Search for new natural sources of morphinans

Codeine, medically the most widely used opiate, is mostly derived from morphine, isolated from opium and poppy straw (Papaver somniferum, opium poppy). Morphine, however, is greatly misused by illegal conversion into its diacetyl-derivative: heroin. The discovery of an efficient alternative medicine or a source for codeine other than opium poppy may contribute to a curtailment of the heroin market. No major adverse properties should be present in such a new medicine or codeine source. In this paper the search for the latter is discussed with regards to the natural occurrence of morphinan derivatives and the biosynthetic pathways in available plants. Economic and social problems connected with the introduction of a new biological source for opiates are reviewed.     

Modification of a Silver Substrate for Advanced Spectro-Electrochemical Applications of SERR Spectroscopy

A substrate for surface-enhanced resonance Raman spectroscopy (SERRS) in the near-ultraviolet (UV) range is presented, extending the potential window for electrochemical applications. Silver nanoparticles were synthesized exhibiting a localized surface plasmon resonance at the excitation wavelength and adsorbed onto a template-stripped silver substrate, whereby the number of particles per unit area was controlled by the adsorption time. Any attempt to employ spectro-electrochemistry on these surfaces, however, was hampered by the anodic dissolution of silver at potentials higher than 300 mV vs. standard hydrogen electrode (SHE). In order to extend the potential window for electrochemistry and still being able to use the resonance effect from silver nanoparticles, a 5-nm thick gold layer was sputtered on top of the Ag/AgNPs substrate. Cyclic voltammetry measurements of cytochrome c (cc) were carried out showing that the electrochemical behavior of gold can extend the potential range of the composite surface significantly. Furthermore, a potentiostatic titration of cc on this substrate by SERRS demonstrated that the resonance Raman effect of silver nanoparticles with the Soret band of the heme had been maintained in the presence of the gold adlayer. The positions of the plasmon resonances measured by reflection spectroscopy method were confirmed by finite-difference time-domain simulations. Gold is the optimal substrate for electrochemistry, whereas silver is the optimal material for plasmonic applications. Combining both metals gives us a surface with good performance for electrochemical applications as well as an enhancement effect sufficient to study redox-active biomacromolecules such as cc.     

The respiratory electron transport system of heterotrophically-grown Rhodopseudomonas palustris

The enzymatic activities and the cytochrome components of the respiratory chain were investigated with membrane fractions from chemoheterotrophically grown Rhodopseudomonas palustris. Whereas the level of electron transfer carriers was not distinctly affected by a change of the culture conditions, the potential activities of the enzymes were clearly increased when the cells were grown aerobically. Reduced-minus oxidized difference spectra of the membrane fractions prepared from dark aerobically grown cells revealed the presence of three b-type cytochromes b ₅₆₁, b ₅₆₀ and b ₅₅₈, and at least two c-type cytochromes c ₅₅₆ and c ₂ as electron carriers in the electron transfer chain. Cytochrome of a-type could not be detected in these membranes. Reduced plus CO minus reduced difference spectra of the membrane fractions were indicative of cytochrome o, which may be equivalent to cytochrome b ₅₆₀, appearing in substrate-reduced minus oxidized difference spectra. Cytochrome o was found to be the functional terminal oxidase. CO difference spectra of the high speed supernatant fraction indicated the presence of cytochrome c′. Succinate and NADH reduced the same types of cytochromes. However, a considerable amount of cytochrome b ₅₆₁ with associated β and γ bands at 531 and 429 nm, respectively, was reducible by succinate, but not by NADH. A substantial fraction of the membrane-bound b-type cytochrome was non-substrate reducible and was found in dithionite-reduced minus substrate-reduced spectra. Cytochrome c ₂ may be localized in a branch of the electron transport system, with the branch-point at the level of ubiquinone. The separate pathways rejoined at a common terminal oxidase. Two terminal oxidases with different KCN sensitivity were present in the respiratory chain, one of which was sensitive to low concentrations of KCN and was connected with the cytochrome chain. The other terminal oxidase which was inhibited only by high concentrations of cyanide was located in a branched pathway, through which the electrons could flow from ubiquinone to oxygen bypassing the cytochrome chain.     

