Oestrogen receptors in chick oviduct. Characterization and subcellular distribution. Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.


Conversion of type III hyperlipoproteinaemia to type IV hyperlipoproteinaemia by a fat-free, carbohydrate rich diet. Hyperlipoproteinaemia type III has been considered a clear cut clinical entity characterized by the presence of cholesterol rich "floating" beta very low density lipoproteins (VLDL) after ultracentrifugation. Carbohydrate inducibility is pronounced in type III. This study was undertaken to elucidate the effect of a high carbohydrate diet in type III as an effort to clarify a suggested association between type III and IV hyperlipoproteinaemia. On an isocaloric fat free carbohydrate rich diet the lipoprotein pattern changed as the VLDL triglycerides increased 31 per cent on average and LDL and HDL cholesterol decreased by 40 and 22 per cent. The high ratio cholesterol/triglycerides in VLDL was normalized. The lipoprotein levels in serum after carbohydrate induction showed all characteristics of a type IV with high VLDL triglycerides, normal cholesterol/triglyceride ratio in VLDL, subnormal cholesterol levels in LDL and HDL with changed relation between LDL1 and LDL2. On electrophoresis the floating beta band disappeared. It is probable that depending on the nutritional situation, the metabolic defect in type III may be expressed either as a type III or a type IV. An explanation of the effects of the carbohydrate rich diet on the lipoprotein pattern is suggested.


Influence of peptides on reduced response of rats to electric footshock after acute administration of morphine. Acute treatment of rats with morphine (10 mg/kg) resulted in a marked reduction of motor response to inescapable electric footshock (EFS). Nalorphine (2mg/kg) antagonized this action of morphine. Pretreatment with synthetic ACTH 1-24 (10 IU) 60 min prior to testing also inhibited this morphine-induced reduction, whereas other ACTH-like peptides, lacking corticotrophic activity, were ineffective. ACTH 1-24 had no effect on the response of adrenalectomized rats to EFS after morphine. In intact rats dexamethasone pretreatment 4 hr prior to testing also antagonized the action of morphine on EFS. Taken together these findings suggest that ACTH 1-24 interferes with the antinociceptive action of morphine and that the integrity of the adrenal is essential for demonstration of this antagonism.


Influence of agents that act on DNA and RNA synthesis on the activity of poly(ADP-Rib) polymerase. The activity of poly(ADP-Rib) polymerase is enhanced in the presence of spermine and spermidine. Among the adenosine-like antibiotics tested, only formycin B and showdomycin cause an inhibition of the enzyme, which is competitive to NAD. The activity of poly(ADP-Rib) polymerase is not reduced by rifamycin, alpha-amanitin and 2-phenylethanol.


Mechanism of urea nitrogen binding by proposed oxidized starch gastrointestinal absorbents. By use of 14-C label it was demonstrated that apparent binding of urea N to polyaldehyde starch was probably preceeded by hydrolysis to ammonium ion. Thus direct urea binding was not the mechanism through which ingested polyaldehyde starch might increase fecal N excretion in uremic patients.


Membrane fractions of rabbit granulocytes. 1. The presence of ouabain-sensitive ATPase and the electrophoretic patterns. In one or two membrane fractions isolated from the homogenate of casein-induced rabbit granulocytes ouabain-sensitive ATPase activity was found definitely. Electrophoretic patterns of these membrane fractions showed no significant differences in staining for carbohydrate and protein.


APUD characteristics and immunocytochemistry of avian pituitary corticotrophs. L-DOPA is taken up and decarboxylated by cells in the avian adenohypophysis, which are situated largely in the cephalic lobe. These APUD cells have been shown by sequential formaldehyde-induced and immunofluorescence to be corticotrophs and the relationship between the two procedures has been found to have a reciprocal quality.


Effect of insulin on the glucose uptake of protozoa. Insulin stimulates the glucose uptake of Tetrahymena pyriformis. This shows the presence of insulin receptors in Tetrahymena, consequently receptors may be present in a level of phylogenesis, where the natural contact between the given hormone and the cell is unnecessary and impossible.


Effects of hypophysectomy, bilateral adrenalectomy and hormone replacement therapy upon organ monoamine oxidase activity. Changes in MAO activity after hypophysectomy (HX) are not due to adrenal insufficiency. ACTH failed to reverse the effects of HX and enhanced the depression of cardiac and spleenic MAO. The data suggests both facilitatory and inhibitory effects of pituitary hormones on MAO activity.


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[Determination of conjugated and free estrogen in male plasma (authors transl)]. Radioimmunoassay for conjugated and free estrone (E1), estradiol-17beta(E2) and estriol (E3) in male plasma was investigated, while no precise determination of conjugated estrogen in male plasma had been established. Each tritiated estrogen glucuronides for determining the recovery was added to 3 ml of plasma samples. These plasma samples were extracted with acetone, hydrolyzed with 15 vol% HCl at 100 degrees C for 60 minutes, and resulting free estrogens were extracted with ether. These extracts were washed with 1% NaOH and 1% NaHCO3, and applied to microcolumn of Sephadex LH-20. Then they were determined by the radioimmunoassay as the same method of evaluation for free estrogen. Each mean values of plasma free and conjugated estrogen in 10 normal adult males was as follows: 36.0 +/- 9.81 pg/ml(free E1), 29.5 +/- 11.0 pg/ml(free E2), 1.80 +/- 1.27 pg/ml (free E3), 31.0 +/- 12.6 pg/ml (conjugated E1), 36.0 +/- 14.2 pg/ml (conjugated E2), 30.6 +/- 8.35 pg/ml (conjugated E3). Conjugated estrogen to free estrogen ratio was 1.45.


[Steroidogenesis in isolated adrenal cells of rat. -Effects of cyanoketone on pregnenolone and corticosterone production- (author's transl)]. In order to study the effect of cyanoketone on steroidogenesis of rat adrenal, the assay technique for corticosteroids released into the incubated media of the rat adrenal cells treated with collagenase was basically investigated. Corticosterone was measured by fluorometric method and pregnenolone by radioimmunoassay. Reliability of radioimmunoassay was satisfactory. About 400,000 cells were obtained from one adrenal gland of male or female rats and sex-dependent difference in pregnenolone and corticosterone production in response to ACTH was not found. Net corticosterone production by isolated adrenal cells was related to the log of the concentration of ACTH by a sigmoid curve over the range 1 to 1000 muU/ml. The half-maximum response was observed at an ACTH concentration of 10 muU/ml, and maximum corticosterone production responding to ACTH (100-1000 muU/ml) was about 5 mug/adrenal/120 min. When cell suspensions were incubated with 1000 muU/ml of ACTH, the conversion from pregnenolone to corticosterone was inhibited 50% by cyanoketone at a concentration of 2 times 10(-8) M. The conversion was completely inhibited at a concentration of more than 10(-7) M. Cyanoketone up to a concentration of 10(-5) M seemed to have no inhibitory effect on cholesterol side-chain cleavage. In the absence of ACTH significant amount of pregnenolone was formed (about 60 ng/adrenal) by isolated adrenal cells obtained from normal adult female rats during incubation with 10(-7) M of cyanoketone for 60 min. To eliminate the possibility of the effect of endogenous ACTH which might be present in incubation medium, cell suspensions were obtained from hypophysectomized female rats. Incubations were carried out in the same condition as mentioned above and significant amount of pregnenolone was formed by cell suspension, which was about 35 ng/adrenal.


Androgen-dependent accumulation of carnitine by rat epididymis after injection of [3H]butyrobetaine in vivo. After i.m. injection of [3H]butyrobetaine into rats, the accumulation of carnitine into the epididymis, prostate gland, seminal vesicles, testis and heart was studied. The concentration of radiolabeled carnitine into the cauda epididymis increased linearly with time up to 72 h after the injection of the precursor, while its level in the prostate and seminal vesicles decreased rapidly. Very low levels of carnitine were found in the testis. Castration reduced the carnitine accumulation by cauda epididymis to 6% of the control levels while treatment of castrated animals with testosterone propionate (500 mug/day) partly restored the carnitine uptake. Similar treatment with 17beta-oestradiol valerate or 17alpha-hydroxyprogesterone had no effect. Surprisingly, cyproterone acetate (5 mg/day) also significantly stimulated carnitine accumulation by the epididymis to a level above that of the castrated controls. Simultaneous injection of both cyproterone acetate and testosterone propionate to castrated animals caused an additive effect of these steroids. This indicated that cyproterone acetate in this system is working as a weak androgen. Treatment of rats with 17beta-oestradiol valerate also reduced carnitine accumulation by the cauda epididymis. This is due to suppression of pituiatry gonadotrophin secretion, since concommitant treatment with testosterone propionate (500 mug/day) caused a normalization of the carnitine uptake. Treatment of intact rats with cyproterone acetate significantly reduced the epididymal weight, but not the carnitine accumulation. 17alpha-Hydroxyprogesterone treatment had no effect either on the epididymal weight or the accumulation of the carnitine. Unilateral orchiectomy reduced the carnitine accumulation by the cauda epididymis to about 40% of that occurring in the non-operated control side. This indicates that the luminal contact between the testis and epididymis or the luminal content of the epididymis itself is of importance for the androgen-dependent metabolic process occurring in the cauda epididymis. Castration or hormone treatment did not change the conversion of butyrobetaine to carnitine, or the carnitine uptake by heart. Carnitine uptake by the testis after [3H]butyrobetaine injection was rather low and this would exclude the possibility of synthesis of carnitine in the testis as a source of epididymal carnitine. Carnitine only accumulated in the cauda epididymis in vivo 4 to 96 h after injection of [3H]butyrobetaine. The presence of radioactively labeled butyrobetaine or methylcholine was not detected.


