Inactivation of VCP/ter94 Suppresses Retinal Pathology Caused by Misfolded Rhodopsin in Drosophila

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (Rh^P23H). Unlike the wild-type Rh, mutant Rh^P23H exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded Rh^P23H and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that Rh^P23H is a substrate of the ER–associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1^P37H (the equivalent of mammalian Rh^P23H) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1^P37H toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1^P37H and further activated the Ire1/Xbp1 ER stress pathway in the Rh1^P37H retina. Despite this, Rh1^P37H flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1^P37H retina. Pharmacological treatment of Rh1^P37H flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration.     

Phylogeography of Asian weaver ants, Oecophylla smaragdina

The phylogeography of Oecophylla smaragdina was studied using the mitochondrial cytochrome b gene (Cytb), cytochrome oxidase subunit I (COI), and nuclear long-wavelength opsin gene (LW Rh). Weaver ants were collected from 35 localities and from one to nine colonies per locality. Neighbor-joining trees inferred from 647 bp of Cytb and 1,026 bp of COI using Oecophylla longinoda as an outgroup indicated that the haplotypes of O. smaragdina were clearly separated into seven groups: group 1 of India excluding West Bengal; group 2 of Bengal, Indochinese Peninsula, Malay Peninsula and Greater Sunda Islands, including Lombok and Sumbawa; group 3 of the Philippines; group 4 of Flores; group 5 of Sulawesi; group 6 of Halmahera; and group 7 of New Guinea and Australia. This grouping was also supported by a strict consensus tree derived from maximum parsimony and maximum likelihood trees. In addition, two haplotypes of LW Rh were found in O. smaragdina: one in group 2 and another in all the other groups. Comparison to haplotypes in other hymenopteran species suggests that group 2 is younger than other groups of O. smaragdina. The clustering of the seven groups was coincident with geological evidence of the distribution of continents, islands, and seas during glacial periods.     

甘肃南部7种高寒杜鹃生物量模拟

精确测定与模拟高山-亚高山灌丛生物量是了解陆地生态系统碳功能的重要基础工作。以甘肃南部高山-亚高山地区常见的7种高寒杜鹃（Rhododendron spp.）灌木为对象,通过标准植株收获法,建立易测因子与各器官生物量及总生物量的方程并检验拟合精度,筛选最优拟合方程。结果表明：（1）自变量和函数的类型对杜鹃生物量的模拟效果影响较大,700组方程中以D和D²H为自变量和以幂函数为模型拟合的R²相对集中、中位数都较高。（2）遴选出的35组单物种最优生物量模型的R²介于0.66-0.99之间、中位数为0.92,除山光杜鹃（Rh.oreodoxa）的茎、叶生物量和地上生物量模型为线性函数、麻花杜鹃（Rh.maculiferum）的所有模型为指数函数外,其余的生物量模型均为幂函数;D和D²H是单物种生物量模型的最佳预测变量,H仅是黄毛杜鹃（Rh.rufum）除根外、美容杜鹃（Rh.calophytum）叶生物量的最佳预测变量。（3）混合物种最优模型是以D²H为自变量的幂函数,除对叶生物量的模拟精度相对较低外,对其它生物量的模拟均较好。甘肃南部7种高寒杜鹃灌木生物量模型的建立为高寒地区灌丛生态系统碳汇功能的研究提供了支撑。     

Reactions of 2-thiopyridone and related N-, S- and C-methylated derivatives with [Rh2Cl2(CO)4]: crystal and molecular structure of fac-[Rh(MeC5H3NS)3] containing 6-methyl-2-thiopyridonato ligands

[Rh₂Cl₂(CO)₄] reacts with the ligands L (2-pyridone, 2-thiopyridone, and the isomers 6-methyl-2-thiopyridone, 2-methylmercaptopyridine, and N-methylthiopyridone) to give initially, when L/Rh = 1, the bridged-cleaved compounds cis- [RhCl(CO)₂L]. Further additions of 2-methyl- mercaptopyridine, N-methylthiopyridone, or 2-pyridone caused no further change, but 2- thiopyridone and 6-methyl-2-thiopyridone gave new cis-dicarbonyl species (L/Rh = 2) and eventually monocarbonyl species (L/Rh > 3). All these solutions are air-sensitive and air oxidation of a solution of [Rh₂Cl₂(CO)₄] with an excess of 6-methyl-2- thiopyridone gave fac-[Rh(MeC₅H₃NS)₃] the X-ray structure of which shows three equivalent chelating 6-methyl-2-thiopyridonato ligands.     

