Effect of phosphate deficiency on growth and protein profile in three strains ofPholiota nameko

Changes in mycelial dry weight and soluble protein amounts and acid phosphatase activities on a mycelial dry weight basis in the mycelia and culture supernatants during the Pi-supplied (P⁺) and Pi-depleted (P⁻) cultures of three strains ofPholiota nameko were examined. Mycelial dry weights of the three strains were lower in the P⁻ culture than in the P⁺ culture. However, soluble protein amounts in the culture supernatants and acid phosphatase activities in the mycelia and culture supernatants of the three strains were higher on a mycelial dry weight basis in the P⁻ culture than in the P⁺ culture. Total proteins of strains N2 and N4 were analyzed by two-dimensional-PAGE. Comparison of electrophoretograms of the P⁺ and P⁻ cultures showed that many polypeptides in the two strains were induced and secreted by Pi deficiency, but more than half of them were specific to each strain. Activity staining of acid phosphatase also revealed that two isozymes with the same molecular weights in the three strains were induced and secreted by Pi deficiency. Adaptive mechanisms for Pi deficiency in the three strains were discussed.     

Electrophoretic analysis of soluble proteins specifically synthesized under phosphate deficiency in the mycelia ofPholiota nameko

Mycelial soluble proteins ofPholiota nameko labeled in vivo during the Pi-supplied (P⁺) and the Pi-depleted (P⁻) cultures were separated by SDS-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis, and visualized by fluorography. A comparison of protein profiles from the P⁺ and P⁻ cultures showed that Pi deficiency induces the synthesis of 15 polypeptides and an increase in the relative amount of 29 polypeptides. These result suggests that many proteins may be specifically synthesized de novo under Pi deficiency as part of the adaptive mechanism for this condition.     

Nuclear selection in monokaryotization of dikaryotic mycelia ofPholiota nameko as described by leading and following nuclei

To examine monokaryotization of dikaryotic mycelia ofPholiota nameko, 18 monokaryotic stocks were used to produce a total of 130 dikaryotic stocks by reciprocal crossing. Monokaryotized mycelium was raised from dikaryotic mycelium in the peripheral zone of the growing colony. The stocks mated with a particular group of monokaryons produced wide-range monokaryotization at higher rates than the other combinations of hybridization. The growth rates of the monokaryotized mycelia exceeded from those of the corresponding parental dikaryons. The monokaryotized mycelium was isolated and back-crossed to parental monokaryotic stocks. Most of the isolates had nuclear types similar to only one of the parental stocks, while the replicates of isolates from two dikaryotic hybrids showed split nuclear type compositions. It is suggested that a relative dominance is active in the selection of one of the two nuclei of the dikaryotic cells in monokaryotization. The hierarchy of relative dominance among nuclei of 18 parental monokaryotic stocks in the monokaryotization of their reciprocal crossing products was estimated. We propose the involvement of a cascade process in dikaryotic cell division, in which the first dividing nucleus (to be found in the monokaryotized cell) may act as the leading nucleus and the other one as the following nucleus.     

Osmotic regulation ofmyo-inositol uptake in primary astrocyte cultures

Uptake ofmyo-inositol by astrocytes in hypertonic medium (440 mosm/kg H₂O) was increased near 3-fold after incubation for 24 hours, which continued for 72 hours, as compared with the uptake by cells cultured in isotonic medium (38 nmoles/mg protein).myo-Inositol uptake by astrocytes cultured in hypotonic medium (180 mosm/kg H₂O) for periods up to 72 hours was reduced by 74% to 8 to 10 nmoles/mg protein. Astrocytes incubated in either hypotonic or hypertonic medium for 24 hours and then placed in isotonic medium reversed the initial down- or up-regulation of uptake. Activation of chronic RVD and RVI correlates with regulation ofmyo-inositol uptake. A 30 to 40 mosm/kg H₂O deviation from physiological osmolality can influencemyo-inositol homeostasis. The intracellular content ofmyo-inositol in astrocytes in isotonic medium was 25.6 ± 1.3 g/mg protein (28 mM). This level ofmyo-inositol is sufficient for this compound to function as an osmoregulator in primary astrocytes and it is likely to contribute to the maintenance of brain volume.     