Some properties of androgen-binding activity in rat testis. High-affinity (Ka approximately equal to 5 X 10(8) M-1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant (t1/2 = 3 min, 0 degrees C, 30% glycerol buffer) using dextran-coated charcoal to separate bound from free hormone. Because of this fact, exchange of endogenous and labeled hormone was complete in the assay incubation time (16 h, 0 degrees C) and Scatchard plots of the high-affinity binding data were shown to measure total as contrasted to available sites. The binding was highly specific for androgens. Polyacrylamide gel electrophoresis separated high-affinity androgen-binding protein (Rf 0.54) from albumin (Rf 0.62). Binding site estimates under saturating conditions or by Scatchard analysis of electrophoresis data utilizing [3H]dihydrotestosterone agreed reasonably well with estimates made by the charcoal technique using [3H]testosterone.


Similarities between 5alpha-dihydrotestosterone-receptor complexes from human and rat prostatic tissue: effects on RNA polymerase activity. Protein macromolecules specifically binding [3H]5alpha-dihydrotestosterone ([3H]DHT) have been identified in cytosol and in nuclei prepared from human benign hypertrophic prostate. These macromolecules have similar properties to receptor proteins from other androgen-dependent tissues, as regards sedimentation coefficients on sucrose gradients and steroid specificity. Cytosol preparations from androgen-dependent tissues were able to transfer [3H]DHT in a recoverable protein-bound form to nuclei of other androgen-dependent tissues but not to nuclei of androgen-independent tissues. No transfer of radioactive steroid from cytosol of these latter tissues to any nuclei could be achieved. Labelled cytosol preparations from androgen-dependent tissues could stimulate the RNA polymerase activity of nuclei from androgen-dependent tissues but not that of nuclei from androgen-independent tissues. Cytosol preparations from these latter tissues could not affect RNA polymerase activity. Under suitable ionic conditions, human cytosol preparations containing DHT could stimulate both alpha-amanitin-sensitive and -insensitive RNA polymerase activities of human prostatic nuclei. However, rat ventral prostatic DHT-cytosol protein complexes were equally as efficient in performing this function, suggesting the possible involvement of specific DHT-receptor complexes in this process. It is therfore suggested that receptor molecules from androgen-dependent tissues may not be species specific but may share properties which would facilitate research into the understanding and aetiology of pathological conditions.


Apparent accelerated aging of human collagen in diabetes mellitus. The chronologic ages of human subjects were determined experimentally by enzymatic digestion of tendon collagen samples. Determined age closely matched actual age for individuals dying with a variety of major diseases. Juvenile diabetics did not fit this pattern; their experimentally determined ages were significantly greater than their actual ages. This raises the possibility of relationships between diabetes mellitus, changes in connective tissue, and accelerated aging.


Androgen-binding proteins in rat epididymis: properties of a cytoplasmic receptor for androgen similar to the androgen receptor in ventral prostate and different from androgen-binding protein (ABP). The cytoplasmic recptor (CR) in rat epididymal 105,000 g supernatant was separated from the androgen-binding protein (ABP) by gel electrophoresis following labeling with [1,2,6,7-3H]-testosterone in vivo. ABP disappeared from epididymal supernatants after castration of hypophysectomy, while CR remained unchanged. CR was evenly distributed between caput and cauda, while much more ABP was present in caput. Properties of CR in epididymis and prostate were similar and distinctly different from ABP. Binding to CR was destroyed by charcoal treatment (1 mg/mg protein) of supernatant for 0 degrees C for 6 h, heating at 50 degrees C for 30 min, or exposure to the sulfhydryl blocking reagent, p-chloromercuriphenylsulfonate (1mM) at 25 degrees C for 30 min, while binding to ABP was unaffected. The isoelectric pH of CR (5.8) was higher than that of ABP (4.6). Dissociation of radioactive 5alpha-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 degrees C is greater than 2 days), while dissociation from ABP was rapid (half-time at 0 degrees C is similar to 6 min). Cyproterone acetate (250 mg/100 g body weight) inhibited binding to CR both in epididymis and ventral prostate but did not affect binding to ABP. Nuclear uptake was inhibited by cyproterone to the same extent as binding to CR, indicating that nuclear uptake and binding are dependent on CR and independent of ABP. The time-course of uptake and binding in epididymal supernatant and nuclear fractions was essentially the same 1 day after bilateral castration when both CR and ABP were present or 8 days after castration when CR alone was present. It is concluded that the cytoplasmic receptor for androgen in rat epididymis has properties very similar to the androgen receptor in ventral prostate but different from ABP.


Deterioration of the microcirculation in diabetes. Studies of the microcirculation in diabetes in the last fifteen years have concentrated heavily on anatomic and biochemical abnormalities of the capillary basement membrane. Greater insights into basement membrane changes have eclipsed the previous picture of widespread progressive deterioration of the entire microcirculation. The history, variety of organ involvement, pattern of circulatory decline, and associated anatomic, physiologic, and biochemical findings are re-examined so that recently described potential mechanisms for the development of diabetic microangiopathy may be understood in a broader perspective. The possible contributions of seven categories of diabetic changes to damage of the microcirculation are outlined. The categories are: (1) altered basement membrane, (2) altered cellular function, (3) cell metabolic changes, (4) altered blood flow properties, (5) distrubed hemostasis, (6) altered oxygen transport, and (7) altered hormone production. The variety of clinical manifestations in long-standing diabetes related to microangiopathy appears to be due to a combination of a widely variable over-all rate of progression and a differing ability of body tissues and organs to accommodate to the sequential circulatory changes. The slow rate of deterioration in most diabetics suggests that several abnormalities must interact to produce the observed progression. A clear understanding of the interactions responsible for diabetic microangiopathy is becoming more important as new options in the management of diabetes become available.


Effect of dioctyl sodium sulfosuccinate on colonic fluid and electrolyte movement. These studies evaluated the effect of dioctyl sodium sulfosuccinate (DSS), a detergent and the active ingredient of some commercially available stool softeners, on intestinal fluid and electrolyte movement. DSS produced water, sodium and chloride secretion into the in vivo rat cecum. This secretion readily reverted to net absorption following replacement of DSS by isotonic saline. In in vitro studies, DSS increased the short circuit current and diminished sodium absorption. The increase in short circuit current was not observed either when HCO3 and chloride were absent from the incubation solution or when theophylline was present. DSS also significantly increased mucosal cyclic adenosine monophosphate levels. These results which are similar to previous studies of the effect of cholera enterotoxin on ileal mucosa and bile salts on colonic mucosa are consistent with the proposal that DSS stimulates fluid and electrolyte secretion which is mediated by increased mucosal cyclic adenosine 3':5' monophosphate.


Control of deoxyribonucleic acid synthesis in normal rabbit colonic mucosa. Although cyclic adenosine 3':5'-monophosphate (cyclic AMP, cAMP) is known to suppress DNA synthesis is cultured cells and experimental tumors, its role in normal intact tissue has been little explored. This study helps to define the influence of modifiers of cyclic AMP levels on DNA synthesis in rabbit colonic mucosa maintained in short term organ culture system. Base line studies showed that incorporation of [3H]thymidine into DNA was linear for 24 hr and predominantly in mucosal cells, as shown by autoradiography. Colon from a normal fed rabbit showed a gradient of DNA synthesis, lowest in the cecum and increasing to a maximum, 3-fold greater, at the splenic flexure. This pattern was obliterated by fasting, at which time no formed stool remained in the colon, and all colon mucosa incorporated thymidine at the lower level of the right colon. Known modifiers of intracellular cAMP were found to depress colonic DNA synthesis. Theophylline inhibited DNA synthesis by 35% at 0.5 mM concentration and increased intracellular cAMP levels. This inhibition took 10 hr to be manifest and was at least partly reversible. It was by far the most active of the methylxanthines, consistent with its potency as a phosphodiesterase inhibitor. N6,02-dibutyryl cyclic AMP inhibited DNA synthesis at concentrations as low as 0.025 mM, whereas adenosine and sodium butyrate were ineffective up to 1.0 mM. 5'-AMP did inhibit DNA synthesis, but only at 0.1 mM or higher and did not elevate intracellular cAMP levels. Other modifiers of cAMP which were effective included prostaglandins E1, E2, and F2alpha (2 times 10(-6) M) and papaverine (1 muM). Thymidine uptake was not affected by any of these drugs. The intrinsic thymidine pool was estimated at 20 muM by isotope dilution, and was not altered by theophylline. DNA synthesis in rabbit colon can be suppressed by increased cAMP levels within the time period allowed by organ culture. Thus, these drugs that elevated cAMP levels did not seem to suppress DNA synthesis by decreasing intracellular thymidine concentrations.


[The present status of the theory of induction of carcinoma of the cervix in man by herpes virus (author's transl)]. A large body of evidence has accumulated during the past decade suggesting that HSV-2 infection of the cervix may be a major precursor of cervical carcinoma. A brief review of the more significant findings to date is presented.


["Chronic mastitis" and carcinoma of the breast seen from the viewpoint of Soviet-russian pathologist (author's transl)]. The group around Prof. D. I. Golowin (Leningrad) distinguishes between the solid epithelial hyperplasia belonging to fibroadenomatosis of the breast and the solid form of lobular carcinoma in situ. The morphological details permitting the differential diagnosis are stressed (see table).


[Phasecontrast cytology for intraoperative diagnosis of malignant breast lesions (author's transl)]. The efficacy of a cytodiagnostic method using a wet preparation of freshly exstirpated suspected tumorous breast tissue and phasecontrast microscopy has been evaluated in 430 cases. 95% of the carcinomas present were found with this method. The rate of wrongly positive cytological findings was less than 1%. It is discussed what advantages of this method can be achieved by a well trained cytologist for the patient and the surgical organisation. Further this new method is a means to check the frozen section findings.


Contractility of the fallopian tube.

Contractility of the fallopian tube. Recordings of intratubal pressure in women reveal a complex pattern of tubal contractions. Overall activity of the fallopian tube is increased during ovulation. During the luteal phase of the menstrual cycle, activity is depressed but never suppressed. The fallopian tube remains active during gestation in contrast with the uterus which is quiescent. The early puerperal tube displays a pattern of activity similar to that recorded during the late luteal phase of the menstrual cycle. Activation occurs during suckling and in response to exogenous oxytocin, prostaglandins, nor-adrenergic compounds, and ergot derivatives. It is concluded that contractility of the fallopian tube is less influenced by ovarian steroids than that of the uterus.


