N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

Two tertiary branched tetraamines, N4-aminopropylnorspermidine and N4-aminopropylsperimidine, are the major polyamines in thermophilic eubacteria, Thermoleophilum

Abstract Two novel polyamines were found as major polyamines of Thermoleophilum album and Thermoleophilum minutum , which are Gram-negative eubacteria obligate for thermophily and n -alkane substrates. They were identified as tertiary branched tetraamines, N⁴-aminopropylnorspermidine ( tris (3-aminopropyl)amine) [NH₂(CH₂)₃N-((CH₂)₃NH₂)(CH₂)₃NH₂ or N(CH₂CH₂CH₂NH₂)₃] and N⁴-aminopropylspermidine [NH₂(CH₂)₃N((CH₂)₃NH₂)(CH₂)₄NH₂] by high-performance liquid chromatography and gas chromatography-mass spectrometry.     

Stability of transgenes and presence of N6 methyladenine DNA in transformed wheat cells

A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months post-selection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common bands had disappeared. Further analysis using a set of methylation-sensitive enzymes revealed that the DNA represented by the common bands was N⁶-adenine methylated (^m6A DNA), and there was even ^m6A DNA in some 30 month samples. Although the source of ^m6A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma-like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpnl, which only cuts ^m6A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.     

Effects of N6-Methyladenosine on the Synthesis of Phenylethanolamine N-Methyltransferase in Cultured Explants of Rat Adrenal Medulla

Abstract: Explants of adrenal medullae were cultured in defined media for up to 48 h, during which time the tissue remained histologically intact. Addition of N ⁶-methyladenosine to the medium led to a diminution in the activity of phenylethanolamine N -methyltransferase (EC 2.1.1.28) in the tissue. The enzyme activity was inversely proportional to the concentration N ⁶-methyl-adenosine in the culture medium. The extent of loss of phenylethanolamine N -methyltransferase, as measured by immunochemical titration, corresponded to the degree of loss in enzyme activity under the same conditions. Furthermore, the decreased amount of enzyme protein was due to a decrease in the rate of synthesis of phenylethanolamine N -methyltransferase. Neither adenosine nor several methylated nucleosides, including 7-methylguanosine, N ²-methylguanosine, and 5-methylcytosine, had an effect on the enzyme. Two other adrenal medullary enzymes, monoamine oxidase (EC 1.4.3.4) and acid phosphatase (EC 3.1.3.2), were not affected by addition of N ⁶-methyladenosine to the medium. The results are consistent with the view that this effect of N -methyladenosine on the concentration of phenylethanolamine N -methyltransferase is due to an inhibition of its biosynthesis rather than to an alteration of its rate of degradation.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

Half-life of tRNA Containing N6(Δ2-isopentenyl)adenosine in Lactobacillus acidophilus ATCC 4963

tRNA containing N⁶-(Δ²-isopentenyl)adenosine may be precursors for the plant hormone cytokinin. To discriminate between tRNA containing and not containing cytokinin nucleotides, double labelling experiments were made by the use of [2¹⁴C]-mevalonic acid and [³H-methyl]-methionine. At a generation cycle of 2 h for Lactobacillus acidophilus ATCC 4963, the half-lives of tRNA labelled with [³H-methyl]-methionine and [2-¹⁴C]-mevalonic acid are similar, namely 3 h. Isopentenylation of tRNA could be measured to be maximally 1:10.     

The Adenosine Analogue N6-L-Phenylisopropyladenosine Inhibits Catecholamine Secretion from Bovine Adrenal Medulla Cells by Inhibiting Calcium Influx

We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine-induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6-L-phenylisopropyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium, or by elevated external K+. The half-maximal effect on 1,1-dimethyl-4-phenylpiperazinium-induced secretion occurred at approximately 5 x 10(-5) M. The inhibition is immediate and reversible. Fura-2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium-evoked secretion from digitonin-permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage-sensitive calcium channel.     

Characterization and Location of Met5-Enkephalin-Arg6-Phe7 Stored in Various Rat Brain Regions

A specific antiserum against met⁵-enkepha-lin-arg⁶-phe⁷ was raised and used to study the distribution and characterization of met⁵-enkephalin-arg⁶-phe⁷-like immunoreactive material in rat brains by radioimmunoassay and immunohistochemical procedures. The antiserum appears to be directed to the COOH-terminus of the peptide, as it fails to cross-react with met⁵-enkeph-alin, met³-enkephalin-arg⁶, met⁵-enkephalin-arg⁶-arg⁷, met⁶-enkephalin-lys⁶, and leu-enkephalin. However, it cross-reacts with phe-met-arg-phe by about 10% and with phe-met-arg-phe-NH₂ to an insignificant degree. The highest content of met⁵-enkephalin-arg⁶-phe⁷ was found in the striatum, which contains a dense network of immunoreactive varicose fibers and terminals, as well as immunoreac tive cell bodies. The met⁵-enkephalin-arg⁶-phe⁷ in striatum can be released in a Ca²⁺-dependent manner by a depolarizing concentration of KC1, raising the possibility of a neu-roregulatory role for met⁵-enkephalin-arg⁶-phe⁷. Characterization of the immunoreactive material by gel filtration and high pressure liquid chromatography revealed the presence of multiple forms of immunoreactive material in some brain regions.     

