Sex- and strain-dependent hepatic microsomal ethylmorphine N-demethylation in mice: the roles of type I binding and NADPH-cytochrome P-450 reductase

The roles of type I binding and NADPH-cytochrome P-450 reductase in ethylmorphine demethylation were investigated in two strains of mice, using sex differences in these activities as a tool. In the CPB-SE strain, females metabolize ethylmorphine faster than males. Sex differences in cytochrome P-450 content and endogenous NADPH-cytochrome P-450 reductase activity were too small to account for this. On the other hand, the differences in the magnitudes of type I spectra and ethylmorphine-induced enhancement of cytochrome P-450 reduction were considerable larger than those in the rates of demethylation. All parameters, except endogenous cytochrome P-450 reduction, were modified in a similar way by testosterone pretreatment: in females they were depressed to the male level, whereas in males they remained unchanged. Castration had no effect in females and enhanced the activities in males. The CPB-V strain exhibited little or no sex differences in ethylmorphine demethylation, cytochrome P-450 content and endogenous cytochrome P-450 reduction. Testosterone pretreatment had little or no influence on these activities. Type I binding and reductase stimulation, however, showed sex differences, comparable to those observed in the CPB-SE strain, which were also abolished by testosterone. A relationship between reductase stimulation and type I binding was observed, which was, apparently, independent of sex or strain. It is concluded that androgen primarily influences the amount of cytochrome P-450-substrate complex formed, but that the reduction of this complex is not rate-limiting in the demethylation of ethylmorphine.     

Skeletal growth in the echinoderm Asterias rubens L. (Asteroidea,Echinodermata) estimated by 45Ca-labelling

The skeletal dry weight of the 4.4 ± 0.2 cm size class of Asterias rubens L. from Kiel Bay in the western Baltic is 0.34 ± 0.08 g. The sum of calcium and magnesium carbonates in the skeleton amounts to 94.0 ± 1.3% while the individual concentrations are 86.9 ± 1.3% CaCO₃ and 7.1 ± 0.7% MgCO₃. The MgCO₃ is 4% lower than expected for a magnesium calcite precipitated under Baltic temperature conditions (8°C). Strontium was not determined but strontium carbonate is known to be in the region of 0.4%. The remainder is organic matter and this gives rise to 2.9 ± 1.3% organic carbon.Animals studied were at the ‘waiting stage’ and their actual growth was minimal. Uptake of ⁴⁵Ca in the skeleton consists of a fast step followed by a slow step. The fast step is attributed to saturation of exchangeable skeletal pools while the slow step is due to net deposition of CaCO₃. Skeletal growth at the waiting stage calculated from the second rate constant was found to be 0.76 μg CaCO₃j-mg skeleton^?1 · day^?1 or 0.09% · day^?1 compared with 9.3 μg CaCO₃ · mg skeleton^t1̄ · day^?1 or 1.1% · day^?1 at log phase. The isotope method is considered superior to size-frequency analysis in that it is capable of detecting differences in growth rate in individuals of the same size class and thus provides an insight into asteroid population structure.     

Chromatin structure in the macronucleus of the ciliate Stylonychia mytilus.

Evidence is presented that macronuclear chromatin in the hypotrichous ciliate Stylonychia mytilus occurs in discrete fragments, each representing at least single genes. The size of these fragments varies between 3 and more than 70 nucleosomes with an average length of about 18 nucleosomes. This observation is discussed with respect to macronuclear structure of hypotrichous ciliates.     

The connection between ionophore-mediated Ca2+-movements and intermediary metabolism in human red cells. I. Relationships between Ca2+-loading,ATP-consumption and glycolytic flux

