Isolation and identification of the principal siderophore of the plant pathogenic fungusBotrytis cinerea

Summary The plant pathogenic hyphomyceteBotrytis cinerea has been shown to produce several trihydroxamate siderophores under conditions of low-iron stress. The total siderophores amounted to approximately 30 mg/l culture filtrate after 5 days of incubation in an asparagine/salt/glucose medium. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) on a reversed phase indicated that ferrirhodin is the predominant siderophore of this fungus. Chemical characterization of the principal siderophore by fast-atom-bombardment (FAB) mass spectrometry, nuclear magnetic resonance (¹H-NMR,¹³C-NMR) and comparison with a reference revealed the identity with ferrirhodin. NMR studies performed on desferrirhodin (desferrirhodin) in dimethylsulfoxide and water revealed the existence of two conformers in D₂O resulting from acis-trans isomerization of the hydroxamic acid groups. Comparative iron-uptake studies showed the following order of uptake inB. cinerea: ferrichrysin (100%), ferrirubin (57%), ferrirhodin (45%), hexahydroferrirhodin (45%), coprogen 6%. Concentration-dependent uptake of ferrirhodin resulted in saturation kinetics only in the low concentration range of 0–30 M (K _m = 2.5 M,V _max = 80 pmol min^–1 mg(^–1). A non-saturable, linear uptake was observed in the high concentration range of 30–80 M. The low concentration range appears to be the physiologically significant range, where siderophore-mediated iron transport inB. cinerea occurs.     

固定化酶的微环境效应—载体离子基团性质的影响

作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。     

Current methods for the laboratory diagnosis of campylobacteriosis]

An analytical review of recent publications of home and foreign authors on the problem of laboratory diagnosis of campylobacteriosis is presented. The commercial nutrient media, methods of creation of the microaerophilic conditions for cultivation of campylobacter are presented. The filtration method is preferable for isolation of these agents from the studied material highly contaminated by accompanying microflora. A special attention is paid to immunodiagnosis of campylobacteriosis: agglutination reaction, coagglutination reaction, passive hemagglutination reaction, immunoenzyme and radioimmune analyses. Seroepidemiological examination of the staff at one of meat-packing factories in the Republic carried out by the method of indirect immunoenzyme analysis has revealed high levels of anticampylobacteriosis antibodies in 17.9% of examinees. The promising trends in perfection of the methods for laboratory diagnosis of campylobacteriosis are outlined.     

A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.

A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.     

Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara)

More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA3 gibberellic acid - NAA -naphthaleneacetic acid - PAS periodic acid Schiff - PCM protoplast culture medium - PCV packed cell volume     

Characteristics of a novel high viscosity polysaccharide,methylan, produced byMethylobacterium organophilum

Summary A new anionic high molecular weight polysaccharide, Methylan, was produced byMethylobacterium organophilum from methanol as a sole source of carbon and energy under the specific culture conditions. By GPC and light scattering, the molecular weight was determined to be 2–4×10⁶ dalton and the distribution of molecular weight was very homogeneous. Methylan was composed of carbohydrate (80%), uronic acid (12%), protein (6%) and pyruvic acid (5%). The sugar composition of Methylan was identified as glucose, galactose and mannose with the approximate molar ratio of 232. Methylan solution showed a pseudoplastic non-Newtonian fluid property at the concentration above 0.05%. At the concentration of 1% Methylan solution, the consistency index was 18,000 centipoise which was almost 10 times higher than that of Xanthan and the flow behavior index was 0.15.     

Supplement of nutrients for effective cultivation of hepatitis B surface antigen-producing recombinant yeast

Summary In the hepatitis B surface antigen (HBsAg)-producing recombinant yeast culture medium, the supply of Bacto-yeast nitrogen base without amino acids was found to be inadequate due to the lack of the several kinds of vitamins and trace elements. When the culture medium for this recombinant yeast was supplemented with sufficient vitamins and trace elements, its growth, HBsAg production and the stability of plasmid were improved.     

Patterns of actin filaments during cell shaping in developing mesophyll of wheat (Triticum aestivum L.).

Young leaves of wheat exhibit a smooth developmental gradient with meristematic cells at the base and highly differentiated cells at the tip. During differentiation, mesophyll cells attain a lobed outline resembling tube-shaped balloons with almost regularly spaced isthmi. Microfilament patterns in developing wheat mesophyll cells were investigated using fluorescent-labeled phalloidin. Various patterns were found, including delicate arrays of transversely oriented microfilaments in the cortex of the cytoplasm. A close correlation between changes in the patterns of cortical microfilaments, microtubules, cell wall microfibrils, and cell shape was observed. The fine arrays of transversely oriented microfilaments coaligned with bands of microtubules occurring during cell elongation. These bands were found beneath sites of intense wall deposition. It has recently been proposed that the resulting hoops of wall reinforcement prevent cell expansion in the corresponding regions and thus give rise to the peculiar cell shape. When cell expansion ceased, and the typical lobed cell shape was attained, a dense network of microfilaments was retained in the cytoplasm, which was in contrast to what has been described for the microtubular arrays.