丛枝菌根真菌侵染势与接种势之间的关系

丛枝菌根(AM)真菌的侵染势(Colonizationpotential,CP)和接种势(inoculumpotential,IP)是菌根学领域非常重要的两个概念。IP已定义为接种物中有活力的真菌繁殖体及结构的数量(Liu&Luo,1994)。而CP的定量描述和测定方法尚未建立。本文将CP定义为单位数量接种物在侵染初期侵染植物根系的能力,其定量测定公式为:CP=N×L/IP×T,其中N为单位根长侵入点数+根内和根外菌丝数+含有丛枝的细胞数+泡囊数;L为每株寄主植物根系总长度;IP为接种物的接种势单位数;T为接种后的天数。用棉花(Gossypiumhirsutum)、大豆(Glycinemax)、红三叶(Trifoliumpratense)和玉米(Zeamays)和3种AM真菌Gigasporamargarita(Gim),Glomusintraradices(Gi),andGlomusversiforme(Gv)不同剂量(100,300,900,2700and8100接种势单位)的接种物进行试验,以定量测定CP、以及CP和IP之间的关系。结果表明,在相同数量的IP条件下,不同AM真菌具有不同的CP,应用该研究…     

Na+/K+ -ATPase regulates tight junction formation and function during mouse preimplantation development

Research applied to the early embryo is required to effectively treat human infertility and to understand the primary mechanisms controlling development to the blastocyst stage. The present study investigated whether the Na(+)/K(+)-ATPase regulates tight junction formation and function during blastocyst formation. To investigate this hypothesis, three experimental series were conducted. The first experiments defined the optimal dose and treatment time intervals for ouabain (a potent and specific inhibitor of the Na(+)/K(+)-ATPase) treatment. The results demonstrated that mouse embryos maintained a normal development to the blastocyst stage following a 6-h ouabain treatment. The second experiments investigated the effects of ouabain treatment on the distribution of ZO-1 and occludin (tight junction associated proteins). Ouabain treatment (up to 6 h) or culture in K(+)-free medium (up to 6 h) resulted in the appearance of a discontinuous ZO-1 protein distribution and a loss of occludin immunofluorescence. The third set of experiments examined the influence of ouabain treatment on tight junction function. Ouabain treatment or culture in K(+)-free medium affected tight junction permeability as indicated by an increase in the proportion of treated embryos accumulating both 4 kDa and 40 kDa fluorescein isothiocyanate (FITC)-dextran into their blastocyst cavities. The results indicate that the Na(+)/K(+)-ATPase is a potent regulator of tight junction formation and function during mouse preimplantation development.     

Intracellular Na+ and K+ contents of Zygosaccharomyces rouxii mutants defective in salt tolerance

Two of five Zygosaccharomyces rouxii mutants defective in salt tolerance, 152S (sat1) and 1717S (SAT3), were inviable in a nutrient medium (YPD) containing more than 1% NaCl. These two mutant cells contained significantly higher amounts of Na⁺ (298 μmol and 285 μmol per g cells of 152S and 1717S, respectively) but lower amounts of K⁺ (242 μmol and 176 μmol per g cells of 152S and 1717S, respectively) than three other mutants, 41S (sat2-1 [98 μmol Na⁺ and 326 μmol K⁺/g cells]), 197S (sat2-2 [103μmol Na⁺ and 336 μmol K⁺/g cells]), 1611S (SAT4 [139 μmol Na⁺ and 294 μmol K⁺/g cells]), as well as a wild-type strain, AN39 (61 μmol Na⁺ and 349 μmol K⁺/g cells), when cultured in YPD medium containing 0.8% NaCl. A KCl supplement, optimally 0.6 M, added to the medium somewhat restored the NaCl-hypersensitivity of 152S and 1717S with a concomitant decrease of intracellular Na⁺. This finding suggests that the NaCl-hypersensitive mutations are due to a defect in the Na⁺-regulating mechanism. The other three mutants showed weak responses to KCl in high NaCl-YPD. These five salt sensitive mutants and the wild-type strain retained the same levels of intracellular glycerol and arabitol when transferred into NaCl (5%)-YPD from YDP medium. This suggests that polyol accumulation is not the only mechanism of salt tolerance in Z. rouxii.     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

