Conformational Preferences of Modified Nucleoside N(4)-Acetylcytidine, ac4C Occur at “Wobble” 34th Position in the Anticodon Loop of tRNA

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac⁴C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac⁴C prefers “proximal” orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2′, and O4′···HC(6). The “proximal” conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA^Met. The solvent accessible surface area (SASA) calculations revealed the role of ac⁴C in anticodon loop. The explicit molecular dynamics simulation study also shows the “proximal” orientation of N(4)-acetyl group. The predicted “proximal” conformation would allow ac⁴C to interact with third base of codon AUG/AUA whereas the ‘distal’ orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon–codon bases revealed that the models ac⁴C₍₃₄₎(Proximal):G₃, and ac⁴C₍₃₄₎(Proximal):A₃ are energetically more stable as compared to models ac⁴C₍₃₄₎(Distal):G₃, and ac⁴C₍₃₄₎(Distal):A₃, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor–acceptor atoms of ac⁴C₍₃₄₎(Proximal):G₃/A₃ base pairs suggesting role of ac⁴C in recognition of third letter of codons AUG/AUA. The “distal” conformation of ac⁴C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac⁴C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.     

Schistosoma mansoni: circulating antigens and immune complexes in infected mice.

Circulating schistosome antigens (CSA) and circulating immune complexes (CIC) were investigated during the course of Schistosoma mansoni infection in mice. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA) with [¹²⁵I]anti-S. mansoni antibodies or [¹²⁵I] anti-antigen “4” antibodies detected, respectively, total CSA and antigen “4” in serum and in 3% polyethylene glycol-precipitated CIC from infected mice. Complement fixation test and [¹²⁵I] C_1q-binding test revealed, respectively, an anticomplementary activity and the presence of C_1q-binding CIC. All these substances appeared in infected mice at approximately the same period, i.e., between the 40th- and the 55th- day postinfection. No correlation was observed between the detection of anticomplementary active substances and C_1q-binding CIC. In contrast, a close relationship was noticed between CSA and complement-activating material during the course of the infection. This suggests that substances with anticomplementary activity in serum from infected mice could be one or various CSA. A close correlation was also observed between C_1q-binding CIC and free or “complexed” antigen “4.” This observation supports well the possibility that antigen “4” is one of the major complexed circulating antigen present in schistosomiasis. The immunoglobulins G₁, G_2a, M, and A were also characterized in 3% PEG-precipitated CIC from infected mice during the period in which we detected C_1q-binding CIC. The roles played by specific S. mansoni CIC in either schistosomal nephropathy or protective mechanisms to a challenge infection in mice are discussed.     

Isolation and Structure of a Novel C20 Gibberellin in Bamboo Shoots

A new gibberellin C₂₀H₂₆O₆, tentatively named “bamboo gibberellin”, was isolated from water extract of bamboo shoots (Phyllostachys edulis) through successive purification procedures: countercurrent distribution, charcoal column chromatography, and silicic acid adsorption and partition chromatography. Its structure was established as lβ-methyl-4aα-fromyl-7α-hydroxy-8-methylenegibbane-lα,10β-dicarboxylic acid (VI) from analysis on infrared, NMR and mass spectra of its methyl ester.     

Identification of S-genotypes in Chinese cherry cultivars (Prunus pseudocerasus Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae, and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility. In the present study, we successfully isolated nine S-RNase alleles from cultivars of Chinese cherry by PCR amplification from genomic DNA and stylar cDNA combining with cleaved amplified polymorphic sequence marker. Analysis of amino acid sequences revealed five novel S-alleles, S ₂ , S ₄ , S ₆ , S ₈ , and S ₉ , with respective accession numbers in the NCBI database of EF541168, EF541173, EF541172, FJ628598, and FJ628599. Results showed that “Dongtang” and “Yinzhu” contained six S-alleles (S ₁ , S ₃ , S ₅ , S ₇ , S ₈ , and S ₉ ); “Taishanganying” contained four S-alleles (S ₁ , S ₂ , S ₄ , and S ₆ ); “Daiba”, “Dayingzui”, and “Xiaomizi” contained four S-alleles (S ₁ , S ₂ , S ₅ , and S ₈ ); “Laiyangduanzhi”, “Shuangquanchangba”, and “Daqingye” contained three S-alleles (S ₁ , S ₂ , and S ₈ ). It is interesting that different cultivars collected from the same place hold the same S-genotypes. Moreover, pollination tests and pollen tube growth assays showed that nine cultivars were self-compatible. Chinese cherry presented in this article are naturally polyploidy, which is a very useful material for the study of self-compatibility, and much of this information will be valuable for further work on self-(in)compatibility of fruit tree in Rosaceae.     

