Cyclopropane-based conformational restriction of GABA by a stereochemical diversity-oriented strategy: Identification of an efficient lead for potent inhibitors of GABA transports

A series of cyclopropane-based conformationally restricted γ-aminobutyric acid (GABA) analogs with stereochemical diversity, that is, the trans- and cis-2,3-methano analogs Ia and Ib and their enantiomers ent-Ia and ent-Ib, and also the trans- and cis-3,4-methano analogs IIa and IIb and their enantiomers ent-IIa and ent-Iib, were synthesized from the chiral cyclopropane units Type-a and Type-b that we developed. These analogs were systematically evaluated with four GABA transporter (GAT) subtypes. The trans-3,4-methano analog IIa had inhibitory effects on GAT3 (IC₅₀ = 23.9 μM) and betaine-GABA transporter1 (5.48 μM), indicating its potential as an effective lead compound for the development of potent GAT inhibitors due to its hydrophilic and low molecular weight properties and excellent ligand efficiency.     

Linkage of adult α- and β-globin genes in X. laevis and gene duplication by tetraploidization

We have used cloned adult X. laevis α- and β-globin cDNAs to analyze globin genes in X. laevis DNA. We detected α¹- and β¹-globin genes which contain intervening sequences and code for the major adult globins, plus additional diverged α²- and β²-globin genes of unknown coding potential. Unlike the case in mammals, the X. laevis α¹- and β¹-globin genes are closely linked and occur in the sequence 5′-α¹-9 kb-β¹-3′. The α²- and β²-globin genes are also linked, and analysis of globin genes in X. tropicalis suggests that this duplication of an α-β-globin gene pair in X. laevis is the result of chromosome duplication by tetraploidization. The close linkage of α- and β-globin genes in Xenopus provides evidence that vertebrate α- and β-globin genes evolved by tandem duplication of a single primordial globin gene.     

Isolation and characterization of substituted 4-hydroxy-cyclohex-2-enones as metabolites of 3,4-dimethoxybenzyl alcohol and its methyl ether in ligninolytic cultures of Phanerochaete chrysosporium

The metabolism of quinones formed in the enzymatic oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) (Ia) and its methyl ether Ib in ligninolytic cultures of Phanerochaete chrysosporium was studied. A metabolite of 2-hydroxymethyl-5-methoxy-2,5-cyclohexadiene-1,4-dione (IIa, formed from Ia by oxidation) was isolated and identified as cis-4-hydroxy-6-hydroxymethyl-3-methoxy-cyclohex-2-en-one (IVa), formally the reduced hydroquinone IIIa. The formation of IVa was also observed when both veratryl alcohol Ia or 2,5-dihydroxy-4-methoxybenzyl alcohol (IIIa), the hydroquinone of IIa, were used as substrates. Analogously, cis-4-hydroxy-3-methoxy-6-methoxymethyl-cyclohex-2-en-one (IVc) was isolated and identified as a metabolite from either 3,4-dimethoxybenzyl methyl ether (Ib) or from its oxidation product 5-methoxy-2-methoxymethyl-2,5-cyclohexadiene-1,4-dione (IIb) as well as from the corresponding hydroquinone 2,5-dihydroxy-4-methoxybenzyl methyl ether (IIIc). The physiological role of these unprecedented conversions is discussed. Correspondence to: H. E. Schoemaker     

Chromosomal arrangement of the chicken β-type globin genes

We have isolated the chicken β-type globin genes from a library of chicken DNA-λ Charon 4A recombinant bacteriophage. There are four β-type genes within this segment of the genome; we believe this represents all of the β-type genes of the chicken. The recombinant λCβG1 contains the embryonic ?- and adult β-globin genes. The hatching β^H and embryonic p-globin genes are found in the recombinant λCβG2. Although λCβG1 and λCβG2 do not physically overlap, we present evidence that all four genes are closely linked and transcribed from the same DNA strand. These experiments demonstrate that the chromosomal regions represented by λCβG1 and λCβG2 lie approximately 1.6 kb apart in the chicken genome. A third recombinant λCβG3 extends the genomic locus studied in the vicinity of the β-type globin genes to approximately 39 kb. The physical order of the chicken β-type globin genes within this segment of the chromosome is 5′ … ?-β^H-β-? … 3′. This arrangement is unique among the vertebrate β-type globin gene clusters thus far examined, in that embryonic genes are located at the 5′ and 3′ ends of the cluster while the hatching and adult genes occupy central positions.     

