Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phasepartition method from the algae Chara corallina and Chara longifolia,algae which differ in their ability to grow in saline environments.Enrichment of plasma membrane and depletion of tonoplast relativeto the microsomal fraction was monitored using phosphohydrolaseassays and crossreactions to antibodies raised against higherplant transporters. Antibodies to the vacuolar ATPase and pyrophosphatasecross-reacted with epitopes in the microsomal fraction, butshowed little affinity for the plasma membrane fraction. Pyrophosphataseactivity also declined in the plasma membrane fraction relativeto the microsomal fraction. The V-type H⁺ -ATPase activity,sensitive to nitrate or bafilomycin, was low in both fractions,though the cross-reaction to the antibody was reduced in theplasma membrane fraction. By contrast, the antibody recognitionof a P-type H⁺-ATPase amino acid sequence from Arabidopsis didnot occur strongly in the anticipated 90–100 kDa range.While there was enhanced recognition of a polypeptide at around140 kDa in the plasma membrane fraction, salt treatment of Charalongifolia resulted in plasma membrane fractions with reducedamounts of this epitope, but no change in vanadate-sensitiveATPase activity, suggesting that it does not represent the onlyP-type ATPase. Microsomal membranes from saltadapted C. longifoliahave higher reactivity with the antibody to the tonoplast ATPase. Key words: Chara, plasma membrane, salt tolerance, ATPase     

Differential Inhibition of Secretagogue-Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors

Abstract: Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca²⁺ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na⁺, which could explain inhibition of secretion and Ca²⁺ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated ²²Na⁺ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 µ M ) and SKF-38393 (100 µ M ) inhibited DMPP-stimulated Na⁺ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 µ M ) inhibited Na⁺ uptake by only 22.9 ± 5.0%. Veratridine-stimulated Na⁺ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µ M CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC₅₀ (∼18 µ M ) for inhibition of both DMPP- and veratridine-stimulated Na⁺ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 m M ) had no effect on either DMPP- or veratridine-stimulated Na⁺ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na⁺ uptake in a cyclic AMP-independent manner.     

Effects of Chronic Ethanol Consumption and Aging on Dopamine, Serotonin, and Metabolites

Abstract: This study examined the hypothesis that chronic ethanol consumption results in significant abnormalities in both the dopaminergic and the serotonergic system of aged rats. Levels of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole-3-acetic acid (5-HIAA) were determined in brain areas of both the nigrostriatal and mesocorticolimbic DA systems in 5-, 14-, and 24-month-old male Fischer 344 rats. Aging was associated with a reduced concentration of DA in the striatum (ST), ventral tegmental area (VTA), and ventral pallidum (VP) and an increased concentration of 5-HIAA in the ST, globus pallidus, nucleus accumbens, frontal cortex, and VP. In addition, there was an increase in the 5-HIAA/5-HT ratio in all brain areas analyzed. Six weeks of ethanol consumption was accompanied by significant changes in mesocorticolimbic brain areas. In the VTA of 5-month-old ethanol-fed rats DA content was decreased to the levels found in aged rats, e.g., 24 months of age. Ethanol also significantly lowered 5-HT and 5-HIAA contents in the VTA and reduced DOPAC and 5-HIAA levels in the VP. In addition, ethanol blunted the normal age-related increase in 5-HIAA/5-HT ratio in the VTA, VP, and substantia nigra. It is interesting that although the age-related changes were found in both nigrostriatal and mesocorticolimbic brain areas, the ethanol-associated effects were found only in brain areas of the mesocorticolimbic system. The changes in DA and 5-HT function that accompany aging and ethanol consumption may contribute to the problems in motor function and ethanol abuse found in the aged.     

Eph Family Receptors and Their Ligands Distribute in Opposing Gradients in the Developing Mouse Retina

The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal–low nasal and high ventral–low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

Opioid receptors: Some perspectives from early studies of their role in normal physiology,stress responsivity,and in specific addictive diseases

The early history of research on the possible existence of specific opioid receptors and on developing a new form of pharmacotherapy for the treatment of heroin addiction in New York City, from 1960–1973, along with the special relationships between two leading scientists conducting these research efforts, Dr. Eric Simon and Dr. Vincent P. Dole Jr., are presented in a historical perspective. The linkage of these early efforts and the subsequent identification and the elucidation of the effects of exogenous opiates acting at specific opiate receptors in human physiology, including some findings from perspective studies of heroin addicts at time of entry to and during methadone maintenance treatment, are presented in the context of the important clues which thereby were provided concerning the possible roles of the endogenous opioids in normal mammalian physiology. From many of these early clinical research findings and studies in animal models, the hypothesis that the endogenous opioids system may play an important role in stress responsivity was formulated along with the related hypothesis, first presented in the early 1970s, that an atypical responsivity to stress and stressors might be involved in the acquisition and persistence of, and relapse to specific addictive diseases, including heroin addiction, cocaine dependency and alcoholism. More recent studies of the possible involvement of the specific opioid receptors in these three addictive diseases—heroin addiction, cocaine addiction and alcoholism—from our laboratory are discussed in a historical perspective of the development of these ideas from the early research findings of not only Dr. Eric Simon, but his numerous colleagues in opioid research in the United States and throughout the world. Special issue dedicated to Dr. Eric J. Simon.     

