Significant year-to-year variation of the response to radiation-induced stress shown by gill fatty acid metabolism in eels (Anguilla anguilla captured from the wild

• 1.1. In eels captured in Roskilde Fjord in 1972 and 1975, a specifically enhanced synthesis was found from ¹⁴C-acetate of ¹⁴C-labelled mono-unsaturated fatty acids (C_16:1 and C_18:1) relative to saturated fatty acids (C_16:0 and C_18:0) in sea water 4 days after irradiation (10 Gy, ⁶⁰Co).
• 2.2. Corresponding experiments in 1976 and 1982 showed rather the opposite: irradiation resulted in more ¹⁴C-labelled saturated fatty acids relative to unsaturated fatty acids, both in fresh and sea water.
• 3.3. The latter effect was less marked than that in 1972 and 1975, but still statistically clearly significant.

     

Role of ADAMs in the Ectodomain Shedding and Conformational Conversion of the Prion Protein

The cellular prion protein (PrP^C) is essential for the pathogenesis and transmission of prion diseases. PrP^C is bound to the plasma membrane via a glycosylphosphatidylinositol anchor, although a secreted, soluble form has also been identified. Previously we reported that PrP^C is subject to ectodomain shedding from the membrane by zinc metalloproteinases with a similar inhibition profile to those involved in shedding the amyloid precursor protein. Here we have used gain-of-function (overexpression) and loss-of-function (small interfering RNA knockdown) experiments in cells to identify the ADAMs (a disintegrin and metalloproteinases) involved in the ectodomain shedding of PrP^C. These experiments revealed that ADAM9 and ADAM10, but not ADAM17, are involved in the shedding of PrP^C and that ADAM9 exerts its effect on PrP^C shedding via ADAM10. Using dominant negative, catalytically inactive mutants, we show that the catalytic activity of ADAM9 is required for its effect on ADAM10. Mass spectrometric analysis revealed that ADAM10, but not ADAM9, cleaved PrP between Gly²²⁸ and Arg²²⁹, three residues away from the site of glycosylphosphatidylinositol anchor attachment. The shedding of another membrane protein, the amyloid precursor protein β-secretase BACE1, by ADAM9 is also mediated via ADAM10. Furthermore, we show that pharmacological inhibition of PrP^C shedding or activation of both PrP^C and PrP^Sc shedding by ADAM10 overexpression in scrapie-infected neuroblastoma N2a cells does not alter the formation of proteinase K-resistant PrP^Sc. Collectively, these data indicate that although PrP^C can be shed through the action of ADAM family members, modulation of PrP^C or PrP^Sc ectodomain shedding does not regulate prion conversion.The prion protein (PrP)³ is the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk (1). In these diseases the cellular form of PrP (PrP^C) undergoes a conformational conversion to the infectious form PrP^Sc that is characterized biochemically by its resistance to digestion with proteinase K (PK) (2). Mature PrP^C is anchored to the extracellular surface of the cell membrane through a glycosylphosphatidylinositol (GPI) anchor and, like most GPI-anchored proteins, is clustered into cholesterol-rich, detergent-resistant membrane rafts (reviewed in Ref. 3). Although the precise subcellular site of conversion remains undefined, conformational conversion of PrP^C to PrP^Sc is believed to occur either at the cell surface or within the endocytic pathway (4–6).A number of studies indicate that modulation of PrP^C levels at the cell surface may represent a possible future disease intervention strategy. For example, the retention of PrP^C at the cell surface and concomitant prevention of its endocytosis through the use of PrP antibodies inhibited PrP^Sc formation (7). Furthermore, the sterol-binding polyene antibiotic filipin reduced endocytosis, and induced cellular release, of PrP^C with a concomitant reduction in PrP^Sc accumulation (8). More recently, it has been shown that modulation of cell surface PrP^C levels by the novel sorting nexin SNX33 can interfere with PrP^Sc formation in cultured cells (9). Nonetheless, the natural processes regulating PrP^C levels at the cell surface remain poorly defined. One such mechanism of regulation is via shedding of the bulk of the ectodomain of PrP^C either through cleavage of the polypeptide close to the GPI anchor or within the GPI anchor itself. Indeed, it has long been established that PrP^C can be shed into the medium of cultured cells and is present as a soluble form in vivo in human cerebrospinal fluid (10, 11).Numerous cell surface proteins can be proteolytically shed by the action of a group of zinc metalloproteinases known collectively as secretases or sheddases (reviewed in Refs. 12, 13). Whereas most proteolytically shed proteins are derived from transmembrane polypeptide-anchored substrates, several GPI-anchored proteins, including the folate receptor (14), the ecto-ADP-ribosyltransferase ART2.2 (15), and a GPI-anchored construct of angiotensin-converting enzyme (16) are shed by the action of metalloproteinases. We have previously shown that PrP^C can also be proteolytically shed from the cell surface through the action of one or more zinc metalloproteinases with similar properties to those of the α-secretases responsible for the shedding of the amyloid precursor protein (APP) of Alzheimer disease (17). This α-secretase-mediated ectodomain shedding of APP from the cell surface is carried out by at least three members of the a disintegrin and metalloproteinase (ADAM) family, namely ADAM9, -10, and -17 (reviewed in Ref. 18). In addition to cleavage by ADAMs, APP is also cleaved by the β-secretase, BACE1 (β-site APP-cleaving enzyme) and the γ-secretase complex to release the neurotoxic amyloid-β peptide (19). BACE1 itself is also subject to ectodomain shedding by as yet unidentified members of the ADAM family (20).The similarities between the ectodomain shedding of APP and PrP^C, in particular the similar profile of inhibition by a range of hydroxamate-based zinc metalloproteinase inhibitors (17), led us to investigate whether the same members of the ADAM family were also involved in the shedding of PrP^C. It should be noted that this ectodomain shedding of PrP^C by cleavage of the polypeptide chain near to the site (Ser²³¹) of GPI anchor addition in the C terminus of the protein is distinct from the so-called α-cleavage between residues 111 and 112 in the middle of the protein (21, 22). This latter “endoproteolytic” cleavage of PrP^C is reported to be carried out by members of the ADAM family (23, 24).To investigate the role of ADAMs in the ectodomain shedding of PrP^C, we used loss-of-function and gain-of-function experiments in cultured cells in which candidate PrP sheddases were either knocked down by siRNA or overexpressed. We have also further characterized the shedding of BACE1 by comparison to the shedding of APP and PrP^C. In addition, we have explored whether proteolytic shedding of PrP^C is of importance in regulating its conversion into PrP^Sc.     

