Reversal of methylcholanthrene-induced changes in mouse prostates in vitro by retinoic acid and its analogues. The influence of vitamin A-related compounds on hyperplasia and metaplasia induced by methylcholanthrene was studied in mouse prostate glands in organ culture. Methylcholanthrene was found to cause extensive hyperplasia and squamous metaplasia of the prostatic epithelium which persisted after withdrawal of the carcinogen. The retinoids included retinoic acid and 6 of its structural analogues synthesized in an attempt to enhance the anticarcinogenic action and reduce the toxicity of the parent compound. These where the cyclopentenyl analogus 7699, A2-retinoic acid, 13-cis-alpha-retinoic acid and 3 aromatic analogues. Administration of the compounds following the carcinogen reduced the extent and incidence of hyperplasia significantly and with the exception of one compound reversed the squamous metaplasia. Two of the aromatic analogues, one with a terminal ethylamide group (1430), and the other with a terminal ethylester group (9369), proved to be the most potent inhibitors, followed by compound 7699 and (9369), proved to be the most potent inhibitors, followed by compound 7699 and retinoic acid. A2-retinoic acid and 13-cis-alpha-retinoic acid showed the lowest activity. The inhibition of hyperplasia appeared to be mediated via a reduction of DNA synthesis. It seemed unrelated to either the biological growth-promoting activity of the compounds or their surface-active properties. It is tentatively suggested that vitamin A and its analogues may act as hormones.


Purification of a cytotoxic factor from human and rat tissues. Human and rat normal tissues and tumours have been studied for the presence of toxic substances, possibly of importance in the development of cachexia in patients with cancer and other chronic diseases. The toxic effect of tissue extracts was gauged by measuring the inhibition of growth of mouse L-cells in 1-ml cultures, as revealed by reduced incorporation of [14C]leucine into cell protein. A common cytotoxic substance of mol. wt. approximately 700 daltons was isolated from all rat and human tissues tested, including tumours. The isolation procedure involved tissue homogenization, followed by pressure dialysis, gel filtration of concentrated pressure dialysates, cation exchange chromatography, and thin layer chromatography. Amounts isolated from different tissues varied by a factor of 3. The purified substance reacted with ninhydrin and a few other reagents for amino groups. It was completely resistant to acid and enzymatic hydrolysis. The evidence thus suggests that the substance is an amine. It is toxic to L-cells, HeLa cells and normal rat fibroblasts in concentrations of 10-20 muM, producing cell death and lysis during incubation overnight.


Factor VIII and human platelet aggregation. I. Evience that the platelet aggregating activity of bovine factor VIII is a property of its 'carrier protein' subunit. Bovine factor VIII is a potent inducer of aggregation of human platelets. Upon gel filtration of five-thousand-fold purified material in 0.5 M CaCl2, bovine factor VIII is separated into high and low molecular weight components; the former contains both a 'carrier protein' and platelet aggregating activity, the latter the procoagulant activity (low molecular weight factor VII, LMW-FVIII). Upon removal of Ca2+ ions, LMW-F VIII recombines with the 'carrier protein'. LMW-F VIII modifies aggregation by 'carrier protein', but not aggregation by undissociated bovine factor VII, adenosine-5'-diphosphate or adrenaline. This finding indicates that the platelet aggregating activity in indeed a property of the 'carrier protein'.


Factor VIII and human platelet aggregation. II. Characteristics of aggregation of human platelets by bovine factor VIII. Bovine factor VIII aggregates human platelets either in a strong single wave at high concentration (10 mug/ml platelet suspension) or in two waves at low concentration (0.2-I mug/ml). The strong single wave of aggregation is not associated with release of [14C]serotonin or beta-glucuronidase; the high concentration does not induce retraction of reptilase-clotted platelet-rich plasma. Wtih the low concentration, relase of [14C]serotonin is observed just prior to the onset of the second wave of aggregation; release of beta-glucuronidase does not occur at any moment. The low concentration of bovine factor VIII induces moderate retraction of reptilase-clotted platelet-clotted platelet-rich plasma, which is inhibited by acetylsalicylic acid, indomethacin and apyrase, indicating that it is a consequence of release of platelet adenosine-5'-diphosphate. It has previously been suggested tht carbohydrate groups are involved in the human platelet-bovine factor VIII interaction, since galactose oxidase and periodate oxidation abolish the platelet aggregating activity of bovine factor VIII. The present study shows that these oxidizing substances also induce a degradation of bovine factor VIII, so that the exact role of carbohydrate groups in the aggregation process remains to be established.


Histoacryl tissue adhesive in some types of retinal detachment surgery. Preserved human scleral graft and histoacryl-blue tissue adhesive were used in four cases of retinal detachment surgery to obtain scleral buckling effect and to protect staphylomatous or necrotic scleral areas. The use of histoacryl produced a strong and resistant adhesion between the host and the preserved scleral patch. The postoperative inflammatory reaction was mild and disappeared within one week.


Molecular topology of the photosynthetic light-harvesting pigment complex, peridinin-chlorophyll a-protein, from marine dinoflagellates. The photosynthetic light-harvesting complex, peridinin-chlorophyll a-protein, was isolated from several marine dinoflagellates including Glenodinium sp. by Sephadex and ion-exchange chromatography. The carotenoid (peridinin)-chlorophyll a ratio in the complex is estimated to be 4:1. The fluorescence excitation spectrum of the complex indicates that energy absorbed by the carotenoid is transferred to the chlorophyll a molecule with 100% efficiency. Fluorescence lifetime measurements indicate that the energy transfer is much faster than fluorescence emission from chlorophyll a. The four peridinin molecules within the complex appear to form two allowed exciton bands which split the main absorption band of the carotenoid into two circular dichronic bands (with negative ellipticity band at 538 nm and positive band at 463 nm in the case of peridinin-chlorophyl a-protein complex from Glenodinium sp.). The fluorescence polarization of chlorophyll a in the complex at 200 K is about 0.1 in both circular dichroic excitation bands of the carotenoid chromophore. From these circular dichroic and fluorescence polarization data, a possible molecular arrangement of the four peridinin and chlorophyll molecules has been deduced for the complex. The structure of the complex deduced is also consistent with the magnitude of the exciton spliting (ca. greater than 3000 cm-1) at the intermolecular distance in the dimer pair of peridinin (ca. 12 A). This structural feature accounts for the efficient light-harvesting process of dinoflagellates as the exciton interaction lengthens the lifetime of peridinin (radiative) and the complex topology increases the energy transfer probability. The complex is, therefore, a useful molecular model for elucidating the mechanism and efficiency of solar energy conversion in vivo as well as in vitro.


Histone redistribution and conformational effect on chromatin induced by formaldehyde. Histone redistributions between endogenous DNA in calf thymus chromatin and exogenous DNA from Clostridium perfringens (69% A + T) or from Micrococcus luteus (30% A + T) induced by 0.6 M NaCl or by 2% formaldehyde were studied by thermal denaturation. The observed redistribution occurred on histone Hl when the exogenous DNA was (A + T)-richer than the DNA in chromatin, and when the mixture was exposed to 0.6 M NaCl or formaldehyde. When a (G + C)-richer DNA was added as the acceptor for histones, no substantial transfer of histones from chromatin DNA to exogenous DNA was found. Thus the activation energy of histone dissociation from chromatin DNA seems to be substantially lowered by 0.6 M NaCl or formaldehyde such that histones (mostly histone Hl) can be dissociated and bind the (A + T)-richer DNA and form a more stable complex. It is suggested that the formaldehyde effect on histones may be due to the loss of positive charges on lysine and arginin residues (probably more on lysine than on arginine) in histones after their rapid reaction with formaldehyde. Formaldehyde treatment of chromatin also distorts the DNA conformation, as revealed by circular dichroism (CD) studies. This structural effect occurs mainly on those base pairs bound by histones other than Hl, or within the chromatin subunit. Histone redistribution is treated as a thermodynamic phenomenon of histone binding to DNA. The validity of using formaldehyde to study chromatin structure is discussed.


Terminal residues of hog pancreatic amylase. Studies on the identification of the terminal residues in protease-free hog pancreatic alpha-amylase, prepared by glycogen precipitation, demonstrated the absence of free amino terminals when four different chemical procedures were used. These methods were based on reaction with fluorodinitrobenzene, trinitrobenzenesulfonic acid, dansyl chloride, and cyanate. In the search for the presence of a possible alpha-N-blocking group, an acetyl group was detected as acetic acid dinitrophenyl hydrazide after hydrazinolysis and dinitrophenylation. Quantitation of acetyl groups by a gas chromatographic or a specific enzymatic method yielded 0.7 mol of acetyl group per 51,000 g of protein. Other acyl groups, such as formyl or propionyl, were not found. Leucine was shown to be the carboxyl terminal residue by hydrazinolysis or by carboxypeptidase A digestion of acid denatured amylase. With either procedure, 0.8 mol of carboxyl terminal leucine was found per 51,000 g of protein. These findings are consistent with the proposal that hog pancreatic alpha-amylase is composed of a single, alpha-N-acetylated chain of molecular weight 50,000. Claims of other investigators for subunit and multichain structures for this enzyme are discussed in view of these end group data.


Comparison of human and hamster mitochondrial transfer RNA. Physical properties and methylation status. We have compared mitochondrial transfer RNA from cultured human (HeLa) and hamster(BHK) cells, giving emphasis to distinctive properties previously noted for the BHK system. Both HeLa and BHK mitochondrial tRNA sedimented slower, and had lower mobilities in "warm" acrylamide gels, than their cytoplasmic counterparts, suggesting that both mitochondrial tRNA populations have unusually loose configurations. HeLa mitochondrial tRNA was found to contain 2.8 methyl groups per 100 nucleotides compared to 8.7 for cytoplasmic, figures similar to previous BHK results. Both mitochondrial tRNA populations were notably deficient in 7-methylguanine, methylribose, and methylated pyrimidines. We propose that mitochondrial tRNA (or perhaps animal mitochondrial tRNA) constitutes a distinct evolutionary class.


