The composition of Erythrosins, Fluorescein, Phloxine and Rose Bengal: a study using thin-layer chromatography and solvent extraction

Synopsis Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4-iodofluorescein, 4,5-di-iodofluorescein, 2,4,5-triiodofluorescein and 2,4,5,7-tetraiodofluorescein, in some instances together with 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4-bromo-4,5,6,7-tetrachlorofluorescein, 4,5-dibromo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tribromo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4-iodo-4,5,6,7-tetrachlorofluorescein, 4,5-di-iodo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.     

Circular dichroism of DNA-proflavine,DNA-ethidium and DNA-distamycine flow-oriented complexes

The circular dichroism (CD) spectra of flow-oriented complexes of DNA with proflavine (PF), ethidium (ET) and distamycine (DS) have been studied in the ultraviolet region. The CD spectra with light propagating in parallel to the flow direction were measured by the method of Chung and Holzwarth [1]. and values have been obtained by this method. It was shown that all the complexes studied exhibit a strong CD anisotropy so that isotropic CD spectra measured with a conventional procedure can be attributed to the mutual compensation of the two components of opposite signs.     

High crossability of wild barley (Hordeum spontaneum C. Koch) with bread wheat and the differential elimination of barley chromosomes in the hybrids

Four bread wheat (Triticum aestivum L.) cultivars, Aobakomugi, Chinese Spring, Norin 61 and Shinchunaga, were pollinated with five barley lines/cultivars consisting of three cultivated barley (Hordeum vulgare L.) lines, Betzes, Kinai 5 and OHL089, and two wild barley (Hordeum spontaneum C. Koch) lines, OUH602 and OUH324. Crossability, expressed as the percentage of embryo formation, varied from 0 to 55.4% among the cross combinations. The two wild barley lines generally had a higher crossability than the previously reported best pollinator, Betzes, and some Japanese wheat cultivars were better as the female parent than Chinese Spring. Ninety four hybrid plants were obtained from 250 embryos cultured, and their somatic chromosome numbers ranged from 21 to 36. Eighteen plants were mosaic in chromosome number. Twenty one-chromosome plants appeared most frequently (45.7%) followed by 28-chromosome plants (14.9%). C-banding analysis revealed that elimination of barley chromosomes was mainly responsible for the occurrence of aneuploid plants. In hypoploids derived from Betzes-crosses, chromosome 5 was preferentially eliminated as previously reported, while in hypoploids derived from OUH602-crosses, chromosome 4 was preferentially eliminated. The wild barley line OUH602 may be a useful parent for producing a new wheat-barley addition set because of its high crossability with wheat and a different pattern of chromosome elimination.     

Wheat chromosome 2D carries genes controlling the activity of two DNA-degrading enzymes

DNA-degrading enzymes of 24.0 kDa and 27.0 kDa were observed to have different activities in two common wheat (Triticum aestivum L.) cultivars, Wichita and Cheyenne. A substrate-based SDS-PAGE assay revealed that these two enzymes were much more active in Wichita than in Cheyenne. Genes controlling the activities of these two enzymes were localized on chromosome 2D by testing DNA-degrading activities in reciprocal chromosome substitution lines between Wichita and Cheyenne. While the allele on Wichita chromosome 2D stimulated the activities of the 24.0- and 27.0-kDa enzymes in Cheyenne, the allele on Cheyenne chromosome 2D did not reduce the activities of the 24-kDa and 27-kDa enzymes in Wichita. Whether these genes code for the DNA-degrading enzymes themselves or for factors that regulate the enzyme activities remains unknown.This work was supported in part by USDA-Competitive Research Grants Office grant No. 90-37140-5426 to P.S.B. Contribution from Agricultural Research Division, University of Nebraska. Journal Series Number 10304     

Differences in the heat-shock response between thermotolerant and thermosusceptible cultivars of hexaploid wheat

Summary Heat-shock protein (HSP) gene expression in two wheat lines cv Mustang (heat-tolerant) and cv Sturdy (heat-susceptible) were analyzed to determine if wheat genotypes differing in heat tolerance also differ in in-vitro HSP synthesis (translatable HSP mRNAs) and steady-state levels of HSP mRNA. Several sets of mRNA were isolated from seedling leaf tissues which had been heat-stressed at 37 °C for various time intervals. These mRNAs were hybridized with HSP cDNA or genomic DNA probes (HSP17, 26, 70, 98, and ubiquitin). Protein profiles were compared using in-vitro translation and 2-D gels. The Northern slot-blot data from the heat-stress treatment provide evidence that the heat-tolerant cv Mustang synthesized low molecular weight (LMW) HSP mRNA earlier during exposure to heat shock and at a higher level than did the heat-susceptible cv Sturdy. This was especially true for the chloroplast-localized HSP. The protein profiles shown by 2-D gel analysis revealed that there were not only quantitative differences of individual HSPs between the two wheat lines, but also some unique HSPs which were only found in the Mustang HSP profiles. The high level of RFLP between the two wheat lines was revealed by Southern blot hybridization utilizing a HSP17 probe. These data provide a molecular basis for further genetic analysis of the role of HSP genes in thermal tolerance in wheat.     