Interrelations between C4 Ketogenesis, C5 Ketogenesis, and Anaplerosis in the Perfused Rat Liver

We investigated the interrelations between C₄ ketogenesis (production of β-hydroxybutyrate + acetoacetate), C₅ ketogenesis (production of β-hydroxypentanoate + β-ketopentanoate), and anaplerosis in isolated rat livers perfused with ¹³C-labeled octanoate, heptanoate, or propionate. Mass isotopomer analysis of C₄ and C₅ ketone bodies and of related acyl-CoA esters reveal that C₄ and C₅ ketogenesis share the same pool of acetyl-CoA. Although the uptake of octanoate and heptanoate by the liver are similar, the rate of C₅ ketogenesis from heptanoate is much lower than the rate of C₄ ketogenesis from octanoate. This results from the channeling of the propionyl moiety of heptanoate into anaplerosis of the citric acid cycle. C₅ ketogenesis from propionate is virtually nil because acetoacyl-CoA thiolase does not favor the formation of β-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA. Anaplerosis and gluconeogenesis from heptanoate are inhibited by octanoate. The data have implications for the design of diets for the treatment of long chain fatty acid oxidation disorders, such as the triheptanoin-based diet.The regulation of the metabolism of C₄ ketone bodies, i.e. β-hydroxybutyrate (BHB)² and acetoacetate (AcAc) has been extensively investigated in vivo in isolated livers, hepatocytes, and subcellular preparations (for reviews, see Refs. 1–4). In contrast, very little information is available on the metabolism of C₅ ketone bodies, i.e. β-hydroxypentanoate (BHP) and β-ketopentanoate (BKP), which are known in the clinical literature as 3-hydroxyvalerate and 3-ketovalerate (5, 6). The C₅ ketone bodies are formed in liver from the partial oxidation of odd-chain fatty acids (see Fig. 1, center column). C₅ ketogenesis uses the same enzymes of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) cycle as C₄ ketogenesis. The counterpart of HMG-CoA in C₅ ketogenesis is 3-hydroxy-3-ethylglutaryl-CoA (HEG-CoA). We only found one report on the formation of [¹⁴C]HEG-CoA in liver extract incubated with propionyl-CoA and [1-¹⁴C]acetyl-CoA (7).Open in a separate windowFIGURE 1.Scheme of C₄ ketogenesis and C₅ ketogenesis in the liver. Numbers refer to the following enzymes: 3-ketoacyl-CoA thiolase (1), HMG-CoA synthase (2), HMG-CoA lyase (3), and β-hydroxybutyrate dehydrogenase (4). The figure also shows the link between propionyl-CoA and the CAC via anaplerosis.Because odd-chain fatty acids are absent from the diet of non-ruminant mammals, body fluids contain only traces of C₅ ketone bodies. However, C₅ ketone bodies and hydroxyethylglutarate are found in body fluids of patients with disorders of the anaplerotic pathway, propionyl-CoA → methylmalonyl- CoA → succinyl-CoA, such as deficiency in propionyl-CoA carboxylase and methylmalonyl-CoA mutase as well as biotin or vitamin B12 deficiency (5, 6, 8). The formation of C₅ ketone bodies in these pathological states involves either the conversion of propionyl-CoA to BKP-CoA via 3-ketoacyl-CoA thiolase (Fig. 1, reaction 1) or the β-oxidation of odd-chain fatty acids synthesized in these patients (9) using propionyl-CoA as a primer (10).Like their C₄ counterparts, the C₅ ketone bodies are interconverted by mitochondrial BHB dehydrogenase (11). In peripheral tissues, C₅ ketone bodies are converted to propionyl-CoA (which is anaplerotic) + acetyl-CoA via 3-oxoacid-CoA transferase (12) and 3-ketoacyl-CoA thiolase. Peripheral tissues have a high capacity to utilize exogenous C₅ ketone bodies (13), especially heart, kidney, and brain, which have high activities of 3-oxoacid-CoA transferase (14, 15).Our interest in C₅ ketone body metabolism arose from an ongoing clinical trial where patients with long chain fatty acid oxidation disorders are treated with a diet containing triheptanoin (16, 17) instead of the classical treatment with the even-chain triglyceride trioctanoin. These patients suffer from muscle weakness and rhabdomyolysis, manifested by the release of creatine kinase in plasma. It was hypothesized that the accumulation of long chain acyl-CoAs and long chain acylcarnitines results in membrane damage with release of large and small molecules from cells. The leakage of small molecules would deplete intermediates of the citric acid cycle (CAC) which carry acetyl groups as they are oxidized. It was further hypothesized that boosting anaplerosis with a suitable substrate would compensate for the chronic cataplerosis and improve heart and muscle function. The catabolism of heptanoate yields propionyl-CoA, which can be used for anaplerosis in most tissues, and C₅ ketone bodies in liver. C₅ ketone bodies are converted to propionyl-CoA, which can be used for anaplerosis in peripheral tissues. The marked improvement of the patients'' conditions after switching from a trioctanoin- to a triheptanoin-based diet supported the hypothesis.After ingestion of meals containing triheptanoin as the only lipid component, both C₅ ketone bodies and C₄ ketone bodies accumulated in the plasma of patients that have been diagnosed with disorders of long chain fatty acid oxidation (16). This suggested that acetyl groups derived from heptanoate can be used for the synthesis of C₄ and C₅ ketone bodies. Alternatively, the accumulation of C₄ ketone bodies after triheptanoin ingestion might result from the inhibition of the utilization of C₄ ketone bodies in peripheral tissues by C₅ ketone bodies.The aim of the present study was to investigate the interaction between C₄ and C₅ ketogenesis in rat livers perfused with octanoate and/or heptanoate. To gain insight on the fates of the acetyl groups of both fatty acids and on the fate of the propionyl-CoA moiety of heptanoate, we conducted the experiments with a series of labeled substrates: [1-¹³C]octanoate, [8-¹³C]octanoate, [5,6,7-¹³C₃]heptanoate, [1-¹³C]heptanoate, and [¹³C₃]propionate. The outcome of the propionyl-CoA moiety of [5,6,7-¹³C₃]heptanoate and [¹³C₃]propionate was traced by measurements of anaplerosis and glucose labeling by mass isotopomer³ analysis (18). In previous studies on the metabolism of odd-chain fatty acids in liver or hepatocytes (19, 20), ketone bodies were assayed with BHB dehydrogenase. This assay does not differentiate C₄ from C₅ ketone bodies. In the present study we used gas chromatography-mass spectrometry to specifically assay C₄ and C₅ ketone bodies (13).     

Carnitine ester hydrolysis in arteries from normal and cholesterol-fed rabbits and the effects of carnitine esters on arterial microsomal ACAT

• 1.1. Carnitine ester hydrolysis was observed in homogenates of normal rabbit (Oryctolagus cuniculus) aortas and in intact aortas from normal and cholesterol-fed rabbits using [¹⁴C]palmitoylcarnitine as a substrate.
• 2.2. Hydrolytic activity was decreased approximately 50% in arterial tissue from cholesterol-fed rabbits and may account for the observation that carnitine esters accumulate in arteries of animals fed atherogenic diets.
• 3.3. Long-chain acylcarnitines (C₁₄–C₁₈) were found to be moderate inhibitors of microsomal acylCoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26); short-chain acylcarnitine (C₂–C₁₀) and carnitine itself were not inhibitors.
• 4.4. The data suggest that the increase in activity of arterial ACAT that characteristically parallels the development of atherosclerosis does not occur as a result of carnitine ester accumulation.