The effect of 5-bromodeoxyuridine on messenger RNA production in cultured cells. Cells of clone B(5)59, a derivative of the murine B16 melanoma, are highly tumorigenic, and produce both melanin and plasminogen activator. Cells of clone C(3)471, a line obtained by continued growth and maintenance of B(5)59 cells in medium containing 5-bromodeoxyuridine are nontumorigenic, amelanotic, and have no plasminogen activator. Differences in the mRNA complement of these two syngenic mouse melanoma clones have been determined by hybridization kinetics between complementary DNA transcribed from mRNA of either B(5)59 OR C(3)471 cells, and mRNA isolated from both sources. To detect the presence of unique mRNA sequences produced in B(5)59 cells, complementary DNA produced using B(5)59 mRNA as a template was exhaustively hybridized to C(3)471 mRNA. The results indicate that (i) polyadenylated mRNA produced by either clone can be divided into three main groups based upon the relative complexity of mRNA species within each group, (ii) more than 98% of the mRNA species produced by the B(5)59 cells are also produced by the cells of the C(3)471 clone, (iii) approximately 25% of the mRNA species produced by the cells of the C(3)471 clone in moderate abundance (500 molecules per cell) are not produced by the B(5)59 clone. Despite the fact that at least two of the proteins synthesized by B(5)59 cells are not detectable in C(3)471 cells, our results support the hypothesis that the major effect of BrdUrd incorporation into DNA is the induction rather than the repression of transcription of polyadenylated mRNA sequences.


Energization of mitochondrial inner membranes caused by L-malate. It was found that 0.06 mug antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c1 completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.


Hydration sites of egg phosphatidylcholine determined by means of modulated excitation infrared spectroscopy. Conventional and modulated excitation infrared spectra of egg phosphatidylcholine are measured in the spectral range of 4000-900 cm-1 by modulation of relative humidity with an amplitude of +/- less than 5% at a mean value of 75% (T=30 degrees C). From the modulated-excitation spectrum, hydration sites are found to be the greater than PO2 group, the greater than C=O group and the choline group.


The manipulation of the fatty acid composition of Dictyostelium discoideum and its effect on cell differentiation. The fatty acid composition of Dictyostelium discoideum has been modified by growing the axenic strain, Ax-2, in media conta-ning long chain polyenoic fatty acids. Large amounts of linoleic and linolenic acids are incorporated into the cellular lipids and further desaturated to two unusual fatty acids, 5,9,12-octadecatrienoic acid and 5,9,12,15-octadecatetraenoic acid, respectively. Arachidonic acid is also extensively incorporated but not further de;aturated. D. discoideum normally contains none of the above polyenoic fatty acids, and the amount incorporated depends upon the concentration of the fatty acid in the growth media. The cells containing large quantities of polyenoic fatty acid grow normally b,t exhibit impaired differentiation when removed from the growth medium. The incorporation of smaller quantities of the fatty acid has no adverse effect on differentiation. Cells grown in the presence of saturated or monoenoic fatty acids exhibit, at the most, only slight changes in the fatty acid composition of the cellular lipid and both grow and differentiate normally.


[Modification and immobilization of beta-galactosidase]. beta-galactosidase from fungus Curvularia inaequalis was modified by a chlortriazin dye active bright-orange KH. The modified enzyme contained two molecules of dye per one molecule of protein. The incorporation of six sulfuric groups with remains of the dye resulted in a slight decrease of the acid protein isoelectric point. The catalytic activity of the modified protein remains practically unchanged. The coloured protein is firmly absorbed on anionites. Preparations of immobilized beta-galactosidase were obtained by adsorption on anionites.


Studies on experimental hypotrophy in the rat. III. Plasma insulin and glucagon. Insulin and glucagon have been measured in the plasma of rats with intrauterine growth retardation (IUGR) and their littermate controls. A lower plasma insulin and a higher plasma glucagon were observed in IUGR term rat fetuses. After birth there was no difference in the circulating level of insulin and glucagon in IUGR rats and their littermate controls. It is concluded that postnatal hypoglycemia of IUGR rat did not result from an impaired glucagon secretion or an increased insulin secretion.


The flexibility of low molecular weight double-stranded DNA as a function of length. I. Isolation andphysical characterization of seven fractions. Sonicated calf thymus DNA was fractionated by rate zonal centrifugation into seven fractions with weight average molecular weights ranging from 0.28 to 1.3 X 10(6) daltons, as determined by sedimentation equilibrium and light scattering measurements (the latter are described in the accompanying paper). Electron microscopy and sedimentation equilibrium analysis revealed these fractions to be narrowly disperse with Mw/Mn ratios averaging about 1.06. Intrinsic viscosities and sedimentation rates were measured and found to vary linearly with molecular weight in double-logarithmic plots in fair agreement with previously published functions relating these parameters for low molecular weight DNA. Thso agreeing with reported estimates of this parameter for short DNA. These data will be used in the second paper of this series to calculate the persistence length of the DNA fragments in each of the seven fractions by light scattering and hydrodynamic theories for the Kratky-Porod worm-like coil.


The flexibility of low molecular weight double-stranded DNA as a function of length. I. Light scattering measurements and the estimation of persistence lengths from light scattering, sedimentation and viscosity. In the preceding paper are described the isolation and physical characterization of seven narrowly disperse fractions of calf thymus DNA in the molecular weight range 0.3 to 1.3 X 10(6) daltons. Herein, we have determined by light scattering the molecular weights and root mean square radii of these fractions in a solvent comprising 0.2 M NaCl, 2 mM EDTA, 2mM Na-PO4,pH7. Measurements were made in a modified Wippler-Scheibling photometer to a 20 degree lower limit of scattering angle on solutions rendered virtually dust-free by procedures described. The optical anisotropies of the DNA fractions were measured permitting the experimental molecular weights and root mean square radii to be corrected to their true values. From these values, with appropriate polydispersity corrections, we calculate a Kratky-Porod persistence length, a, of 54.0 +/- 5.6 nm which is invariant over the molecular range examined. From the sedimentation coefficients (preceding paper) and the theory of Yamakawa and Fujii, we calculate a to be 66 nm, a value found to apply equally well to several DNA samples of various origins whose sedimentation rates are known in themolecular weight range from about 4 X 10(4) to 10(8) daltons. Similarly, from the intrinsic viscosities and the theory of Yamakawa and Fujii, we calculate a to be 59 nm, which again adequately applies to a number of DNA samples whose viscosities have been measured by other workers in the molecular wieght range 3 X 10(5) to 10(8) daltons. The Flory-Mandelkern paramerter, beta, was found to vary with molecular weight in the manner predicted by the theory of Yamakawa and Fujii. The average value of a from the three sets of measurements is 60 +/- 6nm, which we believe applies to double-stranded DNA molecules, independent of chain length, over the whole range of molecular weights from which reliable data exist.


Calorimetric studies of the interaction between DNA and poly-L-lysine. The transition enthalpy deltaH of the helix-random coil transition of the DNA-polylysine complex was measured as a function of the peptide:nucleotide ratio by the help of an adiabatic scanning differential calorimeter. Furthermore the transition enthalpy of a complex with a specific peptide:nucleotide ratio was determined as a function of the cation concentration of the solution. Finally the reaction enthalpy of the interaction of polylysine with native and denatured DNA was measured with the help of a LKB batch calorimeter. From the results of the calorimetric measurements one can conclude that the transition enthalpy of the DNA-polylysine complexes is linearly dependent on the nucleotide: peptide ratio. The extrapolated value for the 1:1 complex is 14.4 kcal per mole base pairs.


Determination of halothane-induced sleeping time in the rat: effect of prior administration of centrally active drugs. A method is described for the determination of halothane-induced sleeping time in the rat. 2 The sleeping time exhibited a diurnal variation which was due, at least in part, to a change in the sensitivity of the central nervous system (CNS) to the anaesthetic. 3 Tolerance to halothane did not develop in rats repeatedly exposed to the anaesthetic over a period of over 48 hours. 4 Repeated sleeping time determinations have been used to follow changes in the sensitivity of the CNS to the anaesthetic occurring with time. 5 A tolerance to halothane was induced by pretreatment of rats with doses of amylobarbitone, pentobarbitone or meprobamate sufficient to keep animals anaesthetized for approximately 12 hours. This tolerance was followed by a period of halothane-hypersensitivity. 6 Halothane-tolerant animals awakened with higher brain halothane concentrations and were also tolerant to intracerebroventricularly administered pentobarbitone. 7 Halothane-hypertensive rats awakened with lower brain halothane concentrations and were also hypersensitivity to intracerebroventricularly administered pentobarbitone. 8 The possibility that the induction of cross-tolerance to halothane may be indicative of a drug's potential to produce dependence is discussed.


Effects of drugs affecting endogenous amines or cyclic nucleotides on ethanol withdrawal head twitches in mice. 1 Twenty-four hours after ethanol withdrawal, dependent mice exhibited frequent head twitching. Naive mice exhibited similar twitching 15 min after treatment with 5-hydroxytryptophan (5-HTP) or 6 h after alpha-methyl-p-tyrosine (AMPT). Ethanol lessened the incidence of head twitches induced by any of these treatments. 5-HTP and AMPT each increased the incidence of head twitches induced by withdrawal of ethanol from dependent mice. 2 Drugs that affect the amount or activity of endogenous amines or cyclic nucleotides modified the incidence of head twitches. Nearly all drugs acted in the same direction on twitching elicited by any of these three treatments. 3 The incidence was lessened by: (a) methysergide, methergoline, MA 1420, p-chlorophenylalanine and p-chloroamphetamine; (b) dopamine, noradrenaline, L-DOPA, amphetamine and apomorphine; (c) hyoscine and nicotine; and (d) adenosine triphosphate, dibutyryl cyclic adenosine-3',5'-monophosphate (db cyclic AMP) and prostaglandins E1 and E2. 4 The incidence was increased by: (a) acetylcholine, carbachol and physostigmine; and (b) guanosine triphosphate, dibutyryl cyclic guanosine monophosphate (db cyclic GMP), theophylline and 3-isobutyl-1-methyl-xanthine. 5 These findings suggest that head twitching induced by these three treatments arises from a common biochemical mechanism, which may ultimately be a change in favour of cyclic GMP of the balance between this nucleotide and cyclic AMP within appropriate neurones. This imbalance appears to be elicited or increased by 5-hydroxytryptamine and acetylcholine and to be decreased by dopamine, noradrenaline and E prostaglandins. 6 Neither actinomycin D nor cycloheximide, given during the induction of ethanol dependence, altered the incidence of head twitches after ethanol withdrawal.