Influence of the nitrogen level on root growth and morphology of two potato varieties differing in nitrogen acquisition

Two potato (Solanum tuberosum L.) cultivars (Astrid and Bodenkraft) differing in their nitrogen acquisition from the soil (Hunnius, 1981) were used in nutrient solutions to study the effect of increasing concentrations of nitrate (0.05; 0.5; 5.0 mol m^-3) particularly on root growth and morphology. In each variety increasing nitrogen concentrations stimulated shoot growth more than root growth. At all nitrate concentrations, the variety with higher nitrogen acquisition (Astrid) had a significantly larger root system. The larger root system of Astrid compared to Bodenkraft was particularly evident when surface area and total length of the roots, instead of root dry weight were used as parameters. The results stress the importance of root length and surface area for nitrogen acquisition from soils.     

Elevated CO2 effects on carbon and nitrogen cycling in grass/clover turves of a Psammaquent soil

Effects of elevated CO₂ (525 and 700 L L^–1), and a control (350 L L^–1 CO₂), on biochemical properties of a Mollic Psammaquent soil in a well-established pasture of C3 and C4 grasses and clover were investigated with continuously moist turves in growth chambers over four consecutive seasonal temperature regimes from spring to winter inclusive. After a further spring period, half of the turves under 350 and 700 L L^–1 were subjected to summer drying and were then re-wetted before a further autumn period; the remaining turves were kept continuously moist throughout these additional three consecutive seasons. The continuously moist turves were then pulse-labelled with ¹⁴C-CO₂ to follow C pathways in the plant/soil system during 35 days.Growth rates of herbage during the first four seasons averaged 4.6 g m^–2 day^–1 under 700 L L^–1 CO₂ and were about 10% higher than under the other two treatments. Below-ground net productivity at the end of these seasons averaged 465, 800 and 824 g m^–2 in the control, 525 and 700 L L^–1 treatments, respectively.in continuously moist soil, elevated CO₂ had no overall effects on total, extractable or microbial C and N, or invertase activity, but resulted in increased CO₂-C production from soil, and from added herbage during the initial stages of decomposition over 21 days; rates of root decomposition were unaffected. CO₂ produced h^–1 mg^–1 microbial C was about 10% higher in the 700 L L^–1 CO₂ treatment than in the other two treatments. Elevated CO₂ had no clearly defined effects on N availability, or on the net N mineralization of added herbage.In the labelling experiment, relatively more ¹⁴C in the plant/soil system occurred below ground under elevated CO₂, with enhanced turnover of ¹⁴C also being suggested.Drying increased levels of extractable C and organic-N, but decreased mineral-N concentrations; it had no effect on microbial C, but resulted in lowered microbial N in the control only. In soil that had been previously summer-dried, CO₂ production was again higher, but net N mineralization was lower, under elevated CO₂ than in the control after autumn pasture growth.Over the trial period of 422 days, elevated CO₂ generally appears to have had a greater effect on soil C turnover than on soil C pools in this pasture ecosystem.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Analysis of the genotypic variation in radiocaesium uptake from soil

Young spinach (Spinacia oleracea L., cv. Subito) and wheat (Triticum aestivum, cv. Tonic) plants were hydroponically grown in eight different nutrient solutions containing ¹³⁷Cs. Ca, Mg, K and NH₄ concentrations were varied whilst anion concentrations were equal in all solutions. The large differences in potassium content between spinach and wheat were not reflected in similar differences in ¹³⁷Cs content at any nutritive treatment.Both crops were also grown in a potted podzolic soil contaminated with radiocaesium. This experiment was conducted in a phytotron at two climatic conditions (summer and winter) which differed in day length and light intensity. Wheat plants had higher ¹³⁷Cs levels than spinach at both conditions. The ¹³⁷Cs levels furthermore decreased during development. The ¹³⁷Cs plant/soil solution concentration ratio was lower at the summer than at the winter conditions.     

