microsomal enzyme (G6Pase) and a loosely bound microsomal enzyme (IDPase). It is demonstrated that by perfusion with glutaraldehyde for three minutes good cellular morphology is obtained and that 50-60% of the initial activity of glucose-6-phosphatase, inosine-diphosphatase and acid phosphatase remains. The localization and deposition of G6Pase activity were distinct and observed throughout the endoplasmic reticulum and the nuclear envelope. For acid phosphatase, the reaction product was confined to various types of lysosomes including presumed autophagic vacuoles. No signs of enzyme diffusion were noted. The present approach seems to offer some advantages: it is simple and requires no extra equipment, penetration of the fixative and incubation enzyme medium is good, and finally freeze artifacts are avoided.


Ultrastructural localization of glucose 6-phosphatase activity in tracheal epithelium of the rat.

Ultrastructural localization of glucose 6-phosphatase activity was studied in the tracheal epithelium of the rat. The reaction product for the enzyme activity was localized in the endoplasmic reticulum and nuclear envelope of all cell types composing the tracheal epithelium, namely ciliated cells, goblet cells, brush cells and basal cells. This pattern of localization is similar to that shown by hepatocytes, proximal convoluted tubule cells of the kidney and a variety of other cell types. The function of this enzyme in tracheal epithelial cells is discussed.


Mitochondrial structure in the rat adrenal cortex.

Two distinct classes of mitochondria are described in the normal adrenal cortex of the Sprague Dawley CFY rat. Polyaminar mitochondria were frequently observed in the zona fasciculata and zona reticularis, particularly after ACTH stimulation of the cortex resulting from cold-stress exposure. It is uncertain whether such organelles are degenerating forms, or whether they have a specific functional role related to steroidogenesis in the normal cortical cell. In both normal and stressed adrenal cortices, protrusions of the outer membrane of mitochondria were evident, and were often seen penetrating lipid droplets. It is suggested that these protrusions may have some significance in the transport of cholesterol from the lipid droplet to the inner mitochondrial memrane 'desmolase complex', thus facilitating side-chain cleavage of cholesterol to pregnenolone.


Influence of cycloheximide on the lung.

We examined the time course of the influence of cycloheximide on descending pressure-volume curves of excised lungs and on protein and lecithin synthesis and oxygen consumption by lung slices. We also looked at the influence of cycloheximide on granular pneumocyte ultrastructure. Excised lungs from cycloheximide-treated animals are more compliant than controls. After ventilation with air, lungs from control and cycloheximide animals show increased retractive forces and a shift to the right of the deflation P-V curve. Incubation at 38 degrees C for 30 min reverses these changes in control lungs, but not in lungs from cycloheximide-treated rabbits. There is no change in liquid delfation P-V curves after cycloheximide. Cycloheximide causes an immediate decrease of 50% in incorporation of radioactive leucine into protein by lung slices. Incorporation of radioactive palmitate into lecithin and oxygen consumption are also decreased by 50% 6 h after cycloheximide. Lamellar bodies in granular pneumocytes are smaller after cycloheximide. Cycloheximide causes a significant increase in the surface density of the lamellar body envelope. Cytoplasmic area of granular pneumocytes is increased after cycloheximide.


Comparison of virus recovery techniques for assessing the virucidal activity of disinfectant spray products.

A comparison was made of virus recovery from inoculated glass surfaces by 3 different viral recovery techniques used in spray test methods. On a total of 7, 5, and 5 observations, for the cover glass crushing, cotton swab rubbing, and microscopic slide shaking techniques, respectively, the log reduction of the median tissue culture infective dose (log TCID-50/ml) averaged 1.48, 2.57, and 2.22, the mean percentage recovery was 77.98, 61.55, and 66.94%, and the coefficient of variation was 4.37, 10.01, and 7.42%. The cover glass technique was found to be the least variable and the most effective and precise of the 3 methods.


Detection of ochratoxin A in barley, using silica gel minicolumns.

A simplified procedure has been developed to detect ochratoxin A in cereals which can be used in the field where equipment for thin layer chromatography is not available. The procedure includes extraction of the acidified sample with chloroform, purification over sodium bicarbonate, and minicolumn chromatography. Under longwave ultraviolet light ochratoxin A appears as a blue-green fluorescent band at the lower end of the column. Contamination levels as low as 12 ppb can be detected by this method.


Quantitative fluorodensitometric determination and survey of aflatoxins in nutmeg.