Effects of contraceptive agents on drug metabolism in various animal species. The effect on liver microsomal enzyme activity of three steroid contraceptive drug (SCD) combinations was compared in rats, mice and guinea-pigs. Lynestrenol plus mestranol, norethisterone plus mestranol and norethynodrel plus mestranol were given orally for 4 consecutive days (acute treatment) or 30 days (chronic treatment) at various doses eliciting an experimentally controlled antifertility activity which varied in its extent. In rats and mice all the combined treatments (with the exception of norethynodrel plus mestranol in mice) were active as inducers of liver microsomal enzymes. This induction seems to be mediated mainly by the progestogenic compounds. Oestrogens showed a very poor effect bordering on significance only in a few cases. No effect on liver microsomal protein or cytochrome P 450 concentration was obtained after treatment with doses capable of increasing the microsomal enzyme activity. The activity of the liver microsomal enzymes did not appear to be reduced immediately (2 h) after the last administration of the SCD given during 4 or 30 days. Contraceptive treatments at doses capable of eliciting complete antifertility activity were inactive on liver microsomal enzyme activity in guinea-pigs.


The behavioural effects of single and combined stressors: a test of arousal theory. Arousal theory assumes that a single physiological dimension underlies the curvilinear (inverted-U) relation of behavioural efficiency to level of stimulation. Traditional tests of this assumption, which involved correlating different physiological measures of arousal, have produced equivocal results. The present study predicted that, if arousal is unitary, then stressors which separately induce it should be additive in their effects on behavioural efficiency. Although curvilinear changes in behavioural efficiency were found for white noise and shock separately, their combination failed to support the prediction.


Combined one- and two-dimensional ultrasound system for monitoring fetal breathing movements. A combined one- and two-dimensional ultrasonic system for monitoring respiratory movements in the human fetus has been developed. A real-time cross-sectional image of the fetal chest at the level of the fetal heart can be obtained, and a time motion recording of fetal respiratory movements can then be written on a strip-chart recorder. Combining the features of one-dimensional and two-dimensional systems produces an accurate means of investigating fetal breathing movements.


Actions of opiates upon single unit activity in the cortex of naive and tolerant rats. The effect of microelectrophoretically and systemically applied opiates on neuronal discharge activity in the sensorimotor cortex of naive and morphine tolerant/dependent rats has been studied. In naive rats depression of spontaneous discharge activity was the predominant effect of low doses of phoretically applied morphine. Higher doses and repeated application frequently converted this effect into excitation. Only the depressant effect was antagonised by naloxone. Naloxone itself had no effect on spontaneous discharge activity when applied at dose-levels sufficient to antagonise the depressant effect of morphine. Levorphanol mimicked the action of morphine whereas dextrorphan was inactive. Morphine depressed the excitatory action of L-glutamate and of acetylcholine by a naloxone-antagonisable mechanism. Systemic application of Fentanyl mimicked the inhibitory effect of phoretically applied morphine upon transcallosally evoked discharge activity. The late response was markedly depressed whereas the primary response was little affected. Phoretically applied naloxone antagonised the effects of systemically applied Fentanyl. In chronically morphinised rats the depressant effect of microelectrophoretically administered morphine was almost lacking and a naloxone-resistant excitation became the predominant effect. In these animals the excitant effect of naloxone was also increased and the anti-glutamate effect and the anti-acetylcholine effect of morphine was abolished. The present data speak in favour of a postsynaptically located stereospecific receptor which mediates the inhibitory effects of opiates and which may be involved in the development of acute and chronic tolerance to these drugs.


Retrograde amnesia after electrical stimulation of the substantia nigra: mediation by the dopaminergic nigroneostriatal bundle. Stimulation of the zona compacta of the substantia nigra (SNC) has been reported to impair the long-term retention of a passive avoidance step-down task. In the present experiments it was observed that previous ipsilateral lesions of the dopaminergic nigro-neostriatal bundle (NSB) with 6-hydroxydopamine blocked the stimulation-induced memory deficit. Unilateral or bilateral lesions of the dopaminergic NSB did not by themselves disrupt acquisition or retention of the passive avoidance task. In addition, the memory deficit observed after electrical stimulation of the caudate-putamen was not reduced by ipsilateral NSB lesions. These results indicate that the retention deficit produced by stimulation of the SNC was mediated by the dopaminergic NSB. However, in view of the fact that lesions of this projection did not affect acquisition or retention of the step-down task, it is suggested that this system is not critically involved in learning and memory. Rather, it is hypothesized that stimulation of the SNC results in an excess release of dopamine in the caudateputamen which interferes with neurochemical and neurophysiological events in this structure which are important substrates for long-term memory.


Comparison of the structure of hyperactive behavior in rats after brain damage from x-irradiation, carbon monoxide and pallidal lesions. Nocturnal hyperactivity, as measured by photocell counts of locomotion in a residential maze, was produced in rats by 3 different kinds of brain damage, X-irradiation at gestational day 14 or 15, exposure to carbon monoxide on the fifth day of postnatal life, or bilateral stereotaxic lesions of the globus pallidus in adult rats. These brain-damaged rats and their controls were photographed at 1 frame/sec during their first exploratory experience in a simple cage. Frequency of 15 motor acts, duration of each occurrence and associations of pairs of acts were calculated. The 15 motor acts were divided into 3 clusters of acts labeled grooming, exploratory and attention behaviors. Hyperactivity was associated with shortened durations and increases in frequency of exploratory acts, while grooming and attention behaviors tended to decrease in duration and frequency. Sequences of behavior acts were less structured in hyperactive animals than in controls. In spite of the marked differences known to be produced on brain structures by the 3 different kinds of damage, no changes in behavior structure were found which were uniquely associated with one kind of brain damage. Hyperactivity appeared to be a continuum in that the intensity of effects produced on behavior as measured by the photographic technique correlated well with the amount of increase in photocell activity.


Endosymbionts of Sitodrepa panicea.

Endosymbionts of Sitodrepa panicea. Morphological and physiological characteristics of seven strains of yeast-like symbionts isolated from Sitodrepa panicea justify their inclusion into the genus Torulopsis as a new species: T. buchnerii. The symbiotic relationship is mutually beneficial: the symbionts obtain some nitrogenous compounds and carbohydrates, such as proline and trehalose from the host's hemolymph, and synthesize and make available to the host all the essential amino acids and vitamins, except biotin.


[An agar medium for direct enumeration of Staphylococcus aureus: pork plasma medium for S. aureus (PPSA)]. A selective agar medium (pork plasma medium for S. aureus (PPSA)) enables the direct enumeration of coagulase-positive staphylococci. This medium is based on the Baird-Parker agar without egg yolk and is supplemented with pig plasma. Colonies of Staphylococcus aureus are surrounded by a halo of precipitated fibrin. When foods such as dairy products contain large numbers of egg yolk-negative strains of S. aureus, the PPSA agar has the advantage over egg yolk containing media such as Baird-Parker agar that fewer suspect colonies have to be confirmed.


Sexual hormone in the cellular slime mould Dictyostelium purpureum. The existence of sexual hormone in Dictyostelium purpureum was revealed when the extracelluar medium from certain strains (Dp6 or Dp7) induced macrocyst formation when added to cells of the opposite mating type (Dp2). Our results suggest that mating in cellular slime moulds may involve a secreter-responder system whereby one mating-type strain (Dp6 or Dp7) releases sexual hormone while the opposite strain (Dp2) responds. However, the existence of a hormone released by the responding strain has not been completely ruled out by our experiments. The sexual hormones of cellular slime moulds appear to be species-specific since hormone from D. purpureum cannot induce macrocyst formation in responder strains of D. discoideum and hormone from D. discoideum has no effect on D. purpureum cells.


Interaction between Pseudomonas pseudomallei and cultured rabbit peritoneal macrophages. The interaction between two strains of Pseudomonas pseudomallei of different virulence with normal and immune rabbit peritoneal macrophages was compared in vitro. Phagocytic activity of macrophages and bacterial survival within macrophages were dependent upon the virulence of the bacterial strain and the immune status of the macrophages. Virulent bacteria were more resistant than the less virulent strain to phagocytosis and destruction. Immune macrophages were more phagocytic and bactericidal than normal macrophages. Specific immune serum facilitated ingestion and destruction of bacteria by both normal and immune macrophages.


Hyperactive behavior of rats after lesions of the globus pallidus. Following bilateral lesions of the globus pallidus, rats living in a residential maze were hyperactive during the 12 hr dark cycle but not during the 12 hr light cycle. Lesioned rats were less exploratory during the light cycle than control rats but not during the dark cycle. Exploratory behavior of rats was photographed for 15 min during the light cycle. The duration of 6 behavior acts was significantly shorter than controls (scratching, grooming, sitting, sniffing, standing and rearing). The number of initiations of grooming, scratching, sniffing and smelling decreased while looking and walking increased in frequency. The linkage of behavior acts into sequences was diminished compared with controls. Similar, but not identical, changes were found when the structure of exploratory behavior of naive rats was compared with the exploratory behavior of experienced rats. It is concluded that naive control rats are hyperactive relative to experienced rats in this exploratory situation and that rats with pallidal lesions display changes in their behavior which are characteristic of hyperactive animals even when other tests under similar conditions, such as exploration during the light cycle in a maze, show the pallidal rats as hypoactive relative to control rats.


Degradation of [14C]photodieldrin by Trichoderma viride as affected by other insecticides. Various soil fungi were tested for their capacity to degrade the insecticide [14C]photodieldrin. Of nine species investigated, Trichoderma viride was the only one which degraded the insecticide to an appreciable extent into water-soluble, non-insecticidal compounds within 4-5 weeks. These products amounted to 32-41% of the radiocarbon applied to the culture media. The degradation was a function of live mycelia, which metabolized the insecticide and excreted water-soluble compounds into the culture media. Since soils usually contain a mixture of pesticide residues, the effects of several chlorinated hydrocarbon insecticides on the capacity of the fungus to degrade [14C]photodieldrin were studied. Thus, in fungal cultures treated with compounds structurally similar to photodieldrin, such as aldrin and dieldrin, only 4-17% of the applied radiocarbon was water-soluble and more photodieldrin remained. In controls, however, 35% of the applied radiocarbon was in the form of water-soluble products and less photodieldrin remained. The degradation of [14C]photodieldrin by T. viride, with time, was associated with a continuous decline of hexane-soluble radiocarbon and a steady increase of water-soluble metabolites, which appeared in the fungal media. The amount of hexane-soluble radiocarbon in mycelia was directly related to the fungal mass.


