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1.
An in-vitro culture system allowing the simultaneous germination of cysts was used to study the early host-independent release of phytoalexin elicitors by Phytophthora megasperma f. sp. glycinea, a soybean pathogen. Significant elicitor activity could be detected in the culture medium as early as 2 h after germination of P.m. f. sp. glycinea, race 1, cysts. The phytoalexin elicitor was heat-stable and heterogeneous in size. The apparent molecular mass ranged from 3 to 80 kDa. Anion exchange and lectin-affinity chromatography followed by sugar analysis confirmed that the elicitor activity resided primarily in glucans. The time course of elicitor release could then be accurately monitored by means of a competitive radioligand-displacement assay using the -glucan elicitor-binding sites of soybean (Glycine max (L.) Merr.) membranes. Linkage-composition analysis of the glucan elicitors showed that they were primarily (1 3)-linked with (1 6)--branches, a composition similar to that of glucans obtained by heat release from mature mycelium but different from that of elicitors obtained by acid hydrolysis or from spontaneous autohydrolytic release by senescent cultures. The naturally released elicitors displayed a biological activity in soybean cotyledon bioassays higher than purified acid-hydrolysed glucan elicitor or than the hepta-(1 3, 1 6)--glucoside, the smallest known carbohydrate elicitor for soybean. The present results demonstrate that elicitor release from the pathogen and perception by the potential host can take place in this system as early as during germ-tube formation and independent of the presence of host-produced endoglucanases.Abbreviations EC50 effector concentration necessary for halfmaximal response - GC-MS gas chromatography-mass spectrometry - HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-1-[hexagluco-syl]-deoxyglucitol - IC50 inhibitor concentration necessary for half-maximal inhibition - P.m. f. sp. glycinea Phytophthora megasperma f. sp. glycinea - V0 void volume Deceased on March 19, 1990This work was supported by the Deutsche Forschungsgemeinschaft (SFB 206). We thank Dr. H. Mayer, C. Warth and D. Borowiak (Max-Planck-Institut für Immunbiologie, Freiburg) for helpful discussions and experienced technical assistance (glucosyl-linkage analysis).  相似文献   

2.
Summary Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione, were accumulated in cultured onion cells. Removal of extracellular Ca2+ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5-trimethoxybenzoate caused similar effects, whereas the addition of the Ca2+ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca2+ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca2+ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells.Abbreviations A23187 4-bromo-calcium ionophore - cAMP adenosine 3,5-cyclic monophosphate - [Ca2+]cyt cytoplasmic Ca2+ concentration - EGTA ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid - EtOH ethanol - Et2O diethyl ether - fr.wt fresh weight - HR hypersensitive response - PIPES piperazine N,N-bis-(2-ethanesulfonic acid) - TMB-8 [8-N,N-(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate - Tsl tsibulin  相似文献   

3.
As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate  相似文献   

4.
Expression of too many co-dominant major histocompatibility complex (MHC) alleles is thought to be detrimental to proper functioning of the immune system. Polyploidy of the genome will increase the number of expressed MHC genes unless they are prone to a silencing mechanism. In polyploid Xenopus species, the number of MHC class I and II genes has been physically reduced, as it does not increase with higher ploidy genomes. In the zebrafish some class IIB loci have been silenced, as only two genomically bona fide loci, DAA/DAB and DEA/DEB, have been described. Earlier studies indicated a reduction in the number of genomic and expressed class II MHC genes in a hexaploid African large barb. This prompted us to study the number of MHC genes present in the genome of an African large barb individual (Barbus intermedius) in relation to those expressed, adopting the following strategy. Full-length cDNA sequences were generated from mRNA and compared with partial genomic class Ia and II sequences generated by PCR using the same primer set. In addition, we performed Southern hybridizations to obtain a verification of the number of class I and IIB genes. Our study revealed three 2-microglobulin, five class Ia, four class IIA, and four class IIB genes at the genomic level, which were shown to be expressed in the hexaploid barb individual. The class Ia and class II data indicate that the ploidy status does not correlate with the presence and expression of these MHC genes.  相似文献   

5.
Summary With the aim of ascertaining the existance of a correlation between in vivo resistance to Fusarium oxysporum f. sp. dianthi and in vitro response to fungal elicitors and toxic substances, phenylalanine ammonialyase and phytoalexin accumulation, on one hand, and resistance to culture filtrate, on the other, were assayed in in vitro cultures of three susceptible and four resistant Dianthus caryophyllus cultivars. Cultivars showing varying degrees of resistance in vivo either tolerated higher culture filtrate concentrations (Niki) or showed high PAL activity and phytoalexin production when treated with Fusarium elicitor (Duca), or responded positively to both treatments (Mei-Ling, Pulcino). No such responses were shown in tissue cultures of susceptible cultivars. The differential response to the fungal elicitor seemed to be highly specific as genetic differences between cultivars were not observed in tissue cultures treated with other biotic (Phytophthora infestans) and abiotic (HgCl2) elicitors.Abbreviations FuCWC cell wall components from Fusarium oxysporum f. sp. dianthi race 2 - PhCWC cell wall components from Phytophthora infestans - PAL phenylalanine ammonia-lyase (EC 4.3.1.5)  相似文献   

