An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.     

Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.     

Depolarization and Agonist-Stimulated Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.     

Endopeptidase-24.11, a Cell-Surface Peptidace of Central Nervous System Neurons, Is Expressed by Schwann Cells in the Pig Peripheral Nervous System

Endopeptidase-24.11 is a 90-kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase-24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and cervical sympathetic trunk in which greater than 99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono- and polyclonal anti-endopeptidase antibodies. Endopeptidase-24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed 125I-insulin B-chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM. Moreover, a 90-kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelin- and non-myelin-forming Schwann cells. Endopeptidase-24.11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase-24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.     

Conserved Organization of γ-Aminobutyric AcidAReceptor Genes: Cloning and Analysis of the Chicken β4-Subunit Gene

A series of genomic clones containing DNA that encodes the chicken gamma-aminobutyric acidA (GABAA) receptor beta 4 subunit have been isolated. These have been restriction mapped and partially sequenced to determine the structural organization and the size of the beta 4-subunit gene. This gene, which comprises nine exons, spans more than 65 kb. The organization of the chicken GABAA receptor beta 4-subunit gene has been compared to that of the murine GABAA receptor delta-subunit gene and to those of the genes that encode other members of the ligand-gated ion-channel superfamily, namely muscle and neuronal nicotinic acetylcholine receptors (AChRs). Although the positions of the intron/exon boundaries of GABAA receptor subunit genes are seen to be highly conserved, there are significant differences between the genes that encode GABAA receptor and AChR subunits. These results are discussed in relation to the proposal that this superfamily of ligand-gated ion-channel receptor genes arose by duplication of an ancestral receptor gene.     

草鱼免疫应答的初步研究

研究了草鱼在不同水温条件下受抗原刺激后其中和抗体的变化。15℃培养条件下中和抗体上升缓慢,9周内滴度低于1:8;20℃时,3周后抗体可上升到1:256,最高达1:5270,而在25℃时,1周中和抗体即达到1:570,最高可达1:20000以上。并探索了从草鱼血清中提纯抗体的条件,研究其抗体的特性。草鱼血清中的抗体为大分子蛋白,容易解离为抗原性相同,分子量近似于人IgG的较小分子,含有较多的二硫键,具有类似IgM的某些特性。     

Effects of Frequency and Pattern of Medial Forebrain Bundle Stimulation on Caudate Dialysate Dopamine and Serotonin

In vivo microdialysis was employed to detect changes in extracellular dopamine and serotonin in the rat caudate in response to electrical stimulation of the medial forebrain bundle. Extracellular dopamine concentrations increased linearly as a function of the frequency (4-33 Hz) of evenly spaced stimuli in both the presence and absence of cocaine added to the dialysate. Because dopamine neurons are known to fire in single-spike and burst patterns, stimulation pulses were also delivered in a bursting pattern. The response of extracellular dopamine was augmented in both the presence and absence of cocaine when the same number of stimuli were delivered in bursts as compared to an evenly spaced pattern. Serotonin, which was only assessed in the presence of cocaine, similarly increased linearly with frequency, but, in contrast to the dopamine response, levels of serotonin were not augmented by stimuli presented in bursts. These results suggest that microdialysis can be used to detect physiological changes in synaptic transmitter concentrations.     

Pattern of sea bass oocyte development after ovarian stimulation by LHRHa

The cyclic pattern of oocyte development in the sea bass, Dicentrarchus labrax L., was studied after induction of spawning by two injections, 24 h apart, of a luteinizing hormone releasing-hormone analog (LHRHa) administered at the end of vitellogenesis. The first difference in the developmental stage of the ovary and in the size-frequency distribution of oocytes between the LHRHa treated group and the control group, was detected 32 h after the first injection, the LHRHa group showing a higher proportion of the 900 μm diameter oocyte class (maturing oocytes) ( P <0.01). At 48 h LHRHa-treated females showed an increase in the 1000 and 1100 μm classes (maturing oocyte and ovulated eggs) ( P <0.01) and at 72 h these females exhibited a bimodal pattern, reaching the highest proportions in the 1100 (27.4%) and the 600 (14.7%) μm classes (ovulated eggs and advanced vitellogenic oocytes, respectively). Bimodal distributions were present in 80% of the LHRHa-treated females. Once oocyte final maturation was triggered by LHRHa the time needed for ovulation was about 48 h and the interval between consecutive ovulations and spawnings seemed to be 48–72 h.     