Ultrastructural localization of N-acetylglucosamine residues in the cell wall of Gigaspora margarita throughout its life-cycle

The cell ultrastructure of the VAM fungus Gigaspora margarita was examined, and its N-acetylglucosamine content and distribution were assessed by means of WGA/ovomucoid-gold labelling. The various ontogenic stages of the fungus were studied with particular reference to cell walls. The results provide new information on chitin incorporation during spore wall development. A decrease of chitin was observed which was correlated to the structural simplification of the fungus wall throughout its life-cycle. This suggests an involvement of chitin in specific biological functions such as mechanical resistance and plasticity.     

Role of endosomal Na+-K+-ATPase and cardiac steroids in the regulation of endocytosis

Plasma membrane Na⁺-K⁺-ATPase, which drives potassium into and sodium out of the cell, has important roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically interact with the pump and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds are present in mammalian tissues, synthesized in the adrenal gland, and considered to be new family of steroid hormones. In this study, the mechanism of Na⁺-K⁺-ATPase involvement in the regulation of endocytosis is explored. We show that the effects of various CS on changes in endosomal pH are mediated by the pump and correspond to their effects on endosomal membrane traffic. In addition, it was found that CS-induced changes in endocytosed membrane traffic were dependent on alterations in [Na⁺] and [H⁺] in the endosome. Furthermore, we show that various CS differentially regulate endosomal pH and membrane traffic. The results suggest that these differences are due to specific binding characteristics. Based on our observations, we propose that Na⁺-K⁺-ATPase is a key player in the regulation of endosomal pH and endocytosed membrane traffic. Furthermore, our results raise the possibility that CS-like hormones regulate differentially intracellular membrane traffic. bufalin; ouabain; endosomal pH     

Identification and function of a cytoplasmic K+ site of the Na+, K+ -ATPase

A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme. The functional roles of this third K(+)/Rb(+) binding site were studied by site-directed mutagenesis, replacing the side chain of Asp(742) donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na(+), K(+)-ATPase, the mutants display a biphasic K(+) concentration dependence of E(2)P dephosphorylation, indicating that the cytoplasmic K(+) site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E(2) to E(1) conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K(+)/Rb(+) site not only enhances E(2)P dephosphorylation but also stabilizes the E(2) dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K(+) by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K(+)/Rb(+) site appears to be conserved among Na(+), K(+)-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E(2) state.     

Heterologous expression of Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) and glycerol dehydrogenase gene (ZrGCY1) in Saccharomyces cerevisiae

We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Deltagpd2Delta). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.     

The PAM1 gene of petunia,required for intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi,encodes a homologue of VAPYRIN

Most terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.     

Reciprocal modulation of function between the D1 and D2 dopamine receptors and the Na+,K+-ATPase

It is well documented that dopamine can increase or decrease the activity of the Na+,K+-ATPase (NKA, sodium pump) in an organ-specific fashion. This regulation can occur, at least partially, via receptor-mediated second messenger activation and can promote NKA insertion or removal from the plasma membrane. Using co-immunoprecipitation and mass spectrometry, we now show that, in both brain and HEK293T cells, D1 and D2 dopamine receptors (DARs) can exist in a complex with the sodium pump. To determine the impact of NKA on DAR function, biological assays were conducted with NKA and DARs co-expressed in HEK293T cells. In this system, expression of NKA dramatically decreased D1 and D2 DAR densities with a concomitant functional decrease in DAR-mediated regulation of cAMP levels. Interestingly, pharmacological inhibition of endogenous or overexpressed NKA enhanced DAR function without altering receptor number or localization. Similarly, DAR function was also augmented by small interfering RNA reduction of the endogenous NKA. These data suggest that, under basal conditions, NKA negatively regulates DAR function via protein-protein interactions. In reciprocal fashion, expression of DARs decreases endogenous NKA function in the absence of dopamine, implicating DAR proteins as regulators of NKA activity. Notably, dopamine stimulation or pertussis toxin inhibition of D2 receptor signaling did not alter NKA activity, indicating that the D2-mediated decrease in NKA function is dependent upon protein-protein interactions rather than signaling molecules. This evidence for reciprocal regulation between DARs and NKA provides a novel control mechanism for both DAR signaling and cellular ion balance.     