Human red cells (RBC) were loaded with moderate amounts of Ca²⁺ by the ionophore A23187. Quantitative relationships between Ca²⁺-loading, ATP consumption and glycolytic flux were established. 1. Ca²⁺-loading is accompanied by ATP depletion. A maximum ATP consumption of approximately 10 mmoles/l RBC/h was estimated. 2. There is a positive correlation between lactate formation and Ca²⁺-loading. This is linear from 1.4 to about 4 mmoles lactate/l RBC/h. 3. Ca²⁺-induced glycolytic stimulation seems not to be mediated by adenine nucleotides. A wide range of energy charges and very different adenine nucleotide patterns were associated with the same stimulation of lactate production. 4. The turnover of the (Ca²⁺-Mg²⁺)-ATPase and its share in the Ca²⁺-stimulated ATP consuming processes were estimated with inhibitors. 1 mM La³⁺ inhibited both Ca²⁺-outward transport and ATP consumption by 80%. The remaining 20% of the ATP consumption was accounted for by the (Na⁺-K⁺)-ATPase. 5. A Ca²⁺ extrusion to ATP consumption molar ratio of 2:1 was found. However, when ATP consumption was due to the breakdown of previously accumulated glycolytic intermediates, the ratio dropped to about 1.     

Membrane modulation in a methylotrophic bacterium methylococcus capsulatus (Texas) as a function of growth substrate

Methylococcus capsulatus (Texas), when grown on methane, undergoes with age a progressive degeneration of internal membrane structure with a simultaneous accumulation of intracellular inclusions. When M. capsulatus is grown on methanol, virtually no internal membranes are present but, instead, cells contain many intracellular droplets morphologically similar to inclusions in old methane-grown cells. Membranes are regenerated by the cells when a methanol-grown culture is transferred back to methane. The oxidative ability of methane- and methanol-grown cells was compared.     

Female secondary sexual coloration and sex recognition in the keeled earless lizard, Holbrookia propinqua

Relationships between male social behaviour and female reproductive coloration in the iguanid lizard Holbrookia propinqua were examined by introduction of tethered non-resident lizards into the territories of adult males. Introduced lizards were plainly coloured females, females having bright reproductive coloration, adult males, plain females painted to resemble brightly coloured females, bright females painted to mimic plain females, and males painted with the bright yellow and orange secondary sexual coloration of females. Resident males courted all unpainted females, despite being aggressively rejected by the brightly coloured ones. They also courted all but two females in each of the painted groups. All unaltered non-resident males were challenged, attacked or subjected to other aggressive behaviour by residents, but all non-resident males painted to resemble bright females were courted. This differential treatment is highly significant. It clearly demonstrates that the bright female pigmentation functions in sex recognition, identifying a female to males. Other stimuli, especially pheromonal and behavioural cues, may contribute to sex recognition. Because sex recognition alone seems insufficient to account for the evolution of bright female coloration in H. propinqua and several other iguanids, several proposed additional functions are discussed.     

Determination of simultaneous turnover of serotonin,dopamine and norepinephrine in the telencephalon of unrestrained behaving rats

Rats received an injection of tritiated tryptophan and tyrosine via a chronic indwelling jugular catheter, and at 60 and 90 minutes post-injection, were killed by near-freezing. The three biogenic monoamines were separated by ion exchange and thin layer chromatographyand were quantitated with respect to radioactivity and content. The decline in specific activities can be used to calculate rate constants and turnover of serotonin, dopamine and norepinephrine concurrently in unrestrained behaving animals.     

Hog thyroid peroxidase: physical, chemical, and catalytic properties of the highly purified enzyme.

Studies are reported on the purity and on the physical, chemical, and catalytic properties of a highly purified, stable, thyroid peroxidase (TPO). The enzyme was solubilized by treatment with deoxycholate and trypsin, and it was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. The final product, designated TPO VII, had a value for A₄₁₀/A₂₈₀ of 0.54, and its specific activity based on the guaiacol assay (794 μmol of guaiacol oxidized/min/mg) was considerably greater than that of any previously described TPO. Specific activity values based on other peroxidase-catalyzed reactions were also higher for TPO VII than for previous TPO preparations. Purity estimates for TPO VII, based on polyacrylamide disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels, ranged from 80 to 95%. The molecular weight, determined by sedimentation equilibrium, was 93,000. Results of sodium dodecyl sulfate-gel electrophoresis also indicated a molecular weight of approximately 90,000. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions indicated that TPO VII is composed of two peptide chains of unequal size, with the larger about 2.5-fold the size of the smaller. Carbohydrate analysis revealed that TPO is a glycoprotein containing about 10% by weight of carbohydrate. The predominant sugars were mannose and N-acetyl glucosamine. A significant amount of glucose was also found, along with small amounts of galactose, fucose, and xylose. The amino acid composition of TPO VII showed a high proline content, a predominance of arginine over lysine, and a ratio of [Asp] plus [Glu] to [Lys] plus [Arg] of over 2. Isoelectric focusing in polyacrylamide gels indicated an isoelectric pH of 5.75. In agreement with observations made on earlier preparations of TPO, heme spectral data showed significant differences between the pyridine hemochromogens of TPO VII and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. Circular dichroism measurements indicated that approximately 40% of TPO VII involves α helix or β structure.     

