Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca²⁺] _o (extracellular Ca²⁺ concentration) is between about 10^–4 to 10^–3 m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca²⁺ _o or increasing [Ca²⁺] _o to 10^–2 m inhibit exocytotic membrane fusion. Veratridine does not release Ca²⁺ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na⁺ _o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca²⁺] _o ) in the somatic, i.e., nonciliary cell membrane and that a Ca²⁺ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4.     

The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells

Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for l min stimulates the bumetanide-inhibitable K⁺ influx fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The K _I for bumetanide was 0.19±0.06 m for the cytoplasts as compared to 0.67±0.11 m for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the 80 kD protein.We are grateful to Marianne Schiødt, Birgit Blytmann Jørgensen, Thomas Krarup and Beverley Dyer for expert assistance. This work was supported by grants from the Danish Natural Science Research Council (11-6835 to E.K.H.) and the National Institutes of Health (DK 33640 to P.B.D.) and by a Carlsberg Foundation research fellowship (to F.J.).     

P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees

The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca²⁺-transporting ATPases; cluster 2: Na⁺- and gastric H⁺-ATPases; cluster 3: plasma membrane H⁺-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K⁺, Cd²⁺, Cu²⁺ and an unknown cation). The one bacterial exception to this general pattern was the Mg²⁺-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques. Correspondence to: M.H. Saier, Jr.     

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins

Abstract: The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M _r PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M _r PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and are DD-carboxypeptidases. All three low M _r , PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic α-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.     

Microelectrode-recorded development of the symplasmic autonomy of the sieve element/companion cell complex in the stem phloem of Lupinus luteus L.

From the cambial stage onwards, the symplasmic autonomy of sieve element/companion cell complexes (SE/CC-complexes) was followed in stems of Lupinus luteus L. by microinjection techniques. The membrane potential and the symplasmic autonomy of the mature SE/CC-complex was measured in successive internodes. A microelectrode was inserted into SE/CC-complexes or phloem parenchyma cells (PPs) and, after stabilization of the membrane potential, the membrane-impermeant fluorescent dye Lucifer Yellow CH (LYCH) was injected intracellullary. The plasmodesmata of the cambial SE/ CC precursor were gradually shut off at all interfaces beginning at the walls to be transformed into sieve plates. In the course of maturation, symplasmic discontinuity was maintained at the longitudinal walls of the complex. In the transverse walls of the SE, wide sieve pores were formed giving rise to longitudinal multicellular symplasmic domains of SE/CC-complexes. Symplasmic isolation of the files of mature SE/CC-complexes was demonstrated in several ways: (i) the membrane potential of the SE/CC-complexes (between -100 mV and -130 mV) was consistently more negative than that of the PPs (between-50 and -100 mV), (ii) No exchange of LYCH was observed between SE/CC-complexes and the PPs. Lucifer Yellow CH injected into the SEs exclusively moved to the associated CCs and to other SE/CC-complexes whereas LYCH injected into the PPs was only displaced to other PPs. (iii) The electrical coupling ratio between adjacent PPs was ten times higher than that between SE/CC-complex and PP. A gradient in the membrane potential of the SE/CC-complexes along the stem was not conclusively demonstrated.Abbreviations LYCH Lucifer Yellow CH - membrane potential - PMF proton-motive force - PP phloem parenchyma cell - SE/CC-complex sieve element/companion cell complex - SR-G sulphorhodamine G     

Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10^?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10^?2 M). Isoporterenol (10^?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E₁ (10^?5 M) produced similar responses in the two strains. G_i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G_i2α and G_qα/G₁₁α were similar in the two strains, while the levels of G_sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ～20% in MHS membranes. The α-subunit of G_i3 was dramatically reduced by ～80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.     