A study is presented for the quantitative fluorodensitometric analysis of aflatoxins in spices, in particular nutmeg (Semen myristicae). Samples were extracted with chloroform, followed by silica gel column cleanup according to the AOAC officail first action method, 26.019(a), and by 2-dimensional thin layer chromatography according to the antidiagonal technique. The method includes a confirmatory test for aflatoxins by hemiacetal formation. The concentrations of aflatoxins in samples were determined by measurement of the fluorescent intensities of the separated aflatoxin spots from sample and standards on the same chromato-plate with a reflectance flying-spot sensitometer. With such a technique, a coefficient of variation value of 5.22 plus or minus 1.24% (P = 99%) was calculated for a series of 5 standard B-1 spots and averaged for 13 TLC plates, demonstrating the precision of the chromatographic and densitometric procedures. An average recovery of 108.4 plus or minus 5.8% (P = 95%) was obtained for 11 spiked nutmeg extracts (5.0-20.0 mu-g B-1 added/kg), whereas an average recovery of 92.6 plus or minus 4.9 (P = 95%) was established for 13 spiked nutmeg samples (5.0-20.0 mu-g B-1 added/kg). The coefficient of variation of the complete analytical procedure for ground nutmeg was 8.80%. In a survey on the occurrence of aflatoxins in 40 commercial nutmeg samples (covering 12 different brands) in The Netherlands, aflatoxins were detected in 30 ground samples (32 ground samples analyzed) in concentrations ranging from 1.0 to 23.2 mu-g B-1/kg or 2.7 to 36.5 mu-g B-1 + B-2 + G-1 + G-2/kg, whereas no aflatoxins were present in whole nutmeg kernels (8 samples analyzed). The lowest level of detection was 1.0 mu-g B-1/kg. In addition, 50 commercial spices consisting of 19 different types of commodities other than nutmeg wer assayed for aflatoxins according to the same procedure. No aflatoxins were detected in these samples, with the exception of 1 sample of bay leaf which contained 5.1 mu-g B-1/kg.


Role of adenylate kinase in the regulation of macromolecular biosynthesis in a putative mutant of Escherichia coli defective in membrane phospholipid biosynthesis.

The regulation of macromolecular biosynthesis was studied in a temperature-sensitive mutant of Escherichia coli previously identified as containing a single mutation causing a thermolabile sn-glycerol-3-phosphate acyltransferase, the first enzyme of the pathway for phospholipid biosynthesis. When this mutant was shifted to a nonpermissive temperature, phospholipid synthesis, as well as ribonucleic acid, deoxyribonucleic acid, and protein synthesis, decreased in a coordinate manner, suggesting the existence of a common regulatory mechanism. During the same time that the rate of macromolecular synthesis was decreasing at the nonpermissive temperature, the intracellular concentration of adenosine 5'-triphosphate dropped dramatically and the concentration of adenosine monophosphate increased. The concentration of adenosine 5'-diphosphate dropped, but not as markedly. The decrease in macromolecular synthesis and the changes in the adenine nucleotide concentrations can now be attributed to a thermolabile adenylate kinase. The inactivation of adenylate kinase prevented the cell from converting adenosine 5'-monophosphate to adenosine 5'-diphosphate and consequently from making adenosine 5'-triphosphate. This in turn caused a decrease in the rate of macromolecular synthesis and cell growth. Adenylate kinase, therefore, is a key enzyme in controlling the rate of cell growth. The nature of the possible relationship between adenylate kinase and glycerol-3-phosphate acyltransferase is discussed.


Multiple forms of cytochrome b in Mycobacterium phlei: kinetics of reduction.

The kinetics of reduction of the b-type cytochromes in the electron transport particles (ETP) from Mycobacterium phlei were studied with nicotinamide adenine dinucleotide, reduced form (NADH) or succinate as electron donors. There appeared to be three active cytochromes b in the ETP,bS563 and bS559, which were reducible by either substrate, and bN563, which was reducible by NADH but not by succinate. In the presence of adenosine 5'-triphosphate, a substantial increase in b563 reduction was observed with succinate at anaerobiosis. This was followed by a decrease in absorption. Adenosine 5'-triphosphate did not effect an increase in cytochrome b563 reduction at transition with NADH, but the occurrence of a secondary decrease in absorption was reflected in a decrease in total enzymatic reduction. The adenosine 5'-triphosphate effect was altered in trypsin-treated ETP, and abolished by uncoupling agents or by removal of the coupling factor-latent adenosine triphosphatase. In the presence of a supernatant fraction obtained during the preparation of the ETP, b563 reduction with succinate was greatly increased. A smaller increase was observed with NADH. Cytochrome b reduction was also studied in ETP inhibited by 2-n-nonylhydroxyquinoline-N-oxide, which appears to inhibit at bS563. On the basis of these data the interrelationships among the b-type cytochromes can be described in relation to the M. phlei electron transport chain.


Replication of R-factor R1 in Scherichia coli K-12 at different growth rates.