The relative extension of alginates having different chemical composition

From light-scattering and viscosity experiments, it was found that the relative extension of the three types of “blocks” in alginate increased in the order “MG-blocks” <“MM-blocks”<“GG-blocks”, both in 0.1M aqueous sodium chloride and in the unperturbed state. From a comparison with calculated free-rotation dimensions, it was concluded that this was due to a difference in the hindrance to rotation around the glycosidic linkage in the different blocks. Calculations by statistical mechanics, using Kitaygorodsky potential functions for the non-bonded interaction between atomic groups in adjacent dimers, yielded unperturbed dimensions which were in agreement with the above order, only when the L-guluronic acid residue was assumed to adopt the 1C (¹C₄) conformation.     

Influence du milieu de culture sur la composition en sterols de Leptosphaeria typhae

C₂₇, C₂₈ and C₂₉ sterols have been isolated from a Leptosphaeria typhae culture grown in vitro in light on a synthetic medium. These products were characterized by GLC and MS. Saturated and mono-, di- and tri-unsaturated sterols are present, both free and esterified. There are significant differences between these sterols and those in the same fungus grown on “oat water”. Unexpectedly, cholesterol was detected in the latter case.     

Structure and Biosynthesis of Cuticular Lipids: Hydroxylation of Palmitic Acid and Decarboxylation of C(28), C(30), and C(32) Acids in Vicia faba Flowers

The structure and composition of the cutin monomers from the flower petals of Vicia faba were determined by hydrogenolysis (LiAlH₄) or deuterolysis (LiAlD₄) followed by thin layer chromatography and combined gas-liquid chromatography and mass spectrometry. The major components were 10, 16-dihydroxyhexadecanoic acid (79.8%), 9, 16-dihydroxyhexadecanoic acid (4.2%), 16-hydroxyhexadecanoic acid (4.2%), 18-hydroxyoctadecanoic acid (1.6%), and hexadecanoic acid (2.4%). These results show that flower petal cutin is very similar to leaf cutin of V. faba. Developing petals readily incorporated exogenous [1-¹⁴C]palmitic acid into cutin. Direct conversion of the exogeneous acid into 16-hydroxyhexadecanoic acid, 10, 16-dihydroxy-, and 9, 16-dihydroxyhexadecanoic acid was demonstrated by radio gas-liquid chromatography of their chemical degradation products. About 1% of the exogenous [1-¹⁴C]palmitic acid was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes, which were identified by combined gas-liquid chromatography and mass spectrometry as the major components of the hydrocarbons of V. faba flowers. The radioactivity distribution among these three alkanes (C₂₇, 15%; C₂₉, 48%; C₃₁, 38%) was similar to the per cent composition of the alkanes (C₂₇, 12%; C₂₉, 43%; C₃₁, 44%). [1-¹⁴C]Stearic acid was also incorporated into C₂₇, C₂₉, and C₃₁n-alkanes in good yield (3%). Trichloroacetate, which has been postulated to be an inhibitor of fatty acid elongation, inhibited the conversion of [1-¹⁴C]stearic acid to alkanes, and the inhibition was greatest for the longer alkanes. Developing flower petals also incorporated exogenous C₂₈, C₃₀, and C₃₂ acids into alkanes in 0.5% to 5% yields. [G-³H]n-octacosanoic acid (C₂₈) was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes. [G-³H]n-triacontanoic acid (C₃₀) was incorporated mainly into C₂₉ and C₃₁ alkanes, whereas [9, 10, 11-³H]n-dotriacontanoic acid (C₃₂) was converted mainly to C₃₁ alkane. Trichloroacetate inhibited the conversion of the exogenous acids into alkanes with carbon chains longer than the exogenous acid, and at the same time increased the amount of the direct decarboxylation product formed. These results clearly demonstrate direct decarboxylation as well as elongation and decarboxylation of exogenous fatty acids, and thus constitute the most direct evidence thus far obtained for an elongation-decarboxylation mechanism for the biosynthesis of alkanes.     