A sensitive spectorophotometric assay for pyruvate dehydrogenase and oxoglutarate dehydrogenase complexes

The activities of pyruvate dehydrogenase and oxo-glutarate dehydrogenase can be reliably measured by coupling the production of NADH to the reduction of added cytochromec. Maximum activities required the addition of NADH-cytochromec reductase activity prepared from rat heart mitochondria. Compared to other spectrophotometric assays this method provides an eight-fold increase in sensitivity and is particularly suitable for use with small tissue samples such as needle-biopsy samples of human skeletal muscle. Measurements of activities in rat tissues showed them to be in the order skeletal muscle < liver < heart ≤brown adipose tissue. Activities in normal human skeletal muscle were similar to those of rat muscle, tn the rat tissues specific differences were seen in the relative activities of the two complexes and cytochromec oxidase suggesting tissue-specific differences in the activities of the dehydrogenases and components of the electron-transport chain.     

Litoribrevibacter albus gen. nov. sp. nov., isolated from coastal seawater,Fujian province,China

A Gram-stain negative, short rod-shaped aerobic bacterium with flagella, designated strain Y32^T, was isolated from coastal seawater in Xiamen, Fujian Province of China. 16S rRNA gene sequence comparisons showed that strain Y32^T is a member of the family Oceanospirillaceae, forming a distinct lineage with species of the genus Litoribacillus. The 16S rRNA gene sequence similarities between strain Y32^T and other strains were all less than 94.0 %. Strain Y32^T was found to grow optimally at 28 °C, at pH 7.0–8.0 and in the presence of 4–5 % (w/v) NaCl. The major fatty acids were identified as Summed Feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c, 49.4 %), C_16:0 (17.7 %), C_14:0 (6.9 %) and C_18:1 ω9c (5.4 %). The major respiratory quinone was identified as ubiquinone-8 (Q-8). The major polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of strain Y32^T was determined to be 55.6 mol%. According to its morphology, physiology, fatty acid composition, polar lipids composition and 16S rRNA gene sequence data, strain Y32^T represents a novel species of a new genus in the family Oceanospirillaceae, for which the name Litoribrevibacter albus gen. nov. sp. nov. is proposed. The type strain of Litoribrevibacter albus is Y32^T (=MCCC 1F01211^T=NBRC 110071^T).     

New target carotenoids for CCD4 enzymes are revealed with the characterization of a novel stress-induced carotenoid cleavage dioxygenase gene from Crocus sativus

Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC–DAD and the volatile products by GC/MS. β-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9′,10′) bonds and generating β-ionone, although β-cyclocitral resulting from a 7,8 (7′,8′) cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates.     

The cytochromes of a marine Beggiatoa

Naturally grown Beggiatoa filaments, occurring in massive near-mono-cultures at a “black smoker” wall of the Guaymas Basin hydrothermal vent site, were harvested and used for the analysis of their cytochromes. The cytochromes have been characterized by gel permeation chromatography, optical spectroscopy and redox potentiometry. Only c-type cytochromes were detected; a small, high potential cytochrome c that seems typical of its class, and a large complex (M_r 210,000) containing at least four thermodynamically distinct c-type hemes, which was partially dissociated by chromatography on DEAE-Sepharose. The hemes of the large complex have appropriate oxidation-reduction midpoint potentials (E_m7 +240 mV, +15 mV,-160 mV,-340 mV) to be involved in the metabolism of sulfide, which is presumed to be the source of reductant for this organism.     

Isolation and properties of membrane-bound cytochrome c-552 from photosynthetic bacterium Chromatium vinosum

A membrane-bound cytochrome c-552 was isolated and purified from the photosynthetic bacterium Chromatium vinosum by treatment with sodium cholate, sodium deoxycholate and bacterial alkaline protease followed by gel filtration. The purified cytochrome c-552, which may have been modified by the protease treatment, was electrophoretically homogeneous. Its minimal molecular weight was estimated to be 19 and 20 kdaltons, respectively by SDS polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-100. Cytochrome c-552 showed the absorption maxima at 419, 523 and 552 nm in the reduced form. Reduced-minus-oxidized difference millimolar absorption coefficient was 10.6 for the wavelength pair, 552 minus 540 nm. The midpoint potential at pH 8.0 was ?130 mV. The polarity in the amino acid composition of cytochrome c-552 was 40.1% and reflected its hydrophobicity. The solubilized cytochrome c-552 was shown to be a different entity from the soluble flavocytochrome c-552 in several respects.     