Purification and characterization of secreted acid phosphatase under phosphate-deficient condition inPholiota nameko

Activity of acid phosphatase secreted by mycelia ofPholiota nameko on cultivation for 30d in Pi-depleted medium was 88-fold higher than the corresponding activity in the Pi-supplied medium. One isozyme of the secreted acid phosphatases was purified from the culture filtrate of Pi-depleted medium by ammonium sulfate fractionation and cation exchange chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed change chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed that the native molecule had a molecular weight of 117,000. The molecular weight on gel electrophoresis with SDS was 52,000, indicating that the native form of the enzyme was a homodimer. The optimum pH and temperature of the enzyme were, 5.5 and 45°C, respectively, and the isoelectric point of the enzyme was pH 6.9. Adsorption on Con A-Sepharose and periodic-Schiff stain suggested that the enzyme is a glycoprotein. The enzyme hydrolyzed a wide variety of phosphate esters, nucleoside phosphates, sugar phosphates, and phosphorylated amino acids. Cu²⁺, Fe²⁺, Hg²⁺, iodoacetate, molybdate, tartaric acid, and SDS inhibited the enzyme activity. Fe³⁺ (1 mM), Triton X-100, methanol, and ethanol activated it. Fifteen residues of the N-terminal amino acid sequence were determined.     

Nuclear selection in monokaryotic oidium formation from dikaryotic mycelia in a basidiomycete,Pholiota nameko

The effect of nuclear dominance in monokaryotic oidium formation from dikaryotic mycelia inPholiota nameko was examined. Over 90% of oidium isolates from dikaryotic mycelia were monokaryotic. Although only one parental nuclear type was recovered from an average of about 80% in these isolates, the nuclear selection process in oidium formation seems essentially to produce split nuclear type composition in oidium products. The hierarchy of relative dominance among the nuclear types of the parental dikaryons in monokaryotic oidium formation was determined. The two hierarchies in nuclear selection between monokaryotic oidium formation and monokaryotic mycelium formation coincided at a level of at least 75%.     

Acid phosphatase isozymes secreted under phosphate-deficient conditions inPholiota nameko

We previously reported the purification of an acid phosphatase (APase52) secreted from the mycelia ofPholiota nameko under phosphate-deficient conditions. In the present study, two other isozymes (APase47 and APase48) were found and their structures were compared with that of APase52. Thirteen amino acid residues at theN-terminus of APase47 were completely identical with those of APase48 and had partial homology with those of APase52. The deglycosylation of proteins indicated that three APase isozymes differ in theN-linked oligosaccharide content. The protease-generated peptide maps of the APases differed from one another in the band pattern. These results suggest that the APases are the products of different genes.     

Morphological and cytological aspects of oidium formation in a basidiomycete,Pholiota nameko

Pholiota nameko produced abundant oidia on aerial hyphae from monokaryotic and dikaryotic test stocks, but oidia were rare on submerged hyphae. The oidia from the former stocks had a layer of hydrophobic protein between the cell wall and the inner cell membrane which was absent in the oidia from the latter. The only remarkable differences in the morphological features of the oidia from monokaryotic and dikaryotic mycelia was the slightly larger size of the latter. Observation of various test stocks on slide cultures revealed that about 80% of oidia were produced from the secondary branched hypha, and about 20% from the terminal hyphal, cell of the main hypha. In the former, the secondary hyphae were segmented to form several oidium cells; in the latter, a single or several oidia were formed at the terminal end of the main hypha. Most oidia from monokaryons and dikaryons had only one haploid nucleus, while the remainders were multinucleate. Among the stocks tested, most oidia had a DNA content with a haploid amount at the G1 phase of the cell cycle, but a few contained twice that amount corresponding to the G2 phase     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

The effect of phytase on the availability of P from myo-inositol hexaphosphate (phytate) for maize roots

The effect of adding phytase to the root medium of maize plants on the P-availability of added myo-inositol hexaphosphate (phytin) has been studied in pot experiments. When 40 mM phytin-P in nutrient solution was incubated in quartz-sand for 15 days in the absence of plants, 80% of it could be recovered from the solution as soluble organic P. Maize plants growing on this mixture assimilated P from phytin at rates comparable to those from inorganic phosphate (Pi). At a lower addition rate (2 mM phytin-P) only 10% was recovered in the soil solution, and plant growth was severely limited by P. At this low phytin level, the addition of phytase (10 enzyme units per kg sand) increased the plants' dry weight yield by 32%. The relative increases of the Pi concentration in the solution and of the amount of P in the plants were even higher, indicating that the observed growth stimulation was due to an increased rate of phytin hydrolysis. The enzyme-induced growth stimulation was also observed with plants growing in pots filled with soil low in P, when phytin was added. However, on three different soils the addition rates of phytin and phytase necessary for obtaining a significant phytase effect were both about 10 times higher than those required in quartzsand. It is concluded that the P-availability from organic sources can be limited by the rate of their hydrolytic cleavage.Abbreviation Pi inorganic phosphate     

Kinetic and structural analysis of a bacterial protein tyrosine phosphatase-like myo-inositol polyphosphatase