Scrutiny of chain-length and N-terminal effects in α-helix folding: a molecular dynamics study on polyalanine peptides

Protein folding remains an unsolved problem as main-chain, side-chain, and solvent interactions remain entangled and have been hard to resolve. Polyalanines are promising models to analyze protein folding initiation and propagation structurally as well as energetically. In the present work, the effect of chain-length and N-terminal residue stereochemistry in polyalanine peptides are investigated for their role in the nucleation of α-helical conformation. The end-protected polyalanine peptides, tetra-alanine, Ac-^LAla₄-NHMe (Ia) and Ac-^DAla-^LAla₃-NHMe (Ib), hexa-alanine, Ac-^LAla₆-NHMe (IIa) and Ac-^DAla-^LAla₅-NHMe (IIb), and octa-alanine, Ac-^LAla₈-NHMe (IIIa) and Ac-^DAla-^LAla₇-NHMe (IIIb), are assessed as chain-length and stereochemical-structure perturbed models. The appreciable variations in the sampling of α-helical conformation, including a sampling of α-helix folds, due to the cooperative effect of chain-length and N-terminal residue stereochemistry have been noted. The electrostatics of α-helical conformation rather than the conformational entropy of the main-chain appear to be decisive in the initiation of α-helix folding. The results of the present work will enhance our understanding on the nucleation of α-helical conformation in short peptides and aid in the design of novel peptides with α-helical structure that can modulate disease-related protein–protein interactions.     

Genomic organization and promoter activity of the maize starch branching enzyme I gene

Starch branching enzymes (SBE) which catalyse the formation of α-1,6-glucan linkages are of crucial importance for the quantity and quality of starch synthesized in plants. In maize (Zea mays L.), three SBE isoforms (SBEI, IIa and IIb) have been identified and shown to exhibit differential expression patterns. As a first step toward understanding the regulatory mechanisms controlling their expression, we isolated and sequenced a maize genomic DNA (−2190 to +5929) which contains the entire coding region of SBEI (Sbe1) as well as 5′-and 3′-flanking sequences. Using this clone, we established a complete genomic organization of the maize Sbe1 gene. The transcribed region consists of 14 exons and 13 introns, distributed over 5.7 kb. A consensus TATA-box and a G-box containing a perfect palindromic sequence, CCACGTGG, were found in the 5′-flanking region. Genomic Southern blot analysis indicated that two Sbe1 genes with divergent 5′-flanking sequences exist in the maize genome, suggesting the possibility that they are differentially regulated. A chimeric construct containing the 5′-flanking region of Sbe1 (−2190 to +27) fused to the β-glucuronidase gene (pKG101) showed promoter activity after it was introduced into maize endosperm suspension cells by particle bombardment.     

Reciprocal effects of change of subunit structure on ligand equilibria of haemoglobin valency hybrids. Attempted correlation with electron paramagnetic resonance spectra

Haemoglobin valency hybrids have been further investigated with a view to evaluating evidence for α-β interactions. Comparison of equilibrium oxygen binding of the compounds α^III_H2Oβ^II, α^III_Fβ^II, α^III_N3β^II and α^III_CNβ^II show that the derivatives possessing the α chain in the (low spin) oxy conformation possess higher oxygen affinity than those possessing the same chain in (high spin) deoxy conformation. On the other hand, equilibrium titration of the aquo derivative by fluoride and azide showed a higher azide affinity and a slightly lower fluoride affinity for α^III_H2Oβ^II_CO compared to the deoxy form α^III_H2Oβ^II. Essentially the same pattern of relations were obtained for the different forms of α^IIβ^II also.Electron paramagnetic resonance spectra of the hybrids at pH 6 showed no change of the g value of 5.85 absorption of the ferri chain on change of spin state of the partner ferro chain. However, this was no longer the case at pH 9; the spectra of the alkaline form gave evidence of α-β interaction for the α^IIIβ^II hybrid only, but not for α^IIβ^III. The electron paramagnetic resonance results suggest that α-β interaction in the hybrids may operate in the β → α direction only.The equilibrium and spectroscopic data are discussed in the general context of haem-haem interaction.     