     

Substrate specificity of acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacterium producing myristoleic acid

Chlorophyll b-containing cyanobacterium Prochlorothrix hollandica is characterized by a high content of esterified fatty acids (FA) with 14 and 16 carbon atoms in the membrane lipids. Depending on the conditions of cultivation, the relative amount of myristic (C_14:0) and myristoleic (C_14:1) acids can reach 35%, and palmitic (С_16:0) and palmitoleic (С_16:1) acids can reach 60% of the sum of all fatty acids in cells. Monounsaturated FAs are represented by C_14:1, and C_16:1 with an olefinic bond presumably located in the Δ⁹ position. We cloned the gene of acyl-lipid Δ9-desaturase, desC1, from Prochlorothrix hollandica and characterized its specificity to the length of the substrate using the heterologous expression in Escherichia coli cells adding C_14:0 or stearic (C_18:0) acids as exogenous substrates. The results show that DesC1 Δ⁹ desaturase generates olefinic bonds in the FAs with a length of 14 to 18 carbon atoms with an approximately equal efficiency. This indicates that the length of the FA chain in P. hollandica is determined by the activity of the FA synthase, and the chain is desaturated at the Δ⁹ position nonspecifically relatively to its length.     

<Emphasis Type="Italic">Arcobacter acticola</Emphasis> sp. nov., isolated from seawater on the East Sea in South Korea

A Gram-stain-negative, facultative aerobic, non-flagellated, and rod-shaped bacterium, designated AR-13^T, was isolated from a seawater on the East Sea in South Korea, and subjected to a polyphasic taxonomic study. Strain AR-13^T grew optimally at 30°C, at pH 7.0–8.0 and in the presence of 0–0.5% (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that strain AR-13^T fell within the clade comprising the type strains of Arcobacter species, clustering coherently with the type strain of Arcobacter venerupis. Strain AR-13^T exhibited 16S rRNA gene sequence similarity values of 98.1% to the type strain of A. venerupis and of 93.2–96.9% to the type strains of the other Arcobacter species. Strain AR-13^T contained MK-6 as the only menaquinone and summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), C_16:0, C_18:1ω7c, and summed feature 2 (iso-C_16:1 I and/or C_14:0 3-OH) as the major fatty acids. The major polar lipids detected in strain AR-13^T were phosphatidylethanolamine, phosphatidylglycerol, and one unidentified aminophospholipid. The DNA G+C content was 28.3 mol% and its mean DNA-DNA relatedness value with the type strain of A. venerupis was 21%. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain AR-13^T is separated from recognized Arcobacter species. On the basis of the data presented, strain AR-13^T is considered to represent a novel species of the genus Arcobacter, for which the name Arcobacter acticola sp. nov. is proposed. The type strain is AR-13^T (=KCTC 52212^T =NBRC 112272^T).     

A simplified method for study of RNA conformation--reaction of formylmethionine transfer RNA with [14C]methylamine-bisulfite

A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNA^fMet in solution. The procedure involves conversion of cytosine residues to N⁴-[¹⁴C]methylcytosines by treatment with ¹⁴CH₃NH₂ and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces ¹⁴C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNA^fMet has been found to contain only six reactive cytosines: C₁, C₁₆, C₁₇, C₃₅, C₇₅ and C₇₆. In addition, slow reaction at C^m₃₃ was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNA^fMet obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.     

<Emphasis Type="Italic">Viridibacterium curvum</Emphasis> gen. nov., sp. nov., isolated from freshwater

A Gram stain-negative, yellowish green-pigmented, facultatively anaerobic, motile, curved rod-shaped bacterium designated as strain JJ016^T was isolated from an artificial lake in South Korea, and characterized using a polyphasic approach. The 16S rRNA gene sequence of strain JJ016^T indicated that the isolate belonged to the family Rhodocyclaceae and exhibited 95.0% identity to Uliginosibacterium gangwonense 5YN10-9^T. The major cellular fatty acids of the novel strain were summed feature 3 (C_16:1 ω6c and/or C_16:1 ω7c), C_16:0, C_14:0, and summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c). The DNA G+C content of strain JJ016^T was 61.9 mol%. The major respiratory quinone and major polar lipid of strain JJ016^T were ubiquinone-8 and phosphatidylethanolamine, respectively. Based on the morphological and physiological properties and the biochemical evidence presented, we concluded that strain JJ016^T represents a novel species of a new genus in the family Rhodocyclaceae, for which the name Viridibacterium curvum gen. nov., sp. nov. is proposed. The type strain is JJ016^T (=KACC 16899^T =JCM 18715^T).     