Pyrimidine nucleotide metabolism in L5178Y murine leukemia cells: deoxycytidine protection from deoxyguanosine toxicity. Deoxyguanosine was found to inhibit reduction of uridine phosphate within L5178Y cells. A correlation of this effect with deoxyguanosine-induced inhibition of radioactive incorporation into DNA and inhibition of L5178Y cell growth suggests that depletion of thymidylate pools is involved in the toxicity induced by deoxyguanosine. Deoxycytidine appears to protect from deoxyguanosine toxicity by metabolism to thymidylate via a dCMP deaminase pathway, thus replenishing the thymidylate pools which have been depleted by deoxyguanosine.


Spontaneous occurrence of tumors in laboratory-reared arvicoline rodents. Data from more than 2500 laboratory-reared rodents of four arvicoline genera raised since 1966 indicated that the incidence of neoplasia increased spontaneously in a nearly linear manner from 1968 to 1974. The incidence was highest in Clethrionomys rutilus, with an age-adjusted rate of 32 tumors/100 animals in 1974. The various species of Dicrostonyx exhibited dissimilar rates (22 tumors/100 animals, average for 1972 to 1973) with lower rates observed in the genera Microtus and Lemmus. Tumors of epithelial origin predominated with four common types accounting for 69% of the 260 tumors observed. A trend toward development of multiple primary tumors corresponded with the increase in incidence. An agent, identified as an adenovirus, was isolated from Harderian and preputial gland adenocarcinomas, and a second virus, tentatively identified as a hamster type H partcle, was observed in mammary and salivary gland tumors of Dicrostonyx stevensoni and C. rutilus, respectively. These and other data presented suggest an infectious etiology.


Regulatory controls of oncotrophoblast proteins and developmental alkaline phosphatases in cancer cells. The two oncotrophoblast proteins, Regan isoenzyme (placental-type alkaline phosphatase) and human chorionic gonadotrophin, are readily studied oncodevelopmantal gene products in human cancer patients and in three experimental model systems. The latter consists of (a) HeLa sublines TCRC-1 and TCRC-2, which produce Regan and non-Regan isoenzymes, (b) HEp-2 and FL amnion cell lines as models for the reciprocal expression of developmental genes, and (c) modulation in vivo of developmental gene expression in HeLa cells. In the case of the third model, for example, HeLa TCRC-1 cells grow in immunosuppressed rats to form a tumor nodule, which expresses a new oncoamnion (FL) isoenzyme, while the Regan isoenzyme disappears. Return of the tumor cells to cell culture medium results in a disappearance of the oncoamnion (FL) species and the reappearance of Regan isoenzyme. This interesting model is expected to bridge the interpretation of experiments done in cell culture with observations made on tumors of cancer patients. Most helpful in interpretation of all these studies has been a chronology of early development. It appears that the counterparts of a number of tumor proteins appear as early as gametogenesis and as late as 10 weeks of gestation.


A mechanism of estrogen action on gene expression at the level of translation. One of the earliest events following the administration of estradiol to ovariectomized rats is an increase in transfer RNA (tRNA) methyltransferase activity activity of the uterus. Ovariectomy diminishes by about one-half the capacity of extracts of uteri to introduce methyl groups in vitro into heterologous tRNA. Administration of estradiol to ovariectomized animals restores the tRNA methyltransferase capacity to normal. Alterations in the population of uterine serine tRNA were also observed following ovariectomy. These findings represent reversible qualitative changes produced by a hormone in the target tissue. Whether the tRNA's are involved in regulating hormone-induced protein synthesis was investigated by studying the estrogen-induced ovalbumin synthesis in immature chick oviducts. tRNA's from oviducts of estrogen-stimulated chicks or from oviducts of laying hens produced an enhanced stimulation of ovalbumin synthesis in vitro compared with tRNA's from oviducts of immature chicks that were withdrawn from estrogen stimulation (these chicks do not synthesize ovalbumin). Some of these hormone-induced alterations may be of importance in understanding the molecular mechanisms of estrogen action on gene expression. During these studies, we have observed a novel effect of the administration of the carcinogen, ethionine, in the immature chicks. Ethionine induces ovalbumin and conalbumin synthesis in immature chick oviducts that were withdrawn from estrogen for 3 to 4 weeks following primary estrogen stimulation.


Inhibitory and cytotoxic effects of Oncovin (Vincristine sulfate) on cells of human line NHIK 3025. The inhibitory effect of Oncovin (vincristine sulfate) on cell division was studied in human cell line NHIK 3025. Oncovin arrested the cells in metaphase at concentrations as low as 10(-3) mug/ml. At 8 X 10(-3) mug/ml and higher concentrations, the arrest was complete after 6 hr of treatment. The arrest was irreversible after exposure for 6 hr to 16 X 10(-3) mug Oncovin per ml. The X-ray radiosensitivity of aerobic cells of the same line pretreated with 16 X 10(-3) mug Oncovin per ml for 6 hr (Oncovin removed before irradiation) was found to be about equal to that of untreated cells. Even when present during irradiation, Oncovin did not exert any modifying effect on the radiosensitivity of either aerobic or extremely hypoxic cells.


Spontaneous cell loss during growth of postconfluent primary cultures from mammary adenocarcinomas. Growth properties of cells cultured from primary mammary tumors of C3H mice have been analyzed. Cells were seeded at 2 different densities (1 X 10(5) and 5 X 10(5)/sq cm) and were supported with a culture fluid containing 10% fetal calf serum and 5 mug insulin per ml. Mitosis continued after confluence was achieved, but cells did not accumulated in the monolayer; rather, certain cells were released into the culture fluid. Very few cells detached in this way from subconfluent cultures. Relased cells multiplied vigorously if replated. The release of these cells was strongly depressed by adrenal steroids, but other manipulations of culture conditions (hormones, culture substratum) influenced the release process much less. Analyses of release kinetics and observations of detachment with the scanning electron microscope suggested that tumor cells that became spheroid (including mitotic cells), and hence partly detached from the culture dish, were unable to reflatten into the monolayer because neighboring nonmitotic cells had spread onto the vacated culture surface. Eventually, such rounded cells apparently lost altogether their attachment to the culture dish. The release process may be related to the "critical phase" transition and to the sarcomatous transformation observed in long-term cultures from mouse epithelial tumors. The event could also reflect the tendency in vivo for cells of mammary tumors to slough into the lymphatics and blood vessels.


Inhibition of ribonucleotide reductase activity and nucleic acid synthesis in tumor cells by the dialdehyde derivatives of inosine (NSC 118994) and inosinic acid. Periodate-oxidized inosine (Inox; NSC 118994) and periodate-oxidized 5'-inosinic acid (PI-IMP) were prepared and studied for their effects on ribonucleotide reductase activity in partially purified extracts from Ehrlich tumor cells and on nucleic acid synthesis in intact tumor cells in culture. Ribonucleotide reductase activity in cell-free extracts from Ehrlich tumor cells was inhibited by Inox and PI-IMP. PI-IMP was more inhibitory to the reductase activity than was Inox. Furthermore, the inhibition of ribonucleotide reductase activity by Inox and PI-IMP was greater for cytidine-5'-diphosphate reductase activity than for adenosine-5'-diphosphate reductase activity. The ribonucleotide reductase activity in cell-free extracts prepared from Ehrlich tumor cells treated with Inox or PI-IMP in culture was decreased compared with the activity in the extracts from untreated cells. Incorporation of labeled cytidine into the RNA and DNA of Ehrlich tumor cells in culture was inhibited by both Inox and PI-IMP. The conversion of cytidine to deoxycytidine nucleotides in the acid-soluble pool was likewise inhibited. These data indicate that Inox and PI-IMP inhibit the ribonucleotide reductase step as one of the sites of action of these compounds. However, the inhibition of RNA synthesis indicates that there must be additional sites of action of these nucleoside analogs.


Studies on the mechanism of diurnal variation of proliferative indices in the small bowel mucosa of the rat. In the rat small bowel mucosa significant variation was found in both the labelling and the mitotic indices with time of day. The zenith and the nadir of labelling and mitotic activity coincided at 15.00 and 02.00 hours respectively. Small changes were found in the "cut-off" position, but this variation in proliferative compartment size was insufficient to account for the comparatively wider fluctuations in proliferative indices. Measurements of the rate of entry into mitosis, using metaphase arrest with vincristine at three widely separated times during the day, showed no significant change. Changes in the growth fraction or in the birth rate as measured cannot account for diurnal variation in the proliferative activity of the small bowel mucosa. We propose a hypothesis which involves diurnal fluctuations in the transit times through G1 and through G2.


A light and electron microscope study on oogenesis in the freshwater pulmonate snail Biomphalaria glabrata. In the freshwater snail Biomphalaria glabrata the formation and composition of yolk granules and the role of the follicle cells were studied by histochemical and electron microscopical techniques. The rough endoplasmic reticulum and the Golgi apparatus appeared to be involved in yolk formation, which is a continuous process throughout oogenesis. From the very beginning of yolk formation two main types of yolk granules were distinguished morphologically. However, with histochemical and enzyme cytochemical methods no differences were observed between these types. The granules acquire lysosomal enzymes after oviposition, indicating that their main function is probably digestion of perivitelline fluid, which contains nutrients for the developing embryo. Yolk formation and the activity of the follicle cells were studied in successive stages of oogenesis by quantitative electron microscopy. The data strongly suggest that the follicle cells are involved in the formation of the follicular cavity and hence in the ovulation process.


The programmed switch in lysine-rich histone synthesis at gastrulation. The pattern of histone synthesis changes during development in sea urchins. One change involves two different lysine-rich histones. In Lytechinus, the late appearing histone, H1g, begins to be synthesized at gastrulation. Preformed ("maternal") mRNA of unfertilized eggs contains sequences that direct the synthesis of a part of the histone made during the period of cleavage. During that interval a different lysine-rich hsitone, H1m, is produced. Maternal mRNA is not, however, the sole source of templates for histone synthesis. Transcription of embryonic genomes provides the major translate both maternal and embryonic messages. A cell-free system derived from wheat germ was used to test RNAs from unfertilized eggs and from post-gastrula embryos for their capacity to direct the synthesis of histones, including the H1 types in vitro. Maternal mRNA includes templates for the synthesis of H1m, but it appears to lack those for H1g, since the cell-free system yields only H1m when challenged wigh egg RNA. Since the cell-free system is capable of translating H1g mRNA when presented with it, the synthesis of H1g is probably a very early developmental event controlled at the level of transcription.


