Isolation and characterization of an ω3-desaturation-defective mutant of an arachidonic acid-producing fungus,Mortierella alpina 1S-4

A mutant considered to be defective in the conversion of n-6 to n-3 fatty acids (3-desaturation) was derived from a 5-desaturation-defective mutant (Mut44) of Mortierella alpina 1S-4, after treating its spores with N-methyl-N-nitro-N-nitrosoguanidine. This mutant cannot produce 8(Z),11(Z),14(Z),17(Z)-eicosatetraenoic acid or any other n-3 fatty acids, of which about 10% was found in its parental strain upon cultivation at 12°C. The mutant's growth rate was comparable to that of the parental strain when grown at 28°C, but it became much slower when the mutant grew at 12°C, at which the lag phase for Mut44 was about 2 d but 5 d for the mutant.Abbreviations 18:33 9(Z),12(Z),15(Z)-octadecatrienoic acid - 18:43 6(Z),9(Z),12(Z),15(Z)-octadecatetraenoic acid - 20:43 8(Z),11(Z),14(Z),17(Z)-eicosatetraenoic acid - AA arachidonic acid - DHGA dihomo--linolenic acid - EPA 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid - GLC gas-liquid chromatography - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PC phosphatidylcholine     

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

The PucC protein of Rhodobacter capsulatus mitigates an inhibitory effect of light-harvesting 2 α and β proteins on light-harvesting complex 1

Rhodobacter capsulatus contains lhaA and pucC genes that have been implicated in light-harvesting complex 1 and 2 (LH1 and LH2) assembly. The proteins encoded by these genes, and homologues in other photosynthetic organisms, have been classified as the bacteriochlorophyll delivery (BCD) family of the major facilitator superfamily. A new BCD family phylogenetic tree reveals that several PucC, LhaA and Orf428-related sequences each form separate clusters, while plant and cyanobacterial homologues cluster more distantly. The PucC protein is encoded in the pucBACDE superoperon which also codes for LH2 α (PucA) and β (PucB) proteins. PucC was previously shown to be necessary for formation of LH2. This article gives evidence indicating that PucC has a shepherding activity that keeps the homologous α and β proteins of LH1 and LH2 apart, allowing LH1 to assemble properly. This shepherding function was indicated by a 62% reduction in LH1 levels in ΔLHII strains carrying plasmids encoding pucBA along with a C-terminally truncated pucC gene. More severe reductions in LH1 were seen when the truncated pucC gene was co-expressed in the presence of C-terminal PucC::PhoA fusion proteins. It appears that interaction between truncated PucC::PhoA fusion proteins and the truncated PucC protein disrupts LH1 assembly, pointing towards a PucC dimeric or multimeric functional unit.     

Isolation,structure and biological activities of platencin A2–A4 from Streptomyces platensis

Natural products serve as a great reservoir for chemical diversity and are the greatest source for antibacterial agents. Recent discoveries of platensimycin and platencin as inhibitors of bacterial fatty acid biosynthesis enzymes supplied new chemical scaffolds for potential antibacterial agents to overcome resistant pathogens. Discovery of natural congeners augment chemical modification in understanding of structure–activity relationship (SAR). Chemical and biological screening of the extracts led to isolation of three hydroxylated analogs of platencin. The C-12, C-14 and C-15 hydroxylated analogs showed attenuated activities which provided significant understanding of functional tolerance in the diterpenoid portion of the molecule. A truncated and oxidized C-13 natural congener was isolated which suggested direct intermediacy of ent-copalyl diphosphate for the biosynthesis of platensimycins and platencins.     

The structure of 5a,6–anhydrotetracycline and its Mg2+ complexes in aqueous solution

Semiempirical molecular orbital theory has been used for a systematic scan of the binding positions for a Mg²⁺ ion with 5a,6–anhydrotetracycline taking both conformational flexibility and possible different tautomeric forms into account. The magnesium ion has been calculated alone and with four or five complexed water molecules in order to simulate the experimental situation more closely. The results are analyzed by comparing the behavior of the title compound with that of tetracycline itself and possible causes for the stronger induction of the Tetracycline Receptor (TetR) by 5a,6–anhydrotetracycline than by tetracycline are considered. Energetically favored 3D -structure of the zwitteranionic 5a,6-anhydrotetracycline magnesium complex in solution Electronic Supplementary Material Supplementary material is available for this article at     

Isolation of sea urchin embryo histone H2Aα1 and immunological identification of other stage-specific H2A proteins

H2A_α1, the principal H2A histone synthesized prior to the blastula stage of the sea urchin, was isolated free of other putative H2A subtypes and other histones. Its amino acid composition provides confirmation that H2A_α1 is the H2A protein encoded in the histone gene cluster carried by pCO2. An antibody prepared against this protein cross-reacts strongly with CS2A (a putative H2A synthesized only during the cleavage stage) as well as with H2A_β, H2A_γ, and H2A_δ (putative H2As synthesized principally after the blastula stage) but not with non-H2A core histones or other nuclear proteins. The data support the view that CS2A, H2A_α1, H2A_β, H2A_γ, and H2A_δ are all H2A proteins.     

