LH-hCG receptors and testosterone content during differentiation of the testis in the rabbit embryo.

The development of gonadotropin receptors for LH and hCG in the fetal rabbit testis from 17-29 days of gestation was followed by quantitative binding studies with [125I]iodohCG and compared with gonadal testosterone content and the histological differentiation of the fetal Leydig cells. The concentrations of gonadotropin receptors and testosterone in the fetal testis were low on days 17 and 18 and increased strikingly on day 19. This time sequence for development of LH-hCG receptors and steroid content of the testis was correlated exactly with the histological appearance of the endoplasmic reticulum characteristic of the differentiated Leydig cell. When fetal testes were examined at 12-h intervals between days 17 and 19, gonadotropin binding and testosterone content were closely correlated at all times studied. Thus, no dissociation between the two functions was demonstrable in the testis at any time during gestation. In the fetal ovary, LH-hCG binding and testosterone content were low or undetectable at all stages of gestation. These observations demonstrate a close temporal relationship between the appearance of the LH-hCG receptor and the synthesis of testosterone by the fetal testis and demonstrate that the histological and functional differentiation of the Leydig cell occurs within a few hours at approximately day 18 of gestation. The simultaneous appearance of LH-hCG receptors and testosterone synthesis in the gonad can be regarded as the biochemical manifestations of Leydig cell differentiation in the testis of the fetal rabbit.


Estradiol receptor content in rat granulosa cells during follicular development: modification by estradiol and gonadotropins.

To examine the relationships between gonadotropic hormones, ovarian follicular development, and estradiol receptor content in rat granulosa cells, follicular growth was stimulated in intact immature rats and hypophysectomized immature rats. In intact rats, both PMSG and oFSH given for 2 days stimulated follicular growth and endogenous estrogen production but caused estradiol receptor content in granulosa cells to decrease markedly. In hypophysectomized rats estradiol (2 mg/day X 4) was capable of increasing the content of its own receptor, stimulated preantral follicular development, and potentiated the responses of granulosa cells to highly purified hFSH. Thus, subsequent treatment with hFSH stimulated antral formation and maintained high levels of estradiol receptor content in granulosa cells. LH, on the other hand, caused a rapid decline in the content of estradiol receptor in nuclei of granulosa cells. In hypophysectomized rats primed with estradiol alone, a high dose of oLH caused follicles to undergo atresia. In hypophysectomized rats primed with hFSH in addition to estradiol, oLH stimulated luteinization. Thus, induction of atresia or luteinization by oLH in these rats appears to be associated (cause or effect?) with a loss of estradiol receptor in granulosa cells. Whether follicles of estrogen-treated, hypophysectomized rats luteinize or undergo atresia in response to LH appears to be determined by their stage of differentiation and the nature of the prior stimulation by FSH.


The effect on gastrin secretion of agents which increase the intracellular concentration of 3',5'-adenosine monophosphate.

An in vitro technique that allows study of gastrin secretion from isolated pieces of rat gastric antrum was used to study the effect on gastrin release of agents known to increase intracellular cAMP levels. Isuprel (10(-5)M), PGE1 (10(-5)M), theophylline (10(-4)M), and dibutyryl cAMP (5 X 10(-4)M) did not affect gastrin release when used alone, but each enhanced arginine-stimulated gastrin release. A biphasic pattern of gastrin release in response to arginine was seen in all experiments. The studies emphasize the close functional similarity between the antral G cells and the B cells of the pancreatic islet.


A sensitive and simple in vitro assay for corticotropin-releasing substances utilizing ACTH release from cultured anterior pituitary cells.

Graded doses of rat hypothalamic extract (HE) were added to dishes containing dispersed, pooled rat adenohypophyseal cells cultured for several days. ACTH secretion into the medium gave a linear log-dose response curve over a 100-fold range between 0.01 and 1 mg of NIH-HE (0.0125-1.25 rat hypothalamus). Forty percent of the maximal ACTH secretion in response to a given dose of HE occurred within 3 min. No decrease in intracellular ACTH occurred at any time or with any dose of HE, indicating that secretion was always balanced by production. ACTH secretion stopped as soon as HE-containing medium was replaced by medium without HE. The same cultured cells could be satisfactorily used in repetitive assays performed on the same or different days.


Inhibition of the response of mouse thyroid to tryrotropin induced by chronic triiodothyronine treatment.

Administration of 1 mU bovine TSH iv to mice resulted, within 1 hour, in the increase of the serum T4 level from 32 +/- 1.4 ng/ml to 53 +/- 2.6 ng/ml (Mean +/- SE, n = 24). Treatment with 1 mug triiodothyronine (T3) per day, for 10 days, abolished the responsiveness of the thyroid to TSH, as measured by thyroxine (T4) release. Thyroidal response to TSH was measured also in vitro. The basal hormonal release was 4.66 +/- 0.55 ng T4 and 0.98 +/- 0.15 ng T3 per thyroid per 3 h (n = 30). In the presence of bovine TSH (0.2 mU/ml) the hormonal secretion increased 3-fold for T4 and 2.5-fold for T3. Thyroids from mice pretreated with T3 for 10 days showed almost no response to TSH. Partial refractoriness to TSH was already significant 5 days after T3 pretreatment. Responsiveness to TSH was restored 3 days after T3 withdrawal or after 3 daily injections of 10 mU bovine TSH, concomitant with the last 3 days of T3 pretreatment. These results indicated that the prolonged absence of an adequate level of trophic hormone may be the cause of thyroidal unresponsiveness to acute TSH treatment. With 20 mU of TSH, cAMP levels rose from 4 +/- 0.5 picomoles to 80 +/- 9.3 picomoles per thyroid (n = 6). In mice subjected to 10 days of T3 pretreatment the response was markedly reduced: 20 +/- 3 picomoles/ thyroid. Thyroids of the T3-treated mice responded normally to 1 mM DBcAMP in vitro. From these results it was concluded that the impaired responsiveness of the thyroids to TSH occurs at a step prior to cAMP accumulation.


Effect of estrogen-progestogen administration on tissue cation concentrations in the rat.

Five studies are reported which were designed to define the effects of an estrogen and a progestogen, alone and in combination, on tissue concentrations (heart, kidney, and liver) of two trace metals (zinc, copper) and four bulk cations (sodium, potassium, calcium, magnesium). Male rats received Premarin (0.08 mg) and/or progesterone (2 mg) in saline intramuscularly 5 days a week for 12 weeks. A significant increase in kidney copper concentrations was the most striking finding in treated, compared to control, animals. A small but significant increase in kidney zinc concentrations also was observed. The changes in cation concentrations in the liver were less striking and consistent, and no changes were observed in cation concentrations of heart muscle. The increase in renal copper was greater with estrogen than with progestogen alone but the two in combination produced an additive effect. Dose-response and time-response changes were documented. Two weeks after discontinuing treatment, renal copper and zinc concentrations returned to control levels.


Calcium transport in isolated bone cells. III. Effects of parathyroid hormone and cyclic 3',5'-AMP.

The effects of parathyroid hormone (PTH) and cyclic 3',5'-AMP (cyclic AMP) on calcium transport were studied in isolated bone cells. Bone cells were isolated by collagenase digestion of 20-21 day old fetal rat calvaria. Calcium transport was measured with 45Ca. PTH (0.2 mug/ml) increased calcium uptake 30--40% over control values at 37 C. At 4 C, the effects were magnified and 70--170% increases in calcium uptake were observed. The effects were present 1--10 minutes after the simultaneous addition of hormone and 45Ca. PTH had no effect on calcium efflux. Neither dibutyryl cyclic AMP (10(-4)-10(-3)M) nor cyclic AMP (10(-7)-3 X 10(-6)M) had any effect on calcium uptake or efflux. Methylisobutylxanthine (0.1 mM) caused no change in calcium uptake although increase in cyclic AMP were noted. The characteristic PTH-induced increase in cyclic AMP seen at 37 C was not observed at 4 C. It is postulated that PTH increases the permeability of bone cell membranes to calcium. At 4 C the membrane is relatively impermeable so the PTH effect is magnified. The PTH-induced increase does not appear to be mediated through cyclic AMP.


Effects of parathyroid hormone on the accumulation of cyclic AMP in bone of vitamin D-deficient rats.

The mechanism of skeletal refractoriness to parathyroid hormone (PTH) in vitamine D-deficient animals was studied in terms of the adenylate cyclase-cyclic AMP system in rat calvaria. In vitamin D-deficient, thyroparathyroidectomized rats, plasma calcium concentration was not elevated by iv administration of PTH, while responsiveness to the hormone was recovered within 24 h after a single dose (2.5 mug) of vitamin D3. In spite of the remarkable dependency of PTH on vitamin D for mobilization of calcium from bone, PTH stimulated adenylate cyclase activity in particulate bone cell fractions in vitro. PTH also enhanced the levels of cyclic AMP in the skeletal tissues of vitamin D-deficient rats in vivo and in vitro to an extent similar to those found in rats given 2.5 mug of D3. Administration of theophylline or dibutyryl cyclic AMP to the vitamin D-deficient rats did not cause any significant hypercalcemic effects, while these drugs enhanced plasma calcium concentration significantly in the rats given vitamin D3. These data strongly indicate that the cause of the skeletal refractoriness to PTH in vitamin D-deficient animals is  not a defective activation of adenylate cyclase, but must be related to a later step or steps in the biochemical events leading to bone cell activation.


Characterization of mouse ACTH in plasma and in extracts of pituitary and of adrenotropic pituitary tumor.