The behaviour of NAD+ and NADH in Acinetobacter calcoaceticus during n-alkane assimilation. The behaviour of the nicotinamide adenine dinucleotides NAD+ and NADH in Acinetobacter calcoaceticus during n-alkane assimilation was studied, acetate and succinate being used as reference carbon sources. The intracellular concentration of the two nucleotides was found to increase during the exponential growth phase, reaching its maximum in the phase of decreasing growth rates. In the exponential phase, the NAD+/NADH quotients were less than 1 and showed only unimportant variations. In the phase of decreasing growth rates, the concentration of NADH showed a distinct decrease, reaching its minimum in the stationary phase. Parallel to this, the concentration of NAD+ showed a continuous increase until the stationary phase was reached. This resulted in an increase, during the phase of decreasing growth rates, of the NAD+/NADH quotients to values greater than 1, similarly as recorded in the stationary phase. There were no fundamental differences in this behaviour between the individual carbon sources.


Penicillinamidohydrolase in Escherichia coli. III. Catabolite repression, diauxie, effect of cAMP and nature of the enzyme induction. Synthesis of penicillinamidohydrolase (penicillin acylase, EC 3.5.1.11) in Escherichia coli is subjected to the absolute catabolite repression by glucose and partial repression by acetate. Both types of catabolite repression of synthesis of the enzyme in Escherichia coli are substantially influenced by cyclic  3',5'-adenosinemonophosphate (cAMP). Growth diauxie in a mixed medium containing glucose and phenylacetic acid serving as carbon and energy sources is overcome by cAMP. cAMP does not influence the basal rate of the enzyme synthesis (without the inducer). Derepression of synthesis of penicillinamidohydrolase by cAMP in a medium with glucose and inducer (phenylacetic acid) is associated with utilization of the inducer, due probably to derepression of other enzymes responsible for degradation of phenylacetic acid. Lactate can serve as a "catabolically neutral" source of carbon suitable for the maximum production of penicillinamidohydrolase. The gratuitous induction of the enzyme synthesis in a medium with lactate as the carbon and energy source and with phenylacetic acid is not influenced by cAMP; however, cAMP overcomes completely the absolute catabolite repression of the enzyme synthesis by glucose.


Cross-resistance of Escherichia coli B/r to cis-platinum (II) diamminochloride, UV light and alkylating agents. Gradual transfers of the strain Escherichia coli B/r on M9 agar with increasing concentrations of cis-platinum (II) diamminochloride (cis-Pt(II)) yielded a resistant strain SM 405 capable of growing on liquid M9 medium containing 250 muM cis-Pt(II). The parent strain Escherichia coli B/r is completely inhibited in both division and growth at cis-Pt(II) concentrations as low as 30 muM. The resistant mutant has a longer doubling time than the parent strain. No other differences were found between the two strains. To elucidate the nature of the resistance, the effect of cis-Pt(II) on the survival of the two strains was compared with that of nitrogen mustard, UV light and ethyl methanesulphonate (EMS). The resistant strain SM 405 was found to be more hardened against the lethal action of UV light and nitrogen mustard but less so against EMS. It had also a higher ability of a host-cell reactivation of UV-irradiated phage T3. The different resistance of the B/r and SM 405 strains is probably due to a mutation increasing the effectiveness of the excision repair in the latter.


Personality characteristics and sleep variables. In a sleep study of 14 normal healthy adults an investigation was made of sleep measurements of a baseline record for its eventual relationship to the percentage of increase of REM percentage of the 1st recovery night following partial differential REM deprivation (PDRD), as well as to personality characteristics. The percentage of change in NREM sleep of the 1st recovery night was compared with the baseline record andthen compared with corresponding values of REM sleep. The results are summarized as follows. No significant correlation exists between the percentage of increase in the REM percentage of the 1st recovery night and sleep measures of the baseline record. An investigation of the relationship between sleep measures of the baseline record and personality characteristics revealed that stage SWS(%) was significantly greater in the introvert than in the extrovert, in the neurotic than in the non-neurotic, and in the nervous than in the optimistic. Comparison of the changes in NREM and REM sleep percentages of the 1st recovery night with the baseline record was made between paired personality characteristics. A significantly high percentage of increase in REM percentage was almost always associated with a significantly high percentage of decrease in stage 2 percentage. From these results it was inferred that an increase in REM percentage occurs at the expense of stage 2 percentage.


Characteristics of poor sleep with the normal human being. 1. To investigate the differences between good sleep and poor sleep, 41 sleep records were observed with nine normal male subjects ranging in ages from 22 to 40 years. A comparison was done of the differences of sleep records between the good and poor sleep estimations for each subject. 2. Poor sleep showed significantly shorter total sleep time (P less than 0.01) and significantly shorter stage 2 (P less than 0.05) than good sleep, but there were no differences in stages 1, 3, 4, stage REM, the percentage of every sleep stage to total sleep time, REM latency, mean REM duration, REM cycle and REM density. 3. Poor sleep showed longer latency for falling sleep (P less than 0.10) and a significant increase in stage changes from stage 2 to stage 3 (P less than 0.01) than with good sleep. 4. Poor sleep showed more prolonged awakenings and significantly longer durations spent for awakenings (P less than 0.05). 5. As to awakening in the morning poor sleep showed longer times required from terminal stage 2 and stage 4 and a significantly shorter time from terminal stage REM (stages 2 and 4, P less than 0.10, REM, P less than 0.01). 6. A certain amount of stage 4 and its decreasing seemed to be delayed in poor sleep, though its initial appearance wasn't. 7. REM periods of good sleep peaked at the third or the fourth REM stage and decreased to the fifth but those of poor sleep showed a gradual increasing from the second to the fifth having almost the same durations. 8. Awakenings in the morning often occurred from stage REM or just after an REM stage in poor sleep but in good sleep, on the other hand, occurred from stage 2. 9. The thresholds of natural wakenings from stages 3, 4 and REM of good sleep during the night might be higher than those of poor sleep.


[Chronobiological aspects of cyclothymia (author's transl)]. The paradoxical effect of sleep deprivation in endogenous depression has renewed the traditional interests of psychiatrists in the rhythmic phenomena associated with the symptomatology and course of cyclothymia. During the last decade, the rapid development of clinical research on sleep, neuroendocrinology, chromobiology and chronopathology in conjunction with important developments of methodology and statistical procedures have enabled a modern reconceptualization of the relationship between cyclothymia and disturbances in biological rhythms. An intensive review of the literature on circadian, ultradian, and infradian rhythms is reported here, and their relationship to the course and individual phases of cyclothymia is explored. The results of traditional analyses of sleep stages which had originally indicated that particular disturbances of sleep may be specific to cyclothymia have been shown to be incorrect. Rather, the abbreviation and fragmentation of sleep and the decrease of del asleep with concomitant increase in shallow stages have been found as well in serious sleep disturbances of other origins. Striking inter-and intra-individual variabilities in REM-sleep parameters characterize not only psychotic depression and severe mania but also a wide range of other acute psychoses...


Metabolism in normal and virus-transformed chick embryo fibroblasts as observed with glucose labeled with 14C and tritium and with tritium-labeled water. Glucose metabolism in normal and virus-transformed chick embryo fibroblast cells in culture was observed by allowing the cells to metabolize [U-14C]glucose plus glucose labeled with tritium in the C-1, C-3, and C-6 positions. Similarities and differences between normal and transformed cells were observed and measured. Both normal and transformed cells are found to metabolize about 20% of the glucose via the oxidative pentose phosphate cycle, with the rates being about twice as much for transformed cells as for normal cells under the chosen conditions. Nevertheless, the ratio of glucose metabolized via oxidative pentose cycle to the net flow of that metabolized directly to fructose 6-phosphate is about the same in normal and transformed cells. Although the rate of flow of [14C]glucose into the tricarboxylic acid cycle intermediates and amino acids derived from them appears to be the same in normal and transformed cells, the rate of tritium incorporation from H3HO into these intermediates seems to be much higher in normal cells.


Purification and properties of cytochrome c-556 from Agrobacterium tumefaciens B2a. Cytochrome c-556 from Agrobacterium mefaciens B2a was isolated in a pure, homoneous state. The best purification procedure volved ammonium sulphate fractionation, delting on Sephadex G-25, column chromatographic fractionation on DEAE- and CM-cellulose, and gel filtration on Sephadex G-75 superfine. Substitution of the CM-cellulose step by isoelectric focusing was successful. The purity of the final preparation is warranted by the purity index value, the electrophoretic patterns in the absence and presence of sodium dodecylsulphate, the sedimentation profile and the N-terminal amino acid analysis (alanine). The absorption spectrum of reduced cytochrome c-556 has maxima at 318, 419, 526 and 555.5 nm. The molar extinction coefficient for the alpha-band is 20 200M-1cm-1. The isoelectric point, determined both by preparative and analytical isoelectric focusing, is 5.55 +/- 0.10. The molecular weight of cytochrome c-556 was determined by gel filtration as 12000 and by dodecylsulphate gel electrophoresis as 11 500.


Extraction of collagenase from the 6000 times g sediment of uterine and skin tissues of mice. A comparative study. An enzyme capable of digesting native collagen in solution at neutral pH was extracted from the 6 000 times g sediment of the involuting uterus of the mouse and of the back skins of mice and rats. The collagenase could be dissociated at cold-room temperature from the sediment in about equal amounts when neutral Tris buffer containing 1.0M NaCl or 5M urea was used for the extraction step. The enzyme has been concentrated by ammonium sulfate precipitation and the activity was measured by using [14C]collagen in solution at pH 7.5. Collagen breakdown products were identified by disc electrophoresis. The amount of enzyme extracted was a function of temperature and salt concentration. As 5M urea extracted collagenase from the sediment in a relatively short time, this method of extraction seems to be a useful tool for serial experiments in the study of collagenase activity in collagen-rich tissues.