Influence of dietary sources of fat on lipid synthesis in mink (Mustela vison) mammary tissue

• 1.1. The fatty acid composition of the triglyceride fraction of mink milk sampled during mid-lactation (day 28 post partum) from two nursing mink was compared to that of plasma samples and to the fatty acid composition of the feed rations used.
• 2.2. Chemical analysis of the triglyceride composition of mink milk demonstrated only minute concentrations of fatty acids with a chain length below C₁₄.
• 3.3. The saturated C_16:0- and C_18:0-unit fatty acids in mink milk made up for 24–40% of the total amount of fatty acids extracted, the remainder being represented by mono and polyunsaturated long-chain (C₁₆-C₂₄) fatty acids.
• 4.4. Preliminary in vitro experiments proved the incorporation of¹⁴C-labelled glucose, acetate or palmitate into triacylglycerols in cultures of mink mammary tissue to be linear for at least 2 hr.
• 5.5. The in vitro capacity for de novo fatty acid synthesis in mink mammary tissue using 14C-labelled glucose or acetate was low, i.e. ranging from 0.096–0.109 nmol/g (fresh tissue)/min, and amounted to only about 5% of that obtained in the case of [¹⁴C]palmitic acid incubation.
• 6.6. Following ¹⁴C-labeIled acetic or palmitic acid incubation of mink mammary tissue neither desaturation nor chain elongation was observed.
• 7.7. In response to long-term feeding on rations with two different sources of animal fat (F = fish oil or L = lard) the influence of compositional changes in dietary neutral lipids on the fatty acid composition of the lipids of mink milk is discussed.

     

The dynamics of the permeation of an analogue of insect juvenile hormone into nematodes

• 1.1. ¹⁴C-dichlorofarnesoate permeated rapidly into Haemonchus contortus (infective juveniles) and Panagrellus redivivus (mixed cultures) and was strongly bound by hydrophobic association (K_s > 10⁻⁴M).
• 2.2. Uptake rose linearly with increases in temperature (5–38°C) and external concentration (C₀; 0.07–2.15 × 10⁻⁴ M). Within 1 hr the internal concentration, C₁ was >C C₀.
• 3.3. The pH of the medium (6–8) did not affect uptake.
• 4.4. Efflux of dichlorofarnesoate was low: the half-time of release was > 18 hr.
• 5.5. The uptake curve approximated to the expression C₁/C₀ = a(1 − e^−bt) with a and b as constants and t in hr.
• 6.6. These results clarify previous work on the inhibitory action of juvenile hormone on the development of nematodes.

     

<Emphasis Type="Italic">Tardibacter chloracetimidivorans</Emphasis> gen. nov., sp. nov., a novel member of the family <Emphasis Type="Italic">Sphingomonadaceae</Emphasis> isolated from an agricultural soil from Jeju Island in Republic of Korea

A pale yellow bacterial strain, designated JJ-A5^T, was isolated form an agricultural soil from Jeju Island in Republic of Korea. Cells of the strain were Gram-stain-negative, motile, flagellated and rod-shaped. The strain grew at 15–30°C, pH 6.0–9.0, and in the presence of 0–1.5% (w/v) NaCl. Growth occurred on R2A, but not on Luria-Bertani agar, nutrient agar, trypticase soy agar and MacConkey agar. The strain utilized alachlor as a sole carbon source for growth. The strain JJ-A5^T showed 16S rRNA gene sequence similarities lower than 95.4% with members of the family Sphingomonadaceae. Phylogenetic analysis showed that the strain belongs to the family Sphingomonadaceae and strain JJ-A5^T was distinctly separated from established genera of this family. The strain contained Q-10 as dominant ubiquinone and spermidine as major polyamine. The predominant cellular fatty acids were summed feature 8 (C_18:1ω7c and/or C_18:1ω6c), summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), 11-methyl C_18:1ω7c, C_16:0 and C_14:0 2-OH. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, and phosphatidylcholine. The DNA G + C content of the strain was 62.7 mol%. On the basis of the phenotypic, genomic and chemotaxonomic characteristics, strain JJ-A5^T is considered to represent a novel genus and species within the family Sphingomonadaceae, for which the name Tardibacter chloracetimidivorans gen. nov., sp. nov. is proposed. The type strain of Tardibacter chloracetimidivorans is JJ-A5^T (= KACC 19450^T = NBRC 113160^T).     

