Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

Summary Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as pnApA in excellent yield (80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5-phosphorimidazolides.Abbreviations P₃! trimetaphosphate - A adenosine - U uridine - EDTA ethylenediaminetetraacetic acid - Ap adenosine 2(3)-phosphate - Ap! adenosine cyclic 2:3-phosphate - pA adenosine 5-phosphate - pA²p adenosine 2, 5-diphosphate - pA³p adenosine 3, 5-diphosphate - pAp! 5-phospho-adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - ImpA adenosine 5-phosphorimidazolide - A²pA adenylyl-[25]-adenosine - A³pA adenylyl-[35]-adenosine - A²pU adenylyl-[25]-uridine - A³pU adenylyl-[35]-uridine - pA²pA 5-phosphoadenylyl-[25]-adenosine - pA³pA 5-phospho-adenylyl-[35]-adenosine - pA²pU 5-phospho-adenylyl-[25]-uridine - pA³pU 5-phospho-adenylyl-[35]-uridine - pApN (N= A, U) 5-phosphate of a dinucleoside phosphate - p_nApN (N = A, U; n = 2, 3, 4.) 5-polyphosphate of a dinucleoside phosphate - ImpA²pA imidazolide of pA²pA - ImpA³pA imidazolide of pA³pA - ImpA²pU imidazolide of pA²pU - ImpA³pU imidazolide of pA³pU - ImpApN imidazolide of pApN     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Monosomic analysis of heading date and spikelet number in the common wheat (Triticum aestivum L.) multispikelet line ‘Noa’

Summary Genetic analysis of heading date and spikelet number was carried out in the common wheat (Triticum aestivum L.) multispikelet line Noa, by using the monosomic series of the regular line Mara. Noa's high number of spikelets was found to be controlled by a recessive major gene on chromosome 2D; a slight reduction in spikelet number was induced by another recessive gene on Noa's 7A chromosome. Noa's late heading date was found to be controlled by two recessive genes, located on chromosome 2D (a major effect) and 6B (a minor effect). The nature of the genes located on Noa's 2D chromosome and the relationship between spikelet number and heading date are discussed.     

Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis

The effect of intraperitoneal administration of tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the tocopherol treated group were not observed. The light emission was significantly higher in the control than in the tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbateFe²⁺ lipid peroxidation. The protector effect observed by tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

Structure and molecular organization of the photosynthetic accessory pigments of cyanobacteria and red algae

Summary Cyanobacteria (blue-green algae) and Rhodophyta (red algae) contain high concentrations of photosynthetic accessory pigments (phycobiliproteins) which trap light energy in the region between 400 and 650 nm. The electronic excitation energy is then transferred along a chain of these pigments to the reaction center chlorophyll of Photosystem II by a radiationless induced resonance process.Unlike the protein-chlorophyll complexes in the photosynthetic lamellae, the phycobiliproteins are readily soluble in aqueous solution, can be isolated in a variety of assembly forms, and crystallize readily. These properties facilitate the study of the structure of these proteins by chemical, physical, and immunological methods, as well as by X-ray diffraction and electron microscopy.The brilliantly colored phycobiliproteins are a homologous family of conjugated proteins of differing spectroscopic properties. The basic structural unit in these proteins is a monomer of 30,000–40,000 daltons made up of two dissimilar polypeptide chains, and . Each subunit carries covalently linked tetrapyrrole prosthetic groups related to the bile pigment biliverdin.The distinctive spectroscopic properties of each phycobiliprotein are a consequence of the chemical structure of the bile pigment it carries, and of the influence of the conformation and aggregation state of the protein on the spectra of these prosthetic groups. In vivo, the phycobiliproteins are organized into particles, phycobilisomes, attached in a regular array to the outer surface of the photosynthetic lamellae. Studies on phycobilisomes, and on intact cells, indicate the following pathway of energy transfer.Phycoerythrin Phycocyanin (_max 560 nm) (_max 620 nm) Allophycocyanin Allophycocyanin B (_max 650 nm)(_max 671 nm) Chlorophyll a (_max 680 nm)The amounts of the various phycobiliproteins in the cell are influenced by the intensity and energy distribution of the incident radiation. The phenomena of intensity adaptation and complementary chromatic adaptation yield insights into the structure of phycobilisomes and the molecular basis of the plasticity of the structure of this light-harvesting system.Invited article.     