PhyA from Selenomonas ruminantium (PhyAsr), is a bacterial protein tyrosine phosphatase (PTP)-like inositol polyphosphate phosphatase (IPPase) that is distantly related to known PTPs. PhyAsr has a second substrate binding site referred to as a standby site and the P-loop (HCX5R) has been observed in both open (inactive) and closed (active) conformations. Site-directed mutagenesis and kinetic and structural studies indicate PhyAsr follows a classical PTP mechanism of hydrolysis and has a broad specificity toward polyphosphorylated myo-inositol substrates, including phosphoinositides. Kinetic and molecular docking experiments demonstrate PhyAsr preferentially cleaves the 3-phosphate position of Ins P6 and will produce Ins(2)P via a highly ordered series of sequential dephosphorylations: D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, and D-Ins(2,4)P2. The data support a distributive enzyme mechanism and suggest the PhyAsr standby site is involved in the recruitment of substrate. Structural studies at physiological pH and high salt concentrations demonstrate the "closed" or active P-loop conformation can be induced in the absence of substrate. These results suggest PhyAsr should be reclassified as a D-3 myo-inositol hexakisphosphate phosphohydrolase and suggest the PhyAsr reaction mechanism is more similar to that of PTPs than previously suspected.     

L-<Emphasis Type="Italic">myo</Emphasis>-inositol-1-phosphate synthase: partial purification and characterisation from <Emphasis Type="Italic">Gleichenia glauca</Emphasis>

A screening for the enzyme L-myo-inositol-1-phosphate synthase [EC 5.5.1.4] has been made first time in both vegetative and reproductive parts of the representative members of pteridophytes: Lycopodium, Selaginella, Equisetum, Polypodium, Dryopteris, and Gleichenia. The enzyme has been partially purified following low-speed centrifugation, streptomycin sulphate precipitation, ammonium sulphate fractionation, chromatography on DEAE-cellulose and gel-filtration through Sephadex G-200, and characterised from the reproductive pinnules of Gleichenia glauca Smith. The enzyme has a pH optimum at 7.5. The K_m for glucose-6-P and NAD⁺ were 0.922 × 10^–3 M and 0.9 × 10^–4 M, respectively. A basal activity of the enzyme has been recorded in absence of exogenous NAD⁺. The enzyme activity was augmented with NH₄Cl, but heavy metals like Hg²⁺, Cu²⁺ and Zn²⁺ inactivated it.     

Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.     

拟南芥中myo-肌醇-多磷酸合成与代谢及其信号

在酵母、真菌、动物和植物等真核生物中, 以myo-肌醇为基石通过不同位点的磷酸化形成各种myo-肌醇-多磷酸及其衍生物。过去10年的研究发现这些肌醇多磷酸参与了膜脂定向转运、蛋白结构稳定、离子通道调控、RNA转运以及DNA修复和染色质重塑等细胞生物学的基本进程。近些年在模式植物拟南芥(Arabidopsis thaliana)的研究中, 许多调控植物生长发育和环境胁迫应答的重要基因被发现, 并证实这些基因参与myo-肌醇-多磷酸的合成与代谢。该文概述了拟南芥中myo-肌醇-多磷酸合成与代谢的基因调控机理, 综述了不同肌醇多磷酸作为信号分子的功能, 提出肌醇多磷酸如同一类信息代码传递着植物细胞有序进程的基本指令。     

Degradation of <Emphasis Type="Italic">myo</Emphasis>-inositol Hexakisphosphate by a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

High-pressure liquid chromatography (HPLC) analysis established myo-inositol pentakisphosphate as the final product of phytate dephosphorylation by the phytate-degrading enzyme from Pantoea agglomerans. Neither product inhibition by phosphate nor inactivation of the Pantoea enzyme during the incubation period were responsible for the limited phytate hydrolysis as shown by addition of phytate-degrading enzyme and phytate, respectively, after the observed stop of enzymatic phytate degradation. In additon, the Pantoea enzyme did not possess activity toward the purified myo-inositol pentakisphosphate. Using a combination of High-Performance Ion Chromatography (HPIC) analysis and kinetic studies, the nature of the generated myo-inositol pentakisphosphate was established. The data demonstrate that the phytate-degrading enzyme from Pantoea agglomerans dephosphorylates myo-inositol hexakisphosphate in a stereospecific way to finally D-myo-inositol(1,2,4,5,6)pentakisphosphate.     

