A novel series of C<Subscript>18</Subscript>–C<Subscript>22</Subscript><Emphasis Type="Italic">trans</Emphasis> ω3 PUFA from Northern and Southern Hemisphere strains of the marine haptophyte <Emphasis Type="Italic">Imantonia rotunda</Emphasis>

A series of novel C₁₈–C₂₂ trans ω3 polyunsaturated fatty acids (PUFA) with a single trans double bond in the ω3 position was found in Northern and Southern Hemisphere strains of the marine haptophyte Imantonia rotunda. The novel ω3 PUFA were identified as 18:3(9c,12c,15t) (0.2–1.8 % of total fatty acids), 18:4(6c,9c,12c,15t) (1.9–4.1 %), 18:5 (3c,6c,9c,12c,15t) (0.7–8.8 %), 20:5(5c,8c,11c,14c,17t) (1.2–4.1 %) and 22:6(4c,7c,10c,13c,16c,19t) (0.3–4.3 %), and were accompanied by larger proportions of the all cis isomers: 18:3ω3(9,12,15) (2.7–3.5 %), 18:4ω3(6,9,12,15) (9.3–14.3 %), 18:5ω3(3,6,9,12,15) (7.8–18.5 %), 20:5ω3(5,8,11,14,17) (3.2–3.9 %), 22:5ω3(7,10,13,16,19) (0.1–0.3 %) and 22:6ω3(4,7,10,13,16,19) (2.3–5.2 %). GC analysis of FAME using a non-polar column did not reveal the trans isomers as they coeluted with the all cis PUFA. However, GC using a polar column resolved the trans PUFA from the all cis PUFA, with the trans isomers eluting before the all cis isomers. GC-MS of FAME fractionated by argentation solid-phase chromatography confirmed the molecular ions of all components. FAME were derivatized to form 4,4-dimethyloxazoline (DMOX) derivatives, and GC-MS revealed the same double bond positions for each trans and cis FAME. The results suggest that the ω3 trans double bond originated from the Δ15/ω3 desaturation of 18:2(9c,12c), suggesting that this desaturase has dual cis/trans activity in these species. These results indicate that 18:3(9c,12c,15?t) was the precursor trans isomer produced for the trans series and further desaturation by the common Δ6 desaturase to produce the trans tetraene and successive elongations and desaturations led to the subsequent series of trans ω3 PUFA isomers. To our knowledge, this is the first report of these trans ω3 isomers occurring in strains of I. rotunda. These trans ω3 PUFA may be used as biomarkers in marine food webs for this species and with their unique structure may be biologically active.     

Modification of fatty acid selectivity of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase A by error-prone PCR

Objective

To generate Candida antarctica lipase A (CAL-A) mutants with modified fatty acid selectivities and improved lipolytic activities using error-prone PCR (epPCR).

Results

A Candida antarctica lipase A mutant was obtained in three rounds of epPCR. This mutant showed a 14 times higher ability to hydrolyze triacylglycerols containing conjugated linoleic acids, and was 12 and 14 times more selective towards cis-9, trans-11 and trans-10, cis-12 isomers respectively, compared to native lipase. Lipolytic activities towards fatty acid esters were markedly improved, in particular towards butyric, lauric, stearic and palmitic esters.

Conclusion

Directed molecular evolution is an efficient method to generate lipases with desirable selectivity towards CLA isomers and improved lipolytic activities towards esters of fatty acids.

     

Function and regulation of tau conformations in the development and treatment of traumatic brain injury and neurodegeneration