Stimulatory effect of calcium chelators on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles

The calcium chelators EGTA, EDTA and cyclohexanediamine tetraacetic acid (CDTA) enhance initial rates of Na_i⁺-dependent Ca²⁺ uptake by cardiac sarcolemmal vesicles. The affinity of the exchanger for calcium is increased in the presence of the chelators to an extent dependent on chelator concentration and on the range of free calcium concentrations over which the phenomenon is measured. For free Ca²⁺ in the range of 4 μM or less, the apparent K_m is lowered to approximately 1 μM. The Ca-chelator complex appears to be the species which causes stimulation. The effect is not due to sequestration of contaminating heavy metal ions in the sarcolemmal membrane preparations or the solutions used in experiments. Caution is suggested in the use of EGTA or EDTA as calcium buffers when measuring calcium dependence of phenomena involving calcium binding and transport, because the added chelator may alter the properties of the system.     

Activation of protein kinase in the guinea pig fundic gastric mucosa by histamine.

The effect of histamine, 1,4-methylhistamine and ethanol on cyclic AMP levels and protein kinase activation was measured in tissue strips from the fundic region of guinea pig gastric mucosa. Histamine induced a significant elevation of tissue cyclic AMP levels and also $\text{in situ}$ activation of the protein kinase. 1,4-methylhistamine, an inactive analog of histamine, and ethanol had no effect on these two parameters. Results suggest that protein kinase activation is involved in the cyclic AMP-mediated action of histamine on the gastric fundic mucosa.     

Effect of tryptophol on pentylenetetrazol and picrotoxin induced convulsion in mice.

The effect of treatment of mice with tryptophol (TOL), a neutral metabolite of tryptophan, on drug-induced convulsion was studied. TOL effectively suppressed both pentylenetetrazol and picrotoxin induced convulsion. Diphenylhydantoin (DPH), a potent inhibitor of brain aldehyde reductase, significantly reduced the anticonvulsant effect of TOL, however, TOL level in brain of DPH-treated mice was rather higher than that of control one. These results strongly suggest that the manifestation of the anticonvulsant effect of TOL requires the conversion of TOL to its active metabolite, indoleacetaldehyde.     

Effects of zomepirac and indomethacin on renal blood flow and electrolyte excretion in anesthetized monkeys

Zomepirac sodium is a new inhibitor of prostaglandin cyclooxygenase with an $\text{in vitro}$ potency equivalent to indomethacin. Since inhibitors of prostaglandin synthesis have marked effects on renal hemodynamics, zomepirac may be expected to reduce renal blood flow (RBF) in a manner similar to indomethacin. This study compares the effects of zomepirac and indomethacin on RBF and electrolyte excretion in anesthetized Rhesus monkeys. Each experiment consisted of a control period followed by 3 or 4 drug treatment periods in which increasing doses of zomepirac (0.5 to 20 mg/kg) or indomethacin (0.5 to 10 mg/kg were given. Indomethacin (5 mg/kg) reduced RBF by 22% and the higher dose (10 mg/kg) reduced RBF by an additional 13%. Zomepirac had little effect on RBF in doses as high as 20 mg/kg. At any given dose the mean plasma concentration of zomepirac was equal to or greater than indomethacin. Peak indomethacin concentration was 48 μg/ml after the 10 mg/kg dose while the peak zomepirac, after 20 mg/kg, was 158 μg/ml. Neither drug had a significant effect on either glomerular filtration rate or excretion rate of sodium or potassium. Thus, zomepirac had only minimal effects on RBF while indomethacin decreased RBF of anesthetized monkeys in a manner qualitatively similar to its effect in other species. The minimal renal effects caused by zomepirac relative to indomethacin in this primate may indicate a therapeutic advantage for zomepirac in man.     