The predominant component of immunoreactive ACTH in the plasma of adrenalectomized normal mice and of mice bearing the adrenotropic mouse pituitary tumor, AtT-20, and in extracts of the normal mouse pituitary and pituitary tumor, has an elution volume on Sephadex G-50 gel filtration approximately midway between the void volume and the elution volume of human ACTH (1-39 peptide). The tumor extracts are shown to contain, in addition to this intermediate ACTH, 2 other components of immunoreactive ACTH, one which coelutes with 131I-labeled albumin (big ACTH) and the other with [125I]hACTH (little ACTH). Big and intermediate ACTH are urea-stable. Controlled tryptic digestion of mouse-tumor big ACTH results within 10 seconds in conversion to an intermediate component followed by continued loss of immunoreactivity. Under the same conditions of tryptic digestion of intermediate ACTH, there is only continuous loss of immuno-reactivity with no change of hormonal form. These findings strengthen the hypothesis that mouse intermediate ACTH is not a precursor for little ACTH.


Epileptogenic effects of several penicillins and penicillin-related compounds in rat neocortex.

Several penicillins and penicillin-related compounds were tested for their ability to produce epileptiform activity in rat neocortex. (1) Alterations in the side chain of penicillin G decreased epileptogenic capability in all the compounds tested in this study: Phenoxymethylpenicillin produced a primary focus but no mirror focus, suggesting a dissociation of the mechanisms underlying these two processes. Ampicillin, 6-aminopenicillanic acid, and potassium 6-aminopenicillanic acid, and potassium 6-aminopenicillanic acid produced little or no epileptiform activity. All these compounds have free amino grounds and are amphoteric. (2) Breaking the beta lactam ring of penicillin G (potassium penicillin G penicilloate) eliminated epileptogenic capability. (3) Potassium salts of penicillin or its derivatives (potassium penicillin G, propicillin, potassium 6-aminopenicillanic acid, potassium penicillin G penicilloate) consistently suppressed cortical activity, regardless of the ability of the compound to produce spike discharges. Thus, mechanisms underlying these two properties can be dissociated. (4) Antibiotic activity of penicillins bears no relationship to epileptogenic capability.


Adenosine-3':5'-monophosphate-dependent and plasma-membrane-associated protein kinase from bovine corpus luteum. Solubilization and properties of solubilized enzyme.

The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3':5'-monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of cyclic [3H]AMP to plasma membranes was increased about 150-200%. Triton X-100 was a better solubilizing agent that Lubrol WX or deoxycholate. In addition to solubilization, 0.1% Triton X-100 also stimulated the protein kinase activity 150-200%. The properties of Triton X-100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co-sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE-cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP STIMULATION THAN Ia and IIa peaks. From these studies it is concluded that the plasma-membrane associated and cytosol protein kinases have similar catalytic properties but differ in some of their physical properties.


Structure and synthesis of a lipid-containing bacteriophage. Chemical modifications of bacteriophage PM2 and the resulting alterations in acyl-chain motion in the PM2 membrane.

The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidlethanolamine residues, were selectively cross-linked in the presence of 0.1-0.5% glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05% toluene 2,4-diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 10(6)-fold in all cases. The spike and coat proteins were selectively cross-linked in the presence of 7.5 mM N,N'-p-phenylenedimaleimide. The biological activity of virus modified by this reagen was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X-100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N'-p-phenylenedimaleimide, however.


Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.


The influence of insulin on glucose permeability and metabolism of human granulocytes.

Viable human polymorphonuclear leukocytes isolated from peripheral blood were incubated for 1 h at 37 degrees C with variable concentrations of insulin in a saline medium buffered at pH 7.4. The hormone increased glucose consumption by about 40% without influencing the permeability of the membranes to glucose, whose uptake followed a passive diffusion process. The measurement of intermediates localized activation of glycolysis by insulin, down to 0.36 nM, at the phosphofructokinase step. However, the spectrophotometric measurement showed no activation of phosphofructokinase after preincubation with insulin of either intact granulocytes or crude or ultracentrifuged homogenates. The level of cyclic AMP, which is known to activate phosphofructokinase, was not modified by insulin; cyclic GMP did not activate the enzyme in the granulocyte extracts: neither of the two nucleotides can therefore be considered as a direct messenger of the action of insulin on phosphofructokinase. An important fraction of the extra glucose consumed under the influence of insulin was recovered as neither glycogen nor lactate, nor was it oxidized in the Krebs cycle. It might be assumed to have been converted into glycerolipids. However, insulin produced no detectable accumulation of triglycerides and activated neither the pentose phosphate pathway nor oxidative decarboxylation of pyruvate. The fate of the extra glucose consumed under the influence of insulin therefore remains questionable.


Sites of action of fusidic acid in eukaryotes. Inhibition by fusidic acid of a ribosome-independent GTPase from Artemia salina embryos.

1. A ribosome-independent GTPase activity has been isolated from the high-speed supernatant fraction of Artemia salina embryos, and some of its properties have been studied. This activity is inhibited by fusidic acid, an antibiotic generally thought to inhibit only EF-2 in eukaryotes. However, several lines of evidence indicate that the GTPase activity, described here, is distinct from EF-2. The results suggest, therefore, that the inhibitory effect of fusidic acid in eukaryotic systems is not restricted to EF-2 (and ribosome)-dependent functions only. 2. The results of other experiments have revealed that, despite its ability to inhibit the GTPase activity mentioned above, fusidic acid is not a non-specific inhibitor of all ribosome-independent GTPase and ATPase activities present in eukaryotic cells.


The allosteric properties of beef-liver fructose bisphosphatase.

1. The activity of beef liver fructose bisphosphatase has been shown to respond cooperatively to increasing concentrations of the activating cations Mg2+ and Mn2+. The allosteric inhibitor AMP caused an increase in this cooperativity and a decrease in the apparent affinity of the enzyme for the activating cation. 2. The cooperative response of the enzyme to AMP is similarly increased by increasing cation concentrations with a concomitant decrease in the apparent affinity. 3. Direct binding experiments indicated that in the absence of either Mg2+ or Mn2+ the enzyme bound AMP non-cooperatively up to a maximum of two molecules per molecule of enzyme, a result that is indicative of half-sites reactivity. The binding became increasingly cooperative as the concentration of the activating cation was increased. 4. The substrate fructose bisphosphate had no effect on any of these cooperative responses. 5. These results may be most simply interpreted in terms of concerted model in which the activating cation functions both as an allosteric activator and as an essential cofactor for the reaction.


Contrasting effects on plasma lipoproteins of intravenous versus oral administration of a triglyceride-phospholipid emulsion.

The effect of fat on plasma lipoproteins was compared when administered by the oral and intravenous routes to healthy control subjects on a low fat diet for 5-6 weeks. During this time each subject underwent two 5 day periods of fat supplementation with a soya bean triglyceride-egg yolk phospholipid emulsion (Intralipid), once via intragastric tube, once intravenously. Changes in plasma lipoproteins were assessed by measurement of their lipid and protein content. Intragastric fat administration significantly decreased the level of very low density lipoprotein (VLDL), whereas intravenous fat administration caused a significant rise in low density lipoprotein (LDL). Morphological evidence of persistence of exogenous phospholipid in plasma following intravenous fat administration was confirmed by alterations in the fatty acid composition of lecithin in the d 1.006-1.063 fraction of plasma. In vitro studies showed that exchange readily occurred between Intralipid and LDL lecithin, causing the latter to assume a more saturated pattern. It is concluded that the occurrence of similar changes in vivo could explain the rise in low density lipoprotein following intravenous fat, possibly by influencing the catabolic rate of the apoprotein. Hydrolysis of ingested phospholipids during absorption presumably explains why none of these changes occurred after oral fat supplementation, and would seem to be an important regulatory function of the small intestine in relation to plasma lipoprotein metabolism.


The effect of blocking dopamine release on synthesis rate of dopamine in the striatum of the rat.

Two experiments were carried out in rats to investigate the mechanisms by which dopamine (DA) synthesis is regulated. First, a unilateral lesion was made in the substantia nigra, thus interrupting the nervous impulse flow of the nigro-striatal pathway. Secondly, the release of DA in the striatum was blocked by means of 1-hydroxy-3-amino-pyrrolidone-2 (HA-966). In both experiments the synthesis rate of DA was accelerated as was shown by analysing the time course of the specific activity of striatal DA after an i.v. injection of 3,5-3H-tyrosine. Furthermore the influence of apomorphine on the rate of DA synthesis, accelerated by HA-966 or by lesion, was investigated. Apomorphine appeared to block the increase of DA synthesis. The results are discussed in the light of a transsynaptic feedback mechanism.


Tricaine (MS-222): effects on ionic conductances of squid axon membranes.

Experiments utilizing internally perfused squid giant axons have been carried out to determine the mode of action of tricaine (MS-222) in producing its anesthetic effects. Tricaine, in concentrations of 1 and 3 mM, was applied to the internal surface of the membrane via the perfusion system. Peak sodium current and steady-state potassium current were measured by the axial-wire voltage clamp technique. Tricaine suppressed both of these membrane ionic currents with the major effect being on the peak sodium current. Its action on nerve membrane appeared quite similar to other local anesthetics. One major difference was noted in that tricaine shifts sodium conductance curves in the direction of depolarization along the potential axis, an effect quite similar to that observed with increasing external Ca2+ concentration and with application of pentobarbital.


Types of neuronal responses in the rat thalamus to peripheral temperature changes.

Recordings were made of spike-trains from 163 neurons of the rostral part of the ventrobasal thalamus complex of the rat while the temperature of the scrotal skin was altered. The following results were obtained: 55 neurons were non-thermosensitive, 7 neurons cold-sensitive and 101 neurons warm-sensitive. In the case of the warm-sensitive cells a definite discrimination was possible: 61.4% of the neurons altered their firing behavior during peripheral cooling, changing from relatively even spike frequency to burst firing. This change could be induced repeatedly by altering the temperature. 38.6% of the neurons, on the other hand, reacted to cooling by slowing down their frequency. By way of statistical methods the firing patterns of the two response-types were analyzed more precisely and the differences in response to temperature stimuli more exactly defined. Likewise, the spatial distribution of the two response-types of warm-sensitive cells exhibited differences; whereas the cells devoid of burst activity occured rather uniformly in the region of the thalamus studied, the cells with bursting activity were confined more to the mediocaudal region. These findings are discussed with regard to the phenomenon of peripheral bursts and to the projection of thermoafferent pathways onto the ventrobasal thalamus complex. The functional interpretation of the various cell reactions and their role in the central processing of thermoafferent signals still remains unexplained and requires comparative studies of peripheral and central parts of the thermoafferent system.