6.
Summary The nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis has been determined to be: G- A C -G-U-A-C-G-G-C-C-A-U-A-C-U-A-C-C-G-G-G-A-A-U-A-C-A-C-C-U-G-A-A-C-C-C-G--U-C-G-A-U-U-U-C-A-G-A-A-G-U-U-A-A-G-C-C-G-G-G-U-U-A-G-G-C-C-C-A-G-U-U-A-G-U-A-C-U-G-A-G-U-G-G-G-C-G-A-C-C-A-C-U-U-G-G-G-A-A-C-A-C-U-G-G-G-U-G-C-U-G-U-A-C-G-C-U-Up. This RNA is 119 nucleotides long and the sequence of a probable tRNA-binding site is GAUU (position 41–44 from the 5-terminus), which is the same as that of a trypanosoma species,Crithidia fasci-culata. TheEuglena 5S rRNA has a pseudouridine residue at position 38 and 3-terminus is phosphorylated. The 5S rRNA sequence ofEuglena resembles those of several other protozoa and higher animals rather than plants.On leave from Department of Zoology, Hiroshima University, Hiroshima, Japan  相似文献   

7.
Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF- -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential ( m) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained m and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of m and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt m or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of m and apoptosis when cells were treated with TNF- . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained m and prevented apoptosis, but could not reduce ROS until four hours after TNF- treatment. According to these data, we suggest that TNF- induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF- -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain m, prevent cytochrome c release and deactivate the caspase cascade pathway.  相似文献   

8.
Summary Each of the three genomes in hexaploid wheat controls the expression of a specific lectin in the embryo. The chromosomes which control their synthesis were determined using nullisomic-tetrasomic and inter-varietal chromosome substitution lines of Chinese Spring. All three wheat lectins were shown to be controlled by the homoeologous group 1 chromosomes. Using ditelosomic lines of Chinese Spring the lectin genes could be localized on the long arms of chromosomes 1A and 1D. Inter-specific addition and substitution lines of Aegilops umbellulata chromosomes to Chinese Spring indicated that chromosome 1U, which is homoeologous to the group 1 chromosomes of wheat, controls lectin synthesis.  相似文献   

9.
Summary A set of transducing phages carrying varying lengths of the E. coli chromosome around the structural gene for initiation factor IF3 (infC) was derived from p2 which is known to cary, besides infC, the structural genes for the subunit of phenylalanyl-tRNA synthetase (pheS), the subunit of phenylalanyl-tRNA synthetase (phetT) and the structural gene for threonyl-tRNA synthetase (thrS). The E. coli coding content of these derived phages was analysed by genetic complementation of a set of mutants and by SDS-polyacrylamide gel analysis of the proteins synthesized in UV irradiated cells infected with these phages. The segregation pattern of the different genes among these derived phages indicates that the order of the genes is pheT-pheS-P12-(infC, thrS) where infC is probably between P12 and thrS. P12 is the structural gene of a 12,000 molecular weight unidentified protein.Abbreviations PRS (EC 6.1.1.20) phenylalanyl-tRNA synthetase - TRS (EC 6.1.1.3) threonyl-tRNA synthetase - IF3 Initiation factor IF3 - SDS Sodium dodecyl sulfate - PPR pyrophosphate resistant - PPS pyrophosphate sensitive  相似文献   

10.
Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. We previously showed that iron-catalyzed ROS in part mediate asbestos-induced AEC DNA damage and apoptosis. Mitochondria have a critical role in regulating apoptosis after exposure to agents causing DNA damage but their role in regulating asbestos-induced apoptosis is unknown. To determine whether asbestos causes AEC mitochondrial dysfunction, we exposed A549 cells to amosite asbestos and assessed mitochondrial membrane potential changes (m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. We show that amosite asbestos, but not an inert particulate, titanium dioxide, reduces m after a 4 h exposure period. Further, the m after 4 h was inversely proportional to the levels of apoptosis noted at 24 h as assessed by nuclear morphology as well as by DNA nucleosome formation. A role for iron-derived ROS was suggested by the finding that phytic acid, an iron chelator, blocked asbestos-induced reductions in A549 cell m and attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell m after 4 h and diminished apoptosis. We conclude that asbestos alters AEC mitochondrial function in part by generating iron-derived ROS, which in turn can result in apoptosis. This suggests that the mitochondrial death pathway is important in regulating pulmonary toxicity from asbestos.  相似文献   