Stimulation of collagen synthesis in a cell-free system by mRNA from chick embryos. Embryonic chick RNA was translated in a cell-free system derived from wheat germ. One of the products synthesized in vitro under the direction of this RNA could be identified as collagen on the basis of collagenase digestion experiments and sodium dodecylsulfate-acrylamide gel electrophoresis. By submitting the RNA to chromatography on oligo(dT)-cellulose, a 26-30-fold enrichment of the mRNA coding for collagen was achieved.


Pathology of the early lesion in multiple sclerosis. Ideas concerning the nature of multiple sclerosis continue to be strongly influenced by the unusual morphology of the disease. A review of classic histology studies, however, reveals that there is less agreement than might be supposed concerning several important histiological features of the early lesion. Electron microscopy of brain biopsies, of immersion fixed autopsy tissue and of autopsy tissue fixed by early in situ brain perfusion suggests that the mechanism of demyelination in multiple sclerosis may be an unusual one that involves a progressive reduction in the number of myelin lamellae around nerve fibers in the vicinity of cells of uncertain origin that contain filamentous and multilamellated cytoplasmic inclusions unlike the usual pleomorphic inclusions seen in myelin phagocytes. Lymphocytes are not directly involved in this process but are observed to contact the inclusion material following its delivery to the Virchow-Robin spaces. The putative neurogenic or viral antigen in multiple sclerosis may be contained in this material. The occurrence of filamentous nuclei in early lesions fixed by immersion is an unrelated phenomenon, which may be an autolytic or drug induced artifact although this has not yet been established.


The gangliosidoses.

The gangliosidoses. The gangliosidoses are hereditary diseases with a recessive mode of inheritance and are caused by a genetically induced enzymatic block, which results in the accumulation of gangliosides in various tissues of the body, mainly in the brain. Although Tay-Sachs disease, the most commonly occurring of the gangliosidoses, has been known for nearly 100 years, additional variants of ganglioside "storage" disorders have been discovered during the past 15 years. Considerable progress in the knowledge of these disorders has been made with the advent of electron microscopy and with the elaboration of new biochemical and enzyme-chemical techniques. At the present the gangliosidoses are not amenable to therapy. Therefore the foreseeable future the pragmatic approach involves identification of the high-risk pregnancy and antenatal diagnosis.


Pathologic axons and synapses in human neuropsychiatric disorders. Ultrastructural studies of cerebral biopsy specimens from patients with various forms of psychomotor retardation and dementia have disclosed pathologic changes in axons and presynaptic or postsynaptic processes. The clinical disorders with lesions in axons and presynaptic terminals are reviewed. Three basic abnormalities have been detected: proliferation of tubulovesicular structures which probably originate from the smooth endoplasmic reticulum, "abnormal" mitochondria, and proliferation of 80 to 100 A filaments. Understanding of the pathogenesis of human disorders associated with axonic or "synaptic" lesions will probably depend on progress in areas of basic biomedical research concerned with the synthesis and turnover of biological membranes and the packaging and secretion of neurotransmitters, elucidation of mechanisms of cytoplasmic streaming and axoplasmic flow, and biophysical and biochemical characteristics and functions of "fibrous" proteins (neurotubules, neurofilaments, pathologic fibrous proteins). In several cases of mental retardation of unknown etiology, abnormal dendritic spines of cortical neurons have been observed with the use of the Golgi method. These dendritic (postsynaptic) disorders have been attributed to defective development ("dysgenesis"). The knowledge provided by ultrastructural analysis of brain tissue from the human disorders of mental retardation or dementia is "still formless, incomplete, lacking the essential threads of connection," and only future developments in lacking neurobiology will make possible the dissection of the primary phenomena from the secretory and probably irrelevant findings.


Lipodystrophy of the extremities. A dominantly inherited syndrome associated with lipatrophic diabetes. A female patient with the following symptoms has been observed: complete absence of subcutaneous fat on the arms and legs, well developed adipose tissue on the trunk and face, severe hyperlipidemia, eruptive xanthomas, insulin resistant diabetes mellitus with lack of ketoacidosis, hepatomegaly and elevated basal metabolic rate. The patient thus exhibited all characteristics of lipatrophic diabetes (Lawrence type of diabetes). The mother and a sister of the patient were found to have the same peculiar appearance and a slight hyperlipidemia but no diabetes mellitus. The combination of this type of partial lipodystrophy with severe hyperlipidemia, insulin resistant diabetes mellitus without ketoacidosis and elevated basal metabolic rate was further observed in 2 unrelated patients without known familial occurrence. Thus partial lipodystrophy of the extremities is another, previously undescribed, syndrome associated with the Lawrence type of diabetes mellitus. In the 1 family the syndrome of lipodystrophy and hyperlipidemia is dominantly inherited. Besides the autosomal recessively inherited syndrome of congenital generalized lipodystrophy there is a heterogenous group of dominantly inherited syndromes with various types of lipodystrophy.


Stability of X chromosomal inactivation in human somatic cells transformed by SV-40. Clones of fibroblasts from a G6PD A heterozygote transformed with SV-40 did not express the G6PD silent allele in the transformed heteroploid cultures. In addition, transformed fibroblasts from a woman heterozygous for both G6PD A and HGPRT deficiency, subjected to selective pressure, did not reveal a single cell expressing either silent allele. Since the incidence of sex chromatin was significantly lower in these cells after transformation, it is likely that the loss of sex chromatin reflects the loss of the inactive X-chromosome at an early stage following transformation.


Red cell uridine monophosphate kinase polymorphism in Japanese. Samples from 635 unrelated Japanese blood doners in Tokyo were examined for their red cell uridine monophosphate kinase phenotypes using starch gel electrophoresis. Three phenotypes were found: UMPK1 (570, 89.76%), UMPK 2--1 (63, 9.92%) and UMPK 2 (2, 0.32%). The corresponding gene frequency for UMPK1 was 0.9472 and for UMPK2 was 0.0528. The UMPK2 frequency in the present study was slightly higher than the previously reported value for Caucasians in the United States, but the difference is not statistically significant. A study of 15 twin pairs and their parents was in agreement with the hypothesis of autosomal codominant inheritance for UMPK.


Chromosome no. 1 of man and chimpanzee: identity of gene mapping for three loci: PPH, PGM1, and Pep-C. Cytogenetic and biochemical analysis of 10 independant Chimpanzee-Mouse cell hybrids and of 18 subclones of one of these showed that PPH, PGM1 and Pep-C are localized on the Chimpanzee chromosome homologous to the human chromosome No. 1.


Use of epithelial cell cultures for studies on the mechanism of transformation by chemical carcinogens. Evidence is reviewed for and against four major theories of chemical carcinogenesis. The development of several normal and transformed epithelial cell lines which should be useful for the analysis of this problem is described. The detection of RNA viral particles in cells transformed with chemical carcinogens is a recurrent finding in studies from our own and other laboratories, but the significance of these particles in terms of the mechanism of chemical carcinogenesis remains to be determined. Finally, we have described the first mutants of chemically transformed epithelial cells which are temperature sensitive in the maintenance of the transformed phenotype. These mutants should be particularly useful for detecting the critical biochemical changes that distinguish a chemically induced tumor cell from its normal counterpart.


Properties of cells transformed by DNA tumor viruses. Transformation by the papovaviruses, SV40 and polyoma, is reviewed briefly, including factors that affect the frequency of transformation. Virus markers useful in the determination of the etiology of virus-free tumors are described, including viral DNA, viral mRNA, virus-induced antigens, and the rescue of infectious virus. Finally, the evidence that viral genes are involved in the maintenance of transformation is presented.


Immunization against experimental Chagas' disease by using culture forms of Trypanosoma cruzi killed with a solution of sodium perchlorate. Protection against infection with virulent blood (trypomastigote) forms of Trypanosoma cruzi was accomplished in mice by immunization with culture (mainly epimastigote) forms killed by treatment with sodium perchlorate. Sodium chloride, used instead of sodium perchlorate, with all other conditions kept the same, failed to kill all the organisms, indicating that the effects of the perchlorate anion were not simply ionic or osmotic, suggesting that they might be chaotropic. A single dose of the immunogen, without adjuvants, was sufficient to significantly protect against the infection. Protection was achieved by either intraperitoneal, intramuscular, or subcutaneous immunization, though the first two routes appeared to be more effective. After challenge, parasitemias were negative in 25, 29, and 17% of the animals immunized intraperitoneally, intramuscularly, and subcutaneously, respectively.


Pathogenesis of simian foamy virus infection in natural and experimental hosts. Inoculation of simian foamy virus type 1 into New Zealand white rabits resulted in an infection which was very similar to that observed in naturally infected nonhuman primates. Both intraperitoneal and intranasal inculations were found to be efficient procedures for the establishment of the infection in rabbits. Infection by the nasal route was found to be the best method, whereas no infection could be established by feeding virus in the drinking water. Once infection was established, virus persisted in the tissues and organs for as long as 264 days after inoculation, during which time the animals maintained significant levels of neutralizing antibody. Infectious virus was recovered from spleen, liver, lung, salivary gland, kidney, and, to a lesser extent, the brain. Virus was isolated from the blood only during early infection and never from the urine. A comparison of the distribution of foamy virus in naturally infected monkeys and baboon with experimentally infected rabbits showed that both groups harbored infectious virus in the same internal tissues and organs. Recovery of infectious virus from both groups of animals was accomplished by cultivation and/or co-cultivation of infected cells onto Vero cells.


Host effect on vesicular stomatitis virus morphogenesis and "T" particle formation in reptilian, avian, and mammalian cell lines. The morphogenesis of vesicular stomatitis virus (VSV) in three reptilian cell lines (turtle heart, gecko lung, and viper spleen) was studied by thin section electron microscopy. Simultaneous growth studies were conducted in reptilian, chick embryo fibroblast, and BHK/21 cells to compare the yields of infectious VSV during serial passages. The mean length of gecko lung cell VSV measured from electron micrographs is statistically significantly shorter than that of VSV replicating in other reptilian systems studied. This observation, along with comparative growth studies, suggests the predilection of gecko lung cells to form "auto-interfering" truncated "T" rather than infectious "B" VS virions.