The crystal and molecular structures of dioxo mo(VI) complexes of tripodal,tetradentate N,S-donor ligands

The crystal and molecular structures of the complexes MoO₂((SCH₂CH₂)₂NCH₂CH₂SCH₃), I and MoO₂((SCH₂CH₂)₂NCH₂CH₂N(CH₃)₂), II, have been determined from X-ray intensity data collected by counter methods. Compound I crystallizes in two forms, Ia and Ib. In form Ia the space group is P2₁/n with cell parameters a = 7.235(2), b = 7.717(2), c = 24.527(6) Å, β = 119.86(2)°, V = 1188(1) ų, Z = 4. In form Ib the space group is P2₁/c with cell parameters a = 14.945(5), b = 11.925(5), c = 14.878(4) Å, β = 114.51(2)°, V = 2413(3) ų, Z = 8. The molecules of I in Ia and Ib are very similar having an octahedral structure with cis oxo groups, trans thiolates (cis to both oxo groups) and N and thioether sulfur atoms trans to oxo groups. Average ditances are MoO = 1.70, MoS (thiolate) = 2.40, MoN = 2.40 and MoS (thioether) = 2.79 Å. Molecule II crystallizes in space group P2₁2₁2₁ with a = 7.188(1), b = 22.708(8), c = 7.746(2) Å, V = 1246(1) ų and Z = 4. The coordination about Mo is octahedral with cis oxo groups, trans thiolates and N atoms trans to oxo. Distances in the first coordination sphere are MoO = 1.705(2), 1.699(2), MoS = 2.420(1), 2.409(1) and MoN = 2.372(2), 2.510(2) Å. The conformational features of the complexes are discussed. Complex I displays MoO and MoS distances which are very similar to those found by EXAFS in sulfite oxidase. This similarity is discussed.     

Synthesis of 1,5-diarylhaloimidazole analogs and their inhibitory activities against PGE2 production from LPS-treated RAW 264.7 cells

A number of 1,5-diarylimidazole analogs were synthesized and evaluated their inhibitory activities of cyclooxygenase-2 catalyzed prostaglandin E₂ production. Reactions of 1,5-diarylimidazoles with halogenating reagents (NCS, NBS, NIS) afforded halogenated analogs. Among the analogs tested, compounds Ib, IIa, IIb and IIe exhibited significantly improved inhibitory activities against COX-2-mediated PGE₂ production from LPS-induced RAW 264.7 cells compared to those of the parent 1,5-diarylimidazoles. Especially, the analogs Ib (IC₅₀ = 0.55 μM) and IIa (IC₅₀ = 0.58 μM) showed best results. Halogenation on the 1,5-diarylimidazole ring enhanced inhibitory activities against COX-2 catalyzed PGE₂ production, however, inhibitory activities were significantly varied by position(s) and species of the substituted halogen(s).     

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.     

Evaluation of andrographolide-based analogs derived from Andrographis paniculata against Mythimna separata Walker and Tetranychus cinnabarinus Boisduval

To discover new natural-product-based pesticides, we structurally modified andrographolide, a labdane diterpenoid isolated from Andrographis paniculata, and stereoselectively prepared a series of 12α-(substituted)benzylamino-14-deoxyandrographolide derivatives (I–V). Three-dimensional structures of compounds 3c, 3d, IIIa and IIIb were further determined by single-crystal X-ray diffraction. Compounds IIa (R¹ = n-C₃H₇, R² = PhCH₂) exhibited more promising insecticidal activity against Mythimna separata than toosendanin. Compounds 3a (R¹ = H), Ib (R¹ = H, R² = 4-ClPhCH₂), and IVa (R¹ = 4-ClPh, R² = PhCH₂) showed potent acaricidal activity against Tetranychus cinnabarinus.     

