Dextrorphan: an antagonist for phencyclidine receptors

Radio-binding assay, bioassay and HPLC detection were used to observe the antagonistic effects of dextrorphan on PCP's actions. Dextrorphan displayed high affinity to PCP receptor in the rabbit mesenteric blood vessels. It had weak PCP-like bioactivity, but could antagonize PCP's action dose-dependently in vitro study with the rabbit ear artery preparation and shifted the dose-response curve of PCP to the right. After PCP administration, the content of norepinephrine in the vascular bath medium was increased, which was reversed by dextrorphan. Thus suggests that dextrorphan is an antagonist with very mild agonistic action for PCP receptors.     

A test using cultured cells with induced mixed-function oxygenase in the unscheduled DNA synthesis assay for detecting promutagens/procarcinogens

The human FL cell line contains very low levels of constitutive AHH activity, but it could be greatly induced by NE, beta-NF and 3-MC, and induced slightly by PB. When two different types of inducer, for example, 3-MC and PB or 3-MC and NE were given in combination, an additive inductive effect was not observed. Both the constitutive and induced AHH in FL cells have characteristics of MFO, namely, NADPH-dependence and CO-sensitivity. The fact that the constitutive and induced AHH in FL cells could be inhibited by a known hydroxylase inhibitor 7,8-BF indicated that the AHH in FL cells belongs to the cytochrome P-448 dependent MFO type. After removal of inducer from the medium, the induced AHH activity remained at a high level for at least 24-36 h. By using AHH-induced FL cells in the UDS assay system for the detection of promutagens/procarcinogens, we found that AFB1 and 3-MC did not induce a UDS reaction in uninduced FL cells, while in beta-NF induced cells, 10(-6)-10(-4) M AFB1 and 10(-7)-10(-6) M 3-MC elicited a very significant UDS reaction, which was concordant with the results obtained in the UDS assay system using HeLa cells or FL cells supplemented with liver microsomes or using primary cultured hepatocytes as indicator cells. B(a)P elicited the UDS reaction at concentrations of 10(-6)-10(-3) M in beta-NF induced cells, whereas 10(-4)-10(-3) M was required in uninduced cells. The results above indicate that this new design is feasible, but further study is needed to assure its accuracy.     

Changing Activity of Glucose-6-Phosphate Dehydrogenase from Pea Chloroplasts during Photosynthetic Induction

Light inactivation of glucose 6-phosphate dehydrogenase is rapid and occurs before photosynthetic O₂ evolution is measureable in intact chloroplasts. Likewise, dark activation is rapid. The major light induced change in the kinetic parameters of glucose 6-phosphate dehydrogenase is in maximal velocity.     

The effect of light and phytochrome on 1-aminocyclopropane-1-carboxylic Acid metabolism in etiolated wheat seedling leaves

While light-grown wheat leaves produced ethylene at a low rate of <0.1 nanomoles per gram per hour and contained 1-aminocyclopropane-1-carboxylic acid (ACC) at low levels of <2.5 nanomoles per gram, etiolated wheat leaves produced ethylene at a rate of 2 nanomoles per gram per hour and accumulated concentrations of ACC at levels of 40 nanomoles per gram. Upon illumination of 8-day-old etiolated wheat seedlings with white light, the ethylene production rate increased initially, due to the activation of ethylene-forming activity, but subsequently declined to a low level (0.1 nanomoles per gram per hour) at the end of the 6-hour illumination. This light-induced decline in ethylene production rate resulted from a decline (more than 35 nanomoles per gram) in ACC level, which was accompanied by a corresponding increase in 1-(malonylamino)cyclopropane-1-carboxylic acid content. These data indicate that illumination promoted ACC malonylation, resulting in reduced ACC level and consequently reduced ethylene production. However, light did not cause any significant increase in the extractable ACC-malonyltransferase activity. The effect of continuous white light on promotion of ACC malonylation was also observed in intermittent white light or red light. A far-red light treatment following red light partially reversed the red light effect, indicating that phytochrome participates in the promotion of ACC malonylation.     

Temperature Effects on Phosphoenolpyruvate Carboxylase from a CAM and a C(4) Plant : A Comparative Study

The effect of temperature in the range from 10 to 35°C on various characteristics of phosphoenolpyruvate carboxylase from the leaves of a CAM plant, Crassula argentea and a C₄ plant Zea mays shows a number of different effects related to the environment in which these distinct types of metabolic specialization normally operate. The Arrhenius plot of V_max for the two enzyme forms shows that the CAM enzyme has a linear increase with temperature while the C₄ enzyme has an inflection at 27°C implying a conformational or aggregational change in the enzyme or a shift in reaction mechanism to one requiring a lower activation energy. The Arrhenius plot of K_m for the two enzymes reveals the startling fact that at temperatures above 20°C an increasing temperature causes an increase in K_mPEP for the CAM enzyme while the C₄ enzyme displays a decreased K_m as the temperature increases. The inhibitory effect of 5 millimolar malate also shows opposite trends for the two enzymes. For the CAM enzyme the percent inhibition by malate increases from essentially none at 15°C to 70% at 35°C. For the C₄ enzyme the percent inhibition drops from about 60% at 20°C to 2% at 30°C. Similar opposite behavior of the two enzymes is found with the K_i for malate. Pretreatment at high temperatures for periods up to 2 hours was found to result in differences similar to those described above if the treated enzyme were subsequently assayed at 25°C.     