Effect to tween 80 and aquasol A on virus plaque formation. The enhancement of vesicular stomatitis virus plaques on human embryonic lung cells in the presence of Tween 80 or Aquasol A was studied to determine the optimal conditions for the enhancement. Enhanced numbers and sizes of vesicular stomatitis virus plaques occurred when Aquasol A or Tween 80 was added to the cell culture 30 min before virus adsorption but not when added after adsorption. These substances did not have a direct effect on the virus and did not have an effect on virus adsorption or penetration. A few other viruses and cell systems were also studied to determine if enhancement would extend to other viruses and cell systems. The cell system seemed to be important since enhancement of vesicular stomatitis virus plating efficiency did not occur on chicken embryo cells. However, the virus was also important since vaccinia virus plating efficiency was not enhanced on the human embryonic lung cell. The greatest enhancement encountered was the increase in the plating efficiency of Friend leukemia virus on S+L- cells.


Candidate cytomegalovirus strain for human vaccination. A strain of human cytomegalovirus called Towne was isolated in WI-38 human fibrolast cell cultures from the urine of an infected infant. It was then passaged 125 times in WI-38, including three clonings, and a pool was prepared in the same cell substrate for use as a potential live attenuated vaccine. The Towne virus has a broad antigenicity and cross-reacts with the AD-169 strain. Several markers of the Towne virus were found which differentiated it from fresh isolates. One of these was resistance of the former to trypsin. The Towne virus was tested for freedom from oncogenicity or other harmful effects in preparation for tests in humans.


Response to primary infection with Herpesvirus saimiri in immunosuppressed juvenile and newborn squirrel monkeys. Immunosuppression of juvenile squirrel monkeys with combined azathioprine, prednisolone, and antilymphocyte globulin resulted in decreased antibody responses to viral antigens after primary infection with Herpesvirus saimiri (HVS). The virus was repeatedly isolated from the oropharynx of immunosuppressed monkeys but not from untreated infected controls. Thus immune factors are important in inhibiting shedding of HVS from the oropharynx. HVS could be isolated from the peripheral blood lymphocytes of infected control monkeys but not from the lymphocytes of immunosuppressed monkeys. Immunosuppressed monkeys also had decreased percentages of lymphocytes capable of forming rosettes with sheep erythrocytes. These results indicate that the  immunosuppressive agents had inhibitory effects on lymphocytes (presumably thymus derived) capable of being latently infected with HVS. Antibody responses in newborn monkeys infected with HVS were delayed compared with juvenile monkeys. Treatment of newborn monkeys with antilymphocyte globulin had no suppressive effect on antibody responses to HVS.


Affinity chromatography purification of Clostridium perfringens enterotoxin. Anti-enterotoxin immunoglobulins immobilized on CH-Sepharose or CNBr-Sepharose were used for affinity chromatography purification of Clostridium perfringens enterotoxin. Cell extracts containing enterotoxin or partially purified toxin preparations were applied to the column and nonspecifically-bound protein was eluted. NaOH was used to elute specifically bound toxin. The purity of enterotoxin purified by  Sephadex G-100 chromatography followed by affinity chromatography appears similar to toxin highly purified by conventional means. The procedure can be used  successfully for the rapid (less than 2 h) purification of small amounts of enterotoxin.


Neutralizing antibody responses to varicella-zoster virus. Neutralization of varicella-zoster (V-Z) virus by human sera and immune rhesus monkey sera was enhanced by fresh guinea pig complement. There was no marked difference in the degree to which complement enhanced neutralization by sera from current V-Z virus infections and sera from long-past varicella infections. Immunoglobulin G neutralizing antibody in sera from varicella cases was enhanced by complement to a slightly higher degree than was immunoglobulin M (IgM) antibody, and immunoglobulin G neutralizing antibody in immune monkey sera was enhanced to a much greater degree than was IgM antibody. There was a rapid decline in the complement requirement of IgM neutralizing antibodies over the course of immunization of the rhesus monkeys. V-Z neutralizing antibody titers in the presence of complement were higher than complement-fixing titers of the same sera in all groups of individuals studied. IgM neutralizing antibody for V-Z virus was demonstrable in all cases of varicella but in only 1 of 22 zoster cases, and V-Z IgM neutralizing antibody was not detectable in primary herpes simplex virus infections in which heterotypic antibody titer rises occurred to V-Z virus. Complement-fixing antibody for V-Z virus was absent in 19S serum fractions which contained IgM neutralizing antibody for the virus.


Increased adhesion of Chinese hamster ovary cells to substratum by cholera enterotoxin. A new, simple assay method using Chinese hamster ovary cells was devised for cholera enterotoxin. The effect of the cholera toxin was measured by an increase in cell adhesion to the substratum. The increase was dependent on the concentration of the toxin used and was effective for a longer period of time than that of adhesion increased by dibutyryl cyclic adenosine monophosphate and theophyline. There was a delay of about 60 min before the increase in adhesion, using the toxin, appeared, whereas the increase caused by dibutyryl cyclic adenosine monophosphate appeared almost at once.


Characterization of monkey peripheral neutrophil granules during infection. Rhesus monkey (Macaca mulatta) neutrophils were shown to contain the azurophilic granule maker enzymes myeloperoxidase and beta-glucuronidase but were deficient in the specific granule markers alkaline phosphatase (AKP) and lysozyme. Isopycnic centrifugation of leukocyte homogenates on linear sucrose gradients resulted in cosedimentation of myeloperoxidase and beta-glucuronidase with an equilibrium density of 1.18. After an intravenous inoculation of monkeys with Salmonella typhimurium AKP activity became marked, whereas that of beta-glucuronidase decreased and myeloperoxidase remained unchanged. Lysozyme was undetected throughout the course of the experiment, but was present in oil-induced peritoneal macrophages and peripheral mononuclear cells. The induced AKP exhibited partial latency and had an equilibrium density of 1.15. It is unclear, however, whether the induced AKP is associated with specific granules or cytoplasmic membranes. Hence, while these data are consistent with the presence of azurophilic granules in polymorphonuclear neutrophils from infected monkeys, the presence of specific granules in polymorphonuclear neutrophils of both uninfected and infected monkeys remains moot.


Ultraviolet inactivation of Epstein-Barr virus: effect on synthesis of virus-associated antigens. The relative sensitivity to ultraviolet (UV) light of genome functions of the P3HR-1 strain of Epstein-Barr virus (EBV) was studied. The formation of viral capsid antigen (VCA) appeared to be more sensitive than that of early antigen (EA), while the synthesis of membrane antigen (MA) was most resistant, as seen on examination in the presence of cytosine arabinoside (Ara-C). However, the appearance of both VCA and EA, but not that of MA, was delayed with UV-irradiated virus, in either the presence or absence of Ara-C. The synthesis of EA and VCA induced by UV-irradiated virus was suppressed in the presence of Ara-C, while that of MA was not.


Production by EBV infection of an EBNA-positive subline from an EBNA-negative human lymphoma cell line without detectable EBV DNA. A continuous lymphoma cell line, BJAB, derived from the tumour of an exceptional African case of Burkitt's lymphoma, has previously been described. Unlike 97% of African BL cases studied, neither the original tumour cells nor the cell line contained detectable amounts of EBV (Epstein-Barr virus) DNA, nor did they express the EBV-determined nuclear antigen EBNA. The cells of the established line had the characteristics of B-type lymphocytes and they carried receptors for EBV. EBNA was induced in the majority of BJAB cells after EBV infection. Usually the cells died within 10 days of infection, but it was possible to establish a permanent EBNA-positive variant (GC-BJAB) of BJAB. The patient from whose tumour the original BJAB line was established was seropositive for EBV antigens, indicating previous exposure to and continuing presence of the virus; yet the tumour had not become infected by EBV. This evidence shows that EBV is not readily "picked up" by the lymphoma.


Vaccination against feline leukaemia virus using a cell membrane antigen system. Cats inoculated with live feline lymphoblastoid cells of the FL74 line developed high titres of antibody to feline oncornavirus-associated cell membrane antigen (FOCMA). Eight cats were subsequently challenged with a large dose of feline leukaemia virus (FeLV) of a highly pathogenic strain. All resisted infection while 10 cats given the challenge virus alone became infected. The FeLV produced by FL74 cells was shown to be of extremely low infectivity in cats and in cultured feline cells. Cats inoculated with either FL74 cells or virus purified from them did not become infected. The purified virus did not induce FOCMA antibody in cats not previously exposed to FeLV. The fact that FL74 cells are highly immunogenic, but produce virus of low infectivity, is of value in devising vaccines against FeLV. Cats were also inoculated with FL74 cells which had been inactivated with paraformaldehyde. They developed FOCMA antibody, reaching a peak titre of 256, and no virus could be cultured either from the vaccine preparations or from the tissues of the cats.


Histological and combined chemoimmunostimulation therapy studies against a murine leukemia. A Graffi murine leukemia was utilized as a model system to investigate the effect of chemoimmunostimulation therapy. Subcutaneous inoculation of approximately 1.0 times 10(6) tumor cells resulted in a rapidly growing tumor at the site of inoculation and subsequent development of splenomegaly and lymphoadenopathy. All animals succumbed to the leukemia within 24 to 30 days. Treatment of diseased animals with two courses of cytoxan over a 2-week period resulted in a remission period of approximately 16 to 18 days before relapse and eventual death of approximately 70% of the drug-treated animals. A significant number of long-term survivors (50 to 83%) was obtained in groups of animals that received combined drug plus BCG or C. parvum therapy. In contrast, the administration of MER (a methanol-extracted residue of BCG) to animals in a drug-induced remission period was no more effective than drug alone. The protective effect afforded by BCG and C. parvum was dependent on the time interval between drug therapy and the administration of the immunostimulators. Treatment of leukemic animals with BCG, C. parvum, or MER alone proved ineffective as all mice died at approximately the same time as untreated control animals. No leukemic cells were observed in any of the histologically examined tissues taken from long-term survivors. The implication of these results for cancer therapy is discussed.