A direct input from amygdaloid complex to caudate nucleus of the rat.

Spontaneous unit activity was recorded from the caudate nucleus in rats anesthetized with urethane. Amygdaloid nucleus stimulation altered the spontaneous activity of 51 units (65% of the population tested); 23 units (29%) showed an increase and 28 (36%) a decrease of discharge rate. Moreover, 25 units (32%) responded to stimulation with stimulus-locked spikes. Post-stimulus time histograms revealed 7 patterns of response to amygdaloid stimulation. In 4 groups of cells, stimulus-locked spikes occurred followed by alteration of spontaneous discharge rate. In another 2 groups there was a change of spontaneous discharge without stimulus-locked spikes and in the final group no effect occurred. The possibility of mono-, oligo-, and polysynaptic connections from amygdaloid nuclear complex to caudate nucleus was discussed. It was suggested that an amygdalofugal connection to basal ganglia may provide a pathway whereby activity of the limbic system may exert a modulatory influence on somatic motor mechanisms.


Adrenal function during chronic danazol administration.

Ten healthy female volunteers received Danazol, 400 mg twice daily for 90 days. Adrenal function testing then showed a normal capacity of the gland to respond to stimulation with synthetic adrenocorticotrophic hormone, and a lack of suppression of central areas controlling adrenal function by a normal response to the metyrapone challenge.


Contraception and cervical colonization with mycoplasmas and infection with cytomegalovirus.

The relationship of cervical colonization of genital mycoplasmas and infection with cytomegalovirus (CMV) was studied in 66 intrauterine device (IUD) users as contrasted to 60 patients using oral contraception and 50 patients using neither an IUD nor oral contraception. No significant increase in colonization or genital mycoplasmas or infection with CMV was noted in IUD users. No CMV was isolated from users of the cooper-t IUD. It is unlikely that genital mycoplasmas are related to either the increased immunoglobulin levels seen in patients using the IUD or the antifertility effects of the IUD.


Treatment of RSV-transformed "poorly" virogenic and non-virogenic mammalian cell lines with inhibitors of protein synthesis: failure to potentiate RSV rescue.

Treatment of the RSV-transformed "poorly" virogenic rat clones LW13-RsK4 and LW13-RsK4 R1 with cycloheximide or puromycin before fusion with chick embryo fibroblasts did not lead to RSV rescue. Neither was RSV rescued from the RSV-transformed non-virogenic mouse line RVP3 treated with 5-bromodeoxyuridine and cycloheximide or puromycin before fusion.


[The relations between lactation and cyclic-AMP -- The influences of adenyl cyclase activity on casein biosynthesis ability in the organ culture of the mouse mammary gland (author's transl)].

The relationships between hormonal action and cyclic AMP as the second messenger of hormones have recently been discussed on many hormones. Lactation is influenced by various hormone, especially, insulin, prolactin, and hydrocortisone. Whether adenyl cyclase activity in the mammary gland of mouse epithelial cells has parallel relations with casein biosynthesis ability or not was examined using the mammary gland organ culture method. Female, mid-pregnant (11-14 days), mice of DDY strain were used. Organ culture was done by the Chen's floating lens' paper method, using the hormone-added MEM media and non-added ones. Casein biosynthesis ability was measured by observing 32P incorporation into the casein molecules. Adenyl cyclase activity was estimated by the amount of 14-C-cyclic AMP produced out of adenine-8-14C by the Kuo and Krishna's method. Radio isotope compounds were pulsed for 4 hours in the medium. The experiments revealed that the added hormones had a remarkable effect on caein biosynthesis ability, but none on adenyl cyclase activity. No parallel fluctuation was observed between adenyl cyclase activity and casein biosynthesis ability, that is, the change of adenyl cyclase activity was found to have nothing to do with casein biosynthesis ability. Consequently, the cyclic AMP addition to the media showed no effect on casein biosynthesis ability.


The influence of hemolymph-binding protein on juvenile hormone stability and distribution in Manduca sexta fat body and imaginal discs in vitro.

The wing discs and fat body of Manduca sexta larvae contain enzymes (i.e. carboxylesterase and epoxide hydratase) that can convert the C18 juvenile hormone (JH) to the acid, diol and acid diol. No evidence of oxidative degradation was noted. In vitro studies suggest that JH can be compartmentalized within the cells of the fat body where it is less accessible to degradative mechanisms. Experiments utilizing a hemolymph-binding protein fraction (BPF) in vitro with fat body and imaginal discs indicate that the BPF retards the uptake of JH by tissues and its subsequent degradation by tissue enzymes. BPF also appears to protect JH from degradation by enzymes released into the medium. By these mechanisms the insect can maintain elevated JH titers for relatively long periods. Binding protein may also keep JH in solution in the hemolymph allowing its rapid distribution throughout the insect. The data suggest that the binding protein plays a key role in maintaining juvenile hormone titers.


Changes in 5alpha- dihydrotestosterone binding to epididymal cytosol during sexual maturation in rabbits: correlation with morphological changes in the testis and epididymis.

We previously demonstrated that the caput epididymis of intact sexually mature rabbits contains a specific high-affinity binding protein for 5alpha-dihydrotestosterone (5alphaDHT). The other anatomical segments (corpus and cauda) of the epididymes of these animals had no detectable 5alphaDHT-binding activity. We have further shown that this binding was due to an androgen-binding protein of testicular origin. In the present study we have investigated 5alphaDHT binding to epididymal cytosol from sexually immature rabbits (20-104 days old). Using sucrose gradient ultracentrifugation, we have detected a unique pattern of binding. The pattern correlated well with testicular and epididymal maturation, but there was little correlation with chronological age or body weight. In the most immature animals (Group I) the seminiferous tubules appeared as solid cords and the epithelium of the ductus epididymis detectable 5alphaDHT-binding activity. In the second group (Group II), there was 5alphaDHT-binding to all three segments. The seminiferous tubules of these rabbits exhibited spermatogenic activity and lumen formation. The height of the epididymal epithelium had increased uniformly throughout the duct. The third group (Group III) had 5alphaDHT-binding only in caput cytosol. Spermatogenesis had progressed to the formation of elongated spermatids in the most immature animals of this group to the release of spermatozoa in the most mature ones. The caput epithelium of this last group of rabbits was fully differentiated. Unilateral orchidectomy of Group II rabbits resulted in a decrease in [3H]5alphaDHT-binding activity on the operated side as compared to the contralateral non-operated control side, suggesting the testicular origin of the binding protein. The failure of cyproterone or cyproterone acetate to inhibit [3H]5alphaDHT-binding to the protein, the lack of effect of N-ethylmaleimide on binding, and the rapid dissociation rate of the [3H]5alphaDHT-binding protein complex suggested that the binding moiety was testicular androgen-binding protein (ABP).


Mechanism of action of progesterone on amphibian oocytes. A possible biological role for progesterone metabolism.

The ability of various steroids to induce maturation of Pleurodeles waltlii oocytes after incubation has been studied. Progesterone is metabolized during the course of maturation. All metabolites isolated are less efficient than progesterone for inducing germinal vesicle breakdown. Progesterone binding to the 'melanosome' fraction has been studied (KD = 4.5 X 10(-8) M at 4 degrees C). 20BETA-Hydroxy-pregn-4-en-3-one, the main progesterone metabolite isolated from the oocyte, also binds to the melanosome fraction, but with a lower affinity (KD = 1.6 X 10(-7) M at 4 degrees C). At high concentration 20beta-hydroxy-pregn-4-en-3-one induces maturation, but at low concentration it is a competitive inhibitor of progesterone. Progesterone metabolism in Pleurodeles oocytes can be interpreted as an inactivation process, and also as a mechanism for inhibitime progesterone action.


In vivo effect of anti-estrogens on the localisation and replenishment of estrogen receptor.

Interactions between estrogen recpetors and triaryl ethylene anti-estrogens (U-11100A, MER 25 and CI 628) were tested on immature rat uteri. The accessible and the total (accessible and occupied) estrogen receptor sites were assayed in the cytosol and nuclear extracts using charcoal adsorption. After in vivo administration of anti-estrogens, the estradiol receptor sites were occupied and subsequently transferred to the nuclear compartment. The nuclear localisation of the receptor induced by the antagonist lasted for several days, during which replenishment of the cytosol receptor occurred. The nuclear receptors transferred by anti-estrogens and labelled in vitro with [3H]estradiol, were similar to the nuclear receptor-estradiol complex formed in vivo as far as their sedimentation constants and extractability from nuclei were concerned. Although these anti-estrogens are capable to translocate the estrogen receptor to the nucleus and to induce the replenishment of the cytosol receptor, the mechanism of their antagonism and of their weak estrogenic activity is still not clear.


Comparison of alpha- and beta-cell secretory responses in islets isolated with collagenase and in the isolated perfused pancreas of rats.

The inhibitory actions of somatostatin (100 ng./ml.) on insulin release, stimulated by high glucose (20 mM), and on glucagon release, stimulated by arginine (15 mM), were studied with two in vitro systems: the isolated perifused rat islets prepared by the collagenase procedure and the isolated perfused rat pancreas. Suppression of arginine-induced glucagon release by glucose (20 mM) and glyceraldehyde (5 mM) was also assessed in both systems. With the perfused pancreas, somatostatin caused 32 per cent inhibition of glucose-mediated insulin release and inhibited arginine-induced glucagon release by 72 per cent. In the same system, glucose and glyceraldehyde were similarly potent inhibitors of arginine-induced glucagon secretion. In contrast to the isolated perfused pancreas, there was no significant somatostation suppression of glucose-induced insulin release or arginine-induced glucagon release whether the inhibitor was present prior to or was added during stimulation by glucose or arginine. Furthermore, glucose was only minimally active and glyceraldehyde ineffective in inhibiting glucagon secretion due to arginine in the perifusion system. The most plausible explanation for the difference in the endocrine response of islet cells in the two types of widely used in vitro systems is that the alpha and beta cells have lost inhibitory receptors in the plasma membrane as a result of the collagenase isolation technic.