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.     

Thalassobius aquaeponti sp. nov., an alphaproteobacterium isolated from seawater

A Gram-stain-negative, aerobic, non-motile and rod-shaped or ovoid bacterial strain, GJSW-22^T, which was isolated from seawater at Geoje island in South Korea, was characterized taxonomically. Strain GJSW-22^T was observed to grow optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain GJSW-22^T grouped with the type strains of Thalassobius species, forming a stable cluster with the type strain of Thalassobius aestuarii (bootstrap value of 83.2 %). Strain GJSW-22^T exhibited the highest 16S rRNA gene sequence similarity value (98.0 %) to the type strain of T. aestuarii. It exhibited 16S rRNA gene sequence similarity values of 95.6–96.1 % to the type strains of the other Thalassobius species. Strain GJSW-22^T was found to contain Q-10 as the predominant ubiquinone and C_18:1 ω7c and 11-methyl C_18:1 ω7c as the major fatty acids. The major polar lipids of strain GJSW-22^T were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G + C content of strain GJSW-22^T is 60.3 mol % and its mean DNA–DNA relatedness value with the type strain of T. aestuarii was 23 %. Differential phenotypic properties, together with the phylogenetic and chemotaxonomic data, confirmed that strain GJSW-22^T is distinct from other Thalassobius species. On the basis of the data presented, strain GJSW-22^T is considered to represent a novel species of the genus Thalassobius, for which the name Thalassobius aquaeponti sp. nov. is proposed. The type strain is GJSW-22^T (=KCTC 42115^T = NBRC 110378^T).     

Biogenesis of mitochondria 20 the effects of altered membrane lipid composition on mitochondrial oxidative phosphorylation inSaccharomyces cerevisiae

1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
6. ATPase activity is apparently unaffected by UFA-depletion, but³²P_i-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.

     

Simiduia aestuariiviva sp. nov., a gammaproteobacterium isolated from a tidal flat sediment

A Gram-stain negative, aerobic, motile and rod-shaped bacterial strain, designated J-MY2^T, was isolated from a tidal flat sediment of the South Sea, South Korea. Strain J-MY2^T was found to grow optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain J-MY2^T forms a cluster with the type strains of Simiduia species. Strain J-MY2^T exhibited 16S rRNA gene sequence similarity values of 97.62–98.77 % to the type strains of four Simiduia species and of <92.95 % sequence similarity to the type strains of the other recognized species. Strain J-MY2^T was found to contain Q-8 as the predominant ubiquinone and summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_16:0, C_18:1 ω7c and C_17:1 ω8c as the major fatty acids. The major polar lipids of strain J-MY2^T were identified as phosphatidylethanolamine, phosphatidylglycerol, three unidentified glycolipids and one unidentified lipid. The DNA G+C content of strain J-MY2^T was determined to be 54.8 mol% and its mean DNA–DNA relatedness values with the type strains of the four Simiduia species were in the range 21–34 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain J-MY2^T is separated from other Simiduia species. On the basis of the data presented, strain J-MY2^T is considered to represent a novel species of the genus Simiduia, for which the name Simiduia aestuariiviva sp. nov. is proposed. The type strain is J-MY2^T ( = KCTC 42073^T = CECT 8571^T).     

Charge interactions of cytochrome c with cytochrome c oxidase

• 1.1. The pyridoxal phosphate (PLP) modification of the lysine amino groups in cytochrome c causes decrease in the reaction rate with cytochrome c oxidase.
• 2.2. The rate constants for (PLP);-cyt. c, PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c (at pH 7.4, 1=0.02) are 3.6 × 10⁻³'sec-', 5.5 × 10⁻³, 5.2 × 10⁻³-'sec⁻¹ and 9.8 × 10⁻³sec⁻¹, respectively.
• 3.3. In spite of the same positive charge of singly PLP-cytochromes c the reaction between PLP(Lys 86)-cyt. c and cyt. c oxidase exhibits the ionic strength dependence that differs from those of the PLP(Lys 79)-cyt. c.
• 4.4. The rate constants at zero and infinite ionic strength for PLP(Lys 86)-cyt. c is 2-fold less than that for PLP(Lys 79)-cyt. c.
• 5.5. The positively charged cytochrome c lysines 86 and 79 form two from four or five predicted complementary charge interactions with carboxyl groups on cytochrome c oxidase.