Immunization against Marek's disease using Marek's disease virus-specific antigens free from infectious virus. Immunity against Marek's disease was conferred by the use of non-infectious materials extracted with non-ionic detergents from cells infected with the attenuated strain of Marek's disease virus (MDV). Antibody-free Rhode Island Red chicks were inoculated at 1 week of age with cell extracts emulsified in Freund's complete adjuvant and were given a second inoculation 1 week later without adjuvant. Protection against natural infection was obtained in groups inoculated with both soluble (not sedimented at 100,000 times g/2h) and insoluble antigens present in Nonidet P40 (NP40) extract, but only with the insoluble fraction of deoxycholate extract. The results suggest that the immunizing antigens can be partially solubilized with 0.5% NP40 and that the growth and spread of MDV are reduced in immunized chickens.


Spleen-cell cytotoxicity for cytomegalovirus-transformed cells. II. Inhibition by cytomegalovirus antiserum. Serum from hamsters immunized repeatedly with cytomegalovirus (CMV) was able to block spleen-cell cytotoxicity for CMV-infected and CMV-transformed cells. Inhibition was observed regardless of whether spleen cells were obtained from hamsters sensitized to the virus or to isografts of the transformed cells (designated Cx-90-3B, T-2). Such serum did not significantly block effector cell response against transformed cells expressing herpes simplex virus or SV40-associated membrane antigens. The mechanism of blocking was investigated. It was found that the blocking activity could be adsorbed out with Cx-90-3B, T-2 cells but not by untransformed hamster embryo fibroblasts. Cx-90-3B, T-2 target cells treated with serum, and then washed, remained resistant to effector cell attack. On the other hand, if serum-treated attacker cells were washed their cytotoxic activity was not significantly impaired. These results suggest that the blocking factor in the serum is antibody-directed against cytomegalovirus-related membrane antigen. This conclusion is supported by the finding that the serum contained antibody specifically reactive with the transformed cell surface in isotopic antiglobulin tests.


Tumour-associated transplantation antigen in sera of rats with large RSV-induced sarcomas. A factor inhibiting tumour growth in syngeneic hosts was found in the sera of inbred Lewis rats carrying Rous sarcoma virus-induced tumour (RSL). The findings presented here suggest that the serum factor is a tumour-associated transplantation antigen (TATA) shed from the neoplasm into the circulation. All the tumour bearers' sera tested with RSL cells were negative in indirect membrane immunofluorescence;however, on passive transfer into syngeneic rats, they protected the animals against the growth of an RSL tumour inoculum. A similar protective effect was also observed after injection of TATA prepared from RSL cell membranes by solubilization with potassium cholate. When incorporated into Freund's adjuvant, tumour-bearers' sera immunized the animals against a subsequent RSL sarcoma graft. Sera collected from immunosuppressed rats bearing large sarcomas which presumably contain neither tumour-specific antibody nor antigen-antibody complexes, transferred inhibition of tumour growth to syngeneic hosts. Intact immunological reactivity of recipients was a necessary prerequisite for the protective effect of sera, since the passive transfer of an inhibitory serum to immunosuppressed rats did not inhibit tumour growth. We assume that the TATA present in tumour-bearers' serum is released from the growing neoplasm as a result of either cell death or membrane metabolic turnover.


Appearance of C-type virus-like particles after co-cultivation of a human tumor-cell line with rat (XC) cells. A serially progagated cell line (L104) was established by co-cultivation of alung adenocarcinoma (L-1) from a patient with concurrent chronic lymphocytic leukemia and XC, a non-producer rat line, known to carry the Rous sarcoma virus (RSV) genome. Karyotype of the L104 cultures revealed predominantly rat-like patterns; however, about 5% of the cells reacted with HLA antibodies and demonstrated human isozyme patterns. Electron microscopy of L104 cells revealed the presence of C-type particles budding from the cell membranes and in cytoplasmic vacuoles. Virus was not detected in any of the other normal lung, lung tumor or XC cells examined after co-cultivation with XC cells. The particles isolated from tissue culture fluids had the biochemical and biophysical characteristics common to other known mammalian C-type particles and were serologically related to the woolly monkey virus (WMV)/gibbon ape leukemia virus (GaLV) complex. Cross-hybridization between viral 3H-DNA transcripts and cellular RNAs from virus-infected cells clearly show the presence of sequences in the L104 cellular RNA related to both the GaLV/WMV group of viruses and rat viruses. Hydroxyapatite chromatography reveals however that the primate-related sequences in the viral RNA are indistinguishable from WMV in thermal elution profile. The host range of L104 virus appears to vary greatly from WMV in being xenotropic and, in the cell lines thus far tested appears, to infect only rat cells. The virus gave positive KC but negative XC assays. Inoculation of whole cells or cell-free supernatants into weaning hamster did not result in either solid tumors or leukemia. Co-cultivation of appropriate cell lines may represent an approach to the detection of latent viruses in human neoplasia.


Temperature-dependent properties of cells transformed by a thermosensitive mutant (TS-121) of polyoma virus. II. Characterization of 121-6 cells. The properties of 121-6 cells were characterized. This cell line is transformed by ts-121, a temperature-sensitive mutant of polyoma virus. Both the morphology and saturation density of the 121-6 cells were affected by temperature; i.e. at 39 degrees C, the cells grew to monolayer sheets and remained contact inhibitied for a long time (at least 25 days), while at 35 degrees C, they grew beyond the monolayer density, like cells transformed by wild-type polyoma virus. These phenotypic changes were reversible, but increased agglutinability by Concanavalin A, which was another phenotype of these transformed cells, was not temperature-sensitive, and even at high temperature, 121-6 cells, like transformed cells, showed high agglutinability.


Genetic control of oncogenesis by murine sarcoma virus Moloney pseudotype. I. Genetics of resistance in AKR mice. Infection of Moloney mouse sarcoma virus (M-MSV) in adult mice of several inbred strains revealed that all strains except AKR are highly susceptible to M-MSV tumor development. F1 hybrids between AKR and CBA, DBA/2 or NIH mice are as resistant (93%) as the parental AKR strain, which indicates that resistance is transmitted as a dominant character. First backcross mice to the susceptible parent show a 3:1 ratio of resistant to susceptible mice. This is the expected ratio for two segregating loci which independently confer resistance. The incidence of resistant F2 mice is somewhat lower than expected. Further support for the two-gene hypothesis was obtained in second backcross mice. None of the major genes affecting MuLV infection (Fv-2 and H-2) seems to play any role in this system and no linkage was found with Thy. 1 and albino, dilute, agouti and brown markers.


Genetic control of oncogenesis by murine sarcoma virus Moloney pseudotype. II. A dominant epistatic susceptibility gene. We have shown in the preceding paper that AKR mice are highly resistant to M-MSV tumor development, and that resistance is transmitted as a dominant character. In the present studies the tumor-response pattern of F1 hybrids between resistant AKR and susceptible strains (C57Bl/6, BALB/c and B10BR) following injection with Moloney mouse sarcoma virus (M-MSV) resembles that of the non-AKR parent. Segregation is observed in first backcross (Bc1) and F2 mice, and the segregation ratios up to Bc3 mice fit a one-gene model. The data of triple cross hybrids suggests that this dominant susceptibility gene inhibits the phenotypic expression of M-MSV tumor resistance in some susceptible Fv1bb strains as well as in their hybrids with AKR. Neither Fv-1 nor H-2 exerts any significant influence on this complex system.


Immunotherapy and vaccination against cancer with non-living BCG and cord factor (trehalose-6,6'-dimycolate). A vaccine containing non-living BCG and living tumour cells prevented the growth of 106 tumour cells introduced distally into the skin of guinea-pigs at the time of vaccination. A similar effect was achieved when living tumour cells of the vaccine were replaced by tumour cells pretreated with mitomycin C. The efficacy of the vaccine was significantly increased when cord factor was added to the vaccination mixture. The percentage of cures was about 88% (17 cases) in experiments in which the vaccine contained living tumour cells, 100% (10 cases) when mitomycin C-treated cells were used. The significance and implications of the above findings are discussed.


Detection of virus-specific DNA sequences by quantitative in situ hybridization during infection with SV40. In situ hybridization has been used as a quantitative method to study the replication of SV40 during lytic infection. Various parameters have been defined such as fixation and denaturation of cytological preparations, concentration of complementary (3H)-cRNA and volume of buffer used for the hybridization reaction. When all these parameters are carefully controlled, a reproducibility of +/-10% can be obtained for the quantitative study of the system SV40-monkey kidney cells (CV-1). In this system, it is evident that the synthesis of viral DNA is not synchronized in confluent cells. Moreover, a proportion of 20 to 30% of cells are not labelled whatever the multiplicity of infection. In SV40-transformed cells, it was not possible to detect significantly the integrated genome due either to the small size of the genome or to the fact that the number of genome-equivalents integrated at the same place is too low to yield a radioactivitity superior to background level for long periods of exposure.


Transformation of mouse cells after infection with ultraviolet irradiation-inactivated herpes simplex virus type 2. A transformed mouse cell line (H238) was obtained following the infection of 238 mouse cells with ultraviolet (UV) irradiation-inactivated herpes simplex virus type 2 (HSV-2). The transformed cells produced tumors with a 100% incidence within 8 weeks in 6-week-old syngeneic BALB/c mice at an inoculum of 1 times 10(6). Indirect immunofluorescence (IF) tests revealed the presence of HSV antigens in the transformed cells. Antibodies to HSV-2 were found in the sera of tumor-bearing animals by neutralization and IF techniques. Neither HSV-2 infectious virus nor viral antigens could be detected by the transfer of transformed-cell DNA into permissive cells.