Fourier transform infrared spectroscopic study of ion binding and intramolecular interactions in the polar head of digalactosyldiacylglycerol

Lipid bilayers composed of digalactosyldiacyl-glycerol (DGDG), that is, Galp1-6Galp1-3DAG, a non-ionic lipid of the thylakoid membrane of chloroplasts, aggregate in aqueous media containing mono- and divalent cations in amounts above a threshold concentration (C_t) of about 1.0, 4.7 and 10.0 mM for Ca²⁺, Mg²⁺ and Na⁺, respectively. In this work, we found that above C_t the DGDG membranes do not undergo fusion and that the aggregation can be reversed, or disrupted. This means that the perturbation induced by the salts results from adsorption, or complexation of the ions in the polar head of DGDG. To investigate this question, we used Fourier transform infrared (FTIR) spectroscopy to identify the molecular sites in DGDG which are modified by interaction, or adduct formation with CaCl₂, MgCl₂ and NaCl. We also determined whether the ions affect the intramolecular hydrogen bonding between the sn₂ ester C = O and the carbon-6 of the -anomer of galactose (Gal). The major conclusions are: (i) the salts do not affect, at least directly, the, ester carbonyl region of DGDG, (ii) the most probable sites of binding, or adsorption, for the ions are the ring oxygen, and (iii) the ring hydroxyls are the sites of either ion complexation or intra- and intermolecular H-bonding in interacting DGDG membranes. Within this framework, the complexation of the ions with Gal might induce total or partial dehydration of the galactolipid headgroup and thus provides the means to overcome the repulsive hydration forces that hinder aggregation of the DGDG membranes.Abbreviations DGDG digalactosyldiacylglycerol - EDTA ethylenediaminetetracetic acid - FTIR Fourier transform infrared - Gal galactose - GIDG D-glucosyldiacylglycerol - Glyc glycerol - LHCII chloroplast light harvesting complex II - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PG phosphatidylglycerol - PS phosphatidylserine - SQDG sulfoquinovosyl-diacylglycerol Correspondence to: M. Fragata     

Special features of phosphatidylcholine vesicles as seen in cryo-transmission electron microscopy

Vesicles of egg yolk phosphatidylcholine (EYPC) were studied by cryo-transmission electron microscopy. The electron micrographs indicate that, despite the rapidity of cooling, membrane undulations are flattened and some vesicles change their shapes before the samples freeze. These artefacts are attributed to the action of the lateral tension that results from the membrane area contraction associated with the temperature drop. Other micrographs represent grainy membranes and angular vesicles. We regard them as the first direct evidence for the superstructure and optically invisible roughness which were recently postulated for these membranes.     

A Protein Extracted from Mouse Sperm That Plays an Important Role in Fertilization

ＡＰｒｏｔｅｉｎＥｘｔｒａｃｔｅｄｆｒｏｍＭｏｕｓｅＳｐｅｒｍＴｈａｔＰｌａｙｓａｎＩｍｐｏｒｔａｎｔＲｏｌｅｉｎＦｅｒｔｉｌｉｚａｔｉｏｎＺＨＵＡＮＧＤａ－ｚｈｏｎｇ;（庄大中）,ＺＨＡＮＧＴｉａｎ－ｙｉｎ（张天荫）,ＣＨＥＮＤａ－ｙｕａｎ;（陈大...     

底物膜技术检测精子顶体酶活性的研究

应用底物膜技术检测１３０例正常精液,精子顶体酶活性百分率的正常值下限为５７％。４５９例不孕症病人精液分析,无精症２５例,其余４３４例中７５％精子顶体酶活性正常。实验表明精子密度对数值与顶体酶活性百分率之间有正相关,ｒ＝０．８４（Ｐ＜０．０１）,回归方程为顶体酶活性百分率ｙ＝４８．４３％＋（８．９％）（ｌｏｇ精子计数）。活动精子百分率与顶体酶活性之间有密切正相关,ｒ＝０．９６７,（Ｐ＜０．０１）,顶体酶活性ｙ＝３８．６％＋０、３６ｘ％。前向活跃直线运动精子百分率与顶体酶活性之间也有密切相关．ｒ＝０．９６,（Ｐ＜０．０１）,顶体酶活性ｙ＝３４．２１％＋０．６１ｘ％。