The conversion of leukotriene C4 to isomers of leukotriene B4 by human eosinophil peroxidase

The smooth muscle contractile and vasoactive mediator leukotriene C₄ (5(S)-hydroxy-6(R)-sulfido-glutathionyl-eicosatetraenoic acid; LTC₄) is converted by phorbol ester-stimulated human eosinophils to two isomers of leukotriene B₄, 5(S),12(R)-6,8,10 trans-14 cis-eicosatetraenoic acid (5(S),12(R)-“all-trans”-LTB₄) and 5(S),12(S)-“all-trans”-LTB₄, which are leukocyte chemotactic factors lacking the humoral functions of LTC₄. Optimal conversion of LTC₄ to the “all-trans” isomers of LTB₄ by intact eosinophils and soluble eosinophil peroxidase requires both H₂O₂ and halide ions. Oxidative metabolism of leukotrienes may represent an important regulatory function of eosinophils in hypersensitivity reactions.     

Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids

An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6–10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C₆–C₁₀ carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C₆–C₁₀ dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C₆–C₁₀ ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.     

Acyl coenzyme A dehydrogenases and electron-transferring flavoprotein from beef hart mitochondria.

Electron-transferring flavoprotein (ETF) and long-chain acyl coenzyme A (CoA) dehydrogenase (LC-AD) have been purified essentially to homogeneity from beef heart (BH) mitochondria and partially characterized. The spectra of the major yellow acyl CoA dehydrogenase from BH mitochondria, both oxidized and when bleached with C₁₆CoA, were found to resemble those of pig liver (PL) LC-AD. The subunit molecular weight was found to be about 38,000 both by Na-dodecyl sulfate gel electrophoresis and by minimal molecular weight based on flavin content (A₄₅₀, ? = 11.3 × 10³ cm^?1m^?1). The enzyme is probably a tetramer with no interchain disulfide bonds. When assayed in the presence of ETF, relative activities are C₈CoA > C₁₆CoA ? C₄CoA. These findings show that physicochemical and specificity characteristics do not coincide in the pig liver and the beef heart enzymes. The BH ETF is similar to the PL ETF in its spectra, in subunit molecular weight determined by minimal molecular weight (based on flavin content as A₄₃₈), by Na-dodecyl-SO₄ gel electrophoresis, the absence of interchain disulfide bonds, V?_p, and the presence of two subunits/molecule. There were some changes in the amino acid composition concomitant with a decrease in apparent isoelectric point. The pig and beef enzymes were reactive with each other. The turnover number of the beef heart system at “saturating” ETF was 100 mol of 1, 6-dichlorophenol indophenol reduced/min/ mol of LC-AD. Abnormally low activity at low ETF concentrations as compared to high ETF concentrations was seen with the beef heart enzymes as with the pig liver system previously studied and again a material obtained during purification of the ETF similar to the “factor” from pig liver (based on chromatographie and disc-gel electrophoretic behavior) stimulated the low activity, while the high-ETF activity was relatively unaffected, permitting linear double-reciprocal plots over wide ranges of ETF concentration. Fatty-acid-free bovine serum albumin (BSA-FAF) could mimic this effect at equivalent protein concentrations (50–100 μg), as could increased LC-AD concentration and, to a lesser extent, limited aging. Studies of activity at very high concentrations of C₁₆CoA revealed a marked high-substrate inhibition with activity peaking at about 4 μm, the reported critical micelle concentration for C₁₆CoA. The addition of BSA-FAF resulted in more “normal” v vs [S] curves, and although the substrate inhibition was still present it was less severe. The K_m for C₁₆CoA in the presence of BSA-FAF is about 1 μm. These results suggest that the inhibitory species may be the C₁₆CoA micelle, and the BSA-FAF may reverse or alleviate the inhibition by binding acyl CoA in a manner analogous to its binding of fatty acid anions.     