Comparative study on the chloroplast,mitochondrial and nuclear genome differentiation in two cultivated rice species,Oryza sativa and O. glaberrima,by RFLP analyses

Summary Restriction fragment length polymorphisms of chloroplast (ct), mitochondrial (mt) and nuclear DNA were investigated using eight cultivars of Oryza sativa and two cultivars of O. glaberrima. Relative variability in the nuclear and cytoplasmic genomes was estimated by a common measure, genetic distance. Based on the average genetic distances among ten cultivars for each genome, the evolutionary variabilities of the mitochondrial and nuclear genomes were found to be almost the same, whereas the variability of the chloroplast genome was less than half that of the other two genomes. Cluster analyses on ct and mt DNA variations revealed that chloroplast and mitochondrial genomes were conservative within a taxon and that their differentiations were well-paralleled with respect to each other. For nuclear DNA variation, an array of different degrees of differentiation was observed in O. sativa, in contrast with little variation in O. glaberrima. As a whole, differentiation between O. sativa and O. glaberrima was clearly observed in all three genomes. In O. sativa, no notable difference was found between the cultivars Japonica and Javanica, whereas a large differentiation was noticed between Japonica (including Javanica) and Indica. In all three genomes, the average genetic distances within Indica were much larger than those within Japonica (including Javanica), and almost similar between Japonica (including Javanica) and Indica. These facts indicate that differentiation in O. sativa was due mainly to Indica.     

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor (TNF) and Interleukin-1 (IL1) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF and IL1. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF (1, 10, 100, 1000 I.U./ml) and IL1 (0.1, 1, 10, 100 I.U./ ml).TNF- and IL1- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF and IL1 and a subsequent down regulation of PDH activity.     

Longer coleoptiles improve emergence through crop residues to increase seedling number and biomass in wheat (Triticum aestivum L.)

Crop residues protect soils from erosion, reduce soil water evaporation and increase soil organic matter. Yet management of stubbles for cropping can be difficult. Surface-retained residue can act as a mechanical barrier to slow emergence and reduce seedling biomass. Longer coleoptiles improve seedling emergence with deep sowing and may assist where stubble load is large. In a glasshouse study, six wheat and barley genotypes were sown at 30 and 50mm depth into pots containing pasteurised soil. Unweathered sorghum, canola and wheat stubble were added at 0, 3 and 6t/ha equivalents to the soil surface and pots watered above or below the stubble. Stubble species and watering regime had little effect on seedling growth. However, deeper sowing and increased stubble mass adversely affected most seedling characteristics particularly slowing seedling emergence and reducing tiller number to decrease plant biomass (environmental correlations (r_e) of –0.98** and 0.88**, respectively). Shorter coleoptile Rht-B1b wheats Banks and Janz, and barley Beecher emerged slower and abnormally with thicker stubble, and had more sterile tillers to reduce total tiller number and biomass. Deeper crowns for these genotypes also resulted in proportionally less biomass located above the stubble. The converse was true of long coleoptile Vigour 18, Halberd, and its Rht8 progeny, HM14bS which were less affected by stubble mass and sowing depth. In a corresponding field study, increasing wheat stubble mass from 0 to 3 and 6 t/ha delayed seedling emergence and decreased plant number to reduce biomass. Short coleoptile wheat genotypes Hartog and Janz emerged slower and produced less biomass at 3 and 6t/ha of stubble than long coleoptile wheat genotypes Halberd and HM14bS. Emergence of seedlings sown at 50mm depth with 6t/ha overlying stubble was similar to that sown at 120mm with no stubble, reflecting the similar impact of retained residues to deep sowing. Genetic variation for coleoptile length and availability of gibberellin-responsive dwarfing genes such as Rht8 will allow development of long coleoptile wheats for deep sowing or where stubble retention is practiced.     

In vitro insertion of the lambda attachment site into the plasmid RP4.

Summary The region of the phage lambda chromosome containing the attachment site (P · P) and the genes int and xis, excised by the action of endonuclease R.EcoRI, has been inserted into the unique site for that enzyme on the promiscuous conjugative plasmid, RP4, generating the recombinant plasmid RP4att. Transformants containing the hybrid plasmid were recognised by their ability to allow efficient lysogenization by phage b2 (Weil and Signer, 1968; Echols et al., 1968) containing the mutant attachment site · P. The construction and properties of the hybrid plasmid RP4att are described.     

Synthesis of N-linked pentasaccharides with isomeric glycosidic linkage

As part of a program to explore the structural requirement of N-glycans in the carbohydrate-mediated biological interactions, N-linked pentasaccharide core structure was stereochemically modified in terms of glycosidic linkage. Three isomers, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, and -D-Man-(13)-[-D-man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, were synthesized. Synthesis of the pentasaccharide with natural linkage is also described.     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.     