Comparison on the genotoxic effects of nuclear vs cytoplasmic irradiation from the alteration of CD59 gene locus

Using microbeam to irradiate human-hamster hybrid AL cells withdefined number of a particles in a highly localized spatial region, this paper showed that cytoplasmic irradiation induced very little toxicity. For example, the cell killing by 4 a particle traversal through the cytoplasm was about 10%, and about 70% cells survived after their cytoplasm was irradiated with 32 a particles. In contrast, the survival fractions for nuclear irradiation at the same doses were 35% and less than 1% respectively. Mutation induction showed that while nuclear irradiation induced 3-4-fold more CD59- mutants than cytoplasmic irradiation at equivalent particle traversal, at an equitoxic dose level of 90% survival, the latter exposure mode induced 3.3-fold more mutants than nuclear irradia-tion. Moreover, using multiplex PCR to analyze five marker genes on chromosome 11 (WT, CAT, PTH, APO-A1 and RAS), the results showed that the majority of mutants induced by cytoplasmic irradiation had retained all of the marker genes analyzed. By comparison, the proportion of mutants suffering loss of additional chromosomal markers increased with increasing number of particle traversal through nuclei.     

Inhibition of the Development of a Cation Accuniulatory System and of Tris-induced Uptake in Storage Tissues by N6-benzyladenine and Kinetin

The effect of N⁶-benzyladenine (BA) and kinetin on cation uptake in disks of beetroot (Beta vulgaris L.) and swede (Brassica napobrassica Mill.) tissue was measured in aerated solutions, containing 1 mM NaCl or 1 mM KCl, and in the presence or absence of trishydroxymethyl ammo methane (tris) buffer at pH 8. This investigation followed a suggestion that the immediate activation of cation uptake in freshly sliced beetroot disks by tris (the tris-effect) may depend on stimulation of hexokinase activity. BA, a competitive inhibitor of hexokinase, caused a complete inhibition of tris stimulated cation uptake in beetroot disks, but the effect was delayed and preceded by a 3–4 hour period of promoted cation uptake. Generally, the effects of BA and kinetin were identical. Use of ¹⁴C-kinetin indicated a rapid incorporation of kinetin into the tissue. BA and kinetin also prevented the development of a cation uptake capability during the aging of the beetroot disks. Swede disks which do not show a tris-effcct and no lag phase in development of cation uptake capability were not affected by BA and kinetin treatments. Mechanisms of tris induced cation uptake are discussed with emphasis on its role as a proton acceptor.     

Identification of N1-acetylnorspermidine in Vibrio parahaemolyticus and an enzyme activity responsible for its formation

Abstract N ¹-Acetylnorspermidine [CH₃CONH(CH₂)₃NH(CH₂)₃NH₃] was identified in Vibrio parahaemolyticus , which contains norspermidine as a major polyamine. This is the first example for the natural occurence of monoacetylated unusual polyamine. The N ¹-acetylnorspermidine content was the highest 4 h after inoculation. Incubation of norspermidine and acetyl CoA with a cell extract from V. parahaemolyticus produced N ¹-acetylnorspermidine. A remarkable increase in specific activity of the acetyltransferase was observed at the exponential phase of growth. Spermidine also served as a substrate for the enzyme, with the formation of two isomers of the acetylspermidines ( N ¹-acetylspermidine was predominant), but the reaction rate was less than 50% of that with norspermidine. These results suggest that norspermidine in V. parahaemolyticus may be associated with the cell growth and its role may be controlled through acetylation, as reported for spermidine in Escherichia coli .     

Comparison of N6-benzyladenine and N-4-pyridyl-N'-phenylurea Effects on Lateral Bud Growth, Flowering and Seed Production of Brussels Sprouts (Brassica oleracea var. gemmifera)

N-4-pyridyl-N′-phenyl urea (NC5392) showed similar activity to N⁶-benzyladenine (BA) in stimulating lateral bud development on Brussels sprout (Brassica oleracea var. gemmifera) seedlings particularly on de-rooted plants, but increased average bud weight far more than did BA. Both compounds increased flowering of young plants given an insufficient cold stimulus and seeds from treated plants showed a higher germination percentage than those from untreated plants.     