Determination of the primary sequence of the duck αD globin mRNA and comparison of all adult duck and chick globin mRNA sequences

The nucleotide sequence of the duck α^D globin mRNA was determined. Its main feature is an exceptionally short 3′ non-coding segment of only 46 nucleotides, placed after the coding sequence of 141 codons. The last of the 6 adult globin mRNA of duck and chicken being thus sequenced, a comparison of all their features has become possible. Comparing the duck α^D mRNA to the related sequence in the chicken, we found greater homology than comparing it to the linked α^A globin sequence in the same species. Extensive homology can be found for a same globin chain α^A, α^D or β in between different avian species including also the goose and the ostrich; the avian α globin chains show a lower degree of sequence conservation in between species than the β chains. In contrast, within one species the three globin sequences have further diverged. The divergence between the α^A and α^D globin within a same species point to individual functional specificity and hence independent evolution and suggest that a mechanism of ‘gene conversion’ did not operate in between the avian α globin genes. Two segments of the amino acid sequence which we named ‘A^α’ and ‘B^α’ remain homologous in all avian α globins; two other regions ‘A^β’ and ‘B^β’ are identical in between the β globins. Segment A is placed at the 5′ end of exon II, and segment B at the 3′ end of the same exon; some amino acids in those segments are involved in the Heme binding site. Being almost identical in all know mammalian and avian globins of the α respectively the β type, regions A and B seem to represent the best conserved sequences in adult globin mRNA maintained during the divergence of species.     

Activation of the progesterone-signaling pathway by methyl-beta-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa Galphas

Treatment of Xenopus laevis oocytes with cholesterol-depleting methyl-β-cyclodextrin (MeβCD) stimulates phosphorylation of mitogen-activated protein kinase (MAPK) and oocyte maturation, as reported previously [Sadler, S.E., Jacobs, N.D., 2004. Stimulation of Xenopus laevis oocyte maturation by methyl-β-cyclodextrin. Biol. Reprod. 70, 1685-1692.]. Here we report that treatment of oocytes with MeβCD increased levels of immunodetectable 39-kDa mos protein. The protein synthesis inhibitor, cycloheximide, blocked the appearance of Mos, blocked MeβCD-stimulated phosphorylation of MAPK, and inhibited MeβCD-induced oocyte maturation. These observations suggest that MeβCD activates the progesterone-signaling pathway. Chemical inhibition of steroid synthesis and mechanical removal of follicle cells were used to verify that MeβCD acts at the level of the oocyte and does not require production of steroid by surrounding follicle cells. Cortical Gα_s is contained in low-density membrane; and treatment of oocytes with progesterone or MeβCD reduced immunodetectable levels of Gα_s protein in cortices and increased internal levels of 45-kDa Gα_s in cortical-free extracts. Dose-dependent increases in internal Gα_s after treatment of oocytes with progesterone correlated with the steroid-induced maturation response, and the increase in internal Gα_s after hormone treatment was comparable to the decrease in cortical Gα_s. These results are consistent with a model in which release of Gα_s from the plasma membrane is involved in activation of the progesterone-signaling pathway that leads to amphibian oocyte maturation.     

The influence of reference radiation photon energy on high-LET RBE: comparison of human peripheral lymphocytes and human–hamster hybrid A<Subscript>L</Subscript> cells

The relative biological effectiveness (RBE) based on the induction of dicentrics in any cell type is principally an important information for the increasing application of high-LET radiation in cancer therapy. Since the standard system of human lymphocytes for measuring dicentrics are not compatible with our microbeam irradiation setup where attaching cells are essential, we used human–hamster hybrid A_L cells which do attach on foils and fulfil the special experimental requirement for microbeam irradiations. In this work, the dose–response of A_L cells to photons of different energy, 70 and 200 kV X-rays and ⁶⁰Co γ-rays, is characterized and compared to human lymphocytes. The total number of induced dicentrics in A_L cells is approximately one order of magnitude smaller. Despite the smaller α and β parameters of the measured linear–quadratic dose–response relationship, the α/β-ratio versus photon energy dependence is identical within the accuracy of measurement for A_L cells and human lymphocytes. Thus, the influence of the reference radiation used for RBE determination is the same. For therapy relevant doses of 2 Gy (⁶⁰Co equivalent), the difference in RBE is around 20% only. These findings indicate that the biological effectiveness in A_L cells can give important information for human cells, especially for studies where attaching cells are essential.     