One of the two common hallmark lesions of Alzheimer’s disease (AD) brains is neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated tau protein (p-tau). NFTs are also a defining feature of other neurodegenerative disorders and have recently been identified in the brains of patients suffering from chronic traumatic encephalopathy (CTE). However, NFTs are not normally observed in traumatic brain injury (TBI) until months or years after injury. This raises the question of whether NFTs are a cause or a consequence of long-term neurodegeneration following TBI. Two conformations of phosphorylated tau, cis p-tau and trans p-tau, which are regulated by the peptidyl-prolyl isomerase Pin1, have been previously identified. By generating a polyclonal and monoclonal antibody (Ab) pair capable of distinguishing between cis and trans isoforms of p-tau (cis p-tau and trans p-tau, respectively), cis p-tau was identified as a precursor of tau pathology and an early driver of neurodegeneration in AD, TBI and CTE. Histological studies shows the appearance of robust cis p-tau in the early stages of human mild cognitive impairment (MCI), AD and CTE brains, as well as after sport- and military-related TBI. Notably, cis p-tau appears within hours after closed head injury and long before other known pathogenic p-tau conformations including oligomers, pre-fibrillary tangles and NFTs. Importantly, cis p-tau monoclonal antibody treatment not only eliminates cis p-tau induction and tau pathology, but also restores many neuropathological and functional outcome in TBI mouse models. Thus, cis p-tau is an early driver of tau pathology in TBI and CTE and detection of cis p-tau in human bodily fluids could potentially provide new diagnostic and prognostic tools. Furthermore, humanization of the cis p-tau antibody could ultimately be developed as a new treatment for AD, TBI and CTE.     

Formation of Diastereoisomeric Piperazine-2,5-dione from <Emphasis Type="SmallCaps">dl</Emphasis>-Alanine in the Presence of Olivine and Water

dl-Alanine (Ala) was heated with/without powdered olivine and water at 120 °C for 8 days to investigate the formation of the diastereoisomers of piperazine-2,5-dione (diketopiperazine, DKP). When only dl-Ala was heated with a small amount of water, 3.0 % of dl-Ala changed to cis- and trans-DKP after 8 days. DKPs were not detected after heating when no water was added. The presence of a small amount of water is important factor controlling peptide production rates under thermal conditions. When DL-Ala was heated with olivine powder for 8 days, the yields of cis- and trans-DKP were 6.8 and 4.9 %, respectively. The high yield of cis-DKP compared with trans-DKP was attributed to greater thermal stability of cis-DKP. After heating for 8 days, the diastereoisomeric excess of cis-DKP without olivine was 7.3 %, whereas a much higher value of 16.3 % was obtained in the presence of olivine. Taken together, these results show that olivine is not only an efficient catalyst for the formation of DKPs but that it also play a significant role in determining the diastereoisomer selectivity of these cyclic dipeptides.     

Proline isomerization in the C-terminal region of HSP27

In mammals, small heat-shock proteins (sHSPs) typically assemble into interconverting, polydisperse oligomers. The dynamic exchange of sHSP oligomers is regulated, at least in part, by molecular interactions between the α-crystallin domain and the C-terminal region (CTR). Here we report solution-state nuclear magnetic resonance (NMR) spectroscopy investigations of the conformation and dynamics of the disordered and flexible CTR of human HSP27, a systemically expressed sHSP. We observed multiple NMR signals for residues in the vicinity of proline 194, and we determined that, while all observed forms are highly disordered, the extra resonances arise from cis-trans peptidyl-prolyl isomerization about the G193-P194 peptide bond. The cis-P194 state is populated to near 15% at physiological temperatures, and, although both cis- and trans-P194 forms of the CTR are flexible and dynamic, both states show a residual but differing tendency to adopt β-strand conformations. In NMR spectra of an isolated CTR peptide, we observed similar evidence for isomerization involving proline 182, found within the IPI/V motif. Collectively, these data indicate a potential role for cis-trans proline isomerization in regulating the oligomerization of sHSPs.     

Investigation of the mechanisms of photo-induced formation of cyclobutane dimers of cytosine and 2,4-diaminopyrimidine

The mechanisms of the formation of cyclobutane dimers (CBD) of cytosine and 2,4-diaminopyrimidine were studied at the CC2 theoretical level and cc-pVDZ basis functions. Four orientations of the two monomers are explored: cys-syn, cis-anti, trans-syn, and trans-anti. The research revealed that in all cases the cyclobutane structures are formed along the ¹ππ* excited-state reaction paths of the stacked aggregates. We localized the S₁/S₀ conical intersections mediating those transformations. The results obtained agree well with the previously reported investigations on the cis-syn cyclodimer formations of other pyrimidines.     