2��-Deoxyriboguanylurea, the primary breakdown product of 5-aza-2��-deoxyribocytidine, is a mutagen, an epimutagen, an inhibitor of DNA methyltransferases and an inducer of 5-azacytidine-type fragile sites

5-Aza-2′-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen, a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly breaks down in aqueous solution to the stable compound 2′-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells. Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by its stable primary breakdown product.     

Isolation and structural analyses of positional isomers of 61,6m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-α-d-mannopyranosyl-cyclomaltooctaose (n=2, 3, 4, and 6)

Eight positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (γCD) (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD (n=2, 3, 4, and 6) in a mixture of products from γCD and d-mannose by condensation reaction of α-mannosidase from jack bean were isolated by HPLC. The structures of four isomers of 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD were elucidated by NMR spectroscopy. On the other hand, four positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-γCD were determined by LC–MS analysis of degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying α-amylase (BSA), and combination of BSA and glucoamylase. Similarly cyclomaltodextrin glucanotransferase also digested these isomers.     

What curves alpha-solenoids? Evidence for an alpha-helical toroid structure of Rpn1 and Rpn2 proteins of the 26 S proteasome

The alpha-helical solenoid proteins adopt a variety of elongated curved structures. They have been examined to identify the interactions that determine their curvature. A sequence pattern characteristic for strongly curved alpha-helical solenoids has been constructed and was found to match protein sequences containing the proteasome/cyclosome repeats. Based on this, a structural model of the repeat-containing domains of the Rpn1/S2 and Rpn2/S1 proteins, which represent the largest subunits of the 26 S proteasome, has been proposed. The model has a novel architecture resembling an alpha-helical toroid. Molecular modeling shows that these toroids have a central pore that would allow passage of an unfolded protein substrate through it. This implies that the Rpn1 and Rpn2 toroids are aligned along the common axial pores of the ATPase hexamer and form an "antechamber" of the 26 S proteasome. The proposed quaternary structure agrees with the available experimental data. It is suggested that the function of this antechamber is assistance to the ATPases in the unfolding of protein substrates prior to proteolysis. An evolutionary link between the PC repeat-containing proteins and tetratricopeptide repeat proteins is proposed.     

The Chriz–Z4 complex recruits JIL-1 to polytene chromosomes,a requirement for interband-specific phosphorylation of H3S10

The conserved band-interband pattern is thought to reflect the looped-domain organization of insect polytene chromosomes. Previously, we have shown that the chromodomain protein Chriz and the zinc-finger protein Z4 are essentially required for the maintenance of polytene chromosome structure. Here we show that both proteins form a complex that recruits the JIL-1 kinase to polytene chromosomes, enabling local H3S10 phosphorylation of interband nucleosomal histones. Interband targeting domains were identified at the N-terminal regions of Chriz and Z4, and our data suggest partial cooperation of the complex with the BEAF boundary element protein in polytene and diploid cells. Reducing the core component Chriz by RNAi results in destabilization of the complex and a strong reduction of interband-specific histone H3S10 phosphorylation.     

Soil–atmosphere exchange of N2O, CO2 and CH4 along a slope of an evergreen broad-leaved forest in southern China

At most sites the magnitude of soil-atmosphere exchange of nitrous dioxide (N₂O), carbon dioxide (CO₂) and methane (CH₄) was estimated based on a few chambers located in a limited area. Topography has been demonstrated to influence the production and consumption of these gases in temperate ecosystems, but this aspect has often been ignored in tropical areas. In this study, we investigated spatial variability of the net fluxes of these gases along a 100 m long slope of a evergreen broadleaved forest in southern China over a whole year. We expected that the lower part of slope would release more N₂O and CO₂, but take up less atmospheric CH₄ than the upper part due to different availability of water and nutrients. Our results showed that the soil moisture (Water Filled Pore Space, WFPS) decreased along the slope from bottom to top as we expected, but among the three gases only N₂O emissions followed this pattern. Annual means of WFPS ranged from 27.7% to 52.7% within the slope, and annual emissions of N₂O ranged from 2.0 to 4.4 kg N ha^?1 year^?1, respectively. These two variables were highly and positively correlated across the slope. Neither potential rates of net N mineralization and nitrification, nor N₂O emissions in the laboratory incubated soils varied with slope positions. Soil CO₂ release and CH₄ uptake appeared to be independent on slope position in this study. Our results suggested that soil water content and associated N₂O emissions are likely to be influenced by topography even in a short slope, which may need to be taken into account in field measurements and modelling.     