11.
Mizuno M  Tada Y  Uchii K  Kawakami S  Mayama S 《Planta》2005,220(6):849-853
In potato (Solanum tuberosum L.) suspension cells, the expression of the gene encoding alternative oxidase (AOX) and H2O2 accumulation were induced by treatment with -glucan elicitor. The inhibition of catalase activity enhanced both AOX mRNA expression and the production of H2O2, whereas the ascorbate peroxidase inhibitor did not have any effect on these responses. Simultaneous inhibition of catalase and AOX activities in elicited cells dramatically increased H2O2 accumulation, leading to the disruption of mitochondrial membrane potential (m) and programmed cell death (PCD). The results demonstrate, for the first time, that not only AOX but also catalase plays a central role in the suppression of mitochondrial m breakdown and PCD induced by -glucan elicitor.  相似文献   

12.
Summary Normal individuals and those homozygous and heterozygous for the gene which determines deficiency in serum 1-antitrypsin (1-at) were given an intravenous injection of typhoid vaccine. Following this injection the trypsin-inhibiting capacity of serum and the concentration of 1-at increases markedly in normal individuals. Heterozygotes for the deficiency gene have an elevation of 1-at and trypsin-inhibiting capacity of their serum but reach only 50 percent of the levels reached by homozygotes for the common gene (normal individuals). Their values are however temporarily in the range of normal men not given typhoid vaccine. Homozygotes for the deficiency gene show only a small elevation of 1-at under these conditions. These findings demonstrate that (1) the increase in trypsin-inhibiting capacity of serum after an injection of typhoid vaccine is largely due to the elevation of the 1-at concentration, and (2) the 1-at deficiency gene inhibits the quantitative response of the 1-at to such a stimulus.Supported in part by USPHS grant HE-06285 from the National Heart Institute.Some of these studies were carried out in the General Clinical Research Center Ward, U. C., FR-79, supported by the Division of Research Facilities and Resources, U.S. Public Health Service.  相似文献   

13.
The Ca2+ channel 1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although N-type Ca2+ channel plays a role in a variety of neuronal functions, 1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca2+ channel 1 or subunit. In this study, we studied the levels of 1A, 1C, 1D, 1E, 1, 2, 3 and 4 mRNAs in adrenal gland of 1B-deficient mice. The 1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of 1C, 1D, 1E, 1, 2, 3 and 4 was found among wild, heterozygous and homozygous mice. The protein level of 1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of 1A gene, we examined lacZ expression in 1B-deficient × 1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of 1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous 1B-deficient × 1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of 1B-deficient mice is that compensation by 1A subunit occurs and that 6.3-kb 5-upstream region of 1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)  相似文献   

14.
Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F1 and F5 progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F1 progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f1 progeny and all F5 progeny from the intrasubspecific crosses, and all F1 progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f1 plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F1 progeny.NRCC No. 38451  相似文献   

15.
Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - Mr relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis  相似文献   

16.
17.
This paper presents evidence that short-term exposure to light increases synthesis of hydroxyei-cosatetraenoic acid (HETE) and prostaglandin D2 (PGD2) and stimulates the uptake and metabolism of 204 in phospholipids and triacylglycerols in rat retina. There was a time-dependent increase in incorporation of 1-14C-204 into glycerolipids in both dark-adapted and light-exposed groups. Exposure to light for 15 or 30 min enhanced the acylation of 204 into phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and triacylglycerols. In the light-exposed groups there was a large increase in the conversion of 204 to leukotriene B4, two diHETEs, 5-HETE, 15-HETE, and PGD2. The stimulation of HETE synthesis by light could be due to early stages of light-induced lipid peroxidation in visual cells. To examine this, we studied peroxidation of 204 in isolated rod outer segments (ROS). There was more oxidation of 204 in light-exposed ROS, as compared to ROS incubated in the dark. Vitamin E and nordihydroguaiaretic acid inhibited the light-induced formation of some of these products. The data indicate that photo-oxidation of 204 in ROS is accompanied by enzymatic oxygenation that is stimulated by light. Increased production of HETEs and PGD2 may be a consequence of the light-induced stimulation of the metabolism of 204 in membrane phospholipids in the retina.  相似文献   

18.
The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (11 and 22). The subunit corresponding to the 11 pair is present inH. petiolaris and in the three populations ofH. annuus studied. The 2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific 2a2 and 44 pairs andH. annuus a specific 33 pair. InH. argophyllus 11 33 or 44 are never observed but are replaced by 13 and 31 pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.  相似文献   

19.
The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 g elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 g ml-1. The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; -methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.Abbreviations PAL phenylalanine ammonia-lyase - PMS Phytophthora megasperma var sojae  相似文献   

20.
Summary Fifteen inbred lines of rye, F1 and F2 progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis  相似文献   

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