Sex allocation in a simultaneous hermaphrodite, the zebra goby Lythrypnus zebra: insights gained through a comparison with its sympatric congener, Lythrypnus dalli

Synopsis The sexual pattern of the zebra goby Lythrypnus zebra, is an apparent exception to sex allocation theory. Most L. zebra are simultaneous hermaphrodites (i.e., have active female and male gonadal tissue), yet it appears they do not reproduce as males and females simultaneously. Understanding the maintenance of simultaneous hermaphroditism in L. zebra could expand sex allocation theory. In this study, I used a comparison with the blue-banded goby, Lythrypnus dalli, a sympatric congener with a qualitatively similar sexual pattern, to investigate the role of male spawning rate, body size and sexual flexibility in determining the sexual pattern of L. zebra and to isolate differences between the species that might explain their differing sexual patterns. Using field measurements of male nesting success, I found no differences between the species in the body size of nesting males suggesting that large size is associated with successful male reproduction in both species. In addition, nesting males spawned at approximately three times the rate of females in both species; thus, reproduction via male function can be equally advantageous relative to adopting the female role. However, the nest longevity of L. zebra males was shorter than that of L. dalli males, suggesting reproduction via male function may be less reliable in L. zebra. Finally, under laboratory conditions, L. zebra females tended to prefer large mates, and L. zebra were able to re-allocate in both directions, exhibiting a greater capacity to switch than L. dalli. Given these results, I suggest that switching between the sexes plays a greater role in maintaining simultaneous hermaphroditism in L. zebra than L. dalli, perhaps because male reproduction is not as consistent in L. zebra. Sexual flexibility may be an important factor affecting patterns of sex allocation, generally.     

Altered drug translocation mediated by the MDR protein: Direct,indirect, or both?

Overexpression of the MDR protein, or p-glycoprotein (p-GP), in cells leads to decreased initial rates of accumulation and altered intracellular retention of chemotherapeutic drugs and a variety of other compounds. Thus, increased expression of the protein is related to increased drug resistance. Since several homologues of the MDR protein (CRP, ltpGPA, PDR5, sapABCDF) are also involved in conferring drug resistance phenomena in microorganisms, elucidating the function of the MDR protein at a molecular level will have important general applications. Although MDR protein function has been studied for nearly 20 years, interpretation of most data is complicated by the drug-selection conditions used to create model MDR cell lines. Precisely what level of resistance to particular drugs is conferred by a given amount of MDR protein, as well as a variety of other critical issues, are not yet resolved. Data from a number of laboratories has been gathered in support of at least four different models for the MDR protein. One model is that the protein uses the energy released from ATP hydrolysis to directly translocate drugs out of cells in some fashion. Another is that MDR protein overexpression perturbs electrical membrane potential () and/or intracellular pH (pH_i) and therebyindirectly alters translocation and intracellular retention of hydrophobic drugs that are cationic, weakly basic, and/or that react with intracellular targets in a pH_i, or -dependent manner. A third model proposes that the protein alternates between drug pump and Cl^– channel (or channel regulator) conformations, implying that both direct and indirect mechanisms of altered drug translocation may be catalyzed by MDR protein. A fourth is that the protein acts as an ATP channel. Our recent work has tested predictions of these models via kinetic analysis of drug transport and single-cell photometry analysis of pH_i, , and volume regulation in novel MDR and CFTR transfectants that have not been exposed to chemotherapeutic drugs prior to analysis. This paper reviews these data and previous work from other laboratories, as well as relevant transport physiology concepts, and summarizes how they either support or contradict the different models for MDR protein function.     

Factors influencing plasma zinc levels in low-income pregnant women

Plasma zinc (Zn) concentrations were measured in 4376 indigent women (86% African-American), at a mean (±SD) gestational age of 15 (±7.8) wk to determine the relationship between various maternal characteristics and plasma Zn levels during pregnancy. Mean plasma Zn levels were lower in African-American women than in Caucasian women, in multiparous women than in primiparous women, and in women with body weight >69.9 kg than in those with body weight ≤69.9 kg (p≤0.001 for each comparison). There were no significant differences related to maternal age, marital status, education, or smoking habit. Multiple regression analysis, including maternal prepregnancy weight, race, age, parity, smoking habit, education, and marital status indicated that race, parity, and pregnancy weight were significantly associated with maternal plasma Zn levels, adjusted for gestational age. Maternal race was the best predictor of plasma Zn concentrations among the population of pregnant women studied A significant proportion of variance in maternal plasma Zn levels remained unexplained after taking into account various maternal characteristics. The reasons for lower plasma Zn levels in African-American women, compared to Caucasian women, during pregnancy are unknown.     

A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana

Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.