Chromatin structure as probed by nucleases and proteases: evidence for the central role of histones H3 and H4. We have examined the role by each histone in forming the structure of the nu-body. When DNAase I, DNAase II, trypsin and chymotrypsin attack chromatin, characteristic discrete DNA and protein digest fragments are produced. Using this restriction of accessibility as diagnostic for chromatin structure, we have examined complexes of DNA with virtually all possible combinations of histones. The results strongly support our previous conclusion (Camerini-Otero, Sollner-Webb, and Felsenfeld, 1976) that the arginine-rich histones are unique in their ability to create, with DNA a structure with many features of native chromatin. Acting together, slightly lysine-rich histones then modify this complex into one very similar to native chromatin. An analysis of the rate constants of staphylococcal nuclease digestion also confirms that the complex of H3, H4, and DNA is crucial to the structure of the nu-body.


Characterization of two different alkaline phosphatases in mouse teratoma: partial purification, electrophoretic, and histochemical studies. Alkaline phosphatase (E.C.3.1.3.1.) has been used as a marker for embryonal carcinoma cells which constitute the multipotential stem cells of the mouse teratoma. Studies by other investigators based on kinetics of thermal inactivation and L-phenylalanine inhibition have shown that the alkaline phosphatase of the teratoma differs from the mouse intestinal and liver isozymes, but resembles the isozymes of kidney and placenta. Since functional characterization of nonpurified enzymes is not the most accurate means for distinguishing different molecular forms of an enzyme, we have partially purified the enzymes from the ascitic (embryoid body) and solid tumor forms of the OTT-6050 teratoma line, and utilized the technique of electrophoresis in polyacrylamide gels to compare the teratoma enzyme with isozymes from kidney and placenta. Covalent 32PO4-labeling of the alkaline phosphatases and polyacrylamide gel electrophoresis in sodium dodecylsulfate was also used to compare the subunit molecular weights of the enzymes. The results indicate that the mouse teratoma enzyme is distinct from the kidney and placental isozymes. Since histochemical studies have localized the enzyme to the stem cell population of the teratoma, the results imply that stem cell alkaline phosphatase is a distinct isozyme. The embryoid bodies contain a second alkaline phosphatase which may correspond to the placental isozyme. This enzyme may be attributed to the outer cell layer of embryoid bodies of the ascitic tumor, since this cell type histochemically demonstrates alkaline phosphatase activity.


On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes. In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972; Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly (A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70 degrees C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly (A)- RNA (7S-14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.


Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei. We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array. Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA. Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.


Prenatal diagnosis of 45,X/46,XY mosaicism with postnatal confirmation in a phenotypically normal male infant. Prenatal detection of chromosome mosaicism has always been a diagnostic dilemma. In 21 reported cases of chromosomal mosaicism in cultured amniotic fluid cells, only two cases had cytogenetic confirmation of the mosaicism. All 21 pregnancies resulted in either phenotypically normal liveborns or grossly normal abortuses. We report a case of XO/XY mosaicism detected prenatally and confirmed postnatally in a grossly normal male infant. The indication for prenatal cytogenetic diagnosis was advanced maternal age (38 years). A diagnosis of XO/XY mosaicism was made from two separate culture flasks of amniotic fluid cells, with 45,X cells predominating (86.4%). The Y chromosome was of normal size but carried no fluorescent band. The parents were counseled and were advised that the phenotype of XO/XY mosaicism can range from relative normality to sexual maldevelopment. They decided to continue this pregnancy. The infant was born at term and was a grossly normal male with normal penis and descended, normal-sized testes. Leukocyte culture from the cord blood and a skin fibroblast culture confirmed the mosaicism of XO/XY. The father's Y chromosome was of identical size and carried a small fluorescent band. It appears that an altered Y chromosome may be predisposed to anaphase lag leading to mosaicism.


HL-A antigens and sacroiliitis.

HL-A antigens and sacroiliitis. Lymphocyte typing for HL-A B27 is useful under conditions in which data based on history and examination are suggestive but not diagnostic of a seronegative spondyloarthropathy. The presence of HL-A B27 would increase the probability of a patient's having one of these entities, but its absence does not rule out such a diagnosis. Furthermore, tissue-typing allows one to predict the probability that a patient with inflammatory bowel disease will develop AS and the likelihood that the family member of a patient with AS will develop a related disease.


Juvenile rheumatoid arthritis.

Juvenile rheumatoid arthritis. JRA is a multisystem disease characterized by exacerbations and remissions. Recognition of the three modes of onset can important in early diagnosis, identification of extra-articular manifestations, and prognosis. This is a chronic illness that requires consideration of the whole patient, his family, and his place in society, both in the present and in the future. With a positive, comprehensive approach and an awareness of the pitfalls of seeking immediate relief, the physician can help to assure a good functional outcome.


High voltage electron microscopical analysis of chromosomal number in the slime mold Echinostelium minutum de Bary. Utilizing serial thick sectioning techniques in combination with high voltage electron microscopy and stereopair analysis a count of 124 chromosomes was determined for a metaphasic plasmodial nucleus of the slime mold E. minutum.


Luteinizing hormone requirements for ovulation in the pentobarbital-treated proestrous rat. The LH requirements for ovulation in the pentobarbital-blocked proestrous CD rat have been studied by increasing serum gonadotropin levels through electrical stimulation of the brain and subsequently comparing the effects of timed hypophysectomy on ovulation and serum LH concentrations. The arcuate nucleus (ARC) or the medial preoptic area (POA) was stimulated unilaterally for 45 min with matched pairs of biphasic rectangular pulses through a coaxial platinum electrode. Serum LH was significantly elevated above basal values at the end of stimulation, but not in sham-stimulated controls. The results of both hormone measurement and hypophysectomy showed that the pituitary continued to release LH after extrinsic stimulation of the hypothalamus had ceased. Animals did not ovulate if they had been hypophysectomized at the end of the 45 min stimulation whereas nearly all ovulated if hypophysectomy was delayed for an additional 20 min. Some evidence suggested that the pituitary could be removed earlier without affecting ovulation if the rate of LH release was increased. The minimum peak LH concentration measured in rats that subsequently ovulated fully was 187 ng/ml, substantially lower than concentrations ordinarily attained in the spontaneous proestrous surge. When serum LH was insufficiently high to cause follicle rupture, there was nevertheless the resumption of meiosis and luteinization of the large ovarian follicles. Attempts were made to restore ovulability in animals presumed to have released a subovulatory quota of gonadotropin. Ovulation was obtained when such animals, prepared by hypophysectomy after the 45 min stimulation, had been bilaterally nephrectomized prior to stimulation. However, multiple injections of progesterone after hypophysectomy were without effect. The results are discussed in relation to variables that affect minimum requirements of LH for ovulation.


Dexamethasone: increased weights and decreased [3H] estradiol retention of uterus, vagina and pituitary in the ovariectomized rat. Ovariectomized rats given 100 mug dexamethasone per day for 5 days had significantly heavier dry weights for uterus, vagina and pituitary, indicating a growth promoting activity of dexamethasone on these tissues in which estrogen normally promotes growth changes. The dexamethasone treated animals also retained significantly less [3H]estradiol per mug dry weight of tissue for uterus, vagina and pituitary. When[3H]estradiol retention was examined in vitro for the nuclear fraction, a significant decrease in retention was found for uterus, vagina and pituitary but not for hypothalamus or cerebral cortex. The decreased ability to bind [3H]estradiol, shown by the estrogen target tissues of the dexamethasone-treated rats, along with the increased growth of the estrogen target tissues, demonstrates that these tissues were able to show trophic responses even when greater levels were one-third of normal. Dexamethasone-treated animals tested for sexual receptivity in the presence or absence of progesterone priming did not show induction of facilitation of sexual receptivity. However, estrogen plus progesterone injections induced sexual receptivity in the presence of dexamethasone. When dexamethasone was combined with a dosage of estrogen, which by itself did not induce sexual receptivity, there was a significant response with 6 to 10 animals showing a low level of receptivity. Thus, dexamethasone can apparently synergize with estrogen to facilitate sexual receptivity.


Gonadotropic responses of male mice to female urine. Five experiments consistently demonstrated the existence of a factor(s) in the urine of female mice which stimulates gonadotropin secretion in male mice. The factor, presumably a priming pheromone, is not found in the urine of intact or castrated males nor is it in the urine of female hamsters. Furthermore, the potency of female mouse urine is apparently independent of ovarian state. In the response of the male, the release of LH is more dramatic than that of Fsh. as little as 10 min of exposure to female urine produced significantly elevated LH levels at 30 min. Finally, the male's responsiveness is largely, and possibly entirely, independent of his sexual experience. These results are discussed within the framework of a two-way pheromonal system by way of which urinary factors of both sexes modulate gonadotropin and gonadal hormone secretion in the opposite sex.


Effect of luteinizing hormone on the pattern of steroid production by preovulatory follicles of pregnant mare's serum gonadotropin-injected immature rats. Preovulatory follicles were explanted on the day before ovulation from immature rats given a single injection of Pregnant Mare's Serum gonadotropin (PMS) 2 days earlier. The follicles were incubated for 4 h in modified Krebs bicarbonate buffer containing glucose and albumin in absence or presence of ovine luteinizing hormone (NIH-LH-S18; 0.1-10 mug/ml). The accumulation of progresterone, androstenedione and 17beta-estradiol in the medium was determined by radioimmunoassay. As in indicator of LH exposure the meiotic stage of the follicle-enclosed oocyte was determined at recovery by interference contrast microscopy. The first group of follicles were explanted in the morning, before the endogenous gonadotrophin surge. In hormone-free medium the oocytes remained in the dictyate stage, whereas addition of LH induced oocyte maturation. These follicles, when incubated in hormone-free medium, secreted predominantly androstenedione and estradiol and only low amounts of progesterone. In the presence of LH the secretion of all steroids was enhanced. The second group of follicles were explanted in the evening, 2-4 h after the endogenous gonadotrophin surge. After incubation in hormone-free medium the follicle-enclosed oocytes had matured. The steroid secretion by the follicles was different from that of the first group. In hormone-free medium they secreted predominantly progesterone and low amounts of androstenedione and estradiol. Addition of LH to the medium caused further enhancement of progesterone secretion, but had no effect on androstenedione and estradiol secretion. The third group of follicles were explanted in the evening from rats in which the preovulatory gonadotrophin surge had been prevented by Nembutal treatment. Oocyte maturation and steroid secretion did not differ from that found for the first group of follicles explanted in the morning. The results are compatible with the hypothesis that LH, after a transitory stimulation, inhibits androgen and estrogen secretion and stimulates progesterone secretion by the preovulatory ovarian follicle.