‘Fine’ and ‘coarse’ mycorrhizal fungi on red clover plants in acid soils: Root colonization and plant responses

Three sterilized acid soils were inoculated with inocula of vesicular-arbuscular mycorrhizal fungi. Soils were limed and/or P fertilized to produce different fertility levels. Most inocula consisted of mixtures of fine + coarse type endophytes. Red clover (Trifolium pratense L.) was seeded in pots and grown in a glasshouse for 4 months. Root colonization by VAM fungi, the relative infection byGlomus tenue compared to that by coarse VAM fungi and the effect of inoculation on red clover growth and mineral nutrition (P, K, Ca and Mg) were studied. Spores were also checked and tentatively identified.Results showed that root colonization by VAM fungi was higher than 50% in most cases, the lower values being found in the soil with the highest P content. Inocula containingG. mosseae + G. tenue infected plant roots only in limed (pH>5.7) soils. A study of the relative colonization by fine and coarse endophytes showed that the competitive ability againstG. tenue followed the orderG. fasiculatum > G. mosseae > G. epigaeum > G. macrocarpum, although soil properties and fertility were crucial factors.Glomus lacteum was tentatively identified in two of the three experimental soils. The inoculum in whichGlommus tenue was most infective was also the most efficient in improving plant growth and nutrient uptake. The effect of inoculation on P and Mg uptake followed a similar pattern.     

Ionic relations of aeroponically-grown olive genotypes,during salt stress

Two olive (Olea europaea L.) genotypes, Frantoio and Leccino, were exposed to increasing concentrations of NaCl (0-30-60-120 mM) in an aeroponic cultivation system for 60 days. Dry weights and sodium and potassium contents of apical and basal leaves, new and old wood, and roots were measured to determine Na uptake rate, Na translocation rate and K-Na selectivity ratio (S_K,Na). Frantoio showed a higher salt resistance than Leccino. Frantoio and Leccino had a similar Na uptake rate, but largely differed for Na translocation to the shoot. Furthermore Frantoio exhibited a higher K-Na selectivity than Leccino at both whole plant level and above all at the level of shoot system. Resistance mechanism of Frantoio is probably related to Na esclusion by roots and to the ability to maintain an appropriate K/Na ratio in actively growing tissues.Research supported by National Research Council of Italy, Special project RAISA.     

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.     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

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.     

High-level Expression of Maize <Emphasis Type="Italic">γ</Emphasis>-zein Protein in Transgenic Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>)

Primers were developed for 118 microsatellites isolated from grape (Vitis vinifera) genomic libraries enriched for (AC)n repeats. Only one microsatellite sequence matched other grape SSR-sequences in the GeneBank database. Genotyping was carried out in the parental lines and four offspring of two pseudo-test-cross populations, Cabernet Sauvignon x Seyval and Chardonnay x Bianca, and a further six other grape genotypes (V. vinifera Sultanina, Merlot, Syrah, Müller-Thurgau, Vitis Regent and V. riparia Gloire de Montpellier). A total of 108 microsatellites showed easily scorable alleles and 100 of them segregated according to a configuration suitable for mapping in either cross. A further 8 SSRs, although unsuitable for mapping in those crosses, showed polymorphism in the other genotypes tested. This set of markers was used, along with 75 microsatellites of other repeat-types, to fingerprint 46 offspring of the cross Chardonnay x Bianca. For each full-sib, individual heterozygosity and distance in repeat units between pairs of alleles at each locus (mean d²) were calculated as a tool for predicting highly outbred recombinant individuals. Six microsatellites with segregation ratios significantly distorted towards the lack of homozygous sibs were identified and mapped to linkage groups LG 3 and LG 5. Estimation of heterozygosity at genome-wide level and genotyping at loci for which homozygous sibs are discriminated against are discussed for marker-assisted background selection in outcrossing grapevines.     

The reduction of tetrazolium salts by plant mitochondria

Summary Data has been obtained concerning the reduction of tetrazolium salts by mitochondria isolated from Jerusalem artichoke tubers with succinate as the substrate using a direct recording spectrophotometric method of assay. ATP was found to increase the rate of reduction of the tetrazolium salts, this being independent of the effect ATP had on the rate of oxygen uptake. The magnitude of the stimulation by ATP depended on the concentration of tetrazolium salts present and under certain circumstances was suppressed by the addition of azide and cyanide. The sites at which the tetrazolium salts were reduced along the electron transport chain were investigated. The role of ATP has been discussed in relation to the mechanism of tetrazolium reduction.Abbreviations TTC 2,3,5-triphenyl-2,1,3,4-tetrazolium chloride - BT 5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-diphenylene)-ditetrazolium chloride - NT 2,2,5,5-tetraphenyl-3,3-(p-diphenylene)-ditetrazolium chloride - MTT 3-(4,5-dimethyl thiozolyl-2)-2,5-diphenyl tetrazolium bromide - INT 2-(p-iodophenyl)-3-p-dinitrophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride - NBT 2,2-dinitrophenyl-5,5-diphenyl-3,3-dimethoxy-4,4-diphenylene)-ditetrazolium chloride - TNBT 2,2-5,5-tetra-p-nitrophenyl-3,3-dimethoxy-4,4-diphenylene) ditetrazolium chloride     

Characterization of the putative α subunit of a heterotrimeric G protein in rice

A recombinant protein with a cDNA that encodes the putative subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice ) with an apparent molecular mass of 45 kDa was bound with guanosine 5-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its Kcat was 0.44. rGrice was ADP-ribosylated by activated cholera toxin.Monoclonal antibodies raised against rGrice reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice . A 45 kDa polypeptide in the plasma membrane, as well as rGrice , was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the subunits of heterotrimeric G proteins in animals, suggesting that the putative subunit is truly the subunit itself.     