Changes of enzyme activities in spleen lymphocytes from tumor-bearing rats.

The induction of concomitant immunity was studied in Donryu strain rats with Yoshida ascites sarcoma cells. The changes of enzyme activity in spleen lymphocytes were also examined in normal and tumor-bearing rats. The concomitant immunity was detected 1 week after transplantation of tumor cells. Extended metastases were found 2 weeks after transplantation. The enzyme activities of ATPase and acid phosphatase were definitely higher than that of normal rat 1 week after the transplantation but decreased to lower level than normal 2 weeks later. On the other hand, alkaline phosphatase activity increased 3 times at 1 week after the transplantation and remained at the same level even at 2 weeks later.


Mucopolysaccharides of rat ascites hepatoma, AH-100B.

The present study was undertaken to determine the mucopolysaccharide content of cells, AH-100B, and in the ascites, liver, and kidney of a rat bearing this hepatoma. AH-100B is similar to other mixed-type cells in having heparan sulfate as the main component in its mucopolysaccharides but its minor component was different in containing heparin. The main mucopolysaccharide in the ascites of AH-100B bearing rat was hyaluronic acid, which was similar to that of control ascites induced by injection of polypeptone, but minor component contained heparin derived from the cells. The content of hyaluronic acid in the liver of a rat bearing this hepatoma was higher than those of control liver and liver of a rat bearing other tumors. There were no significant differences between control kidney and kidney of a rat bearing this hepatoma.


Effect of oil-attached BCG cell wall on the kinetics of lymphocytes in the tumor-draining node.

The lymphocyte distribution into the tumor-draining node was studied with AH-130 hepatoma cells and Donryu strain rats in relation to the adjuvant activity of the oil-attached BCG whole cell wall. The mixed inoculation of the oil-attached BCG cell wall with tumor cells resulted initially in further augmentation of increased distribution of 51Cr-labeled lymphocytes into the draining-node induced by inoculation of the tumor cells alone, and secondarily in the systemic stimulation of response of the lymph  node lymphocytes to phytohemagglutinin. Suppression of the inoculated tumor growth and lymph node metastasis was finally observed. These results were discussed in connection with the therapeutic effect of BCG and its cell wall fraction.


Effect of serum concentration on adenylate cyclase activities of untransformed BHK cells and polyoma virus-transformed derivatives.

Adenylate cyclase activities of untransformed hamster BHK 21/13 cells and their polyoma virus-transformed derivatives were compared using the cells cultured in low and high serum concentrations. The untransformed cells that were cultured in a medium containing 10% serum had a slightly higher activity than the transformed cells cultured under the same conditions. When the cells were cultured in a medium containing 0.5% serum that stopped proliferation of untransformed cells, more significant difference was observed in the adenylate cyclase activity; the untransformed cells had a two-fold higher activity than the transformed cells. The full activity measured in the presence of fluoride was two-fold higher in the untransformed than in the transformed cells. The low serum concentration increased the enzyme activity of both untransformed and transformed cells, but the enzyme activity of transformed cells in the presence of 0.5% serum did not exceed the activity of untransformed cells in 10% serum. Intracellular cyclic AMP levels were in parallel with the activity of adenylate cyclase, whereas no correlation was found between cyclic AMP levels and cyclic AMP phosphodiesterase activities.


Effect of gastro-entero-pancreatic endocrine hormones on the histogenesis of gastric cancer in rats induced by N-methyl-N'-nitro-N-nitrosoguanidine; with special reference to development of scirrhous gastric cancer.

One of five gastro-entero-pancreatic hormones, gastrin, serotonin, histamine, glucagon, and insulin, was intraperitoneally administered for a long period to the rats that received N-methy-N'-nitro-N-nitrosoguanidine. A frequent development of scirrhous carcinoma was demonstrated in the group treated with gastrin.


Age-dependent arginine phosphokinase activity changes in male vestigial and wild-type Drosophila melanogaster.

The activity of arginine phosphokinase, an important muscle enzyme in insects, was investigated with age in vestigial-winged and wild-type Drosophila melanogaster. Identical patterns of age-dependent activity changes were observed in the vestigial-winged flies as in the wild-type, even though vestigial-winged flies exhibit a 50% mortality approximately two thirds that of the wild-type as well as being incapable of flight. Results indicate that the age-dependent changes in arginine phosphokinase activity are intrinsically regulated within the cells of the flight muscle.


Instability of the duration of G1 phase of Yoshida sarcoma and ascites hepatomas.

The durations and coefficients  of variance of G1, S and G2 phase were obtained for 8 cell lines of ascites tumours originated from hepatomas in rats, and these were compared. Although changes in the durations of S and G2 phases were small, the duration of G1 phase increased with the cell cycle time. The coefficient of variance of G1 phase was larger than that of S and G2 phases, most remarkedly at short cycle time. It was concluded that the G1 phase is the most unstable as well as the most variable in its duration.


[Selective pancreatic angiography and retrograde pancreatic cholangiography as a combined examination].

The diagnostic value of selective pancreatic angiography and retrograde pancreatic cholangiography used in combination was studied in 24 patients with chronic pancreatitis or pancreatico-duodenal carcinomas. This was done as a prospective study. The accuracy of the individual methods is 75% and 79% respectively; used in combination, a correct diagnosis is obtained in 91% of cases. The advantage of combining the techniques depends on the fact that they supplement each other.


[Computerized axial tomography of the skull - diagnostic possibilities and clinical results (author's transl)].

Computerized axial tomography is a new method of tissue examination with x-rays whereby a picture can be produced which is a representation of a slice of the skull. This is done by irradiating the skull from 180 or 225 incremental angles and measuring the absorption at each of these angles. Then with the aid of a computer a tomogram is produced which can be displayed on a screen. These tomograms are representations of a cross-section of the skull composed of 160 X 160 points showing the various intracranial structures with great detail. The present study demonstrates the diagnostic possibilities of the high definition matrix with reference to brain disorders in a large sample of patients for the first time. Some tumours are shown as areas of decreased absorption compared with normal brain tissue. Others, however, have been found to have higher absorption values. With glioblastomas very contrasting pictures are produced with coexistng areas of decreased, increased and similar values to brain tissue. The most important finding is the visualization of brain oedema which appears as a low density area. A grading system of brain oedemas is proposed. The brain oedema associated with tumours has been found to propagate mainly in the white matter producing finger-like shapes. Out of 209 intracranial tumours 203 were recognized in the plain scan, a further five after contrast enhancement. In patients who have suffered from a stroke the differentiation between haemorrhage and infarction is made simple due to the contrasting appearance between the two types of lesion. Location, size and propagation direction of a haematoma as well as rupture of a haemorrhage into the ventricular system can be defined exactly. With brain infarction the hypoxically damaged tissue is well delineated and readily attributable to a given vascular area. In head injuries, for the first time it is possible to differentiate brain contusion with oedema from intracerebral haematoma. Coup and contre-coup are demonstrated. All types of acute intracranial haematomas may be diagnosed easily. With chronic subdural haematomas the new method fails if the contents of the haematoma shows the same absorption values as brain tissue. Late sequelae of head trauma such as contusion defects and necrosis of tissue after oedema can be seen in the tomogram. With infantile hydrocephalus, subdural effusions and malformations of the brain, computerized axial tomography offers a complete diagnosis so that other invasive, neuroradiological investigations may be avoided. Orbital tumors are picked out with great clarity. The high definition matrix allows the demonstration of the optic nerve, the lense and the suspension of the eyeball. Without doubt in the coming years computerized axial tomography will play a dominant role in the diagnosis of brain disorders.


In vitro studies of calcitonin release in man.

The influence of various agents on calcitonin release from human thyroid was studied in vitro. Under the condition of this investigation, calcium, magnesium and phosphate did not stimulate calcitonin release from short-term incubated slices of human thyroid. However, pentagastrin and USP glucagon were potent stimulators of calcitonin release. Theophylline and dibutyryl cyclic AMP were also potent stimuli. A highly purified preparation of pancreatic glucagon was without an effect. Those agents which stimulated calcitonin release were associated with augmented cyclic AMP accumulation. Although maximal discharge of calcitonin required the presence of calcium, out in vitro experiments raise the question as to whether a gastrointestinal hormone, rather than calcium, might not be the principal agent affecting calcitonin release.


Primary tissue culture of normal adult human decapsulated adrenal cortex: radioautographic studies on the metabolic effects of ACTH1-24.

ACTH-deprived primary normal adult human adrenocortical cells on the 23rd day in vitro were found to lack any DNA synthetic and proliferative activity, but incorporated uridine-3H and 1-leucine-3H actively. In cortical cells of the same age treated for 2 and 7 days with ACTH1-24 (3 X 10(-6) M) the incorporation of the precursors for RNA and DNA synthesis and the cell proliferation were found to be markedly stimulated. The apparent uptake of leucine-3H was instead reduced in 2-day and, less still, in 7-day treated cells. In parallel with ultrastructural studies, these data suggest that ACTH orderly activates the template activity of chromatin while eliciting the differentiation and hypertrophy of human adrenocortical cells.


Electron spin resonance studies on the lipid-protein interaction between cardiolipin and anti-cardiolipin antibodies.