Steroid metabolism in fetal tissues. III. Conversion of testosterone to 5alpha-dihydrotestosterone in human fetal gonads. Testes and ovaries of 25 human fetuses obtained between the 17th--40th week of gestation were examined in vitro for 5alpha-reductase activity by a double isotope method. In 14 fetal testes, conversion of testosterone to 5alpha-dihydrotestosterone ranged between 1.04 to 4.26% (2.80% +/- 0.31 SEM) and in 11 fetal ovaries a conversion rate of 0.20 to 2.91% (1.09% +/- 0.22 SEM) was found. The difference was statistically significant (p less than 0.001). This was also confirmed by studying the gonads of twins. The data support previous studies indicating that human fetal testes and ovaries are different in their steroid metabolic activities. Age dependency could not be demonstrated.


Crosslinking with bifunctional reagents as a means for studying the symmetry of oligomeric proteins. A method based upon the principle that unlike domains of bonding are reflected in different reactivities and distribution of residues that can be crosslinked, has been elaborated for the determination of symmetry of oligomeric proteins. The derivation of theoretical curves for the prediction of crosslinking patterns of tetramers produced by reaction with a bifunctional reagent and subsequent sodium-dodecylsulphage-gel electrophoretic analysis, is presented. Based upon the theory the symmetry properties of a tetramer, to the extent whether it is an isologous or heterologous association, can be deduced by a simple calculation. Crosslinking patterns obtained with rabbit muscle aldolase and pig muscle lactate dehydrogenase after treatment with a series of diimidoesters of increasing chain length are evaluated and shown to be consistent with the expectations for isologous tetramers. From the patterns obtained with the various reagents the distances between lysyl residues located nearest to each other in different subunits in the two proteins could also be determined.


Organisation of genes for ribosomal RNA in Physarum polycephalum. Physarum polycephalum nucleolar satellite DNA has been analysed by restriction enzyme digests and hybridisation to ribosomal RNA. The nucleolar DNA is isolated as molecules of molecular weight 39 X 10(6). The positions of sites of endo R-EcoRI and endo R-HindIII digestion have been determined accurately in the whole molecule. The endo R-EcoRI sites and one endo R-HindIII site are within the26-S rRNA complementary sequence and the other endo R-HindIII site is within the 19-S rRNA complementary sequence. The sites are arranged symmetrically about the centre of the molecule as if it were a palindrome.


D-penicillamine therapy in ABO hemolytic disease of the newborn infant. This study comprises 120 full-term infants with ABO hemolytic disease admitted to the neonatal department during a period of 60 months from 1970-1975. During the first 30 months newborns (n = 61) received no D-penicillamine therapy, whereas all infants (n = 59) received this treatment (300-400 mg/kg/day, divided into 4 equal doses, for 2-5 days) during the last 30 months. The patients were further subdivided into two groups according to the point of time when D-penicillamine treatment was begun, viz. group I (34 treated and 34 control infants) within the first 24 h of life; group II (25 treated and 27 control infants) after the third day of life. In group I D-penicillamine therapy caused a marked decline of serum bilirubin concentrations at a time when such levels were rising in the control infants. The number of exchange transfusions per infant was 1.32 in the control and 0.11 in the D-penicillamine-treated infants. In group II D-penicillamine considerably reduced the number of exchange transfusions (0.70:0.24 = control:treated) but the difference was statistically not significant. In the latter patients the mean bilirubin values showed a smaller difference compared to the controls than in group I. Since group I represented the results of early or preventive treatment, while group II those of late or therapeutic treatment, it is obvious that, for ensuring success, D-penicillamine treatment should be begun as early as possible in ABO hemolytic disease of the newborn.


Chondrodysplasia punctata--rhizomelic form. Pathologic and radiologic studies of three infants. Pathologic, ultrastructural and radiologic studies are described on 3 infants with the rhizomelic form of chondrodysplasia punctata. Radiologic criteria in the young infant include radiolucent coronal clefts dividing all or most of the thoracic and lumbar vertebral bodies, short humeri with flared metaphyses and punctate calcifications commonly present adjacent to the ossified ischial and pubic bones and less commonly in other locations. In late infancy and childhood the radiologic criteria include demineralization in all bones with slow maturation, flat vertebral bodies, short humeri and femora, metaphyseal flaring, especially in the distal humerus, proximal femur and proximal tibia, immature shape of pelvis, and disappearance of the punctate calcifications with advancing age. The histologic changes of the resting cartilage include areas of degenerating cartilage which had become partially calcified, cystic changes with severe disturbance of the maturation of the cartilage at the physial plate, and the formation of cancellous bone directly on resting cartilage. Ultrastructural changes are characterized by degeneration of chondrocytes, delicate collagenous fibrils without visible periodicity, and the presence of flocculent material within greatly distended endoplasmic reticulum.


Effect of the female sexual cycle of the activity of delta-aminolaevulinate synthase in rat liver. The activity of delta-aminolaevulinate synthase was determined in the livers of fed female rats and was found to vary with the phase of their sexual cycle. The highest enzymic activity was obtained during preoestrus (38.0 +/- 3.2 nmol delta-aminolaevulinateg.g liver.-1 hour-1), the lowest during dioestrus (17.4 +/- 3.4 nmol delta-aminolaevulinate.g liver.-1 hour-1). During oestrus and metoestrus the activity of the enzyme was intermediate. The oscillation of the enzymic activity might be significant in the occurence of the ovulocylic form of acute intermittent porphyria.


Effect of cholingeric drugs on methadone-induced catalepsy and stereotypies in rats treated chronically with methadone. The effects of antimuscarinic (atropine, scopolamine, methylscopolamine), muscarinic (RS86, pilocarpine), antinicotinic (mecamylamine, hexamethonium) and nicotinic (nicotine) cholinergic drugs on the catalepsy and stereotypies induced by acute methadone in rats treated chronically with methadone were studied. The antimuscarinic drugs potentiated and the muscarinic drugs antagonized the cataleptic effect of methadone, whereas the antimuscarinic drugs tended to antagonize and the muscarinic drugs potentiated the methadone-induced stereotypies. Nicotine initially slightly potentiated, and mecamylamine antagonized the cataleptic effect of methadone. The actions of the cholinergic drugs on the extrapyramidal motor effects of methadone were most probably central, because methylscopolamine and hexamethonium had only very weak actions. These results show that the effects of antimuscarinic and muscarinic drugs on the catalepsy and stereotypies induced by methadone are opposite to their effects on the catalepsy and stereotypies produced by drugs which are thought to act on the postsynaptic dopaminergic receptors.


Characterization of the estrogen receptors in the uterine and blood eosinophil leukocytes. Estrogen receptors are found in the rat uterine and in the eosinophil-rich human blood leukocyte 24,000 g fractions, but not in the low-eosinophil count human blood leukocyte 24,000 g fraction. The total number of binding sites per blood eosinophil leukocyte is 7,400 sites per cell, and the Kp=5.6 X 10(-10) M.


Increased aggression in rats after withdrawal of long term used oxazepam. The withdrawal of oxazepam (5 mg/kg i.p.) applied for 1 year in rats, increased shock-induced aggression of animals. This phenomenon is interpreted as a sign of abstinence and suggests that long-term treatment causes dependence to oxazepam in rats.


Changes in the concentration of adenohypophyseal prolactin and morphological manifestations in the adenohypophysis of lactating rats after administration of D-6-methyl-8-[beta-isopropylaminoethyl] ergoline-I. The four days administration of D-6-methyl-8-[beta-isopropylaminoethyl]ergoline-I (VUFB-10726) to nursing rats decreases adenohypophyseal prolactin as determined with disc electrophoresis, and produces changes in the histological appearance of adenohypophyses, which indicate the inhibition of prolaction production and secretion.


The supraependymal cells of the rat hypothalamus: changes in their morphology and cell number during the ovarian cycle. The number of SEC in the hypothalamus of the rat change during the ovarian cycle (5-8 cells in oestrus, 100 cells in dioestrus per ventricular surface). The changes in the number as well the morphology of the SEC support the hypothesis that they are of mesenchymal nature.


Influence of melatonin, constant light, or blinding on reproductive system of gerbils (Meriones unguiculatus). Male and female gerbils were implanted s.c. with a pellet containing 2 mg melatonin/23 mg beeswax every 2 weeks for a total of 3 implants. A significant depression of ovarian and uterine weight was noted in female gerbils receiving melatonin implants. In the melatonin-treated male gerbils, growth of the accessory organs was significantly inhibited although testis size was not depressed.


Proestrous gonadotropin levels in thyroparathyroidectomized female rats. Intact and TPTx animals showed the expected afternoon increase in serum LH,FSH and prolactin levels. But the afternoon increase in serum LH levels in TPTx rats was less than that observed for intact animals (p less than 0.001). Neither serum prolactin nor FSH levels were altered by TPTx.


[Purification of subunits from human chorionic gonadotropin and radioimmunoassay]. A crude hCG with an activity of about 3,000 IU per mg was purifed to 10,000--15,000 IU per mg of dry weight using Amberlite CG-50 chromatography combined with DEAE-Sephadex A-50 and Sephadex G-75. The alpha and beta subunits of hCG were prepared by urea-treatment of the hormone and isolated by DEAE-Sephadex A-50 chromatography. Further purification of the subunits was achieved by gel filtration on a Sephadex G-76 column. For radioimmunoassay hCG was iodinated by the DMSO-chloramine T method. Iodination of hCG with non-radioactive iodine revealed that the addition of DMSO to the iodination mixture seemed to reduce the iodination damage to the antigenic activity of the hormone. Non-radioactive iodine substituted hCG accomplished by the DMSO-chloramine T method showed 1.5 times more immunoreactive in the hCG radioimmunoassay than hCG iodinated by the usual chloramine T method. The radioimmunoassay of the hCG-beta subunit developed in our laboratory was satisfactory with respect to specificity; hLH, hFSH, hTSH and hCG-alpha subunit tested were cross-reacted very poorly in our assay system. Desialylated-hCG and subunits, whose biologic potency was almost zero, exhibited also decreased immunoreactivity, about 30% of the native hormones with grossly unimpaired parallelism in their respective homologous radioimmunoassays. The concentrations of hCG and the subunits were determined on human sera from pregnant patients during the course of pregnancy. The hCG levels reached to the peak at the first trimester of the pregnancy, however, the hCG-beta subunits varied in their concentrations poorly throughout the pregnancy periods. The hCG-alpha levels, on the other hand, depicted two distinct peaks, at the early period and the term of pregnancy.