Isolation and Characterization of an Unusual Nucleoside, 1-α-D-Ribofuranosyl-4-pyridone-3-carboxamide,from the Urines of Normal Human Individuals and Leukemic Patients

Abstract

A novel modified nucleoside, 1-α-D-Ribofuranosyl-4-pyridone-3-carboxamide, has been isolated from the urines of one normal human individual and two CML patients. The structure was assigned on the basis of UV, NMR and mass spectral data and confirmed by comparison with an authentic sample synthesized in our laboratory. This constitutes the first example of the occurrence of a nucleoside with an α-riboside linkage in human urine.     

Preferential activation by galanin 1–15 fragment of the GalR1 protomer of a GalR1–GalR2 heteroreceptor complex

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1–15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1–GalR2 heteromers which may play a significant role in mediating galanin fragment (1–15) signaling. In HEK293T cells evidence for the existence of GalR1–GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET² assays. PLA positive blobs representing GalR1–GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1–15) was more potent than galanin (1–29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1–GalR2 transfected cells. The inhibition of CREB by 50 nM of galanin (1–15) and of galanin (1–29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (1–15) vs galanin (1–29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1–29). In contrast, in NFAT reporter gene assays galanin (1–29) shows a higher efficacy than galanin (1–15) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1–GalR2 heteroreceptor complexes induced by galanin (1–15) may contribute to depression-like actions since GalR1 agonists produce such effects.     

TPA-induced cell transformation provokes a complex formation between Pin1 and 90?kDa ribosomal protein S6 kinase 2

The antioxidant properties of organoselenium compounds have been extensively investigated because oxidative stress is a hallmark of a variety of chronic human diseases. Here, we reported the influence of substituent groups in the antioxidant activity of β-selenoamines. We have investigated whether they exhibited glutathione peroxidase-like (GPx-like) activity and whether they could be substrate of thioredoxin reductase (TrxR). In the DPPH assay, the β-selenium amines did not exhibit antioxidant activity. However, the β-selenium amines with p-methoxy and tosyl groups prevented the lipid peroxidation. The β-selenium amine compound with p-methoxy substituent group exhibited thiol-peroxidase-like activity (GPx-like activity) and was reduced by the hepatic TrxR. These results contribute to understand the influence of structural alteration of non-conventional selenium compounds as synthetic mimetic of antioxidant enzymes of mammalian organisms.     

The WASH complex,an endosomal Arp2/3 activator,interacts with the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα

Vesicle biogenesis machinery components such as coat proteins can interact with the actin cytoskeleton for cargo sorting into multiple pathways. It is unknown, however, whether these interactions are a general requirement for the diverse endosome traffic routes. In this study, we identify actin cytoskeleton regulators as previously unrecognized interactors of complexes associated with the Hermansky–Pudlak syndrome. Two complexes mutated in the Hermansky–Pudlak syndrome, adaptor protein complex-3 and biogenesis of lysosome-related organelles complex-1 (BLOC-1), interact with and are regulated by the lipid kinase phosphatidylinositol-4-kinase type IIα (PI4KIIα). We therefore hypothesized that PI4KIIα interacts with novel regulators of these complexes. To test this hypothesis, we immunoaffinity purified PI4KIIα from isotope-labeled cell lysates to quantitatively identify interactors. Strikingly, PI4KIIα isolation preferentially coenriched proteins that regulate the actin cytoskeleton, including guanine exchange factors for Rho family GTPases such as RhoGEF1 and several subunits of the WASH complex. We biochemically confirmed several of these PI4KIIα interactions. Of importance, BLOC-1 complex, WASH complex, RhoGEF1, or PI4KIIα depletions altered the content and/or subcellular distribution of the BLOC-1–sensitive cargoes PI4KIIα, ATP7A, and VAMP7. We conclude that the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo PI4KIIα interact with regulators of the actin cytoskeleton.     