Restriction fragment analysis of ‘null’ forms at the Gli-1 loci of bread and durum wheats

Summary Wheat accessions lacking some of the - and -gliadin components encoded by the Gli-1 loci on the short arm of chromosome 1D in bread wheat and chromosome 1A in durum wheat were studied by two-dimensional polyacrylamide gel electrophoresis and restriction fragment analysis. Digested genomic DNAs of normal and null forms were probed with a cDNA clone related to -/-gliadins and with a genomic clone encoding an LMW subunit of glutenin. The hybridisation patterns with the -/-gliadin probe were similar to those of cvs Chinese Spring and Langdon used as standards for bread and durum wheats, respectively, but several restriction fragments located on the 1D chromosome of bread wheat and the 1A chromosome of durum wheat were absent in the null forms. In addition, specific LMW glutenin fragments encoded by the same chromosomes were also absent in the null forms, suggesting that simultaneous deletions of blocks of genes for both -/-gliadins and LMW glutenins had occurred. Comparisons of the protein and RFLP patterns enabled some proteins to be mapped to specific restriction fragments.     

Enhanced power of QTL detection for Fusarium head blight resistance in wheat by means of codominant scoring of hemizygous molecular markers

Based on different marker information content mapping of QTLs for Fusarium head blight resistance in wheat was compared with regard to number and consistency of detected QTLs as well as QTL positions and effects. Therefore, two linkage maps, obtained by dominant and codominant genotyping of hemizygous markers, were constructed with 211 AFLPs, 37 SSRs and the barley RGA marker XaACT/CAA. The codominant marker set comprised 59% codominant markers, whereas the dominant map consisted of only 13%. A segregating wheat population of 94 F₄-RILs was used for QTL analysis. Fusarium head blight resistance was estimated in field trials in six environments. Conventional dominant marker scoring found seven QTLs. The phenotypic variations explained by QTLs detected in single environment analyses ranged from 11.1 to 44.6%. QTL analysis performed with the codominant marker set confirmed not only all QTL positions as revealed by dominant QTL analysis', but also 12 additional QTLs were found. QTLs in single environments explained 36.3 up to 55.7% of the phenotypic variation. In the QTL analysis across all environments, none of the QTLs could be confirmed using dominant marker scoring. However, by codominant QTL analysis' environment-specific QTLs were retrieved. STS marker XaACT/CAA was found to be significantly associated with FHB resistance only by codominant scoring. Support intervals of QTLs commonly found in both marker sets averaged to 10.3 cM in the dominant QTL analysis', whereas the length was shortened to 8.9 cM by codominant genotyping. The advantages of extracting codominant information from dominant markers are discussed.     

Bacterial regeneration of ammonium and phosphate as affected by the carbon:nitrogen:phosphorus ratio of organic substrates

The effect of carbonnitrogenphosphorus (CNP) ratio of organic substrates on the regeneration of ammonium and phosphate was investigated by growing natural assemblages of freshwater bacteria in mineral media supplemented with the simple organic C, N, and P sources (glucose, asparagine, and sodium glycerophosphate, respectively) to give 25 different substrate CNP ratios. Both ammonium and phosphate were regenerated when CN and NP atomic ratios of organic substrates were 101 and 161, respectively. Only ammonium was regenerated when CN and NP ratios were 101 and 10–201, respectively. On the other hand, neither ammonium nor phosphate was regenerated when CN and NP ratios were 151 and 51, respectively. In no case was phosphate alone regenerated. As bacteria were able to alter widely the CNP ratio of their biomass, the growth yield of bacteria appeared primarily dependent on the substrate carbon concentration, irrespective of a wide variation in the substrate CNP ratio.     

Green manures of oat,rape and sweet corn for reducing common root rot in pea (Pisum sativum) caused by Aphanomyces euteiches

Green manure crops of sweet corn, soybean, alfalfa, snap bean, rape, pea and of the two oat cultivars Dane and Troy were incorporated into the same soil and containers in which the crop had grown for five weeks. The soil was then evaluated for suppression of common root rot (Aphanomyces euteiches) of pea grown in infested pasteurized and non-pasteurized soils in the greenhouse. Pea biomass reduction and a plant bioassay for A. euteiches were used to measure the green manure suppression of disease. Green manures of sweet corn cv.Jubilee, oat cv.Troy, and rape cv.Humus significantly reduced pea biomass losses over the non-amended control soil treatments. Oat cv.Troyand sweet corn cv.Jubilee green manures significantly reduced inoculum density of A. euteiches over the corresponding fallow controls in inoculated pasteurized soil by 87% and 76%, respectively, and in inoculated non-pasteurized soil by 67% and 66%, respectively. Only the green manure of oat cv. Troy reduced inoculum density significantly below fallow.     

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Summary Long-range physical maps of the small multigene family of the malt -amylase genes (-Amy-1) located on the long arms of wheat chromosomes 6A (the -Amy-A1 locus) and 6B (-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an -Amy-1 cDNA probe and four gene-specific genomic probes from the -Amy-B1 locus, the size of the -Amy-B1 locus was estimated to be about 700 kb and of the -Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of CpG islands are apparent in -Amy-B1, and three in -Amy-A1. Correlation between recombination frequency and physical distance within the -Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.