[Effects of coenzyme Q on plasma aldosterone concentrations in dogs (author's transl)]. The renin-angiotensin system, potassium and adrenocorticotropin (ACTH) are well known as control mechanism for aldosterone secretion. However, the precise mechanism of these factors for aldosterone secretion remain still unclear. Several interesting evidences related to the effects of Coenzyme Q on the secretion and biosynthesis of aldosterone have been demonstrated. Biochemical action of Coenzyme Q is generally accepted as a component of the electron transfer process of respiration in mitochondria. Fabre et al demonstrated that significant reduction of plasma aldosterone concentration in adrenal venous by the Coenzyme Q infusion. Weinstein et al observed that urine sodium excretion decreased after infusion of Coenzyme Q into renal artery. Kumagai et al suggested that Coenzyme Q inhibited the activity of 18-hydroxylase in the adrenal cortex. The present study was designed to evaluate the effects of Coenzyme Q on the secretion of aldosterone. 24 cases of male among el and beagle dogs were subdivided into 4 groups. 1st group were administered intravenous infusion of Coenzyme Q, 2nd group were orally  administered Coenzyme Q for 7 weeks, 3rd group were administered simultaneous infusion of Coenzyme Q and angiotensin II and 4th group were administered furosemide orally under the condition of continuous Coenzyme Q administration. Then, plasma concentrations of aldosterone, 11-OHCS and angiotensin I were determined during the time course. Plasma aldosterone concentration was significantly increased after intravenous infusion of angiotensin II and decreased 45 minutes after the beginning of infusion. However, the concentration still remained higher than control level. By the simultaneous infusion of Coenzyme Q with angiotensin II, the decreased concentration again increased significantly. It seems that above mentioned results suggest possibility that Coenzyme Q potentiate the action of angiotensin II on aldosterone secretion. By the intravenous infusion of Coenzyme Q, plasma aldosterone concentration increased significantly and concentrations of plasma 11-OHCS and angiotensin I did not affected. This result suggests that Coenzyme Q may stimulate aldosterone secretion from adrenal cortex without increase of ACTH and renin-angiotensin. 4 hours after the oral administration of Coenzyme Q, plasma aldosterone concentration was increased significantly. Na/K in 24 hours' excreta was decreased by Coenzyme Q administration. The decrease of Na/K in excreta may be reflection of the increase of aldosterone secretion. Although, plasma aldosterone concentration increased for the short duration by the Coenzyme Q, it decreased gradually and returned to the control level after 7 days under the condition of continuous oral administration. By the oral administration of furosemide under prolonged Coenzyme Q administration plasma aldosterone concentration increased significantly and remained higher than that of control.


[Analysis of thyroid hormones in serum and urine by mass fragmentography using GC-MS]. The first successful analysis of iodine compounds in serum and urine by mass fragmentography using GC-MS combined system was performed. The equipment used was Shimadzu LKB 9000 GC-MS (MID-PM). The TMSi derivatives of the compounds were analyzed by GC-MS system equipped with a 3 ft X 3 mm column packed with 1% OV-1 and the temperature was programmed from 200 degrees to 320 degrees C at 10 degrees C/min. The mass specturm showed molecular ions at m/e 523, 649, 741, 867 and 993 which correspond to the TMSi derivatives of MIT, DIT, T2, T3 and T4 respectively. The base peak at m/e 218 was applied to establish the precise quantitative evaluation of T4 and related compounds by mass fragmentography. The following results were obtained: 1) The minimum detectable limits of the compounds injected into the column were ca. 10 pg for MIT, DIT, 20pg for T2, 50pg for T3, and 500 pg for T4 respectively. 2) The sensitivity was of the order of ng or pg which enables quantitation with 1 ml of human serum and urine sample. 3) West's method was very convenient for extracting the compounds from biological fluids, and procedure can be carried out easily in a short time. The recovery rates were ca. 16.0% for MIT, 26.6% for DIT, 61.5% for T2, 71.6% for T3, and 85.1% for T4 respectively. 4) The ability to simultaneously analyze various iodoaminoacids is sure to be effectively utilized in studies to elucidate the relative importance of these hormones and their metabolic changes in various physioloigcal and pathological states of human beings.


[The relationship between the timing of the Korotokoff sound (QKd) and the serum thyroid hormone concentrations]. The interval between the onsets of the QRS complex and of the brachial Korotokoff sounds at the diastolic pressure was termed QKd and it was already known that QKd was shortend in hyperthyroidism and prolongad in hypothyroidism distinctly. In the present study, simultaneous measurements of QKd, serum thyroxine (T4) and triiodothyronine (T3) concentrations, free T4 index (FT4I) and free T3 index (FT3I) were undertaken and attempts were made to examine the possible correlation of QKd to these parameters of thyroid function in sera. The QKd values of 24 euthyroid subjects had a normal range of 190 to 230 msec., 10 patients with hypertyroidism had Qkd values ranging from 145 to 180 msec. and 9 hypothyroid patients had QKd values ranging from 230 to 305 msec. Plots of QKd agaist serum T4 and T3 concentrations denoted statistically significant inverse relations in the T4 and T3 concentrations denoted statistically significant inverse relation in the T4 and T3 concentrations approximately less than 20 mug/dl and 400 ng/dl respectively (4 = 0.59 and -0.62 respectively) and above these concentrations of T4 and T3, QKd was nearly constant at the level of 165 msec irrepective of T4 and T3 concentrations. The statistically significant inverse relations were also observed between QKd and FT4I and FT3I in the FT4I and FT3I values less than 18 and 280 respectively (r= -0.80 and -0.74 respectively) and above these values of FT4I and FT3I, QKd was remained constant. In 18 hyperthyroid subjects receiving antithyroid medication, QKd, FT4I and FT3I were measured. QKd values from the patients whose FT4I and/or FT3I were above normal were significantly shorter than the QKd from the patients with normal FT4I and ft3i (183 +/- 9.8 msec vs 211 +/- 19 msec, p less than 0.01). In 16 patients with primary hypothyroidism receiving L-thyroxine for replacement therapy more than 6 months of period, QKd, FT4I and FT3I were measured and TRH-test was performed. FT4I and FT3I in these patients were all in normal range and 8 out of 16 patients had normal response to TRH-test and others were of no response to TRH-test. No difference in QKd values was obtained between patients with normal response and with no response to TRH-Test. These results indicate that QKd is more closely related to serum free T4 and free T3 concentrations and the extent of prolongation of QKd in hypothyroidism reflects the decreased serum thyroid hormone concentrations, whereas the shortend QKd in hyperthyroidism does not parallel the increased serum thyroid hormone concentrations. QKd is useful index not only for the rapid assessment of thyroid function as proposed by previous investigaters, but also for the rpaid evaluation of efficacy of antithyroid drugs administered in hyperthyroidism.


Postmaturity in the rat: impairment of insulin, glucagon, and glycogen stores. Prolonged gestation (2 extra days in utero) was obtained by daily subcutaneous injection of progesterone (2.5 mg) to pregnant rats from day 20.5 post coitum (p.c.) throughout day 22.5 p.c. after reduction of the litter to 6 fetuses on day 14.5 p.c. Exogenous progesterone per se or litter reduction were without effect of fetal pancreas or fetal liver. Plasma insulin, insulin and glucagon in the pancreas, and liver glycogen stores have been systematically measured in postmature animals and in controls during the perinatal period. In 23.5 day-old postmature as compared to 21.5 day-old normal fetuses, the intrauterine mortality was increased (26%), the body weight was increased by 30%, the liver weight was decreased by 20%, the glycogen content of liver was dramatically depleted (1.1 +/- 0.2 mg/g body weight on day 23.5 p.c. against 6.7 +/- 0.3 on day 21.5 p.c.), the plasma insulin was lowered by 63% and the blood glucose level was normal. In postmature neonates during the first day of life the mortality rate was considerable (40%) and a dramatic fall of blood glucose was observed 6 hours after birth. The accumulation of insulin and glucagon in the pancreas, which normally occurs in the two first days after birth, was much lower in the postmature fetuses: in 23.5 day-old fetuses as compared to 2 day-old normal newborns of the same gestational age the insulin content was only 50% and the glucagon content 69%. The deficit of insulin accumulation in the postmature pancreas lasted at least five days. The ability of the endocrine pancreas to recover from this alteration as well shown by the lack of diabetes when the animals were examined three weeks later by a glucose tolerance test. These findings suggest that the drop of plasma insulin is a prime factor in causing the lack of glycogen stores in prolonged fetuses and the impairement of glycogen stores appear to be an important feature of postmaturity, since neonates exhibit, in these conditions, a lethal drop of blood glucose as glycogenolysis operates on very low glycogen stores.


DNA and RNA synthesis by fertilised, cleavage arrested sea urchin eggs. Eggs of Lytechinus variegatus which have been fertilised and maintained at 10 degrees C  do not undergo segmentation, but do replicate DNA. It has been found that RNA synthesis is initiated in these eggs following a period of DNA synthesis resulting in amounts of DNA/egg comparable to those amounts found in late blastulae (1000 genome equivalents). The RNA synthesised has been studied by autoradiographic and sucrose density gradient centrifugation. RNAse-sensitive grains appear in autoradiographs of cleavage-arrested eggs exposed to tritiated uridine beginning two days after fertilisation at 10 degrees C. Incorporation of labelled uridine into the 18 and 25 s region of sucrose gradients parallels the appearance of acid-insoluble radioactivity in the uridine autoradiographs. Localisation of the RNA in the autoradiographs was shown to be in close association with Feulgen-positive areas of the egg periphery. Since differentiation is generally thought to involve changing patterns of transcription of the zygote genome, transcription patterns in the absence of cleavage and normal morphogenesis is of considerable interest. The study of molecular events occurring in a time span during which there is no cell division may have bearing on the role of cell division in differentiation.