The R-factor R1drd-19 mediates resistance to beta-lactam antibiotics via a beta-lactamase. A strain of Escherichia coli K-12 carrying R1drd-19 was grown at different growth rates by using different carbon sources. The specific rate of production of the R1 beta-lactamase increased linearly with the growth rate and with the gene dosage. The content of R1 deoxyribonucleic acid was estimated by alkaline sucrose gradient centrifugation and by analysis of the specific rate of beta-lactamase synthesis in nutritional shift-up experiments and was found to decrease fivefold when the growth rate was increased from 0.4 to 1.8 doublings per h. The number of R1 molecules per cell decreased from six to two in the same growth range. The presence of the plasmid affected the mean cell size significantly; at a growth rate of 0.4 doublings per h the R-+ cells were on the average 50% bigger than the R-minus cells, whereas the effect was less than 10% at a growth rate of 1.8 doublings per h. Several reports in the leterature state that the initiation mass of chromosome replication is constant. In this paper it is shown that the initiation mass of R1 replication is proportional to the growth rate. Thus, the replication of the plasmid R1 and of the chromosome are independently regulated processes. It is argued that plasmid replication is under negative control.


Organization and control in the arginine biosynthetic pathway of Neurospora.

Eight enzymes involved in the conversion of acetylglutamate to arginine in Neurospora crassa were studied. The data indicate that of three enzymes early in the sequence, only the first, acetylglutamate kinase, is a nonorganellar enzyme. The next two, N-acetyl-gamma-glutamyl-phosphate reductase and acetylornithine aminotransferase, are in the mitochondrion, which was previously shown to contain the subsequent enzymes: acetylornithine-glutamate acetyltransferase, ornithine carbamyltransferase, and carbamyl-phosphate synthetase A (arginine specific). The last two enzymes of the pathway, argininosuccinate synthetase and argininosuccinate lyase, were previously shown to be cytosolic. All enzymes but one have low amplitudes or repression. Their levels respond little to arginine excess and are about twofold elevated (threefold for ornithine carbamyltransferase) as a result of arginine limitation in the arg-12-8 strain. No restriction of the incorporation of mitochondrial enzymes into mitochondria could be detected when the levels of these enzymes were elevated. Two enzymes, acetylglutamate kinase and carbamyl-phosphate synthetase A, which initiate the synthesis of the ornithine and guanidino moieties of arginine, respectively, show the lowest specific activities in crude extract. These enzymes display special regulatroy features. Acetylglutamate kinase, which has a typically low amplitude of repression, is subject to feedback inhibition. Carbamyl-phosphate synthetase A is wholly insensitive to arginine or citrulline in vitro or in vivo, but displays a very large amplitude of repression (about 60-fold). It is unique in that it can be almost completely repressed by growth of mycelia in excess arginine. These data suggest that mitochondrial localization may be incompatible with a mechanism of feedback inhibition by a cytosolic effector, arginine. Further, they suggest that the high repressibility of carbamyl-phosphate synthetase A compensates for its feedback insensitivity.


Ultrastructal morphology of some prokaryotic microorganisms associated with the hindgut of cockroaches.

Scanning electron microscopy and transmission electron microscopy have been used to visualize the morphology and ultrastructure of two types of microorganisms in the hindgut of the cockroach Blaberus posticus. Both organisms, designated as either short or long rods, are attached to chitinous projections from the gut wall. Micrographs suggest that the organisms are prokaryotic with a cell wall complex characteristic of gram-negative bacteria. However, certain differences were noted between the cell wall complex of the two types. Two forms of the long-rod type were noted, with one form appearing to be a "degenerate" or "transitional" cell. In the degenerate cells, vesicles are observed that often are contiguous with the cytoplasmic membrane. There are indications that the long-rod type may divide by longitudinal fission. Neither the short- nor long-rod type has been cultivated in its respective recognizable form.


Mechanism of the isomerization of isopentenyl pyrophosphate in Rhodotorual rubra-1.

Stereospecifically labeled mevalonic acid was incorporated into the carotenoids of Rhodotorula. The randomized results are discussed in relation to mechanisms proposed for the conversion of isopentenyl pyrophosphate to dimethylallyl pyrophosphate and the prenol transferase enzyme system to Rhondotorula rubra.


Increased ouabain-sensitive 86Rb+ uptake and sodium and potassium ion-activated adenosine triphosphatase activity in transformed cell lines.

During the log phase of growth both the active, ouabain-sensitive K+ uptake, measured as 86Rb+, and the sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase) activity of SV40-transformed 3T3 cells were 2.5-and 5,5-fold higher, respectively, than in untransformed 3T3 cells. A similar higher active K+ uptake was found for Rous sarcoma virus and SV40-transformed baby hamster kidney cells compared with untransformed BHK cells. The active K+ uptake in SV403T3 and normal 3T3 cells decreased when the growth rate of both cell types diminished. Reduction in ouabain-sensitive ATP hydrolysis only occurred later, however, when appreciable decreases in cell viability were seen. Arrhenius plots of the (Na+ + K+)-ATPase activity of SV403T3 cells indicated a discontinuity at 24 degrees, whereas no similar discontinuity was indicated for 3T3 cells. The consequences of elevated K+ transport and (Na+ + K+)-ATPase activity in transformed cells and the possibility that the increased activity might be related to differences inphospholipid fatty acyl chain fluidity are discussed.