Characterization of lecithin:Cholesterol acyltransferase from human plasma: II. Physical properties of the enzyme

The physical properties of purified human plasma lecithin:cholesterol acyltransferase (LCAT) were investigated by techniques including analytical ultracentrifugation, ultraviolet spectroscopy, electrofocusing, and circular dichroism. The partial specific volume of LCAT was determined by sedimentation equilibrium ultracentrifugation experiments in H₂O and D₂O solutions (0.702 ml/g). The M_r was 67,000 by sodium dodecyl sulfate (SDS)-gel electrophoresis and 60,000 by sedimentation equilibrium ultracentrifugation. The discrepancy between the two sets of data presumably arose from the glycoprotein nature of the enzyme. Studies of the ultraviolet spectrum indicated that LCAT contained 6.5% ($\text{w}\text{w}$) tyrosine which corresponds to approximately 18 tyrosine residues/mol of LCAT (polypeptide M_r 45,000). Spectrophotometric titration of the ionizable phenolic side chains indicated that nearly all the tyrosine residues were buried at neutral pH while they became gradually exposed at higher pH. The apparent pK of this transition was about 12.0 contrasted with 9.8, the apparent pK of ionization of the free tyrosyl groups.     

Effect of hypophysectomy on estrogen conjugation and on plasma and tissue concentrations of tritium after administration of 3H-estradiol-17beta.

Estradiol-17beta-6,7-3H was injected into ovariectomized (control) and ovariectomized, hypophysectomized (hypox) rats in order to study the binding of estradiol in the brain. Hypophysectomy resulted in a significant increase in the concentration of tritium in the hypothalamus, and preoptic area, as well as cortex, muscle, plasma and liver. However, since the liver was lighter in hypox rats, the total tritium content in the liver was unchanged from controls. Part of the weight reduction in the liver was due to a loss of stainable glycogen, which took place within 24 hours of hypox. The increase in circulating tritiated estradiol after hypox led us to investigate hepatic metabolism of estradiol. In vitro studies on liver slices from control and hypox rats demonstrated a significant reduction in the formation of conjugates of estrogen. Specifically, estradiol glucuronide and estrone sulfate formation were reduced in hypox rats and conversely the unmetabolized estradiol concentration was higher. Hypophysectomy for 24 hours results in a significant decrease in hepatic metabolism of estradiol-17beta.     

Susceptibility of brain aldehyde metabolizing enzymes to pargyline, disulfiram and diethylmaleate in vivo in rats.

$\text{In vivo}$ susceptibility of mitochondrial (m)- and soluble (s)-aldehyde dehydrogenase (AlDH) and aldehyde reductase (AIR) to three compounds, i.e., pargyline, diethylmaleate and disulfiram in rat brain was studied. In all experiments using the compounds tested, m-AlDH was more significantly inhibited when the low concentration of the substrate (50 μM) was used, as compared with the inhibition of the enzyme in use of high substrate concentration (3.3 mM). Under same condition, little or no inhibition of s-AlDH and AIR was observed. These findings strongly suggest that there are at least two forms of AlDH with different Kms and they have different susceptibility to AlDH inhibitors.     

Distribution and shifts of ingested marker particles in residual bodies and other lysosomes. Studies on in vitro cultivated human glia cells in phase II and 3

Human glia cells approaching phase III in vitro were exposed to thorium dioxide particles. The intracellular distribution of the markers was studied by electronmicroscopy after intervals ranging between 15 min and 3 days. In a second experiment glia cells in phase II were labelled and kept at confluence for time periods varying between 1 day and 22 weeks before they were studied ultrastructurally.The findings showed that secondary lysosomes form part of a vacuome which, by fusion and fission, allows material within membrane-limited vacuoles to spread in the system, although it is in any moment discontinuous. The results further indicated that residual bodies form an integrated part of this vacuome and probably regularly receive lytic enzymes by fusion with other types of lysosomes. The rate of extrusion of residual bodies or exocytosis of indigestible material was found to be low, if it occurred at all in the cell strains examined.