Active-site labelling of kallikreins by chloromethylketone derivatives. Ala-Phe-Lys-CH2-Cl is a chloromethylketone derivative which is able to promote the inhibition of several proteolytic enzymes. In this paper the inhibition of horse urinary and plasmatic kallikreins is described and this inhibition is compared to that produced in human plasma kallikrein. This compound was designed based upon the structure of bradykinin. This enzyme substrate system can provide a model for the study of the interactions between bradykinin and its receptor. The inhibition of the enzymes was achieved both for its esterase and kinin-releasing activities.


Kinetics of the hydrolysis of synthetic substrates by horse urinary kallikrein and trypsin. The kinetic constants for horse urinary kallikrein and trypsin hydrolysis of BAEE, TAME, bradykinin methyl ester and bradykinyl-Ser-Val-Gin-Val-Ser were determined. The values of the ratio kcat/Km show that (1) kallikrein is catalytically less efficient than trypsin for all the substrates (2) the three esters are equally good substrates for trypsin while horse urinary kallikrein is 100-fold more effective on bradykinin methyl ester than on the other substrates (3) for both enzymes the ester of bradykinin is a better substrate than the tetradecapeptide.


[Effects of fusaric acid and its derivative on the cardiovascular system]. The cardiovascular effects of fusaric acid, a dopamine-beta-hydroxylase (DBH) inhibitor and a hypotensive agent, and 5-(4'-chlorobutyl) picolinic acid, one of the most potent DBH inhibitors in fusaric acid derivatives, were investigated in anesthetized dogs. Fusaric acid (10-30 mg/kg) given intravenously caused immediately and dose-dependently a fall in blood pressure, an increase in heart rate, a stimulation of respiration, a marked increase in the rate of superior mesenteric arterial flow, and a decrease in the rate of femoral arterial flow. Fusaric acid (0.3-3 mg) given close-arterially caused a dose-dependent decrease in the sinus rate and in the developed tension in isolated blood-perfused sinoatrial node and papillary muscle preparations, respectively. For the superior mesenteric, renal, and femoral circulations which were perfused with blood, a close-arterial injection of fusaric acid (10-30 mg) increased in the rates of flow in a dose-dependent manner. 5-(4'-Chlorobutyl) picolinic acid showed similar responses as fusaric acid quantitatively or qualitatively. These results indicate that hypotension induced rapidly after intravenous administration of fusaric acid or 5-(4'-chlorobutyl) picolinic acid is not due to the enzyme inhibition, but rather to the direct depression of cardiac function and decrease in peripheral vascular resistance.


[Electroencephalographic effects of lopramine]. Electroencephalographic (EEG) effects of lopramine, a new antidepressant, were investigated in rabbits with chronic electrode implants, and compared with those of imipramine and amitriptyline. All drugs were administered i.v. Lopramine (10, 20 mg/kg) induced a drowsy pattern of spontaneous EEG consisted of high voltage slow waves in the cortex and amygdala, and desynchronization of hippocampal thetha waves. Imipramine and amitriptyline (1-5 mg/kg) also elicited similar EEG changes but were much more potent than lopramine in this effect. Lopramine (10, 20 mg/kg) failed to suppress the EEG arousal responses induced by not only auditory stimulation but also electrical stimulation of the mesencephalic reticular formation, centromedian thalamus and posterior hypothalamus, whereas imipramine and amitriptyline (1 approximately 5 mg/kg) markedly inhibited these responses. The EEG arousal response induced by i.v. injection of physostigmine 0.1 mg/kg showed no change after lopramine (20 mg/kg), while the response was significantly suppressed by imipramine (2, 5 mg/kg) and amitriptyline (1, 2 mg/kg). Lopramine showed no effect on the recruiting response induced by electrical stimulation (8 Hz)  of the centromedian thalamus and slightly enhanced the limbic afterdischarges elicited by either hippocampal or amygdaloid stimulation, while imipramine (2, 5 mg/kg) and amitriptyline (1--5 mg/kg) caused an initial depression followed by sustained enhancement of these afterdischarges. These results demonstrate lopramine to be an antidepressant of a new type which has no effect on the ascending reticular activating system and no central anticholinergic action.


[Antispasmodic mechanisms of actions of isoproterenol and papaverine with special reference to their relation to Ca (experiments using the isolated rat rectum)]. Antispasmodic mechanisms of isoproterenol and papaverine were investigated in relation to the mobilization of Ca in the rat's isolated rectal muscle. The findings on the relaxing effects of isoproterenol and papaverine on the contractions induced by increasing concentrations of K and on the modifications in the shape of K- and ACh-induced contractions by these two agents suggest that the antispasmodic mechanism of isoproterenol may be related to inhibition of the transmembrane influx of Ca as well as release of Ca, whereas that of papaverine may be related to not only inhibition of the transmembrane influx of Ca and release of Ca but also to inhibition of the ability of contractile elements responding to Ca. Furthermore, analysis of the concentration-action curves of K and acetylcholine concerning the phasic and the tonic contractions affected by isoproterenol and papaverine (Table 1) provide additional evidence for such a hypothesis.


[Influence of removal of Na from the bath solution on the antispasmodic actions of isoproterenol and papaverine, with special reference to their relations to Ca (experiments using the isolated rat rectum)]. Influence of the Na removal from bath solution on the antispasmodic actions of isoproterenol and papaverine against the phasic contractions by K and acetylcholine were investigated in relation to the mobilization of Ca in isolated rectal strip from the rat. Effect of the Na removal on the uptake of intracellular Ca into storage sites was also examined. The Na removal attenuated the inhibitory actions of isoproterenol and papaverine on the release of Ca, whereas the Na removal did not affect the inhibitory action of isoproterenol on Ca influx and that of papaverine on the response of the muscle contractile elements. Furthermore, the Na removal inhibited the uptake of intracellular Ca into storage sites. Thus, it is suggested that isoproterenol and papaverine may generate the inhibitory action on the release of Ca by accelerating Ca uptake into storage sites, therefore the antispasmodic actions of these agents are depressed by the deprivation of Na from the bath solution which attenuates the Ca uptake.


[Pharmacodynamics of 2-(4-benzyl-piperidino)-1-(4-hydroxyphenyl)-1-propanol (Ifenprodil). (3) Effects on the autonomic, peripheral and central nervous systems]. Effects of ifenprodil tartrate, a potent vasodilator, on the autonomic, peripheral and central nerve system were studied in experimental animals. In isolated vas deferens of guinea pigs, the contraction in response to noradrenaline and sympathetic nerve stimulation was competetively antagonized by ifenprodil 10(-7)--10(-5) M (pA2: 7.69 against noradrenaline). Ifenprodil (50 approximately 1,000 mug/kg i.v.) inhibited the contraction of cat nictitating membrane and dog urinary bladder induced by sympathetic nerve stimulation. Ifenprodil (250 approximately 1,000 mug/kg i.v.) lowered adrenaline-induced lethality (ED50: 360 mug/kg). The drug produced a hypermotility of guinea pig uterus, and showed a transient hypertonus of dog gut which was abolished by atropine. Ifenprodil (10 approximately 20 mg/kg i.v.) inhibited the propulsion of charcoal meal in mice. In Shay rats, more than 10 mg/kg i.m. of the drug inhibited the secretion of acid gastric juice and the ulceration. Ifenprodil showed a potent local anesthetic action in the guinea pig cornea and skin. The spontaneous EEG of rabbits showed a resting pattern (0.25 approximately 2 mg/kg i.v.) followed by an arousal pattern (5 approximately 10 mg/kg). Ifenprodil (20 approximately 100 mg/kg p.o.) potentiated a hypnosis induced by barbital, and potentiated pentylenetetrazol, strychnine and picrotoxin induced convulsion. The drug (20 and 100 mg/kg p.o.) lowered the body temperature of rats. From these results it is concluded that ifenprodil produces a blocking action of alpha-adrenoceptors in various smooth muscle preparations and a direct relaxation of the smooth muscle itself without affecting the motor and central nerve systems.


[Pharmacological comparison between enflurane and halothane]. General pharmacological properties of enflurane (E) and halothane (H) were investigated. Maximum blood concentrations of both drugs reached 30 min after inhalation. E showed lower maximum blood concentration, initial velocity of uptake and shorter half life than H. Neither drug had any effect on neuromuscular junction, but E increased N-M effect of succinylcholine. Both drugs decreased tension of uterine and intestine muscles. Poly- and monosynaptic reflexes were inhibited more by H. ED50's of E and H for intestine muscles. Poly- and monosynaptic reflexes were inhibited more by H. ED50's of E and H for righting reflux were 1.25 and 1.40%, respectively. Tonic and clonic convulsions and death induced by electric shock were inhibited more by E. Almost equal anticonvulsive potency was observed for chemoshocks. Death due to electric and chemoshock was remarkably inhibited by both drugs. Spontaneous EEG was altered in a different manner, although flattened with spikes at 4% concentration of both drugs. E altered rhythmicity of recruiting response and both drugs inhibited this response. H inhibited more remarkably the augmenting response and E inhibited the arousal response. E increased negative potentials of the primary response evoked by sensory stimulation, while H decreased these potentials. Both drugs completely inhibited the secondary response.


[Effects of fasting on the renal function of rats: Clearance experiments and Henle's loop microperfusion]. Results of a previous experiment indicated that osmotic pressure and sodium concentration in distal tubular fluid of the nephron constantly microperfused were increased during natriuresis of fasting rats. The present experiments were performed in order to clarify whether or not water reabsorption and urea movement in Henle's loop, which may modify osmotic pressure and sodium concentration in distal fluid, change in the fasting rat kidney. Non-fasting, 18 approximately 24 hours and 4 approximately 5 days-fasting rats were used. After surgical procedures, the animal was infused with 2% saline at 33.3 mul/min. The loop of Henle was perfused with 1% saline containing 0.05% lissamine green, 3H-inulin and 14C-urea at 29.1 nl/min. In the fasting groups, urinary flow and sodium excretion were increased, whereas urea excretion was depressed and inulin clearance showed a tendency to decrease. Tubular fluid to plasma ratio of osmotic pressure in the distal tubule was elevated without change of water reabsorption. From present and previous results, the increases of osmotic pressure and sodium concentration in distal tubular fluid of fasted rat kidney cannot be explained from an increase of water reabsorption in the Henle's loop. Consequently, natriuresis with fasting may contribute partially to an inhibition of sodium reabsorption in Henle's loops, possibly in the thick ascending limbs. Recovery of C-urea perfused into Henle's loops of fasted rats increased.