Photoconversion kinetics of chloroplast Photosystems I and II. Effect of Mg2+

The effect of divalent cations on the primary photoconversion kinetics of chloroplast Photosystems (PS) I and II was investigated by absorbance difference spectrophotometry in the ultraviolet (ΔA₃₂₀) and red (ΔA₇₀₀) regions and by fluorescence at room temperature. Three main chlorophyll (Chl) a fluorescence emission components were identified. Addition of 5 mM MgCl₂ to unstacked chloroplasts caused a 5–7-fold increase in F_vα, the variable fluorescence yield controlled by the α-centers. The fluorescence yield F_vβ controlled by the β-centers and the nonvariable fluorescence yield F₀ were only slightly changed by the treatment. The absolute number of α- and β-centers remained unchanged and independent of divalent cations. The rate constants K_α, K_β and K_P-700 determined from the photoconversion kinetics of Q_α, Q_β and P-700 were also unchanged by divalent cations, suggesting a constancy of the respective absorption cross-sections. Evidence is presented that the Mg²⁺ effect on Chl a fluorescence is not due simply to unstacking. Conclusion: (1) In the absence of divalent cations from the chloroplast suspending medium, the variable fluorescence yield is not complementary to the rate of PS II photochemistry. (2) A spillover of excitation from PS II to PS I in the absence of Mg²⁺ cannot account for the 7-fold lowering of the variable fluorescence yield F_vα at room temperature. The results are discussed in view of a model of excitation transfer and fluorescence emission in the pigment bed of PS II_α and PS II_β.     

The exon/intron organization of the globin gene of Scapharca inaequivalvis homodimeric hemoglobin: unusual intron homology with other bivalve mollusc globin genes

In this study, we have investigated the positions of introns in the globin gene of Scapharca inaequivalvis homodimeric hemoglobin. We found the three exon/two intron organization typical of vertebrate globin genes, with the two introns in highly conserved positions, as it occurs in the A and B globin genes of the tetrameric hemoglobin from the same organism, confirming the absence of the so-called `central intron' found in the globin genes of plants and of some invertebrates. We identified two homodimeric globin genes (3207 and 2723 bp) that differ only with respect to the size of the first intron. Sequence analysis of the two first introns (1668 and 1364 bp) has revealed that they are highly homologous, except for a 569- and 296-bp insertion in each intron I. Interestingly, the two first introns contain regions with an unusually high identity (∼80%) with regions of the first intron of the congeneric clam Anadara trapezia and the related clam Barbatia reveana globin genes, suggesting that these uncoding regions may have played a regulatory role that has subsequently been lost during the course of the evolution.     

Multivalent human blood group ABH and Lewis glycotopes are key recognition factors for a lFuc>Man binding lectin from phytopathogenic Ralstonia solanacearum

Ralstonia solanacearum lectin (RSL), that might be involved in phytopathogenicity, has been defined as lFuc?Man specific. However, the effects of polyvalency of glycotopes and mammalian structural units on binding have not been established. In this study, recognition factors of RSL were comprehensively examined with natural multivalent glycotopes and monomeric ligands using enzyme linked lectin-sorbent and inhibition assays. Among the glycans tested, RSL reacted strongly with multivalent blood group A_h (GalNAcα1–3[Fucα1–2]Gal) and H (Fucα1–2Gal) active glycotopes, followed by B_h (Galα1–3[Fucα1–2]Gal), Le^a (Galβ1–3[Fucα1–4]GlcNAc) and Le^b (Fucα1–2Galβ1–3[Fucα1–4]GlcNAc) active glycotopes. But weak or negligible binding was observed for blood group precursors having Galβ1–3/4GlcNAcβ1- (I_β/II_β) residues or Galβ1–3GalNAcα1- (T_α), GalNAcα1-Ser/Thr (Tn) bearing glycoproteins. These results indicate that the density and degree of exposure of multivalent ligands of α1–2 linked lFuc to Gal at the non-reducing end is the most critical factor for binding. An inhibition study with monomeric ligands revealed that the combining site of RSL should be of a groove type to fit trisaccharide binding with highest complementarity to blood group H trisaccharide (H_L; Fucα1–2Galβ1–4Glc). The outstandingly broad RSL saccharide-binding profile might be related to the unusually wide spectrum of plants that suffer from R. solanacearum pathogenicity and provide ideas for protective antiadhesion strategies.