The role of perfusion washout in limb revascularization procedures

Amputated rat hindlimbs were subjected to either normothermic (26 degrees C) or hypothermic (4 degrees C) ischemia. Experimental limbs had their microcirculation washed out (either before or after the ischemic insult) with a physiologic acellular plasma substitute previously reported to enhance flap survival following extended periods of warm ischemia. Control limbs were not washed out; i.e., stagnant blood remained in these limbs. Following the ischemic interval, amputated limbs were replanted. Monastral blue B, a colloidal pigment capable of labeling leaky blood vessels, was administered systemically to all rats just prior to vascular declamping. Limb biopsies of skin and muscle were harvested 30 minutes following revascularization in order to assess Monastral labeling and, therefore, the functional integrity of the microcirculation. Results confirm that stagnant blood under conditions of warm ischemia is detrimental to the functionality of the microcirculation in both skin (p less than 0.03) and muscle (p less than 0.007). Accordingly, perfusion washout, when performed prior to the ischemic period, enhances limb survival following 6 hours of warm ischemia (p less than 0.01). Hypothermia protects against the detrimental effects of stagnant blood; perfusion offers no benefit if hypothermic conditions prevail. Physiologic mechanisms responsible for these findings are discussed.     

Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.     

Plant regeneration from cultured immature embryos of Sorghum bicolor (L.) Moench

Summary Immature embryos of 20 sorghum genotypes were cultured on MS 5 medium containing MS mineral salts supplemented with 2,4-D, zeatin, glycine, niacinamide, Ca-pantothenate, L-asparagine, and vitamins. For regeneration, calli were transferred onto the same medium with the exception that IAA was substituted for 2,4-D. In general, immature embryos obtained 9–12 days after pollination resulted in the best redifferentiation. Ability of calli to regenerate varied among genotypes; cultivars C401-1 and C625 had the highest redifferentiation frequencies. Ability to redifferentiate was heritable and acted as a dominant trait. At least two gene pairs were involved. Regenerated R₀ plants were planted in a greenhouse and their selfed (R₁ and R₂) progenies were planted in the field and examined for morphological and cytological variations. The majority of the phenotypic variations noted in R₀ were not transmitted to later generations. However, variants for plant height, degree of fertility, and midrib color persisted in R₁ and R₂ generations. A variation in tallness was attributable to one dominant mutant gene. Short stature and male sterility variants appeared to be consequences of recessive mutant genes controlling those traits. Minor variations in peroxidase banding patterns were found among R₀ plants.This study was supported by a research grant from Kansas Sorghum Commission and by a Research Fellowship to the senior author from the Ministry of Agriculture, Animal Husbandry, and Fisheries, China. Contribution 86-456-J from the Kansas Agricultural Experiment Station     

Differences in monoamine oxidase activity between cultured noradrenergic and cholinergic sympathetic neurons

Two types of monoamine oxidase activity (MAO-A and MAO-B) help regulate the levels of biogenic amines such as catecholamines and serotonin. Although MAO-A has greater activity toward most catecholamines than MAO-B, no direct experiments have determined the types and levels of MAO activity that are normally expressed in noradrenergic neurons. Noradrenergic neurons from neonatal rat superior cervical ganglia were isolated and cultured under conditions that permit either continued expression of the noradrenergic phenotype or promote a transition to a predominantly cholinergic phenotype. After 14-21 days in vitro, neurons from both types of cultures were assayed for the type and amount of monoamine oxidase activity using tryptamine, a common substrate for both MAO-A and MAO-B. Neurons cultured under noradrenergic conditions expressed sevenfold greater MAO activity than neurons cultured under cholinergic conditions. Essentially all MAO activity in the noradrenergic cultures was inhibited by preincubation with 10(-8)-10(-9) M clorgyline, which indicated that this activity was primarily MAO-A. Cultures grown under cholinergic conditions exhibited 6- to 10-fold lower MAO-A activity and an 8- to 10-fold lower level of catecholamine synthesis from labeled precursors compared to neurons grown under noradrenergic conditions. These results directly demonstrate that high MAO-A activity is expressed in noradrenergic neurons in vitro. The corresponding decreases in both MAO-A specific activity and catecholamine synthesis as neurons become cholinergic in vitro suggest that the expression of the noradrenergic phenotype involves the coordinate regulation of degradative as well as synthetic enzymes involved in catecholamine metabolism.     

The reaction of cytochromes c and c2 with the Rhodospirillum rubrum reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.