Description of <Emphasis Type="Italic">Hymenobacter daejeonensis</Emphasis> sp. nov., isolated from grass soil,based on multilocus sequence analysis of the 16S rRNA gene, <Emphasis Type="Italic">gyr</Emphasis>B and <Emphasis Type="Italic">tuf</Emphasis> genes

A polyphasic taxonomic study was carried out on strains PB105^T and PB108 isolated from a grass soil in Korea. The cells of the strains were Gram-stain negative, non-spore-forming, non-motile, and rod-shaped. Comparative 16S rRNA gene sequence studies showed a clear affiliation of these strains with Bacteroidetes, which showed high pairwise sequence similarities with Hymenobacter algoricola VUG-A23a^T (99.2%), Hymenobacter fastidiosus VUG-A124a^T (97.4%), and Hymenobacter daecheongensis Dae14^T (96.9%). The phylogenetic analysis based on 16S rRNA gene sequences showed that the strains formed a clear phylogenetic lineage with the genus Hymenobacter. The major fatty acids were identified as C_15:0 iso, C_15:0 anteiso, C_16:1 ω5c, C_15:0 iso 3-OH, C_17:0 iso 3-OH, summed feature 3 (C_16:1 ω6c and/or C_16:1 ω7c/t), and summed feature 4 (C_17:1 anteiso B and/or C_17:1 iso I). The major cellular polar lipids were identified as phosphatidylethanolamine, an unidentified aminolipid, and two unidentified lipids. The respiratory quinone was identified as MK-7 and the genomic DNA G+C content was determined to be 64.5 mol% for strain PB105^T and 64.1 mol% for strain PB108. DNA–DNA hybridization value of type strain PB105^T with H. algoricola VUG-A23a^T was 32.3% (reciprocal 39.2). Based on the combined genotypic and phenotypic data, we propose that strains PB105^T and PB108 represent a novel species of the genus Hymenobacter, for which the name Hymenobacter daejeonensis sp. nov. is proposed. The type strain is PB105^T (=?KCTC 52579^T?=?JCM 31885^T).     

Microwave electrode discharge in nitrogen: Structure and characteristics of the electrode region

The parameters of the electrode region of an electrode microwave discharge in nitrogen are studied by emission spectroscopy. The radial and axial distributions of the intensities of the bands of the second (N₂(C ³Π _u → B ³Π _g )) and first (N₂(B ³Π _g → A ³Σ _u ⁺ )) positive systems of molecular nitrogen and the first negative system of nitrogen ions (N ₂ ⁺ (B ²Σ _u ⁺ → X ²Σ _g ⁺ )), the radial profiles of the electric field E and the electron density N _e , and the absolute populations of the vibrational levels v _C = 0–4 of the C ³Π _u excited state of N₂ and the vibrational level v _Bi = 0 of the B ²Σ _u ⁺ excited state of a molecular nitrogen ion are determined. The population temperature of the first vibrational level T _V of the ground electronic state X ¹Σ _g ⁺ of N₂ and the excitation temperature T _C of the C ³Π _u state in the electrode region of the discharge are measured. The radius of the spherical region and the spatially integrated plasma emission spectra are studied as functions of the incident microwave power and gas pressure. A method for determining the electron density and the microwave field strength from the plasma emission characteristics is described in detail.     

Stable Carbon Isotope Discrimination Is under Genetic Control in the C4 Species Maize with Several Genomic Regions Influencing Trait Expression