Methyl-end desaturases with ∆12 and ω3 regioselectivities enable the de novo PUFA biosynthesis in the cephalopod Octopus vulgaris

The interest in understanding the capacity of aquatic invertebrates to biosynthesise omega-3 (ω3) long-chain (≥C₂₀) polyunsaturated fatty acids (LC-PUFA) has increased in recent years. Using the common octopus Octopus vulgaris as a model species, we previously characterised a ∆5 desaturase and two elongases (i.e. Elovl2/5 and Elovl4) involved in the biosynthesis of LC-PUFA in molluscs. The aim of this study was to characterise both molecularly and functionally, two methyl-end (or ωx) desaturases that have been long regarded to be absent in most animals. O. vulgaris possess two ωx desaturase genes encoding enzymes with ∆12 and ω3 regioselectivities enabling the de novo biosynthesis of the C₁₈ PUFA 18:2ω6 (LA, linoleic acid) and 18:3ω3 (ALA, α-linolenic acid), generally regarded as dietary essential for animals. The O. vulgaris ∆12 desaturase (“ωx2”) mediates the conversion of 18:1ω9 (oleic acid) into LA, and subsequently, the ω3 desaturase (“ωx1”) catalyses the ∆15 desaturation from LA to ALA. Additionally, the O. vulgaris ω3 desaturase has ∆17 capacity towards a variety of C₂₀ ω6 PUFA that are converted to their ω3 PUFA products. Particularly relevant was the affinity of the ω3 desaturase towards 20:4ω6 (ARA, arachidonic acid) to produce 20:5ω3 (EPA, eicosapentaenoic acid), as supported by yeast heterologous expression, and enzymatic activity exhibited in vivo when paralarvae were incubated in the presence of [1-¹⁴C]20:4ω6. These results confirmed that several routes enabling EPA biosynthesis are operative in O. vulgaris whereas ARA and docosahexaenoic acid (DHA, 22:6ω3) should be considered essential fatty acids since endogenous production appears to be limited.     

Studies on monoacylglycerols from Gordona lentifragmenta,Gordona bronchialis and Gordona rubropertincta

Monoacylglycerols containing α-branched-β-hydroxylated fatty acids (mycolic acids) ranging from C₄₂ to C₅₀ and from C₆₀ to C₆₆, were isolated from Gordona lentifragmenta and from G. bronchialis, respectively. On the other hand, G. rubropertincta showed only a monoacylglycerol fraction which released non-hydroxylated fatty acids; they were identified as C_16:0-, C_16:1,- C_18:1- and branched C_19:0-fatty acids. This last component was identified as 10-methyl octadecanoic acid (tuberculostearic acid).     

Venom and Dufour's gland secretions in an Australian species of Camponotus

Constituents of the venom (1) and Dufour's gland (25) have been characterized in an Australian representative of the highly evolved ant subfamily Formicinae. The venom reservoir of this ant, Camponotus intrepidus, contains formic acid, identified as the benzyl ester. The Dufour's gland contains a major hydrocarbon and a minor fatty acid fraction. Hydrocarbons include the normal alkanes, C₁₀ to C₁₇ (82 per cent); two series of monomethylalkanes, C₁₂, C₁₃, C₁₄, C₁₆, and C₁₇, the 3-methyl derivatives comprise approximately 16 per cent, and the 5-methylalkanes 2 per cent of the total; there are trace proportions of the n-alkenes, C₁₂, C₁₃, and C₁₅. The minor fatty acids, myristic, pentadecanoic, palmitic, and stearic are present in the ratio 2 : 2 : 12 : 11.     

Salinicoccus halitifaciens sp. nov., a novel bacterium participating in halite formation

Strain JC90^T was isolated from a soda lake in Lonar, India. Strain JC90^T maintains its external pH to 8.5 and participates in halite formation. Based on 16S rRNA gene sequence similarity studies, strain JC90^T was found to belong to the genus Salinicoccus and is most closely related to “Salinicoccus kekensis” K164^T (99.3 %), Salinicoccus alkaliphilus T8^T (98.4 %) and other members of the genus Salinicoccus (<96.5 %). However Strain JC90^T is <36 % related (based on DNA–DNA hybridization) with the type strains of “S. kekensis” K164^T and S. alkaliphilus T8^T. The DNA G+C content of strain JC90^T was determined to be 46 mol %. The cell-wall amino acids were identified as lysine and glycine. Polar lipids were found to include diphosphatidylglycerol, phosphatidylglycerol, phosphatidyl ethanolamine, an unidentified glycolipid and unidentified lipids (L1,2). Major hopanoids of strain JC90^T were determined to be bacterial hopane derivatives (BHD1,2), diplopterol, diploptene and two unidentified hopanoids (UH1,2). The predominant isoprenoid quinone was identified as menaquinone (MK-6). Anteiso-C_15:0 was determined to be the predominant fatty acid and significant proportions of iso-C_14:0, C_14:0, iso-C_15:0, C_16:0, iso-C_16:0, iso-C_17:0, anteiso-C_17:0 and C_18:02OH were also detected. The results of physiological and biochemical tests support the molecular evidence and allowed a clear phenotypic differentiation of strain JC90^T from all other members of the genus Salinicoccus. Strain JC90^T is therefore considered to represent a novel species, for which the name Salinicoccus halitifaciens sp. nov. is proposed. The type strain is JC90^T (=KCTC 13894^T =DSM 25286^T).     