Structure–function analysis and genetic interactions of the Yhc1, SmD3, SmB,and Snp1 subunits of yeast U1 snRNP and genetic interactions of SmD3 with U2 snRNP subunit Lea1

Yhc1 and U1-C are essential subunits of the yeast and human U1 snRNP, respectively, that stabilize the duplex formed by U1 snRNA at the pre-mRNA 5′ splice site (5′SS). Mutational analysis of Yhc1, guided by the human U1 snRNP crystal structure, highlighted the importance of Val20 and Ser19 at the RNA interface. Though benign on its own, V20A was lethal in the absence of branchpoint-binding complex subunit Mud2 and caused a severe growth defect in the absence of U1 subunit Nam8. S19A caused a severe defect with mud2▵. Essential DEAD-box ATPase Prp28 was bypassed by mutations of Yhc1 Val20 and Ser19, consistent with destabilization of U1•5′SS interaction. We extended the genetic analysis to SmD3, which interacts with U1-C/Yhc1 in U1 snRNP, and to SmB, its neighbor in the Sm ring. Whereas mutations of the interface of SmD3, SmB, and U1-C/Yhc1 with U1-70K/Snp1, or deletion of the interacting Snp1 N-terminal peptide, had no growth effect, they elicited synthetic defects in the absence of U1 subunit Mud1. Mutagenesis of the RNA-binding triad of SmD3 (Ser-Asn-Arg) and SmB (His-Asn-Arg) provided insights to built-in redundancies of the Sm ring, whereby no individual side-chain was essential, but simultaneous mutations of Asn or Arg residues in SmD3 and SmB were lethal. Asn-to-Ala mutations SmB and SmD3 caused synthetic defects in the absence of Mud1 or Mud2. All three RNA site mutations of SmD3 were lethal in cells lacking the U2 snRNP subunit Lea1. Benign C-terminal truncations of SmD3 were dead in the absence of Mud2 or Lea1 and barely viable in the absence of Nam8 or Mud1. In contrast, SMD3-E35A uniquely suppressed the temperature-sensitivity of lea1▵.     

Activity of steroid 4 and derivatives 4a–4f as inhibitors of the enzyme 5α-reductase 1

It is known that the growth of prostate metastatic bone tumor depends on androgens, and tumor formation can start from migratory malignant cells produced in that organ. These cells exhibit grater type 1 5α-reductase (5α-R1) activity than type 2 5α-reductase. Noteworthy, both isozymes convert testosterone (T) to the more active androgen dihydrotestosterone (DHT) in the target tissues.Thus, in order to potentially improve the prognosis of this disease, in this work, seven derivatives of 17-(1H-benzimidazol-1-yl)-16-formillandrosta-5,16-dien-3β-yl benzoate (4a–f) and 17-(1H-benzimidazol-1-yl)-3-hydroxy-16-formylandrost-5,16-diene (4) were synthesized, characterized and identified as inhibitors of type 1 5α-reductase (5αR1). These derivatives having the advantage of improved plasma half-life.The inhibitory activity of the compounds towards 5α-R1 isoenzyme was determined by conversion of T into DHT in the presence or absence of compounds 4, 4a–f. Further, in vivo experiments were also carried out, treating gonadectomized hamsters with T and/or 4, 4a–f and evaluating their effect on the diameter of hamster flank organs and on the weight of the prostatic and seminal vesicles. Results indicated that compounds 4, 4b, 4c, served as in vitro inhibitors of the enzyme 5α-R1 and pharmacological experiments showed that 4 and derivatives 4a–f decreased the diameter of the flank glands, the weight of the prostate and seminal vesicles of treated hamsters without any appreciable toxicity during observation. Noteworthy the fact that compound 4 is the product, in all cases, of the hydrolysis of the series of esters 4a–f, thus they can serve as precursors (prodrugs) of the active form 4.     

Insight into the modulation of Shaw2 Kv channels by general anesthetics: Structural and functional studies of S4–S5 linker and S6 C-terminal peptides in micelles by NMR

The modulation of the Drosophila Shaw2 Kv channel by 1-alkanols and inhaled anesthetics is correlated with the involvement of the S4–S5 linker and C-terminus of S6, and consistent with stabilization of the channel's closed state. Structural analysis of peptides from S4–S5 (L45) and S6 (S6c), by nuclear magnetic resonance and circular dichroism spectroscopy supports that an α-helical conformation was adopted by L45, while S6c was only in an unstable/dynamic partially folded α-helix in dodecylphosphocholine micelles. Solvent accessibility and paramagnetic probing of L45 revealed that L45 lies parallel to the surface of micelles with charged and polar residues pointing towards the solution while hydrophobic residues are buried inside the micelles. Chemical shift perturbation introduced by 1-butanol on residues Gln320, Thr321, Phe322 and Arg323 of L45, as well as Thr423 and Gln424 of S6c indicates possible anesthetic binding sites on these two important components in the channel activation apparatus. Diffusion measurements confirmed the association of L45, S6c and 1-butanol with micelles which suggests the capability of 1-butanol to influence a possible interaction of L45 and S6c in the micelle environment.