Anti-inflammatory activity of polysaccharide from <Emphasis Type="Italic">Pholiota nameko</Emphasis>

Pholiota nameko polysaccharide (PNPS-1) has been isolated and purified by enzymatic hydrolysis, hot water extraction, ethanol precipitation, and ion-exchange and gel-filtration chromatography. The anti-inflammatory activity of PNPS-1 was evaluated in rodents using xylene-induced ear edema, egg albumin-, carrageenin-, and formaldehyde-induced paw edema, cotton pellet granuloma test, adhesion of peritoneal leukocytes in vitro, and ulcerogenic activity. The results showed that PNPS-1 (5 mg/ear) inhibited topical edema in the mouse ear and at 100, 200, and 400 mg/kg (intraperitoneally) it significantly suppressed the development of egg albumin-, carrageenin-, and formaldehyde-induced paw edema in the animals. PNPS-1 (100, 200, and 400 mg/kg, per oral) significantly inhibited the growth of granuloma tissues induced by subcutaneously implanted cotton pellets in rats by 10.96, 18.07, and 43.75%, respectively. PNPS-1 also inhibited spontaneous and phorbol-12-myristate-13-acetate-activated adhesion of peritoneal leukocytes in vitro. Further, both acute as well as chronic administration of PNPS-1 (100, 200, and 400 mg/kg, per oral) did not produce any gastric lesion in rats. In conclusion, these data indicated that PNPS-1 possesses significant anti-inflammatory activity suggesting its potential as an anti-inflammatory agent for use in the treatment of various inflammatory-related diseases.     

Uptake of auxotrophic cells of a heterocyst-forming cyanobacterium by tobacco protoplasts,and the fate of their associations

During imbibition, exogenous myo-inositol (MI) was readily introduced into the free MI pool of germinating wheat (Triticum aestivum L.). Maximum uptake, 70 g per caryopsis or 1.5 mg g^–1 of caryopsis, was reached at 0.05 M MI. Movement of free MI within the germinating caryopsis was traced with [2-³H]MI by two procedures, uptake by imbibition and injection into softened endosperm. The former procedure was useful during initial stages of germination; the latter provided a means of tracing the metabolic fate of MI generated by hydrolysis of phytate during mobilization of reserves within the caryopsis. In both procedures, the bulk of the added label was transferred to the seedling where it appeared in uronosyl and pentosyl units of 80% ethanol-insoluble polysaccharides, 2-O, C-Methylene-MI, an inhibitor of the MI oxidation pathway, blocked the utilization of [2-³H]MI as well as d-[1¹⁴C]glucose for biogenesis of pentose-and uronic-acid-containing polysaccharides.Abbreviations MI myo-inositol - OCM-MI 2-O, C-methylene-myo-inositol     

Organic substances in xylem sap delivered to above-ground organs by the roots

Squash (Cucurbita maxima) xylem sap, an apoplastic fluid, contains t-zeatin riboside, glutamine, methylglycine, myo-inositol, fructose, oligosaccharides of arabinogalactan, glucan, galacturonan, and pectins (rhamnogalacturonan-I and rhamnogalacturonan-II), as well as various proteins, including arabinogalactan and pathogen-related proteins. These substances are mainly produced in stele (xylem) parenchyma and the pericycle in the root-hair zone where ion transporter genes are expressed. Glycine-rich protein genes (CRGRPs) cloned by antiserum raised against whole xylem sap of cucumber (Cucumis sativus) were abundantly expressed in the parenchyma cells surrounding xylem vessels in the root-hair zone. CRGRP proteins accumulated and immobilized in the lignified walls of metaxylem vessels and perivascular fibers in shoots, suggesting a systemic delivery mechanism of wall materials via xylem sap. A major 30-kDa protein (XSP30) found in cucumber xylem sap was homologous to the B chains of a lectin (ricin) and bound to a nonfucosylated core N-acetylglucosamine dimer of N-linked glycoproteins abundant in leaf parenchyma cells. XSP30 gene expression, abundant in root xylem parenchyma and pericycle, and the level of XSP30 protein fluctuated diurnally under the control of a circadian clock, and the amplitude was up-regulated by gibberellic acid produced in young leaves, suggesting a long-distance control system between organs.     

Arabidopsis UDP-sugar pyrophosphorylase: Evidence for two isoforms

Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (USP1) and 66 kDa (USP2) in crude extracts of Arabidopsis tissues. The 66 kDa isoform was not the result of proteolytic cleavage of USP1 during extraction. Trypsin digestion of bands on SDS gels corresponding to the location of the two isoforms followed by tandem mass spectrometry confirmed that USP peptides were present in both bands. Both USP isoforms were detected in the cytosol as determined by immunoblots of cellular fractions obtained by differential centrifugation. However, some USP1 was also detected in the microsomal fraction. Immunoprecipitation assays demonstrated that AtUSP antibodies removed USP activity (UDP-GlcA→GlcA-1-P) measured in floret extracts. These results indicate that USP is the only pyrophosphorylase that utilizes UDP-GlcA as a substrate and suggest that it serves as the terminal enzyme of the myo-inositol oxidation pathway.