Electron spin resonance measurements were performed in order to investigate the influence of anti-cardiolipin antibodies on cardiolipin-containing liposomes. The physical state of the lipid structures and the alterations caused by the interaction with specific antibody were determined by measuring the freedom of motion of spin-labeled stearic acid derivatives incorporated into the lipid structures. The interaction of the cardiolipin-containing liposomes with the anti-cardiolipin antibodies reduced the mobility of the spin-labeled stearic acid probe I (12, 3), whose nitroxide group is assumed to be located near the polar region of the lipid bilayer. The restricted mobility, which qualitatively resembles the interaction of cardiolipin liposomes with calcium ions, is probably the result of a tighter packing of the polar groups in their crystalline array. The binding sites of the cardiolipin structures for anti-cardiolipin antibodies and Ca2 ions seem to be identical. As indicated by the spin-labeled stearic acid probe I (1, 14), the apolar region of the lipid bilayer is not affected by the interaction of the cardiolipin-containing liposomes with the anti-cardiolipin antibodies.


[Isolation and separation of nucleic acids from Streptomyces hydrogenans (author's transl)].

Different methods for homogenization of cells of Streptomyces hydrogenans, for extraction of nucleic acids and for fractionation of the RNA and DNA obtained were critically examined. The only way to prepare high molecular weight rapidly labelled RNA and polysomes was to grind freeze-dried cells together with kieselguhr with a mortar and pestle. The best results for extraction of nucleic acids from the cell homogenate were obtained in the presence of diethyl pyrocarbonate (diethyl oxydiformate), yielding nucleic acids of considerable purity in a minimal amount of time. The best resolution of extracted nucleic acids was achieved by electrophoresis in 2% agarose acrylamide gels. This technique proved that during the cell homogenization and extraction procedure the bulk of nucliec acids was not degraded to low molecular weight material. An improved device for the registration of the profile of the absorption after gel electrophoresis is described.


Purification and characterization of neuraminidase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. Neuraminidase secreted by the cells into the culture medium was purified 380 000-fold by: precipitation with ammonium sulfate between 50 and 85% saturation, filtration on Sephadex G-75, electrophoresis on polyacrylamide gel, and by isoelectric focusing. Three enzyme fractions with different migration rates were obtained by preparative disc electrophoresis in polyacrylamide gel, and five fractions with isoelectric points between pH 4.7 and 5.4 were observed after isoelectric focusing. This microheterogeneity disappeared after denaturation of the enzyme in 0.1% sodium dodecylsulfate or 8M urea. The isoelectric point of the denatured enzyme corresponded to pH 4.3. All enzyme fractions were identical with regard to their immunological and kinetic properties; they had the same molecular weights. The origin of the different "conformers" of neuraminidase is discussed. The existence of genuine isoenzymes could largely be excluded. The yield of neuraminidase was 65%, which corresponded to about 10 mg of pure enzyme from 100 l of culture medium. The enzyme was free of protease and various other glycosidase activities. The neuraminidase preparation appeared not to be contaminated by other proteins as judged by electrophoretic analysis using either the native enzyme or the enzyme denatured by sodium dodecylsulfate or urea; ultracentrifugation; chromatography on Sephadex G-200; and immunological methods. The molecular weights of the native or denatured enzyme were found to be in the range between 60 000 and 69 000 (on an average 63 750) using four independent methods. The existence of subunits of neuraminidase was excluded. The neuraminidase exhibited a spec. act. of 580 or 615 U/mg protein with glycopeptides from edible birds' nests or sialyllactose, respectively, as substrates. Additional kinetic properties and the UV-absorption spectrum of the enzyme are described.


[15-Hydroxyprostaglandin dehydrogenase from human placenta, II. Steady state kinetics and influence of prostaglandin F2alpha analogues (author's transl)].

A complete initial rate analysis of the forward reaction catalyzed by 15-hydroxyprostaglandin dehydrogenase from human term placenta was carried out at pH 7.4 (100mM triethanolamine) with the substrates NAD, and the prostaglandins E1, E2 and F2alpha. The limiting Michaelis constants, the dissociation constants, and the limiting maximum velocities for these substrates were calculated by fitting the obtained data by weighted linear regression analysis to the complete rate equation. The product inhibition of the reaction by NADH and 15-oxoprostaglandin was studied and the inhibition constants were graphically determined. The initial rate and inhibition patterns obtained indicate that the reaction follows kinetically an ordered Bi Bi mechanism. The prostaglandin F2alpha analogues ICI 81,008 and ICI 79,939 were not utilized by the enzyme. With ICI 81,008 a slight inhibition of the enzymatic reaction with prostaglandin F2alpha was observed, whereas ICI 79,939 showed no effect. The results are discussed with respect to their possible biological significance.


The fine structure of so-called minute pulmonary chemodectomas.

Electron microscopy was performed on several minute tumors of the type called chemodectomas, all from the lung of a single patient. The cells had a whorling pattern with extensive interdigitating cytoplasmic processes joined by desmosomes. Except for tangles of cytoplasmic fibrils, the tumor cells had few distinctive organelles. They had no endocrine-like granules and were not associated with nerves or basement membranes. The tumors had little resemblance to paragangliomas, but displayed a puzzling similarity to meningiomas. Our observations permit no definite conclusions as to the histogenesis of these lung tumors. Viewed in the light of recent physiologic studies, they cast doubt on the presence of special chemoreceptive paraganglia in the lung.


Prenatal diagnosis of I-cell disease.

A pregnancy from a family in risk of I-cell disease was monitored. The fetus was diagnosed as having I-cell disease based on the findings that (1) lysosomal enzyme activities except for acid phosphatase and alpha glucosidase were clearly elevated in amniotic fluid and were reduced in cultivated amniotic fluid cells, and (2) cytoplasmic inclusions were seen in cultivated amniotic cells by phase contrast microscopy. The accuracy of prediction was confirmed by cultured skin fibroblast of the aborted fetus.


[Studies on the glucocorticoid control of zinc metabolism].

In the present paper a possible connection between ACTH stress, cortisol release and serum zinc level is studied. It could be shown that under ACTH stress the reaction of cortisol release is physiological, whereas the zinc level in serum decreases significantly. These reactions recur under insulin stress in a weaker form. On the basis of these results a possible connection between stress situations and hypozincemia was discussed. At the same time a possible misinterpretation with regard to a general zinc deficiency under the condition of a reduced serum zinc level in stress situations is pointed out. In accordance with other authors it can, however, be said that the zinc metabolism probably depends on glucocorticoidal controls.


Immunisation trials with live attenuated cytomegalovirus TOWNE 125.

Trials with live cytomegalovirus (TOWNE 125 strain), which was attenuated by 125 passages exclusively on WI-38 cells, were done in adult volunteers. No virus take occured after oral/nasal application. When 10(3) TCD50 was given intramuscularly an IgG antibody response was detected at four weeks in all of ten volunteers tested by immunofluorescence; an IgM response was found in seven. Only mild local side-effects and relative lymphocytosis were observed. No virus excretion was found. Many questions remain to be answered by further trials before a cytomegalovirus vaccine can be given to adolescent girls.


Pathogenicity of entamoeba histolytica.

The pathogenicity of Entamoeba histolytica is discussed from an immunologic point of view. The evidence that there is some "trigger" mechanism which converts a commensal dwelling organism into a tissue invasive pathogen is rejected as inadequate. The number of liver abscess cases in comparison with the number of intestinal amebic infections in a population is so low that this in itself suggests that tissue invasion is a rare event in the life history of the ameba. A review is made of the experimental evidence that some type of sensitization is necessary before ameba can invade tissue. In postulating an immunologic basis for the pathogenicity of ameba, a parallel between the behavior of malignant cells in the body and an amebic infection in the gut is made. An appealing hypothesis which deserves further research effort is that an altered immune response is the basis for the pathogenic mechanism in the host.


Adjuvant activity of the histamine-sensitizing factor of Bordetella pertussis in different strains of mice.

The effect of an extract of histamine-sensitizing factor (HSF) of Bordetella pertussis on the immune response of different strains of mice to ovalbumin (OA) was investigated with regard to optimal dose of antigen and adjuvant. It was observed that all strains of mice treated with HSF during immunization with OA demonstrated enhanced production of hemagglutinating antibodies, as compared to animals treated with antigen alone. This enhancement was generally not as great as that demonstrated when Al(OH)3 was the adjuvant. HSF also stimulated a reaginic antibody response (IgE) to OA, but not in all strains of mice. In reagin responders optimal responses were observed with high doses of both antigen and adjuvant, whereas low doses of both produced little or no response. Maximal reagin production occurred usually 14-28 days after immunization and persisted for long periods of time. An anamnestic reagin response was elicited upon secondary immunization with antigen alone, not only in mice immunized with OA and HSF but also in animals treated with OA alone. These studies demonstrate the profound effect that a microbial substance such as HSF can have on reaginic antibody production and suggest that the stimulation of IgE antibody production is the net result of a number of factors including genetic capabilities of the host, environmental influence such as adjuvants, and prior exposure to an antigen.


Effect of exogenous cyclic AMP and other adenine nucleotides on neutrophil chemotaxis and motility.

Cyclic AMP placed in the bottom of the modified Boyden chemotaxis chamber is weakly chemotactic for rabbit peritoneal neutrophils. Dibutyryl-cyclic AMP under similar conditions gives a biphasic response, being weakly chemotactic at low concentrations and inhibiting cell movement at higher concentrations. When mixed with the cells in the upper compartment of the chemotaxis chamber, cyclic AMP and dibutyryl-cyclic AMP enhance neutrophil spontaneous motility; on the other hand, dibutyryl-cyclic AMP mixed with the cells in the upper compartment consistently inhibits neutrophil chemotactic responsiveness, while cyclic AMP has a variable effect on chemotaxis. The effect of ADP and ATP was to inhibit chemotaxis and spontaneous motility. AMP is totally without effect.


Nasopharyngeal carcinoma. IX. Antibodies to EBNA and correlation with response to other ebv antigens in chinese patients.

Ninety-five sera from Chinese NPC patients in different stages of the disease, 38 sera from Chinese patients with other cancers, and 50 normal Chinese sera, were titrated for EBNA, VCA, EA and complement-fixing (CF/S) EBV-specific antibodies. The geometric mean (GMT) EBNA antibody titre of patients with NPC at stage I was found to be four times higher than that of normal individuals and increased in parallel with clinical deterioration. Antibody titres against EBNA did not correlate with either VCA or EA antibodies but, in general, correlated with CF/S antibodies. EA antibodies correlated relatively well with VCA antibodies and discriminated better than EBNA titre between NPC at stage I and controls.


