Characterization of Microtubule-Associated Protein 2 from Mouse Brain and Its Localization in the Cerebellar Cortex

Microtubule-associated protein (MAP) 2 was purified from the microtubule fraction of mouse brain by heat treatment and BioGel A-5m gel filtration. The purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis using both a gradient gel (3.75-12.5%) and a low-percentage gel (5%), a finding indicating that MAP2B was absent under the conditions used. Amino acid analysis revealed that mouse MAP2 was an acidic protein with an isoelectric point (pI 4.5) and amino acid composition similar to those of porcine brain MAP2. Immunoblot analysis indicated that the antigens that reacted with MAP2 antiserum were present in large quantities in mouse brain. However, we also found a weak reaction in various tissues other than brain, and the major antigens involved were recognized to be common molecular species with the same molecular mass, 162 and 170 kilodaltons. Using antiserum against mouse brain MAP2, the developmental localization patterns of MAP2 in the mouse cerebellar cortex were studied by immunohistochemistry. MAP2 was mainly localized in the neuronal cells throughout development, with the expression in Purkinje cell dendrites being especially remarkable in the growth of arborization from postnatal day 3 to day 20. At the mature stage, the reaction was strong in the dendritic tree but very weak in the proximal dendrites and cell bodies.     

Autolysis of the cell wall. Its possible role in endogenous and IAA-induced growth in epicotyls of Cicer arietinum

Indoleacetic acid (IAA), a factor that induces growth in epicotyls of cicer arietinum L. cv. Castellana, increases the autolytic capacity of the cell walls by 50%, suggesting that autolysis is related to the processes of cell wall loosening that accompany growth. IAA promotes an increase in the specific activities of the enzymes involved in autolysis, mainly α-galactosidase (EC 3.2.1.22). This relationship autolysis-growth. was also observed in a study of the autolytic capacity of cell walls from regions of the epicotyl with different growth capacity. The sugars released and the level of enzymatic protein were higher in the subapical region that towards the base.     

Ca2+-dependent Cl− efflux in tonoplast-free cells ofNitellopsis obtusa

Summary In order to demonstrate the presence of a Ca²⁺-activated Cl^–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca²⁺ concentration was modified by internal perfusion. An increase in the Ca²⁺ concentration caused a large Cl^– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca²⁺ concentration was about 4.0 m. Neither the Cl^– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl^–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl^– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca²⁺ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca²⁺-dependent Cl^–-channel.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

Mechanisms of fusicoccin action: A dominant role for secondary transport in a higher-plant cell

Fusicoccin (FC) is commonly thought to promote electrogenic H⁺ extrusion through its action on the H⁺-ATPase of the plant plasma membrane. Nonetheless, essential support from rigorous electrophysiological analysis has remained largely absent. The present investigation surveys the effects of FC on the charge transport properties at the membrane of a higher-plant cell — stomatal guard cells of Vicia faba L. — for which the electrical geometry is defined, and from which the voltage-dependent kinetic characteristic for the pump has been identified. Current-voltage (I-V) relations of the guard cells were determined before and during treatments with FC, and during brief exposures to NaCN plus salicylhydroxamic acid. Responses of the pump and of the ensemble of secondary transport processes were identified in the whole-membrane conductance-voltage relations and in the difference-current-voltage (dI-V) characteristic for the pump. In 0.1 mM K⁺, exposure to 10 M FC shifted guard-cell potentials negative by 29–61 mV. Current-and conductance-voltage profiles indicated limited changes in the pump I-V characteristic, an observation which was confirmed through explicit kinetic analysis of pump dI-V relations. However, the voltage response was accompanied by a 1.5-to 2.6-fold fall in membrane conductance. These results challenge conventional views of fusicoccin action by ascribing the electrical responses to reduced current passage through secondary transport pathways as well as to enhanced electrogenic ion pumping.Abbreviations and symbols Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SHAM salicylhydroxamic acid - FC fusicoccin - V _m free-running membrane potential - G _m membrane slope conductance at V _m - (d)I-V (difference) current-voltage (relation) - G-V slope conductance-voltage (relation)     

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.)

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p culture media; see Material and methods - cv chltivar - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

限制性内切酶诱发的姊妹染色单体互换

用限制性内切酶PstⅠ,SalⅠ,PvuⅡ和BamHⅠ处理CHO细胞后,发现其SCE率升高,与对照相比,前三种酶具有显著性差异。但这些酶诱导SCE的效应与其致染色体畸变效应相比则较弱,提示引起DNA双链断裂的限制性内切酶不是SCE的强刺激物。实验结果表明,BrdU取代胸苷不能消除限制酶对底物DNA的识别及裂解。