Fatty acids of some cornaceae,hydrangeaceae, aquifoliaceae,hamamelidaceae and styracaceae

Seed, kernel or fruit oils of the Cornaceae (nine species). Hydrangea, Hamamelis, Ilex (Aquifoliaceae) and the Styracaceae (two species) were analysed for fatty acid composition. Special attention was paid to the occurrence of petroselinic acid (18: 1Δ6c). In the species investigated. C₁₈ acids were always present in greater quantities than C₁₆ fatty acids; C₂₀ and C₂₂ acids were only minor components. The Cornaceae show differing fatty acid patterns which correlate well with anatomical, morphological and other chemical data. In Cornus, Curtisia, Mastixia and Corokia linoleic acid predominates, whereas linoleic and linolenic acid form the major components in Davidia and Nyssa. 18 : 1Δ6c, an aralioid type, occurs in large amounts in Aucuba and Griselinia. Hamamelis, Hydrangea and Ilex show a common fatty acid pattern with linoleic acid as the dominant component in all cases. Classification currently based on morphological and anatomical differences between the two species of Styracaceae which were investigated here should include their different fatty acid compositions: in Halesia linoleic acid predominates over oleic acid, whereas in Styrax equal amounts of these two acids are found.     

DIFFERING ONTOGENETIC ORIGINS OF PCR (“KRANZ”) SHEATHS IN LEAF BLADES OF C4 GRASSES (POACEAE)

The origin and early development of procambium and associated ground meristem of major and minor veins have been examined in the leaf blades of seven C₄ grass species, representing different taxonomic groups and the three recognized biochemical C₄ types (NAD-ME, PCK, and NADP-ME). Comparisons were made with the C₃ species, Festuca arundinacea. In “double sheath” (XyMS+) species (Panicum effusum, Eleusine coracana, and Sporoboìus elongatus), the procambium of major veins gives rise to xylem, phloem, and a mestome sheath; associated ground meristem differentiates into PCA (“C₄ mesophyll”) tissue and the PCR (“Kranz”) sheath. Development in the C₃ species parallels this pattern, except that associated ground meristem differentiates into mesophyll and a parenchymatous bundle sheath. In contrast, major vein procambium of “single sheath” (XyMS–) species (Panicum bulbosum, Digitaria brownii, and Cymbopogon procerus) differentiates into xylem, phloem and a PCR sheath; associated ground meristem gives rise to PCA tissue. These observations of major vein development support W. V. Brown's hypothesis that the PCR sheaths of “double sheath” (XyMS+) C₄ grasses are homologous with the parenchymatous bundle sheaths of C₃ grasses, while in “single sheath” (XyMS–) C₄ species they are homologous with the mestome sheath. Although there are some similarities in the development of the major and minor vascular bundle procambium in the C₄ species examined, the ontogeny of the smaller minor veins is characterized by a precocious delineation of the PCR sheath layer that may even precede the appearance of the distinctive cytological features of ground meristem and procambium. This contracted development in minor veins appears to be related to their close spacing in mature leaves and to their comparatively late appearance during leaf ontogeny.     

Final structure of caulerpicin,a toxin mixture from the green alga Caulerpa racemosa

Caulerpicin from the green alga Caulerpa racemosa is shown to consist of a mixture of ceramides derived from 2S, 3R-sphinganine with C₁₈ (32%), C₂₀(2%), C₂₂(6%), C₂₄(35%) and C₂₆(25%) saturated fatty acid