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.     

Predominance and tissue specificity of adenine methylation in rice

Summary Using A and C methylation-specific restriction enzymes, namely, MboI, Sau3AI, DpnI, MspI, and HpaII, total rice cv Basmati 370 DNA, repetitive DNAs, and a specific repeat sequence indicated an abundance of adenine methylation. Although cytosine methylation in 5-CCGG-3 sequences suggested more CpC methylation than CpG, the C methylation in sequence 5-GATC-3 was comparatively less than A methylation. Furthermore, the presence of adenine methylation was tissue specific; it was predominant in rice shoot DNA as compared to embryo DNA. This pattern was also observed in two other cultivars of rice, i.e., R-24 and Sona, and was again confirmed using a cloned probe of a specific repeat sequence. Besides the changes in adenine methylation, there was also a qualitative change in 5mC from CpG to CpC dinucleotides in these two tissue systems.     

Aırborne pollen calendar of Bartın, Turkey

Our study comprises airborne pollen analysis of the years 1995–1997 of Bartn (40°38 N–32°21 E). The aims of the study were: to determine the pollen types present in the atmosphere, to calculate their actual concentrations, and to establish the pollen calendar for Bartn. For this purpose, pollen slides of Durham apparatus, which was located in four different locations in Bartn, were examined on a weekly basis. In this study, 19062 pollen grains were counted and 18484 of those were determined on the slides in the air of Bartn during three years of period. Results show that 13758 of 18484 pollen grains belong to arboreal, 4726 belong to non-arboreal taxa. 3698 of determined non-arboreal taxa belong to Gramineae.     

Disruption of the Gi2α locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair

We have used an insertion vector-based approach to target the G_i2 gene in AB-1 embryonic stem cells. 105 bp located 0.8–0.9 kb upstream of a disrupting Neo marker in exon 3 were deleted and replaced with an engineeredNot I site, that served to linearize the vector. The 105 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homologous recombination. Both target conversion and vector insertion events were obtained (hit step). Clones that had inserted the entire targeting vector were taken into FIAU (1-[2-deoxy, 2-fluoro--d-arabinofuranosyl]-5-ioduracil) counterselection to select against a thymidine kinase (TK) marker flanking the homologous genomic sequences and thus for cells that had excised the plasmid and the TK marker by intrachromosomal recombination (run step). Additional selection in G418 reduced the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more specific selection of the desired intrachromosomal recombination event in tissue culture. This modified hit and run strategy represents a novel approach for vector design and the use of the polymerase chain reaction to detect targeting. It may be particularly useful for targeting genes that display a low frequency of homologous recombination. Germ line transmission of the mutated G_i2 allele is also demonstrated.     

Relative levels of guanosine 5′-diphosphate 3′-diphosphate (ppGpp) in some N2 fixing bacteria during derepression and repression of nitrogenase

Addition of ammonium to N₂ fixing cultures of Azotobacter vinelandii, Klebsiella pneumoniae and Clostridium pasteurianum rapidly reduced the intracellular levels of guanosine 5-diphosphate 3-diphosphate (ppGpp) by 70–90%. This change might reflect a regulatory role of ppGpp in nitrogen metabolism.Abbreviations ppGpp guanosine 5-diphosphate 3-diphosphate     

Tumourigenesis: the subterfuge of selection

Variation or rearrangement of regulatory genes is responsible for cellular malignant change. These types of chromosomal variations also produce heterochrony or paedomorphic evolution at the organismal level. Analogously, neoplasia represents a cellular macroevolutionary event, and a tumour can be said to be an evolved population of cells. To understand this cellular evolution to malignancy, it may be necessary to go beyond a clonal selection (adaptationist) explanation of neoplastic alteration. In the pericellular environment natural selection consists of the organizational restraints of surrounding cells as well as the host's immunological surveillance and non-specific monocyte-macrophage systems. Indirect evidence suggests that success for the neoplasm depends not upon clonal selection, but solely upon a genetic methodology—the function of which is to elude selection.The author has coined the term cellular heterochrony to illustrate analogic similarities in the molecular modes of speciation between anaplastic cancer cells and the heterochronic evolution of organisms. By reverting to a juvenile (embryonic) repertoire of cellular behaviour a tumour secures its own tenure or niche by usurping the host's armamentarium of selection forces, employing many of the same or similar methods by which implanting and invading tissues of the mammalian embryo forestall maternal detection and rejection. A number of ways by which the tumour blocks, subverts or evades selection are discussed.