Colcemid-induced polyploidy and aneuploidy in normal and tumour cells in vitro.

The frequency of colcemid-induced genome mutations (aneuploidy and polyploidy) in normal and SV40-transformed cultures of Djungarian hamster embryonic cells was studied. Genome mutations were easily induced by the drug in transformed but not in normal cultures. Elevation of colcemid concentration and prolongation of the incubation period did not substantially increase the frequency of genome mutations in normal cells. An attempt was made to study the causes of the differences in sensitivity to colcemid-mutagenicity of normal and transformed cells. Transformed cells did not include more 3H-colchicine than normal cells, and binding of the drug to cell homogenates was similar in both kinds of cultures. According to these data the higher sensitivity of transformed cells to colcemid is not connected either with increased permeability of the cell to the drug or with changes in its binding to tubulin.


Interactions between endogenous baboon type-C virus and oncogenic viruses. I. Syncytium induction and development of infectivity assay.

Cells releasing the endogenous baboon virus (BV) can interact with human KC cells containing the Rous sarcoma virus (RSV) genome, resulting in cell fusion and syncytium formation. This interaction has been utilized in the development of a sensitive infectivity assay for BV. The titration pattern is of a one-hit type, demonstrating a linear relationship between virus concentration and number of syncytial plaques obtained in the KC co-cultivation assay. Endpoint titration comparisons indicate that the KC test is as sensitive as the immunofluorescence or the RNA-directed DNA-polymerase assays. Attempts to develop an XC test for BV failed, indicating that while BV can interact with the RSV genome it will do so in the human KC cells and not in the rat XC cells. Syncytia are also induced when KC cells are directly exposed to cell-free BV; however, a linear dose relationship is not obtained. When syncytium-positive KC cultures are passaged, the syncytia disappear and a chronic BV infection is established. These KC-BV cells then lose the ability to interact with either the endogenous cat RD-114 virus or the Mason-Pfizer virus which are known to form syncytia with KC cells.


Characterization of non-producer human cells induced by Kirsten sarcoma virus.

Non-producer (NP) human cells induced by the Kirsten sarcoma virus were characterized. These morphologically altered NP cells produced neither infectious virus nor complement-fixing antigens of the murine sarcoma-leukemia virus complex. The NP cells did not release RNA-dependent DNA polymerase and type-C virus particles with a density of approximately 1.15 g/ml in sucrose gradients by 3H-uridine labelling. The NP cells produced tumors when transplanted subcutaneously into athymic nude mice. The tumor cells re-established in culture resembled the orginal NP cells, were confirmed as human cells by karyological analysis and were also found to be "non-producer". The sarcoma virus genome in NP cells could be rescued not only by co-cultivation with "helper virus"-releasing cells but also by superinfection with helper type-C viruses. Murine (Rauscher, Ki-MuLV, AT-124 and two other xenotropic viruses), feline, RD-114 and Simian (woolly monkey and baboon) type-C viruses possessed the ability to rescue the sarcoma genome from NP cells but not AKR leukemia virus. In addition, the feline leukemia virus titer obtained by the rescuing technique in NP cells was the same as those obtained in feline embryo and NP cells by CF induction assay.


Popper's philosophy for epidemiologists.

This paper discusses the application of Popper's philosophy to epidemiological research, examining in particular the problems of replication without risk of refutation, of mistaking statistical sophistication for deductive inference, and of dealing with causality at a general level. An example is given of a Popperian approach to the test of a causal hypothesis concerning cancer of the cervix.


Adrenocorticotropic (ACTH) cells of rats after head irradiation.

The heads of intact rats and of rats 15 days after adrenalectomy were irradiated with 1200 R. Some of the adrenalectomized animals with irradiated heads were treated with 5 mg of deoxycorticosterone acetate. In adrenocorticotropic cells of adrenalectomized rats 15 and 30 days after irradiation, the decreased amount of the granular endoplasmic reticulum and the Golgi regression imply a considerably decreased protein synthesis. The finding of an increased number of granules and of granules larger than normal suggests that they are the result of slowed down transport and secretion of the products of these cells' activities. After head irradiation an increased number of degenerative cells was evident in the pituitary of both intact and adrenalectomized rats. The degree of the degenerative changes and the number of degenerative cells was higher 30 than 15 days after irradiation and both were decreased if the adrenalectomized rats were treated with deoxycorticosterone acetate.


In vivo characteristics of temperature-sensitive host range mutants of herpes simplex virus type 2.

The in vivo properties of four host range temperature-sensitive (ts) mutants of herpes simplex virus type 2 (HSV-2) were correlated with known in vitro characteristics. Mutants and parental virus were inoculated into newborn and weanling mice and weanling hamsters by intracranial and subcutaneous routes, and into weanling rabbits by corneal scarification. In all cases the pattern of attenuation of mutants in vivo correlated with their stability in vitro at 39 degrees. The most attenuated mutant (mutant 69) was also the most consistent in its inability to induce cytopathic effects (CPE) in vitro or to replicate under nonpermissive conditions. Conversely, the most virulent mutant (mutant 41) was the least stable under nonpermissive conditions.


Heterogeneity of surface antigens on endogenous type C virus-producing cell sublines derived from a clonal line of BALB/3T3 cells.

When viral envelope antigens (VEAs) and virus-associated cell surface antigens (CSAs) are used as markers of type C viruses, more heterogeneity of virus populations can be demonstrated than by using host range alone. In the present study, four CSAs (PC1, X.1, GCSA, and MEV-SA1) and three VEAs (xVEA, x1-VEA, and sub-gsVEA) were used as markers of virus populations present in various sublines of the BALB/c embryonic fibroblast BALB/3T3 clone A31 line. Of four spontaneously transformed sublines, two released N-tropic endogenous type C viruses spontaneously after long-term culture and each had distinct antigenic patterns. Treatment with 5-bromodoxyuridine (BrdU) resulted in the detection of X-tropic viruses in all four sublines. The expression of these X-tropic viruses was associated with two different antigenic patterns. When the clonal rabbit corneal SIRC cell line was infected with the X-tropic viruses obtained after BrdU treatment, the cells acquired detectable amounts of only one of the antigenic markers.


The effect of alpha1-24-corticotropin on growth hormone release in prepubertal children.

The effect of alpha1-24-corticotropin-Zn on the results of pituitary stimulation tests (moderate standardized physical exercise, arginine infusion, insulin-induced hypoglycemia) was studied in 27 prepubertal children with non-endocrine retardation of growth and development. After administration of 1 mg alpha1-24-corticotropin, the basal blood glucose and plasma cortisol levels rose significantly. However, the growth hormone increase after the three stimulation tests was significantly lower than without the corticotropin injection. The results demonstrate the inhibiting effect of alpha1-24-corticotropin on growth hormone secretion.


Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.


Cytochemical correlates of structural sexual dimorphism in glandular tissues of the mouse. I. Studies of the renal glomerular capsule.

The parietal epithelium of Bowman's capsule has been analyzed by enzyme cytochemistry in kidneys of mice (C57BL/6J) from birth to 50 days of age. There is a greater tendency for cells in the central portions of the capsular crescent to be cuboidal in post-pubertal males than in pre-pubertal mice of either sex or in post-pubertal females where they are generally squamous; moreover, these heightened capsular cells have a distinct microvillous border. Cytochemical procedures were selected which might confirm the morphological suggestion that the cuboidal parietal epithelium possesses an absorptive capacity. The oxidoreductase activity of the mitochondria of the cuboidal cells of this layer is comparable to that of the columnar cells of the proximal convoluted tubule. The cytochrome oxidase activity of the mitochondria in both of these segments of the nephron is intense. This is in sharp contrast to the unreactive mitochondria in the squamous cells of the parietal epithelium. Furthermore, a striking heterogeneity in the degree of cytochrome oxidase activity is evident in the mitochondria of the cuboidal parietal cells as well as in the cells of the proximal tubules. In the former cells, active mitochondria were generally found near microvilli at the apical ends and in the areas of the basal infoldings whereas those in a central position were more frequently unreactive. The brush border of the cuboidal capsular epithelium had prominent alkaline phosphatase and aminopeptidase activities as has previously been observed in other brush borders. Functional capacity corresponding to the morphological and cytochemical specialization of the cuboidal capsular cells was demonstrated by their uptake of horseradish peroxidase. This exogenous protein tracer could be seen in apical vacuoles and phagosomes in the cuboidal parietal epithelium. The cytochemical resemblance of the cells of this epithelium to those of the proximal convoluted tubules suggests a similar involvement in resorption and perhaps in active transport. A possible relationship of this differentiation of the capsular epithelium to the proteinuria normal for adult male mice is discussed.


The inhibition of alkaline phosphatase by L-p-bromotetramisole.

A levamisole analogue, the L-p-bromotetramisole is introduced as a potent inhibitor of non-specific alkaline phosphatase. Complete inhibition is achieved cytochemically at a concentration of 0.1 mM in various rat tissues except the intestine, which is not affected. The D-p-bromotetramisole does not influence the alkaline phosphatase activities. Since no effect of the inhibitor is seen on the activities of specific phosphatases, this drug is recommended also as an additive for specific phosphatase media in order to yield the specific activity only.


Electron microscopic observations on the cholesterol distributed in the epithelial cells of the gallbladder.

Epithelial cells of eight human gallbladders with cholesterosis were examined. In the supranuclear portion of the epithelial cells of one case, many spicular, circular and plate-like crystalline structures are present. Spicular and circular structures have not a limiting membrane, but plate-like structures are apparently bounded by a limiting membrane that clearly shows trilamellar structure. After digitonin treatment, the spicular and circular crystalline structures become denser. On the other hand, the plate-like structures do not become denser by digitonization. In the epithelial cells that contain no crystalline structure, there also occur many reaction precipitates after digitonization. These findings may suggest that free cholesterol is highly present in the epithelial cells of gallbladder with cholesterosis and that it, in some case, precipitates in the form of spicular or circular structure for the rapid fixation process.