Role of Protein Phosphatase in the Regulation of Na+-K+-ATPase by Vasopressin in the Cortical Collecting Duct

In the cortical collecting duct (CCD), arginin vasopressin (AVP) has been shown to increase the number and activity of basolateral Na⁺-K⁺-ATPase by recruiting or activating a latent pool of pumps. However, the precise mechanism of this phenomenon is still unknown. The aim of this study was to investigate whether this AVP-induced increase in basolateral Na⁺-K⁺-ATPase could depend on a dephosphorylation process. To this purpose, the effect of protein serine/threonine phosphatase (PP) inhibitors was examined on both the specific ³H-ouabain binding (to evaluate the number of pumps in the basolateral membrane) and the ouabain-dependent ⁸⁶Rb uptake (to evaluate pump functionality) in the presence or absence of AVP. In addition, the activity of two PP, PP1 and PP2A, was measured and the influence of AVP was examined on both enzymes. Experiments have been performed on mouse CCD isolated by microdissection. Results show that inhibition of PP2A prevents the AVP-induced increase in the number and activity of Na⁺-K⁺-ATPases, independent of an effect on the apical cell sodium entry. In addition, AVP rapidly increased the activity of PP2A without effect on PP1. These data suggest that PP2A is implied in the regulation of Na⁺-K⁺-ATPase activity by AVP in the CCD and that the AVP-dependent increase in the number of Na⁺-K⁺-ATPases is mediated by a PP2A-dependent dephosphorylation process. Received: 22 March 1996/Revised: 21 June 1996     

Roles of transmembrane segment M1 of Na+,K+-ATPase and Ca2+-ATPase, the gatekeeper and the pivot

In this review we summarize mutagenesis work on the structure–function relationship of transmembrane segment M1 in the Na⁺,K⁺-ATPase and the sarco(endo)plasmic reticulum Ca²⁺-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca²⁺ interaction/occlusion in Ca²⁺-ATPase and K⁺ interaction/occlusion in Na⁺,K⁺-ATPase. Leu⁶⁵ of the Ca²⁺-ATPase and Leu⁹⁹ of the Na⁺,K⁺-ATPase, located at homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu³⁰⁹/Glu³²⁹. A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the extracytoplasmic gate in both the Ca²⁺-ATPase and the Na⁺,K⁺-ATPase. All numbering of Na⁺,K⁺-ATPase amino acid residues in this article refers to the sequence of the rat α₁-isoform.     

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al³⁺, Ca²⁺, Fe³⁺, and Yb³⁺ and the borderline metal ion Cu²⁺, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the rangepH 5.5–8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized M ⁿ +-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe³⁺-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe³⁺-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metalion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surfaceexposed aspartic and glutamic acid residues on the protein with the immobilized M ⁿ +-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu²⁺-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number and extent of clustering of the surfaceaccessible aspartic and glutamic acid residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.Abbreviations IDA iminodiacetic acid - IDA-Mⁿ⁺ iminodiacetic acid chelated to metal ion - IMAC immobilized metal affinity chromatography - DHCC dog heart cytochrome c - HHCC horse heart cytochrome c, THCC, tuna heart cytochrome c - HMYO horse skeletal muscle myoglobin - SMYO sheep skeletal muscle myoglobin - HEWL hen egg white lysozyme