Pheromone composition,the mtDNA <Emphasis Type="Italic">COI</Emphasis> locus variation,and phylogenetic relationships in leafrollers of the tribe Archipini (Lepidoptera: Tortricidae)

The groups of leafroller species belonging to the tribe Archipini (Archips crataegana, A. rosana, A. xylosteana-Choristoneura diversiana, Ch. fumiferana-A. podana, Pandemis cerasa, Hedya nubiferana-Cydia pomonella) were divided based on the sequences of the mtDNA COI locus. Within these groups, the pheromone blend composition in the species studied reflected the patterns of using cis-, cis/trans-, or only trans-isomers of the pheromone components in the process of chemocommunication. It was demonstrated that polymorphism of the locus investigated in A. podana was associated with inbreeding, while in A. rosana it was associated with the geographic distribution of the species.     

Expression quantitative trait loci analysis of the Rubisco activase gene in maize

Expression quantitative trait loci (eQTL) analyses were applied in order to identify genetic factors that are relevant to the expression of a β-isoform Rubisco activase gene in maize, namely ZmRCAβ, in this study. During two years, a maize recombinant inbred line population was measured for ZmRCAβ expression levels at the grain filling stage. Based on a genetic map containing 916 molecular markers, we detected five eQTLs, namely qRCA2.1 on chromosome 2, and qRCA4.1, qRCA4.2, qRCA4.3, and qRCA4.4 on chromosome 4. These eQTLs explained the phenotypic variation ranging from 6.14% to 7.50% with the logarithm of the odd values ranging from 3.11 to 4.96. Based on the position of the eQTLs and ZmRCAβ on the chromosome, qRCA4.2 was inferred as a cis-eQTL and the remaining as a trans-eQTL, suggesting that a combination of both cis- and trans-acting elements might control ZmRCAβ expression. qRCA4.2, qRCA4.3, and qRCA4.4 were repeatedly detected during two years.     

Molecular cloning and characterization of genes encoding FK506-binding proteins (FKBPs) in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The family of FK506-binding proteins (FKBPs) consists of several members, which show peptidyl prolyl cis–trans isomerase (PPIase) activity. PPIases facilitate the conversion of peptidyl prolyl bonds from cis to trans conformation, a rate-limiting step in protein folding. In the present study, we carried out cloning of cDNAs encoding three different wheat FKBPs viz., TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1. In silico analysis suggested their likely localization to nucleus, cytosol and endoplasmic reticulum, respectively. Biochemical analyses demonstrated that none of the three purified FKBP proteins possesses detectable PPIase activity. Several putative interacting partners of TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1were identified using online software tools. The results of this study provide further evidence that PPIase activity in plant FKBPs is not conserved, and these proteins may be playing important roles in the cell through interaction with target proteins.     

Effect of Cytokinin and Auxin Treatments on Morphogenesis,Terpenoid Biosynthesis,Photosystem Structural Organization,and Endogenous Isoprenoid Cytokinin Profile in <Emphasis Type="Italic">Artemisia alba</Emphasis> Turra In Vitro

Developmental pattern modification in essential oil bearing Artemisia alba Turra was obtained by exogenous plant growth regulator (PGRs) treatments in vitro. Enhanced rooting (in PGR-free and auxin-treated plants) led to elevation of the monoterpenoid/sesquiterpenoid ratio in the essential oils of aerials. On the contrary, root inhibition and intensive callusogenesis [combined cytokinin (CK) and auxin treatments] reduced this ratio more than twice, significantly enhancing sesquiterpenoid production. Both morphogenic types displayed sesquiterpenoid domination in the underground tissues, which however differed qualitatively from the sesquiterpenoids of the aerials, excluding the hypothesis of their shoot-to-root translocation and implying the possible role of another signaling factor, affecting terpenoid biosynthesis. Inhibited rooting also resulted in a significant drop of endogenous isoprenoid CK bioactive-free bases and ribosides as well as CK N-glycoconjugates and in decreased trans-zeatin (transZ):cis-zeatin (cisZ) ratio in the aerials. Marked impairment of the structural organization of the photosynthetic apparatus and chloroplast architecture were also observed in samples with suppressed rooting. It is well known that in the plant cell monoterpenoid and transZ-type CKs biogenesis are spatially bound to plastids, while sesquiterpenoid and cisZ production are compartmented in the cytosol. In the present work, interplay between the biosynthesis of terpenoids and CK bioactive free bases and ribosides in A. alba in vitro via possible moderation of chloroplast structure has been hypothesized.     