Cell-mediated cytotoxicity to chemically-induced rat tumours. Cell-mediated immune reactions against carcinogen-induced rat tumours were demonstrated by the in vitro microcytotoxicity test. Lymph-node cells from syngeneic rats immunized against individual aminoazo-dye-induced hepatomas or 3-methylcholanthrene-induced sarcomas were cytotoxic for cells of the immunizing tumour compared with the effects of normal lymph-node cells. Only a low level of cross-reactivity was detectable against target cells derived from otherr tumours. Conversely, lymph-node cells from tumour -bearing rats were cross-reactive against a range of tumour cells, suggesting that the tumour-bearer was sensitized against a common antigen(s) as well as the individually specific antigen associated with these tumours. Lymph-node cells were reactive at an early time (from day 2) after tumour implantation and their cytotoxic potential persisted until terminal stages of tumour growth. Spleen cells from tumour-bearer and tumour-immune donors also exhibited comparable cytotoxic reactivities of the same specificity as that shown by lymph-node cell preparations. These findings are discussed in relation to the development of cell-mediated immune responses in the tumour-bearing rat and in immunized rats which are capable of rejecting transplanted tumour cells.


Strain-dependent expression of endogenous mouse-tropic leukemia viruses in chemically induced murine leukemias. The effects of two chemical carcinogens, nitrosobutylurea and 7,12-dimethylbenz-(a) anthracene, on the expression of endogenous N- and B-tropic viruses were studied. The mice used were from two inbred strains, C57BL/6 and DDD-Fvr, and from 13 partially inbred strains derived from the cross of C57BL/6 with DDD-Ffr. These mouse strains are classified into three groups by the pattern of expression of endogenous viruses in normal, aged mice: (1) negative for both viruses; (2) positive for N-tropic virus only; and (3) positive for both viruses. Mice were given the carcinogens, and were tested at various intervals by the UV-XC procedure for the viruses in the spleens and other enlarged organs. Irrespective of the presence or absence of leukemias, the carcinogen-treated mice showed the same pattern of expression of endogenous viruses as that of non-treated normal mice. It can be concluded that the activation of the endogenous viruses is dependent on the mouse strains and that the growth of the activated viruses is not a necessary step for the chemical induction of leukemias.


Isozyme pattern of fructose diphosphate aldolase during hepatocarcinogenesis induced by 2-acetylaminofluorene in rat liver. The biosynthesis of aldolase A and B subunits has been studied in rat liver during the administration of carcinogen AAF4. Transition from a predominance of aldolase B to A was observed during carcinogenesis in rat liver. Changes in isozymic pattern and FDP to F-1-P cleavage activity ratio were observed before histological alterations typical of hepatoma could be detected. Our data support the hypothesis of dedifferentiation during hepatocarcinogenesis which in an early stage results in switching on of the gene for aldolase A with simultaneous continuation of biosynthesis of aldolase B within single cells.


Narcotic antagonists: a reasonable method of treating narcotic dependency. Historically, narcotic dependence has been treated primarily by substituting one narcotic drug for another. One alternative chemical treatment for narcotic dependency is through the use of a narcotic antagonist. The present study involves the observed side effects and outcome results of treating 40 male heroin addicts with the narcotic antagonist, cyclazocine, in the context of a comprehensive psychotherapeutic program. It is concluded that the side effects of cyclazocine are minimal, that cyclazocine is not diverted to the streets, and that it is an effective tool in the context of a comprehensive treatment program for narcotic-dependent individuals.


Liver disease in drug addicts: clinical course-toxicological and clinical pharmacological aspects. Drug addicts admitted to the hospital had acute hepatitis in 44%, chronic hepatitis in 34%, toxic liver injury in 16%. About 50% of the patients with acute hepatitis were HAA positive. Patients with intravenous drug abuse acquired acute hepatitis after a mean period of 1.8 years. Acute hepatitis in drug addicts shows a prolonged course in the majority of the cases. Biochemically increases in serum bilirubin and transaminase levels are less pronounced as compared to non-addicted persons. 30-35% of the patients develop chronic hepatitis. Speed abuse promotes the tendency to cholestasis in acute episodes of liver disease. A toxic effect of the drug preparations upon the liver cells is assumed. Chronic hepatitis in drug addicts mainly develops from earlier acute hepatitis with prolonged course. In our material, the percentage of chronic hepatitis at present is 47.5%. Besides toxic effects, also repeated infections have to be discussed with regard the etiology and pathogenesis of the disease.


Clinical pathologic observations in pseudoxanthoma elasticum. Skin biopsy material obtained from 2 patients with pseudoxanthoma elasticum associated with congenital hyperphosphatasia was compared with skin biopsy material obtained from 3 patients with pseudoxanthoma elasticum unassociated with any other disease. Clinically normal skin showed calcification of normal-appearing elastin as the initial change in pseudoxanthoma elasticum. Elastin fibers become more ravelled as the disease progresses. A previously unreported early transient calcification of collagen was demonstrated. Other abnormal collagen forms may also be present. The Von Kossa stain appears to be the stain of choice for identifying these earliest histologic changes.


Sialography with continuous measurement of pressure outside and inside the gland. In almost any method of sialography the results depend on a sign given by the patient. In order to eliminate this very uncertain factor or to limit its effect, a procedure was established to determine the pressure in the gland after the injection of contrast medium, as well as the injection pressure during injection. The difference between injection pressure and gland pressure was minimized through the use of special cannulas. A special syringe was used. Pressure was recorded by a membrane transducer connected to electronic equipment. It appears that the pressures are below or about the same as the diastolic blood pressure. Pressure values do not correlate with the volumes injected. The standard deviation of the pressure is too large to make entirely superfluous the sign given by the patient; nevertheless, the method improves the quality and reliability of sialogram readings and extends the field of application to patients unable to give reliable subjective information.


[The Rothmund-Thomson-syndrome. A case report]. This case of Rothmund-Thomson Syndrome is presented because of its association with hitherto unrecognized disorders, especially internal malignancy. The position and linkage of these associated symptomes with the genetically determined syndrome of Rothmund and Thomson is discussed.


Wildlife and environmental health: raccoons as indicators of zoonoses and pollutants in southeastern United States. Inasmuch as terrestrial fauna are an integral part of our natural environment and are directly exposed to disease and pollutants, it follows that certain wild populations could serve to detect subtle alterations within ecosystems. A collection of studies on raccoons is presented to stimulate other researchers to develop the potential of our wildlife resources as monitors of environmental health. Raccoons have been used as serologic sentinels for St Louis encephalitis and Venezuelan equine encephalomyelitis. Studies suggest that the raccoon may be used as an indicator of leptospirosis, tularemia, and some enteric bacteria and viruses. Base line surveys have defined (1) residue levels of organochlorine and organophosphate compounds and (2) body burdens of mercury, cesium-137, and strontium-90. Physiologic responses, parasite burdens, and reproductive processes are also considered as measures that may reflect pertinent information about environmental health.


EM49, a new peptide antibiotic IV. The structure of EM49. EM49 is a family of similar peptide antibiotics, each an octapeptide acylated with a beta-hydroxy fatty acid. This paper described the determination of the structure of the fatty acyl residue, the selective removal of this residue from the peptide portion of the molecule, and the sequential analysis of the peptide by the EDMAN method. The structure of EM49, 1, is derived by this degradation.


A teaching evaluation questionnaire for postmyocardial infarction patients. The above report is primarily a methodologic presentation of a questionnaire designed to assess the knowledge of patients with a recent myocardial infarction (MI) concerning the nature of their disease, emergency treatment, diet and smoking, resumption of physical activity, psychological factors important in heart disease, and problems encountered when returning home and to work. This questionnaire was designed to asses an in-hospital teaching program for post-MI patients in these areas. Test, re-test results from our first group of 24 patients indicated they significantly increased their knowledge about problems surrounding their return home and to work during their time in the hospital. The failure of these patients to demonstrate a learning effect in the other areas covered has stimulated changes in the teaching program.


Sleep patterns afted graded exercise.

Sleep patterns afted graded exercise. The effects of six graded and measured exercise activities on sleep patterns were investigated in two healthy young men. Electrophysiological recording were made continuously throughout the night to distinguish sleep states. This experiment was designed to test the hypothesis of a relation between physical activity and slow-wave sleep (SWS: stages 3 and 4 of non-REM sleep). A progressive increase in SWS over the whole-night sleep record was found with progressively increasing physical fatigue. A fall in rapid-eye-movement (REM) sleep and at higher exercise levels, of stage 2 sleep, was found. The results support the hypothesis that SWS is involved in the recovery process from fatigue.


A new gas lesion syndrome in man, induced by "isobaric gas counterdiffusion". Normal men have been found to develop pruritus and gas bubble lesions in the skin, and disruption of vestibular function, when breathing nitrogen or neon with oxygen while surrounded by helium at increased ambient pressure. This phenomenon, which occurs at stable ambient pressures, at 1 or many ATA, has been designated the "isobaric gas counterdiffusion syndrome." In a series of analyses and experiments in vivo and in vitro the cause of the syndrome has been established as due to gas accumulation and development of gas bubbles in tissues as a result of differences in selective diffusivities, for various respired and ambient gases, in the tissue substances between capillary blood and the surrounding atmosphere. The phenomenon here described in man is an initial stage of a process shown later in animals to progress to continuous, massive, lethal, intravascular gas embolization.


Respiratory capacity, cyclic adenosine 3',5'-monophosphate, and morphogenesis of Mucor racemosus. A variety of cultural conditions were examined to determine the relationship between respiratory capacity and the growth of Mucor racemosus in the yeast and mycelial form. The results show that both yeasts and hyphae can develop when the respiratory capacity is low (e.g., in N2). In addition, the yeast form of the fungus could be grown in air in the presence of cyclic adenosine 5'-monophosphate with high respiratory rates characteristic of air-grown mycelia. These results indicate that their is not an obligatory relationship between respiratory capacity and morphogenesis in M. racemosus. Low intracellular levels of cyclic adenosine 5'-monophosphate, however, were correlated with aerobic mycelial development, whereas yeast development under CO2 was characterized by higher cyclic adenosine 5'-monophosphate levels.