大麦与油菜种皮特异启动子表达模式的比较

启动子位于转录起始位点上游并能特异性地结合RNA聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得Gerb和Bntt两个种皮特异性启动子,并对其进行生物信息学分析,构建了Gerb::GUS和Bntt::GUS植物表达载体并转化拟南芥,通过组织化学染色观察了GUS的表达情况。结果表明:两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件;转基因拟南芥幼苗期,大麦Gerb种皮特异启动子驱动GUS全株表达且子叶和下胚轴较真叶和根中表达量高;油菜Bntt种皮特异启动子表达较弱;成株期,Gerb在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性;Bntt仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb表达模式未发生显著变化,而Bntt仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子Gerb和油菜种皮特异性启动子Bntt在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G₆-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH₂-terminal amino acid sequence analysis of the G₆-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102598. Identity of the NH₂-terminal amino acid sequences among each component of the multiform G₆-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G₆-amylase gene showed no homology with those of other bacterial α-amylases although the consensus amino acid sequences of the active center were well conserved.     

Some expression of heterosis in F1-hybrid barley

Small and large scale trials of F₁ barley hybrids were done in a glasshouse in the United Kingdom and in the field in the United Kingdom and Europe in 1970, 1971 and 1972. The results showed that there were few instances of significant positive heterosis in yield with either the small or field scale experiments. The hybrids studied were all derived from American varieties as male-sterile parents and therefore the hybrids were not adapted to European countries, thus the case for or against F₁-hybrids cannot be assessed solely on this material.     

Comparison of measured and EF5-r-derived N2O fluxes from a spring-fed river

There is considerable uncertainty in the estimates of indirect N₂O emissions as defined by the Intergovernmental Panel on Climate Change's (IPCC) methodology. Direct measurements of N₂O yields and fluxes in aquatic river environments are sparse and more data are required to determine the role that rivers play in the global N₂O budget. The objectives of this research were to measure the N₂O fluxes from a spring‐fed river, relate these fluxes to the dissolved N₂O concentrations and NO₃‐N loading of the river, and to try to define the indirect emission factor (EF5‐r) for the river. Gas bubble ebullition was observed at the river source with bubbles containing 7.9 μL N₂O L^?1. River NO₃‐N and dissolved N₂O concentrations ranged from 2.5 to 5.3 mg L^?1 and 0.4 to 1.9 μg N₂O‐N L^?1, respectively, with N₂O saturation reaching 404%. Floating headspace chambers were used to sample N₂O fluxes. N₂O‐N fluxes were significantly related to dissolved N₂O‐N concentrations (r²=0.31) but not to NO₃‐N concentrations. The N₂O‐N fluxes ranged from 38 to 501 μg m^?2 h^?1, averaging 171 μg m^?2 h^?1 (±SD 85) overall. The measured N₂O‐N fluxes equated to an EF5‐r of only 6.6% of that calculated using the IPCC methodology, and this itself was considered to be an overestimate because of the degassing of antecedent dissolved N₂O present in the groundwater that fed the river.     

Comparison of measured and EF5-r-derived N2O fluxes from a spring-fed river

There is considerable uncertainty in the estimates of indirect N₂O emissions as defined by the intergovernmental panel on climate change's (IPCC) methodology. Direct measurements of N₂O yields and fluxes in aquatic river environments are sparse and more data are required to determine the role that rivers play in the global N₂O budget. The objectives of this research were to measure the N₂O fluxes from a spring‐fed river, relate these fluxes to the dissolved N₂O concentrations and NO₃‐N loading of the river, and to try and define the indirect emission factor (EF5‐r) for the river. Gas bubble ebullition was observed at the river source with bubbles containing 7.9 μL N₂O L^?1. River NO₃‐N and dissolved N₂O concentrations ranged from 2.5 to 5.3 mg L^?1 and 0.4 to 1.9 μg N₂O‐N L^?1, respectively, with N₂O saturation reaching 404%. Floating headspace chambers were used to sample N₂O fluxes. N₂O‐N fluxes were significantly related to dissolved N₂O‐N concentrations (r²=30.6) but not to NO₃‐N concentrations. The N₂O‐N fluxes ranged from 38–501 μg m^?2 h^?1, averaging 171 μg m^?2 h^?1 (±SD 85) overall. The measured N₂O‐N fluxes equated to an EF5‐r of only 6.6% of that calculated using the IPCC methodology, and this itself was considered to be an overestimate because of the degassing of antecedent dissolved N₂O present in the groundwater that fed the river.