     

Molecular genetic diversity and association mapping of morphine content and agronomic traits in Turkish opium poppy (<Emphasis Type="Italic">Papaver somniferum</Emphasis>) germplasm

As the sole plant source of many potent alkaloids, opium poppy (Papaver somniferum L.) is an important medicinal crop. Nevertheless, few studies have characterized opium poppy germplasm with crop-specific molecular markers. Because Turkey is a diversity center for opium poppy, Turkish germplasm is a valuable genetic resource for association mapping studies aimed at identifying QTLs controlling morphine content and agronomic traits. In this study, the morphological diversity and molecular diversity of 103 Turkish opium poppy landraces and 15 cultivars were analyzed. Potentially useful morphological variation was observed for morphine content, plant height, and capsule index. However, the landraces exhibited limited breeding potential for stigma number, and seed and straw yields. Both morphological and molecular analyses showed distinct clustering of cultivars and landraces. In addition, a total of 164 SSR and 367 AFLP polymorphic loci were applied to an opium poppy association mapping panel composed of 95 opium poppy landraces which were grown for two seasons. One SSR and three AFLP loci were found to be significantly associated with morphine content (P < 0.01 and LD value (r ²) = 0.10–0.32), and six SSR and 14 AFLP loci were significantly associated with five agronomic traits (plant height, stigma number, capsule index, and seed and straw yields) (P < 0.01 and LD value (r ²) = 0.08–0.35). This is the first report of association mapping in this crop. The identified markers provide initial information for marker-assisted selection of important traits in opium poppy breeding.     

Molecular evidence to reconcile taxonomic instability in mahseer species (Pisces: Cyprinidae) of India

The mahseers are an important group of fishes endemic to Asia with most species considered threatened. Conservation plans to save declining wild populations are hindered by unstable taxonomy, and detailed systematic review could form a solid platform for future management and conservation. D-loop and cytochrome c oxidase I (COI) mtDNA sequences were examined in nine mahseer species of Tor, Neolissochilus, and Naziritor. Pseudogenes amplified in a portion of the species limited the utility of the D-loop region. ABGD analysis, NJ, ML, and MP methods and genetic distance (TrN?+?I?+?G) using COI data revealed concordant species delimiting patterns. The three genera were monophyletic, separated as distinct clades (TrN?+?I?+?G 0.064 to 0.106), and Naziritor was flagged as a separate genus, distinct from Puntius (TrN?+?I?+?G 0.196). Out of seven nominal species known for Tor cogeners from India, only five were recovered with mtDNA data (TrN?+?I?+?G 0.000 to 0.037) and two species could not be distinguished with the molecular data set employed. Tor mosal, synonymized as Tor putitora, was rediscovered as a distinct species (TrN?+?I?+?G 0.031) based on its type locality. Tor mussulah was confirmed as a separate species (TrN?+?I?+?G 0.019 to 0.026). Two valid species, Tor macrolepis and T. mosal mahanadicus, were not distinct from T. putitora (TrN?+?I?+?G 0.00). The high divergence with mtDNA data failed to validate T. mosal mahanadicus as a subspecies of T. mosal (TrN?+?I?+?G 0.031). Morphological outliers discovered within the distribution range of Tor tor (TrN?+?I?+?G 0.022 to 0.025) shared the same lineage with T. putitora (TrN?+?I?+?G 0.002 to 0.005), indicating a new extended distribution of the Himalayan mahseer T. putitora in the rivers of the Indian central plateau. The findings indicate the need for integrating molecular and morphological tools for taxonomic revision of the Tor and Naziritor genera, so that taxa are precisely defined for accurate in situ and ex situ conservation decisions.     

Horse heart ferricytochromec: Conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochromec in thepH range 6–0.8 and at the lowest possible ionic strengths, in water, and at 30°C are reported. The neutralpH form, state III, changes to the acidicpH form, state I, through a three-step process: state III ? state IIIa ? state II ? state I, with pKa's of 3.6±0.3, 2.7±0.2, and 1.2±0.2, depending on the monitoring probe, respectively. State IIIa ferricytochromec is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochromec is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 Å.