In plants with C₄ photosynthesis, physiological mechanisms underlying variation in stable carbon isotope discrimination (Δ13C) are largely unknown, and genetic components influencing Δ13C have not been described. We analyzed a maize (Zea mays) introgression library derived from two elite parents to investigate whether Δ13C is under genetic control in this C₄ species. High-density genotyping with the Illumina MaizeSNP50 Bead Chip was used for a detailed structural characterization of 89 introgression lines. Phenotypic analyses were conducted in the field and in the greenhouse for kernel Δ13C as well as plant developmental and photosynthesis-related traits. Highly heritable significant genetic variation for Δ13C was detected under field and greenhouse conditions. For several introgression library lines, Δ13C values consistently differed from the recurrent parent within and across the two phenotyping platforms. Δ13C was significantly associated with 22 out of 164 analyzed genomic regions, indicating a complex genetic architecture of Δ13C. The five genomic regions with the largest effects were located on chromosomes 1, 2, 6, 7, and 9 and explained 55% of the phenotypic variation for Δ13C. Plant development stage had no effect on Δ13C expression, as phenotypic as well as genotypic correlations between Δ13C, flowering time, and plant height were not significant. To our knowledge, this is the first study demonstrating Δ13C to be under polygenic control in the C₄ species maize.During photosynthesis, plants use light energy to convert atmospheric CO₂ and water into carbohydrates. For the element carbon, there are two stable isotopes, ¹²C and ¹³C. Due to the physical and chemical properties of photosynthetic CO₂ fixation, plants are depleted in ¹³C compared with atmospheric CO₂. In C₃ plants, this discrimination of stable carbon isotopes (Δ13C) has long been used to detect genetic differences of water use efficiency and has been shown to be an accurate predictor for yield under drought (Rebetzke et al., 2002). As Δ13C is linearly related to the ratio of intercellular to atmospheric CO₂ partial pressure (Farquhar et al., 1982), stomatal closure under drought stress is associated with reduced Δ13C. For C₄ plants, our knowledge about the mechanisms underlying Δ13C and about its association with water use efficiency is much more limited. Differences in Δ13C between genotypes of C₄ species have been reported, among others, for sorghum (Sorghum bicolor; Hubick et al., 1990) and maize (Zea mays; Monneveux et al., 2007). However, comprehensive studies analyzing the inheritance of Δ13C have not been performed to date.In C₃ plants, the important steps of CO₂ uptake include the diffusion of atmospheric CO₂ through the boundary layer and the stomata. Subsequently, CO₂ is taken up by the cell and enters the chloroplast, where carboxylation by Rubisco takes place. During photosynthetic carbon fixation, the strongest fractionation of carbon isotopes occurs during the carboxylation reaction of Rubisco (Roeske and O’Leary, 1984). A theoretical model of Δ13C in C₃ photosynthesis has been described by Farquhar et al. (1982), in which Δ13C depends linearly on the ratio of intercellular to ambient partial pressure of CO₂ (p_i p_a−1), and thus provides an indication of stomatal conductance and photosynthetic capacity. Additionally, the model includes the dependency of Δ13C on the fractionation of carbon isotopes during CO₂ diffusion in the air and on the enzymatic properties of the Rubisco enzyme.For rice (Oryza sativa), tomato (Solanum lycopersicum), and wheat (Triticum aestivum), it has been shown that genetic variation for Δ13C is quantitative, genotype-by-environment interaction is small, and the trait heritability is high (Condon and Richards, 1992; Rebetzke et al., 2002; Comstock et al., 2005; Impa et al., 2005). Quantitative trait loci (QTL) for Δ13C have been mapped (Handley et al., 1994; Price et al., 2002; Rebetzke et al., 2008), and in the model plant Arabidopsis (Arabidopsis thaliana), four genes have been identified that are associated with Δ13C. Two are involved in stomatal patterning and thus influence stomatal conductance (Masle et al., 2005; Nilson and Assmann, 2010), and one of them influences photosynthetic capacity as well (Masle et al., 2005). One gene plays a role in cuticular wax composition and is also associated with stomatal