Quantum-chemical study of interactions of <Emphasis Type="Italic">trans-</Emphasis>resveratrol with guanine-thymine dinucleotide and DNA-nucleobases

Trans-resveratrol, a natural phytoalexin present in red wine and grapes, has gained considerable attention because of its antiproliferative, chemopreventive and proapoptotic activity against human cancer cells. The accurate quantum-chemical computations based on the density functional theory (DFT) and ab initio second-order Møller-Plesset perturbation method (MP2) have been performed for the first time to study interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-derived nitrogenous bases: adenine, guanine, cytosine and thymine in vacuum and water medium. This compound is found to show high affinity to nitrogenous bases and guanine-thymine dinucleotide. The electrostatic interactions from intermolecular hydrogen bonding increase the stability of complexes studied. In particular, significantly strong hydrogen bonds between 4′-H atom of trans-resveratrol and imidazole nitrogen as well as carbonyl oxygen atoms of nucleobases studied stabilize these systems. The stabilization energies computed reveal that the negatively charged trans-resveratrol-dinucleotide complex is more energetically stable in water medium than in vacuum. MP2 method gives more reliable and significantly high values of stabilization energy of trans-resveratrol-dinucleotide, trans-resveratrol-guanine and trans-resveratrol-thymine complexes than B3LYP exchange-correlation functional because it takes into account London dispersion energy. According to the results, in the presence of trans-resveratrol the 3′-5′ phosphodiester bond in dinucleotide can be cleaved and the proton from 4′-OH group of trans-resveratrol migrates to the 3′-O atom of dinucleotide. It is concluded that trans-resveratrol is able to break the DNA strand. Hence, the findings obtained help understand antiproliferative and anticancer properties of this polyphenol.     

<Emphasis Type="Italic">ZmFKBP20-1</Emphasis> improves the drought and salt tolerance of transformed Arabidopsis

FK506-binding proteins (FKBPs), which belong to the peptidyl-prolyl cis/trans isomerase superfamily, are involved in plant response to abiotic stresses. A number of FKBP family genes have been isolated in plants, but little has been reported of FKBP genes in maize. In this study, a drought-induced FKBP gene, ZmFKBP20-1, was isolated from maize and was characterized for its role in stress responses using gene expression, protein subcellular localization, transformation in Arabidopsis, expression patterns of the stress-responsive genes, and physiological parameter analysis. During drought and salt stresses, ZmFKBP20-1 transgenic Arabidopsis plants exhibited enhanced tolerance, which was concomitant with the altered expression of stress/ABA-responsive genes, such as COR15a, COR47, ERD10, RD22, KIN1, ABI1, and ABI2. The resistance characteristics of ZmFKBP20-1 overexpression were associated with a significant increase in survival rate. These results suggested that ZmFKBP20-1 plays a positive role in drought and salt stress responses in Arabidopsis and provided new insights into the mechanisms of FKBP in response to abiotic stresses in plants.     

Membrane engineering via <Emphasis Type="Italic">trans</Emphasis>-unsaturated fatty acids production improves succinic acid production in <Emphasis Type="Italic">Mannheimia succiniciproducens</Emphasis>

Engineering of microorganisms to produce desired bio-products with high titer, yield, and productivity is often limited by product toxicity. This is also true for succinic acid (SA), a four carbon dicarboxylic acid of industrial importance. Acid products often cause product toxicity to cells through several different factors, membrane damage being one of the primary factors. In this study, cis–trans isomerase from Pseudomonas aeruginosa was expressed in Mannheimia succiniciproducens to produce trans-unsaturated fatty acid (TUFA) and to reinforce the cell membrane of M. succiniciproducens. The engineered strain showed significant decrease in membrane fluidity as production of TUFA enabled tight packing of fatty acids, which made cells to possess more rigid cell membrane. As a result, the membrane-engineered M. succiniciproducens strain showed higher tolerance toward SA and increased production of SA compared with the control strain without membrane engineering. The membrane engineering approach employed in this study will be useful for increasing tolerance to, and consequently enhancing production of acid products.     

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR)

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as “visual cycle” involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.     