Studies in lipid histochemistry. XIII. The OPA (osmiumtetroxide-periodic acid-alpha-naphthylamine) method for the detection of apolar lipids.

A new procedure for the detection of apolar lipids is described. It is a modification of the OTAN method (Adams, 1959) using periodic acid which oxidatively removes lower osmium derivatives from polar sites only, leaving those in apolar lipids intact and demonstrable with alpha-naphthylamine. Control steps for the exclusion of the possible interference of some less polar complex lipids and of lipopigments are described. The described technic is superior to the conventionally used sudan dyes due partly to the fact that only aqueous solutions are employed thus excluding any extraction of lipids, partly to the more distinct coloration.


Effect of bredinin and its aglycone on L5178Y cells.

The aglycone of the nucleoside antibiotic, bredinin, was as strongly cytotoxic to L5178Y cells as bredinin. The cytotoxic properties of the aglycone were very similar to those of bredinin and the minimum inhibitory concentrations of both were 10(-5)M. The growth inhibitory effects of both agents regardless of their concentrations, were reversed by guanylic acid, guanosine or guanine. However, on increasing the concentrations of these agents, the reversing effect of guanylic acid decreased gradually, the dose-response curves for the two agents being similar. Both agents inhibited the incorporation of thymidine and uridine, but not leucine into macromolecules in L5178Y cells and their inhibitory effects were reversed to similar extents by guanylic acid. On the other hand, the growth inhibitory effect of the aglycone on L5178Y cells was prevented by adenine only, though not be adenosine or adenylic acid while the effect of bredinin was not prevented by adenine. These results suggest that the aglycone itself does not inhibit growth, but that its effect is due to its conversion to bredinin by an enzyme such as adenine phosphoribosyl transferase. For recovery of growth, three moles of adenine were required per mole of the aglycone. When the aglycone was administered orally to rats, bredinin was administered orally to rats, bredinin was recovered in their serum and urine.


Degradation of phospholipid in Pseudomonas aeruginosa induced by polymyxin B.

Effects of polymyxin B on the synthesis and degradation of lipid, ribonucleic acid (RNA) and protein in Pseudomonas aeruginosa were investigated. It was found that polymyxin B caused a marked degradation of the lipid fraction which was prelabeled with (3H-2)-glycerol. Thin-layer chromatographic analysis indicated that the main degraded lipids were phosphatidylethanolamine and phosphatidylglycerol, which constituted 80% and 15% of the total phospholipids of this organism, respectively. Polymyxin B also inhibited synthesis of RNA and protein in vivo. The severe inhibition of the uptake of labeled amino acids by polymyxin B indicated that the observed inhibition of RNA and protein synthesis possibly occurred at the level of substrate transports. The degradation of phospholipid might account for the defective membrane activities.


Carbon monoxide-induced localized toxic anoxia in the rat brain cortex.

A new method has been developed for the determination of maximal reduction of NAD in the rat cerebral cortex. NADH fluorescence (450 nm) induced by 366-nm light and UV reflectance were measured by a time-sharing light pipe fluorometer. The redox state of the cortical surface was altered by perfusion of oxygen or carbon monoxide through a Teflon chamber adjacent to the dura. This study examines changes caused by local perfusion with the two gases in normoxia, hypoxia, and anoxia. Alternation of topical carbon monoxide and oxygen becomes effective in altering the intracellular redox state at 15% inspired oxygen and caused 20% changes at zero inspired oxygen. Conversely, topical application of oxygen to the systemically anoxic tissue causes oxidation of reduced NAPH in the cells within the field of fluorometric observation equivalent to that caused by breathing approximately 8% oxygen systemically.


Dicarboxylic acid transport in membrane vesicles from Bacillus subtilis.

Membrane vesicles isolated from Bacillus subtilis W23 catalyze active transport of the C4 dicarboxylic acids L-malate, fumarate, and succinate under aerobic conditions in the presence of the electron donor reduced beta-nicotinamide adenine dinucleotide or the non-physiological electron donor system ascorbate-phenazine methosulfate. The dicarboxylic acids are accumulated in unmodified form. Inhibitors of the respiratory chain, sulfhydryl reagents, and uncoupling agents inhibit the accumulation of the dicarboxylic acids. The affinity constants for transport of L-malate, fumarate, and succinate are 13.5, 7.5, and 4.3 muM, respectively; these values are severalfold lower than those reported previously for whole cells. Active transport of these dicarboxylic acids occurs via one highly specific transport system as is indicated by the following observations. (i) Each dicarboxylic acid inhibits the transport of the other two dicarboxylic acids competitively. (ii) The affinity constants determined for the inhibitory action are very similar to those determined for the transport process. (iii) Each dicarboxylic acid exchanges rapidly with a previously accumulated dicarboxylic acid. (iv) Other metabolically and structurally related compounds do not inhibit transport of these dicarboxylic acids significantly, except for L-aspartate and L-glutamate. However, transport of these dicarboxylic amino acids is mediated by independent system because membrane vesicles from B. subtilis 60346, lacking functional dicarboxylic amino acid transport activity, accumulate the C4 dicarboxylic acids at even higher rates than vesicles from B. subtilis W 23. (v) A constant ratio exists between the initial rates of transport of L-malate, fumarate, and succinate in all membrane vesicle preparations isolated from cells grown on various media. This high-affinity dicarboxylic acid transport system seems to be present constitutively in B. subtilis W23.


Altered hexose transport and salt sensitivity in cyclic adenosine 3',5'-monophosphate-deficient Escherichia coli.

A cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutant strain of Escherichia coli K-12 was studied to determine the effect this cyclic nucleotide has on the overall growth and metabolism of this organism. Deficient cells were found to be more susceptible to growth inhibition by salts than were their cAMP-sufficient counterparts. The deficient cells transported alpha-methylglucoside by passive diffusion, whereas the parental cells or mutant cells grown in the presence of exogenous cAMP were able to take up alpha-methylglucoside by the normal active transport process. When viewed together with earlier studies conducted on cAMP-deficient cells, these findings support the view that cAMP plays a key role in regulating the construction and operation of the E. coli membrane system.


Reduced nicotinamide adenine dinucleotide-activated phosphoenolpyruvate carboxylase in Pseudomonas MA: potential regulation between carbon assimilation and energy production.

Comparison of enzyme activities in crude extracts of methylamine-grown Pseudomonas MA (ATCC 23319) to those in succinate-grown cells indicates the involvement of an acetyl coenzyme A-independent phosphoenolpyruvate carboxylase in one-carbon metabolism. The purified phosphoenolpyruvate carboxylase is activated specifically by reduced nicotinamide adenine dinucleotide (KA = 0.2 mM). The regulatory properties of this enzyme suggests that phosphoenolpyruvate serves as a focal point for both carbon assimilation and energy metabolism.


Identification of an outer membrane protein of Escherichia coli, with a role in the coordination of deoxyribonucleic acid replication and cell elongation.

Protein G of molecular weight 15,000 is the fourth commonest protein in the outer membrane of Escherichia coli B/r. From experiments described here on the relationship of protein G production to cell elongation and septation, the hypothesis is proposed that protein G is a structural protein of cell elongation. Furthermore, a surplus of protein G is produced when deoxyribonucleic acid synthesis is arrested and septation is thereby prevented. Thus protein G may be an important coordination protein in E. coli for integration of deoxyribonucleic acid synthesis, cell envelope elongation, and septation. Inhibition of normal cell elongation in a rod configuration in E. coli B/r by the novel amidinopenicillanic acid FL1060 was accompanied by changes in the rate of appearance of protein G  and several other outer membrane proteins. The rate of appearance of protein G decreased some 70% within 60 min, in parallel with termination of rounds of normal cell elongation. Filament-inducing concentrations of nalidixic acid increased dramatically the rate of appearance of protein G. After 30 min a plateau level some 250% higher than the control value was reached. Similar kinetics were observed in parallel with filament formation induced by incubation of a dnaB mutant of E. coli at the nonpermissive temperature. No change in the rate of appearance of protein G was observed during cephalexin- or benzylpenicillin-induced filament formation, indicating that increased protein G production was not a secondary consequence of filamentation. Cells treated with FL1060 lost their ability to be induced for protein G formation, with nalidixic acid, in parallel with their loss of ability to initiate rounds of normal cell elongation. A pulse-chase experiment demonstrated that the protein G appearing in the outer membrane as a consequence of inhibition of deoxyribonucleic acid synthesis was the result of de novo synthesis rather than of interconversion from previously synthesized protein species. A preliminary characterization of protein G revealed several similarities with the well-characterized lipoprotein of the outer membrane of E. coli. A comparison of the incorporation of several 14C-labeled amino acids into protein G and the lipoprotein revealed substantial differences, however, perhaps ruling out a simple relationship between these two proteins.


Demonstration of acid phosphatase-containing vacuoles in hyphal tip cells of Sclerotium rolfsii.

A lysosomal system was demonstrated in hyphal tip cells of Sclerotium rolfsii by light and electron microscopy observations of the sites of acid phosphatase activity visualized by a modified Gomori lead nitrate method. The cytochemical reaction product was found to be present in numerous vacuoles, each aout 0.5 mum in diameter, which were seen as chains of spheres when viewed with the light microscope. They usually did not occur in the first 30 to 40 mum of the hyphal tip cell, but were concentrated in a zone extending from 30 to 200 mum from the hyphal apex. As shown by the electron microscope, the vacuoles were sometimes interconnected by narrow channels. Acid phosphatase reaction product was also occasionally localized in vacuoles of the older hyphal cells, but never in apical vesicles, lipid bodies, or microbodies. It is proposed that this vacuolar system may orginate from the endoplasmic reticulum.