Operator-promoter functions in the threonine operon of Escherichia coli. The prophage curing properties of secondary-site lysogens of coliphage lambda have been studied. The site of integration in the original lysogen (L79) is within the ooerator-promoter region of the thr operon. As a result, expression of the thr enzymes is reduced, and the strain is a leaky threonine auxotroph. Heat pulse curing of strain L79 and a thr+ lysogenic revertant (L79-20) showed that heat pulse curing of both lysogens was int and xis dependent and occurred by correct excisions of the prophage. The heat pulse curing restored strain L79 to prototrophy whereas strain L79-20 synthesized the thr enzymes constitutively and at high levels. This indicates that the reversion mutation in strain L79-20 occurred outside of the prophage and within the operator-promoter region of the thr operon. In contrast, spontaneous curing of both lysogens occurred by both correct and incorrect excisions. Spontaneously cured derivatives of strain L79-20 gave rise to three classes of regulatory mutants affecting operator and promoter functions to the thr operon.


Neurospora crassa conidial germination: role of endogenous amino acid pools. The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and lambda-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.


Genetic analysis of succinate utilization in enzyme I mutants of the phosphoenolpyruvate: sugar phosphotransferase system in Escherichia coli. Studies on the reversion characteristics of Escherichia coli strains carrying various mutations in the pts region have led to the recognition of a mutation, suc-1, with a previously undescribed phenotype. Strains carrying the suc-1 mutation grow normally on most sources of carbon but are unable to utilize succinate effectively. The suc-1 mutation can be separated genetically from the tightly linked ptsI6 mutation. Reversion of suc-1 mutants for growth on succinate yields interesting classes of suppressor mutations.


Independent constitutive expression of the aerobic and anaerobic pathways of glycerol catabolism in Klebsiella aerogenes. Klebsiella aerogenes dissimilates glycerol aerobically via an inducible pathway initiated by an adenosine triphosphate-linked kinase that converts the substrate to sn-glycerol 3-phosphate. Phosphorylated glycerol is then dehydrogenated to dihydroxyacetone phosphate by an enzyme characteristic of a flavoprotein. Anaerobically, the organism dissimilates glycerol via an inducible pathway initiated by a nicotinamide adenine dinucleotide-linked dehydrogenase that converts the substrate to dihydroxyacetone. The keto product is then phosphorylated by another adenosine triphosphate-linked kinase. Two kinds of constitutive mutants have been isolated: one affecting the aerobic and the other the anaerobic pathway.


Metabolism of cyclic adenosine 3',5'-monophosphate and induction of tryptophanase in Escherichia coli. The relationship between cyclic adenosine 3',5'-monophosphate (cyclic AMP) metabolism and the induction of tryptophanase and beta-galactosidase was studied in several strains of Escherichia coli grown with succinate, acetate, glycerol, or glucose as the carbon source. No consistent relationship between the intracellular concentration of cyclic AMP in the several strains cultured and the various carbon sources was discerned. In E. coli K-12-1 the induction of tryptophanase was found to vary in the order: succinate greater than acetate greater than glycerol greater than glucose, and that of beta-galactosidase was found in the order: glycerol greater than acetate greater than succinate greater than glucose. Rate of accumulation of cyclic AMP in the culture filtrate was in the order: succinate greater than acetate greater than glycerol greater than glucose. The addition of glycerol to E. coli K-12-1 grown in acetate caused inhibition of tryptophanase and slight inhibition of accumulation of extracellular cyclic AMP. These same conditions caused beta-galactosidase induction to be stimulated. The addition of exogenous cyclic AMP to cultures grown with four different carbon sources had an effect characteristic for each of the two enzymes studied as well as each individual carbon source. The results suggest that there are control elements distinct from cyclic AMP and its receptor protein which respond to the catabolic situation of the cell.


Inhibition of growth by erythritol catabolism in Brucella abortus. The growth of Brucella abortus (US-19) in a complex tryptose-yeast extract medium containing D-glucose is inhibited by 10 mM erythritol. The enzymes of the erythritol pathway, except for D-erythrulose 1-phosphate dehydrogenase (D-glycero-2-tetrulose 1-phosphate:nicotinamide adenine dinucleotide (NAD+) 4-oxidoreductase) were detected in the soluble and membrane fractions of cell extracts. Glucose catabolism by cell extracts was inhibited by erythritol, whereas, phosphorylated intermediates of the hexose monophosphate pathway were converted to pyruvic acid with oxygen consumption. Erythritol kinase (EC 2.7.1.27; adenosine 5'-triphosphate (ATP): erythritol 1-phosphotransferase) was found to be eightfold higher in activity than the hexokinase in cell extracts. In vivo, ATP is apparently consumed with the accumulation of D-erythrulose 1-phosphate (D-glycero-2-tetrulose 1-phosphate) and no substrate level phosphorylation. ATP levels dropped 10-fold in 30 min after addition of erythritol to log phase cells in tryptose-yeast extract medium with D-glucose as the carbon source. These data suggest bacteriostasis in the presence of erythritol results from the ATP drain caused by erythritol kinase.


CO2 production from galactose in galactose-1-phosphate uridyl transferase-deficient Escherichia coli. Escherichia coli K-12 deficient in galactose-1-phosphate uridyl transferase is capable of converting significant amounts of d-[1-(14)C]galactose to (14)CO(2), whereas strains deficient in other enzymes of the Leloir pathway cannot do so.


Mutants defective in binding activity for cyclic adenosine 3',5'-monophosphate in Vibrio parahaemolyticus. Mutants of Vibrio parahaemolyticus defective in binding activity for adenosine 3',5'-cyclic monophosphate are described. They were selected with medium containing the nucleotide together with starch.


Role for deoxyribonucleic acid ligase in deoxyribonucleic acid polymerase i-dependent repair synthesis in toluene-treated escherichia coli. In a toluene-treated mutant of Escherichia coli K-12 having a temperature-sensitive, conditionally lethal mutation in the structural gene for deoxyribonucleic acid (DNA) ligase, an extensive DNA repair synthesis occurred in X-irradiated cells at the nonpermissive temperature, 42 C. At the permissive temperature, 30 C, nearly normal semiconservative synthesis and limited repair synthesis were observed when DNA ligase was activated by the addition of nicotinamide adenine dinucleotide.


Induction of alpha-glucosidase and synthesis during the cell cycle of Myxobacter AL-1. Alpha-Glucosidase of Myxobacter AL-1 was induced by the addition of maltose to a defined medium as the sole carbon source. This induction takes place during lag-phase conditions without concomitant growth; it seems not to be regulated by the mechanism of catabolite repression. Using the method of density labeling with deuterated amino acids and subsequent analysis by equilibrium density gradient centrifugation in metrizamide-2H2O gradients it could be shown that the activity increase of the enzyme during induction is the the result of a de novo synthesis of the enzyme protein. After a short pulse of induction with maltose, alpha-glucosidase exhibited a pattern of variation in enzyme activity during the cell cycle that was similar to the one observed after growth of the cells on maltose for several generations.


Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae. Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.


A comparative study on the removal of cellular lipids from Landschütz ascites cells by human plasma apolipoproteins. The effects of human plasma lipoprotein-proteins on the removal of cellular lipids from Landschütz ascites cells were studied. Cellular lipids were labeled by injecting mice previously injected with ascites with either [3H]cholesterol or [3H]choline. Apoproteins from very low density (apoC-I, C-II, and C-111) and high density (apoA-I and A-II) lipoproteins were used. Each of the apoproteins alone was ineffective in removing cellular [3H]cholesterol. However, when synthetic phosphatidylcholines of known composition were added to each apoprotein and the experiments were repeated using either apoprotein-lipid mixtures or ultracentrifugally isolated complexes, the removal of sterol was considerably enhanced. Complexes of saturated phosphatidylcholines with apoA-II, apoC-I, or apoC-III were the most effective in releasing cellular sterol. Apoprotein-phospholipid complexes were much less effective in removing cellular [3H]phosphatidylcholine than the free apoproteins; apoA-I and apoC-I were the best of the five apoproteins studied. When a comparison was made of the adsorption of iodinated apoproteins to ascites cells, 3 to 4 times more apoA-II and apoC-III were bound than apoA-I. The binding of apoproteins was time and temperature dependent. Approximately 50% of the radioactivity that remained in the washed cells was removed with trypsin. To determine if the counts remaining in the trypsin-treated cells were internalized, identical experiments were performed using human erythrocytes, cells that do not exhibit pinocytosis. Again, approximately 50% of the radioactivity of the iodinated apoproteins was not released by trypsin. Succinylation of apoA-II not only destroys its phospholipid-binding properties but also its adsorption to red cells. These results suggest that the plasma apoproteins differ in their ability to remove cellular lipids and bind to both ascites and red cell membranes, and possibly to specific phospholipids, in such a way that only a part of the apoprotein is degraded with proteases.


Lactate dehydrogenase-catalyzed stereospecific hydrogen atom transfer from reduced nicotinamide adenine dinucleotide to dicarboxylate radicals. The dicarboxylate radical -OOC--CH--CH(OH)COO- was generated in an N2O-saturated fumarate solution by high energy ionizing radiation. When NADH was present in the solution, product analysis indicated a stoichiometry of 2 molecules of the radical reacted with 1 NADH molecule to form 2 malate and 1 enzymatically active NAD+ molecules. In a similar experiment using tritium label on position A of NADH, due to an isotope effect, only 10% of the label was transferred to malate; most of the remaining tritium was found in the NAD+ formed. When lactate dehydrogenase was added, however, no la bel was detectable in NAD+, and over 80% of the tritium lost from NADH was found in malate. The stereospecific transfer of the hydrogen atom from lactate dehydrogenase-bound NADH to the dicarboxylate radical suggested that the free radical reaction must have taken place at the active site. The hydrogen atom transfer was inhibited by oxamate. Results from flow experiments in which an irradiated fumarate solution was mixed with a solutionof lactate dehydrogenase and NADH are in support of a mechanism in which the hydrogen atom transfer occurs in the first oxidation step.