Adenylate kinase of human erythrocyte. Isolation and properties of the predominant inherited form.

Adenylate kinase exists in the human erythrocyte in a number of molecular forms with two allels at a single polymorphic locus coding for most of the enzyme forms. The predominant enzyme form, AK a, was purified to constant specific activity in excess of 3000 and appeared homogeneous by chromatography, electrophoresis, and ultracentrifugation. Sedimentation velocity and partial specific volume measurments of AK a yielded values of s20,w = 2.1 S and 0.722 cm-3per g. The molecular weight of the native enzyme was estimated to be 22,500 by sedimentation equilibrium and gel filtration analyses. The molecular weight of the denatured enzyme did not differ, indicating an absence of subunit structure in confirmation of genetic evidence of a single locus coding for the enzyme. The isolated enzyme demonstrated remarkable stability to denaturants (heat, guanidine HCl, urea) in the presence of appropriate stabilizing agents and could not be distinguished from rabbit muscle enzyme on this basis (as well as by a number of other kinetic and physicochemical parameters). The erythrocyte adenylate kinases have a common molecular size but differ in their charge properties. They demonstrate anomalous electrophoretic behavior, migrating anionic to hemoglobin in starch gel, yet exhibit isoelectric points considerably alkaline to hemoglobin (e.g. AK a, pI = 9.0) by isoelectric focusing.


Thf complete amino acid sequence of C-I (apoLp-Ser), an apolipoprotein from human very low density lipoproteins.

C-I was prepared from very low density lipoproteins of patients with familial type V hyperliporproteinemia. Peptides from tryptic digests of unmodified and succinylated C-I, chymotryptic peptides, and the products of cayanogen bromide cleavage were isolated and characterized. Sequence analysis of tryptic peptides was performed by the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) technique and hydrolytic regeneration  of the amino acid residues from the phenylthiocarbamyl derivatives. Alignment of the tryptic fragments within the cyanogen bromide and succinyl-tryptic peptides was confirmed by the overlap chymotryptic peptides. The complete amino acid sequence of C-I, 57 residues in length, does not reveal any obvious basis for its lipophilic properties.


Piericiden A sensitivity, site 1 phosphorylation, and reduced nicotinamide adenine dinucleotide dehydrogenase during iron-limited growth of Candida utilis.

It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir th


The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues.

In crude extracts of adipose tissue the protein kinase dissociates slowly at 30 degrees into regulatory and catalytic subunits in the presence of 700 mug per ml of histone or 0.5 M NaCl. If the kinase is first dissociated by adding 10 muM adenosine 3':5'-monophosphate (cAMP), reassociation occurs instantaneously after removal of the cAMP by Sephadex G-25 chromatography. In contrast, in crude xtracts of heart, the protein kinase dissociates rapidly in the presence of 700 mug per ml of histone or 0.5 M NaCl and reassociates slowly after removal of cAMP. These differences are accounted for by the existence of two types of protein kinases in these tissues, referred to as types I and II. DEAE-cellulose chromatography of extracts of adipose tissue produces only one peak of cAMP-dependent protein kinase activity (type II) which elutes between 0.15 and 0.25 M NaCl. Similar chromatography of heart extracts resolves enzyme activity into two peaks; a type I enzyme which elutes between 0.05 and 0.1 M and predominates (greater than 75% of total activity), and a type II enzyme which elutes between 0.15 and 0.25 M NaCl. The dissociation properties of the types I and II enzymes from heart and adipose tissue are retained after partial purification by DEAE-cellulose and Sepharose 6B chromatography. Rechromatography of the separated peaks of the cardiac enzymes does not change the elution pattern. Sucrose density gradient centrifugation and gel filtration studies indicate that the molecular weights of these enzymes are very similar. The type II enzyme isolated by DEAE-cellulose chromatography of heart extracts resembles the adipose tissue enzyme, i.e. it undergoes slow dissociation at 30 degrees in the presence of histone or 0.5 M NaCl. The adipose tissue kinase and the heart type II kinase are not identical, however, since they do not elute at exactly the same point on DEAE-cellulose columns. A survey of several tissues indicates the presence of type I and II protein kinases similar to the enzymes in adipose tissue and heart as determined by DEAE-cellulose chromatography of crude extracts and by dissociation of the enzymes with histone. The presence of MgATP prevents dissociation of type I enzyme from heart by 0.5 M NaCl or histone. The profile of the enzyme on DEAE-cellulose, however, is not changed...