conductance (Lü et al., 2012), whereas the fourth gene encodes a cellulose synthase subunit, and mutations in this gene lead to decreased Δ13C. Presumably, this is the result of a decreased cell turgor due to a decreased water transport capacity of the xylem (Liang et al., 2010).For C₄ plants, our knowledge about the genetic mechanisms and physiological processes underlying Δ13C is much more limited, due to the more complex mechanism of CO₂ fixation. The first carboxylation step in C₄ plants takes place in mesophyll cells, in which CO₂ is fixed by phosphoenolpyruvate carboxylase (PEPC). During this reaction, combined with the fractionation of carbon isotopes during HCO₃⁻ formation, carbon is actually enriched in ¹³C (Farquhar, 1983). The C₄ organic acid formed by PEPC is transported to the bundle sheath cells, where CO₂ is released to be fixed by Rubisco in the second step. However, a fraction of CO₂ released in the bundle sheath can diffuse back to the mesophyll cells. The proportion of carbon fixed by PEPC that subsequently leaks out of the bundle sheath cells is termed leakiness (ϕ) and reduces the opportunity of Rubisco to discriminate against ¹³C in C₄ plants. According to the theoretical model by Farquhar (1983), Δ13C and p_i p_a−1 are also linearly related in C₄ plants, but the regression slope is determined by ϕ. Consequently, there can be a positive or a negative correlation of Δ13C and p_i p_a−1 depending on ϕ (Hubick et al., 1990). Regarding the entire fixation process, discrimination against ¹³C in C₄ plants is not as strong as in C₃ plants, and so far there have been few studies reporting a genetic variation of Δ13C in C₄ plants. In sorghum, small but significant differences in Δ13C have been observed among 12 cultivars (Hubick et al., 1990), and similar to C₃ plants, Δ13C has been shown to be correlated with transpiration efficiency (Henderson et al., 1998). Additionally, it has been shown for maize and sugarcane (Saccharum officinarum) that stress conditions lead to an increase in Δ13C (Bowman et al., 1989; Meinzer et al., 1994; Ranjith et al., 1995; Buchmann et al., 1996). Experiments under drought and under well-watered conditions showed higher values for Δ13C in drought-tolerant maize hybrids than in susceptible checks (Monneveux et al., 2007).The use of Δ13C as an indirect trait in breeding for drought tolerance in C₄ species would be highly beneficial, given a stable trait expression and high heritability similar to that in C₃ plants. To assess whether Δ13C can also be used in C₄ plants as an indirect selection trait for drought-tolerant lines, it needs to be shown that Δ13C is under genetic control, although the physiology and molecular mechanisms of Δ13C are not yet fully understood. In this study, we used an introgression library (IL; Eshed and Zamir, 1994) derived from two elite parents to analyze the genetic variation in Δ13C under well-watered conditions. ILs have been successfully used in genetics to identify QTL for various qualitatively and quantitatively inherited traits. An IL is a defined set of nearly isogenic inbred lines derived from repeated backcrosses with one of the parents (recurrent parent [RP]) and marker-assisted selection for single fragments (Supplemental Fig. S1). Ideally, each IL line carries a single chromosome fragment of a donor parent (DP) in the genetic background of an RP. Taken together, the different segments cover the whole donor genome, allowing estimation of the effects of single donor fragments in an otherwise identical genetic background (Eshed and Zamir, 1994). The RP of the IL under investigation originates from southeastern Europe and is an elite inbred line of the maize dent pool. As DP, we chose an unrelated maize line representative of the European flint pool. The IL (IL_01–IL_89) was genotyped using the Illumina MaizeSNP50 Bead Chip (Ganal et al., 2011) carrying 56,110 single-nucleotide polymorphism (SNP) markers.Kernel Δ13C of 77 IL lines was measured in the field and in the greenhouse (Δ13C is genetically controlled in the C₄ species maize. Our specific goals were (1) to characterize the genetic architecture of Δ13C (i.e. to determine the number of genomic regions associated with Δ13C), (2) to localize genomic regions influencing Δ13C, and (3) to assess the extent to which genotypic variation in Δ13C might be the result of differences in plant development.