Extractive compounds of birch buds (<Emphasis Type="Italic">Betula pendula</Emphasis> Roth.): I. Composition of fatty acids,hydrocarbons, and esters

This is the first report devoted to study of the hydrocarbon composition of the extract of buds of European birch Betula pendula (family Betulacea). We have identified saturated (C₁₆ to C₂₈, even number of carbon atoms) and unsaturated (linoleic and linolenic) fatty acids, β-caryophyllene, α-humulene, and the components of epicuticular waxes of cover scales, such as n-alkanes (C₂₁ to C₂₆), esters of fatty acids (C₁₆ to C₂₈, even number of carbon atoms), and fatty alcohols (C₁₈ to C₃₀, even number of carbon atoms). The gas chromatographic retention indices of all identified compounds have been determined.     

Photo-sensitized Isomerization of Retinal by Dyes

THE initial reaction following absorption of light in the retina is the isomerization of the 11-cis retinal chromophore of the visual pigment¹. Isolated 11cis retinal will undergo the same isomerization to the all-trans form when excited by light of wavelength shorter than about 450 nm and this reaction can be sensitized to light of longer wavelengths by the addition of trace amounts of iodine to the solution².     

An efficient method for the sequential production of lipid and carotenoids from the Chlorella Growth Factor-extracted biomass of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>

Efficient methodology for simultaneous extraction of multiple bioactive compounds from microalgae still remains a major challenge. The present study provides a method for the sequential production of three major products: Chlorella Growth Factor (CGF, a nucleotide-peptide complex enriched with vitamins, minerals, and carbohydrates), lipid, and carotenoids from Chlorella vulgaris biomass in an economically feasible manner. After protein-rich CGF was extracted, the spent biomass was found to contain 12% lipid and 3% carotenoids when extracted individually, compared to that of the un-utilized (fresh) biomass (lipid, 14%; carotenoids, 4%). When extracted simultaneously using conventional methods, the yield of lipid from “CGF and carotenoids-extracted biomass,” and carotenoids from “CGF and lipid-extracted biomass” were significantly reduced (50%). However, simultaneous extraction using different solvent mixtures such as hexane:methanol:water and pentane:methanol:water mixture-augmented lipid yield by 38.5% and carotenoids by 14%, and additionally retained chlorophyll and its derivatives. Column chromatographic approach yielded sequential production of lipid (18%), lutein (9%) with better yields as well as without chlorophyll interference. Different geometric isomers of lutein all-E-(trans)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol, 9Z(cis)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol, and 13Z(cis)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol were purified by HPLC and elucidated by CD, UV, NMR, FT-IR, and Mass spectra. In conclusion, the study provides an efficient and economically viable methodology for sequential production of lipid and lutein along with its geometrical isomers without chlorophyll influence and yield loss from the protein-rich CGF-extracted spent biomass of marine microalga, Chlorella vulgaris.     

The <Emphasis Type="Italic">trans</Emphasis> and <Emphasis Type="Italic">cis</Emphasis> zeatin isomers play different roles in regulating growth inhibition induced by high nitrate concentrations in maize

Abscisic acid (ABA), auxins, and cytokinins (CKs) are known to be closely linked to nitrogen signaling. In particular, CKs control the effects of nitrate availability on plant growth. Our group has shown that treatment with high nitrate concentrations limits root growth and leaf development in maize, and conditions the development of younger roots and leaves. CKs also affect source-sink relationships in plants. Based on these results, we hypothesized that CKs regulate the source-sink relationship in maize via a mechanism involving complex crosstalk with the main auxin indole-3-acetic acid (IAA) and ABA. To evaluate this hypothesis, various CK metabolites, IAA, and ABA were quantified in the roots and in source and sink leaves of maize plants treated with high and normal nitrate concentrations. The data obtained suggest that the cis and trans isomers of zeatin play completely distinct roles in maize growth regulation by a complex crosstalk with IAA and ABA. We demonstrate that while trans-zeatin (tZ) and isopentenyladenine (iP) regulate nitrate uptake and thus control final leaf sizes, cis-zeatin (cZ) regulates source and sink strength, and thus controls leaf development. The implications of these findings relating to the roles of ABA and IAA in plants’ responses to varying nitrate concentrations are also discussed.