Assembly of the mitochondrial membrane system. Cytoplasmic mutants of Saccharomyces cerevisiae with lesions in enzymes of the respiratory chain and in the mitochondrial ATPase.

Mutants of Saccharomyces cervisiae with defects in enzymes of the electron transfer chain and in the rutamycin-sensitive ATPase have been isolated. Some of the mutants are specifically affected in either cytochrome oxidase, coenzyme QH2-cytochrome c reductase or ATPase. Other strains are deficient in both cytochrome oxidase and coenzyme QH2-cytochrome c reductase but still have rutamycin-sensitive ATPase. All the mutants reported in this study fail to be complemented by a rho0 tester derived from a respiratory competent strain. The meiotic spore progeny obtained by mating the mutants to a respiratory competent haploid yeast, when scored for growth on glycerol, show a non-Mendelian segregation of the phenotype. These two genetic tests indicate the mutations to be cytoplasmically inherited.


Insulin-induced reduction of membrane receptor concentrations in isolated fat cells and lymphocytes. Independence from receptor occupation and possible relation to proteolytic activity of insulin.

Incubation of cultured human lymphocytes or isolated rat fat cells at 37 degrees with insulin results in a time-dependent fall in the ability of the thoroughly washed cells to bind 125I-labeled insulin. Although this effect is dependent on the concentration of insulin used in the preincubation period, the concentration dependence pattern varies directly with the length of this incubation, and kinetically (i.e. at early times) it is virtually impossible to demonstrate saturability with respect to insulin concentration. Significant effects are demonstrable at very early times (e.g. 1 to 3 hours) provided sufficiently high insulin concentration (i.e. 5 mug/ml or more) are used...


Structural analyses of mammalian ribosomal ribonucleic acid and its precursors. Nucleotide sequence of ribosomal 5.8 S ribonucleic acid.

The nucleotide sequence of ribosomal 5.8 S RNA (also known as 7 S or 5.5 S rRNA) from Novikoff hepatoma ascites cells has been determined to be (see article). Estimations of the secondary structure based upon maximized base pairing and the fragments of partial ribonuclease digestion indicate that there may be five base-paired regions in the molecule, three forming a folding of the termini and two forming secondary hairpin loops. The sequence of Novikoff hepatoma 5.8 S rRNA is about 75% homologous with that of yeast 5.8 S rRNA (Rubin, G.M. (1973) J. Biol. Chem. 248, 3860-3875) and similar models for secondary structure are proposed. Both models contain a very stable G-C rich hairpin loop (residues 116 to 138), a less stable A-U-rich hairpin loop (residues 64 to 91) and two symmetrical bulges (residues 15 to 25 and 40 to 44).


Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.


Interaction of human plasma low density lipoprotein with concanavalin A and with ricin.

Native human plasma low density lipoprotein (LDL) interacts with concanavalin A but not with ricin; apOLDL reacts with both lectins. Each reaction is inhibited by the appropriate lectin-specific carbohydrate. The "receptors" on LDL for these two lectins are not destroyed by digestion by proteolytic enzymes. Peptide hydrolysis does not influence the reactivity of LDL toward concanavalin A. It does, however, substantially enhance the ability of the lipoprotein to interact with ricin. The data strongly suggest that the carbohydrate protion of a glycoprotein component of LDL is bound at the saccharidespecific active site on the lectin.


Isolation and characterization of a pyrophosphate-dependent phosphofructokinase from Propionibacterium shermanii.

A pyrophosphate-dependent phosphofructokinase (pyrophosphate; D-fructose-6-phosphate-1-phosphotransferase) has been purified and characterized from extracts of Propionibacterium shermanii. The enzyme catalyzes the transfer of phosphate from pyrophosphate to fructose 6-phosphate to yield fructose-1,6-P2 and phosphate. This unique enzymatic activity was observed initially in Entamoeba histolytica (Reeves, R.E., South, D.J., Blytt, H.G., and Warren, L. G. (1974) J. Biol. Chem. 249, 7734-7741). This is the third pyrophosphate-utilizing enzyme that these two diverse organisms have in common. The others are phosphoenolpyruvate carboxytransphosphorylase and pyruvate phosphate dikinase. The PPi-phosphofructokinase from P. shermanii is specific for fructose-6-P and fructose-1,6-P2, no other phosphorylated sugars were utilized. Phosphate could be replaced by arsenate. The Km values are: phosphate, 6.0 X 10(-4) M; fructose-1, 6-P2, 5.1 X 10(-5) M; pyrophosphate, 6.9 X 10(-5) M; and fructose-6-P, 1.0 X 10(-4) M. The S20w is 5.1 S. The molecular weight of the native enzyme is 95,000. Sodium dodecyl sulfate electrophoresis of the enzyme showed a single band migrating with an Rf corresponding to a molecular weight of 48,000. Extracts of P. shermanii have PPi-phosphofructokinase activity approximately 6 times greater than ATP-phosphofructokinase and 15 to 20 times greater than fructose diphosphatase activities. It is proposed that (a) PPi may replace ATP in the formation of fructose-1-6-P2 when the organism is grown on glucose and (b) when the organism is grown on lactate or glycerol the conversion of fructose-1,6-P2 to fructose-6-P during gluconeogenesis may occur by phosphorolysis rather than hydrolysis.


Steroid-protein interactions. Stopped flow fluorescence studies of the interaction between steroid hormones and progesterone-binding globulin.

Stopped flow fluorometry, measuring changes in the intrinsic fluorescence of progesterone-binding globulin (PBG), was used to determine the association and dissociation rates of the interaction of PBG with seven delta4-3-ketosteroids. The rates of formation and dissociation of the PBG-progesterone complex were measured as a function of concentration and temperature. At 20 degrees, kon = 8.7 X 10(7) M-1 S-1 and koff = 0.060 S-1. The association rate constants for progesterone, deoxycorticosterone, testosterone, testosterone acetate, and medrogestone were found to be the same within experimental error. The different affinities of PBG for these steroids result from the dissociation rate constants of the steroids which ranged from 0.43 S-1 for testosterone to 0.024 S-1 for medrogestone. Two corticosteroids, corticosterone and cortisol, were both bound somewhat more slowly (approximately 5 X 10(7) M-1 S-1). Reflecting their very low affinity for PBG both steroids dissociate very rapidly: corticosterone at 1.4 S-1 and cortisol at 90 S-1. The ratio of association to dissociation rate constants gave affinity constants in agreement with independently determined constants.


Mechanism of action of uridine diphosphoglucose dehydrogenase. Evidence for a second reversible dehydrogenation step involving an essential thiol group.

Although the enzyme UDP-glucose dehydrogenase from beef liver (E.C. 1.1.1.22) is known to abstract the pro-R hydrogen stereospecifically at carbon 6 of the glucose moiety of the substrate by a reversible step in converting UDP-glucose to UDP-alpha-D-gluco-hexodialdose (UDP-Glc-6-CHO), prolonged incubation of the enzyme with UDP-glucose and tritium-labeled NADH, under conditions favoring hydrogen exchange between the two, results in equivalent labeling of both hydrogens at carbon 6. This shows that the pro-S hydrogen at carbon 6 is also abstracted by a reversible process which must then involve a derivative of the carboxyl group of UDP-glucuronic acid (UDP-GlcUA) that is capable of reversible hydrogenation-dehydrogenation. It is the hydrolysis of this derivative that accounts for the well known irreversibility of the overall reaction. Derivatization of the enzyme's essential thiol group with 5,5'-dithiobis-(2-nitrobenzoate) eliminates the ability of the enzyme to either oxidize or reduce UDP-Glc-6-CHO. Replacement of the 5-thio-2-nitrobenzoate group with cyanide fully restores the enzyme's capacity to reduce UDP-Glc-6-CHO but has no effect on the inhibition of the oxidation to UDP-GlcUA. This indicates that the essential thiol group is involved in the second reversible dehydrogenation step and serves to form a thiol ester with the carboxyl of the product, UDP-GlcUA. It is suggested that thiol ester intermediates are a general characteristic of all 4-electron NAD-linked dehydrogenase reactions.


Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.

The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.


Studies on the mechanism of adenosylcobalamin-dependent ribonucleotide reduction by the use of analogs of the coenzyme.

A series of 17 analogs of 5'-deoxy-5'-adenosylcobalamin(adenosylcobalamin) have been synthesized with modifications in the base or ribose moiety of the nucleoside ligand. These analogs have been examined for their effects on reactions catalyzed by the ribonucleotide reductase of Lactobacillus leichmannii. All the analogs are inhibitors of ATP reduction in the presence of adenosylcobalamin as coenzyme, and hence all are bound to the catalytic site. Only the 3-beta-D-ribofuranosyladenine analog (isoadenosylcobalamin) showed substantial activity as a coenzyme in ATP reduction, giving a rate of 59% of that obtained with the adenosylcobalamin. Lesser rates of reduction were obtained with nebularyl-, 2'-deoxyadenosyl-, tubercidyl-, isopropylideneadenosyl-, L-adenosyl-, and ara-adenosylcobalamin, coenzyme activity decreasing in that order. Other analogs showed no significant coenzyme activity. The rate of hydrogen exchange into water from the 5'-methylene group of the nucleoside ligand appeared to parallel the coenzyme activity in those analogs examined, but only the four cobalamins with highest coenzyme activity (adenosyl, isoadenosyl, nebularyl, 2'-deoxyadenosyl) gave detectable amounts of "active coenzyme B12," THe rapidly formed paramagnetic intermediate of ribonucleotide reduction. The enzyme system produced the slowly formed paramagnetic species characterized by a doublet EPR spectrum only with adenosyl- and isoadenosylcobalamin. By contrast the enzymic degradation of analogs to cob(II)alamin and 5'-deoxynucleoside occurred not only with those analogs active as coenzymes and in the exchange reaction but also with a number of coenzymically inactive analogs, and the rate of degradation was unrelated to the rate of ribonucleotide reduction for those analogs with coenzyme activity.