Table I.

Overview of the experiments and experimental designs

<Emphasis Type="Italic">Uliginosibacterium sangjuense</Emphasis> sp. nov., isolated from sediment of the Nakdong River

A Gram-negative, aerobic, motile by flagella, and light yellow bacterium, designated SS1-76^T, was isolated from sediment of the Nakdong River in Sangju-si, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate SS1-76^T belongs to the genus Uliginosibacterium of the family Rhodocyclaceae, exhibiting high sequence similarity with the type strains of Uliginosibacterium gangwonense 5YN10-9^T (96.0%) and Uliginosibacterium paludis KBP-13^T (94.9%). Strain SS1-76^T contains ubiquinone-8 as a respiratory quinone and summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_16:0, and C_14:0 as major fatty acids. The cellular polar lipids are composed of phosphatidylethanolamine, phosphatidylglycerol, and unidentified aminophospholipids. The DNA G+C content was 65.3 mol%. Phenotypic, chemotaxonomic, and phylogenetic evidence clearly indicated that strain SS1-76^T represents a novel species of the genus Uliginosibacterium, for which the name Uliginosibacterium sangjuense sp. nov. is proposed. The type strain is SS1-76^T (= KCTC 52159^T = JCM 31375^T).     

Dual Role of Methionine in the Biosynthesis of Diacylglyceryltrimethylhomoserine in Chlamydomonas reinhardtii

Biosynthesis of the polar group of diacylglyceryl-O-4′-(N,N,N-trimethyl)homoserine (DGTS) was studied in intact cells of Chlamydomonas reinhardtii Dangeard. Among the three C₄ amino acids tested, only l-methionine could specifically inhibit the photosynthetic incorporation of [¹⁴C]NaHCO₃ into the polar group of DGTS. The radioactivity in l-[¹⁴C]methionine, which was labeled at either the C3 + C4, the C1, or the methyl carbon, was efficiently incorporated into the polar group of DGTS. These results suggest that the C₄ backbone and the S-methyl group of l-methionine are precursors to the C₄ backbone and the N-methyl groups of DGTS, respectively.