Cellular mechanisms of paired electrical stimulation in ferret ventricular myocardium: relationship between myocardial force and stimulus interval change

Summary The subcellular mechanisms of twitch-force potentiation with paired electrical stimulation was studied in ferret ventricular myocardium using the bioluminescent calcium indicator aequorin. It is demonstrated for the first time that interpolation of an extrasystole in a train of conditioned twitches results in a beat-to-beat change in [Ca²⁺]_i and force. Steady-state twitch force and Ca _i ²⁺ were increased with paired stimulation. Increased [Ca²⁺]₀ in the setting of paired stimulation resulted in an increase in the amplitude of the postextrasystole and associated Ca²⁺ transient. Verapamil, a Ca²⁺ channel antagonist, had the opposite effect of increased [Ca²⁺]₀. Postextrasystole potentiation was still present, but diminished in amplitude. These results indicate that postextrasystole potentiation is in part due to a verapamil-depletable store (Ca²⁺). Postextrasystole potentiation is therefore predominantly dependent on sarcoplasmic reticulum (SR) Ca²⁺ loading. Ryanodine, an alkaloid which induces Ca²⁺ leakage from the SR, abolished postextrasystole potentiation; however, in the presence of ryanodine the extrasystole was potentiated. Caffeine, a phosphodiesterase inhibitor which induces SR Ca²⁺ release and impairs uptake, also abolished postextrasystole potentiation. As with ryanodine there was resultant potentiation of the extrasystole. In the case of caffeine the calcium transient consisted of a second slow component associated with extrasystole twitch potentiation. The results are consistent with sarcolemmal Ca²⁺ influx playing a role in potentiation of the extrasystole in the presence of an impaired SR. These data indicate that transsarcolemmal Ca²⁺ influx in the presence of impaired intracellular Ca²⁺ buffering can directly activate the myofilaments in agreement with reports on human myocardium.Abbreviations C conditioned stimulus - ESI extrasystolic interval - L_max active tension - PES postextrasystole - PESI postextrasystolic interval - SR sarcoplasmic reticulum - T test stimulus     

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta)

Summary Chloride-stimulated K⁺ secretion by Manduca sexta midgut (5th-instar larvae) was measured as K⁺-carried short-circuit current of the tissue mounted in an Ussing chamber. Microscopic parameters, such as single-channel current and channel density for the rate-determining passive transport step across the basolateral goblet cell membrane (i.e. K⁺ channels), were estimated by means of current-fluctuation analysis of the K⁺ channel blockade by haemolymph-side Ba²⁺ ions. Ba²⁺ was equally effective with Cl^- or gluconate (Glu^-) as the principal ambient anion. The Ba²⁺-induced K⁺ channel conduction noise is reflected by a Lorentzian, or relaxation, noise component in the power spectrum of the K⁺ current fluctuations. A reduced Lorentzian plateau value, but an unchanged corner frequency, were observed when Cl^- was replaced by Glu^-. The results from the analysis of a two-state model of K⁺ channel block by Ba²⁺, with respect to the anion-replacement effects, suggest that the observed changes in K⁺ current and Lorentzian plateau value mirror a complex change of the underlying parameters: Cl^- omission reduces single channel current but increases channel density so that the product of single channel current and channel density is smaller in Glu^- than in Cl^-. It seems likely that basolateral K⁺ channels (1) are subject to anionic gating ligands, and (2) depend on anions with respect to the rate of K⁺ transfer through and open K⁺ channel.Abbreviations a.c. alternating current - single-channel conductance - E _K K⁺ Nernst potential - f frequency contained in current noise - f _c corner frequency - Glu^- gluconate - G _t transepithelial conductance - I _sc short-circuit current - I _K K⁺ current - I _K(max) maximal K⁺ current - i single-channel current - K _Ba barium inhibition constant - K _m Michaelis constant of saturating K⁺ current - k ₀₁ and k ₁₀ barium association and dissociation rate constant, respectively - M K⁺ channel density - S _f power density - S _o Lorentzian plateau value - P _o channel-open probability - P _K K⁺ permeability - V _sc cellular potential at short-circuit These results have already been presented in part, at the 1989 joint meeting of the German and Israel Physiological Societies in Jerusalem (Zeiske et al. 1990).