Analysis of Conserved Glutamate and Aspartate Residues in Drosophila Rhodopsin 1 and Their Influence on Spectral Tuning

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Studies of amphioxus G_o-opsin have demonstrated that Glu-181 functions as the counterion in this pigment. This finding has led to the proposal that Glu-181 may function as the counterion in other invertebrate visual pigments as well. Here we describe a series of mutagenesis experiments to test this hypothesis and to also test whether other conserved acidic amino acids in Drosophila Rhodopsin 1 (Rh1) may serve as the counterion of this visual pigment. Of the 5 Glu and Asp residues replaced by Gln or Asn in our experiments, none of the mutant pigments shift the absorption of Rh1 by more than 6 nm. In combination with prior studies, these results suggest that the counterion in Drosophila Rh1 may not be located at Glu-181 as in amphioxus, or at Glu-113 as in bovine rhodopsin. Conversely, the extremely low steady state levels of the E194Q mutant pigment (bovine opsin site Glu-181), and the rhabdomere degeneration observed in flies expressing this mutant demonstrate that a negatively charged residueat this position is essential for normal rhodopsin function in vivo. This work also raises the possibility that another residue or physiologic anion may compensate for the missing counterion in the E194Q mutant.     

Phosphatidylserine exposure and aminophospholipid translocase activity in Rh-deficient erythrocytes

Endogenous phosphatidylserine (PS) exposure and lipid transport activity have been investigated for seven unrelated cases of Rh_null erythrocytes. Endogenous PS exposure was measured by prothrombinase activity. Out of six cases studied, two Rh_null samples exhibited abnormal aminophospholipid exposure, as suggested by the measurement of a lower Km of factor Xa for prothrombin. Aminophospholipid translocase activity was measured through the transbilayer redistribution of spin-labelled analogues of phospholipids. Provided that incubation conditions allow the maintainance of intracellular ATP level, no difference was observed between Rh_null and control erythrocytes, clearly indicating that the aminophospholipid translocase and Rh polypeptides are different molecular species.     

Localization of Rh1(D), 2(C), 3(E), 4(c), 5(e) and 25(LW) antigens of human Rh blood groups in fetal erythrocyte membranes

The fetal erythrocyte membranes were partially solubilized with Triton X-100 at the low concentration (0.5%). The localizations of Rh1(D), 2(C), 3(E), 4(c), 5(e) and 25(LW) were investigated. Using hemagglutination inhibition assay, Rh1(D) antigen activity was observed in the Triton-treated membrane (Triton shell) containing mainly band 1, 2 (spectrin), band 5 (actin), band 4.1 and a part of band 3, while Rh2(C), 3(E), 4(c), 5(e) and 25(LW) antigens were detected in the supernatant containing band 3, 6, 2.2, 2.3 and 4.2. It is suggested that: Rh1(D) antigen would associate with cytoskeleton matrix of fetal erythrocyte membranes; Rh1(D) and Rh25(LW) antigens might be integral membrane proteins, while Rh2(C), 3(E), 4(c) and 5(e) antigens would be surface membrane proteins which are easily released from membranes by EDTA, mercaptoethanol and alkaline treatments.     

Covalent Binding and Conformational Change of pUC19 DNA by Rhodium (II) Metal Complex

Abstract

Binding of Rhodium (II) acetate [Rh₂(O₂CCH₃)₄] (Rh1) compound with plasmid pUC19 DNA has been studied using different molar ratio of Rh1. After incubation for 24hr at 37 °C, binding of the Rh1 to pUC19 DNA was confirmed by agarose gel electrophoresis. The electrophoretic results indicated the slower migration speed for the linearized pUC19 DNA. Conformation change of the DNA after Rh1 binding was also indicated at higher molar ratio of Rh1. The atomic force microscopy images showed that the Rh1 induced the conformation change to unwind pUC19 DNA. The Rh1-DNA complexes are observed very stable due to covalent bond. This study clearly demonstrates that [Rh₂(O₂CCH₃)₄] reacts with pUC19 DNA and covalently binds to be stable Rh1-pUC19 DNA as interstrand adducts.     

Synthetic, electrochemical and structural aspects of a series of ferrocene-containing dicarbonyl β-diketonato rhodium(I) complexes

Treatment of [Rh(β-diketonato)(cod)] with CO resulted in better yields of [Rh(FcCOCHCOR)(CO)₂] than by treating [Rh(Cl)(CO)₂]₂ with FcCOCH₂COR, R = CF₃ (Hfctfa), CH₃ (Hfca), Ph (Hbfcm, Ph = phenyl) and Fc (Hdfcm, Fc = ferrocenyl). The single crystal structure of the fctfa rhodium(I) complex [C₁₆H₁₀F₃FeO₄Rh], monoclinic, C 2/c(15), a = 13.266(3) Å, b = 19.553(3) Å, c = 13.278(3) Å, β = 100.92(2)°, Z = 8 showed both rotational and translational displacement disorders for the CF₃ group. An electrochemical study revealed that the formal reduction potential, E⁰′, for the electrochemically reversible one electron oxidation of the ferrocenyl group varied between 0.304 (for the fctfa complex) and 0.172 V (for the dfcm complex) versus Fc/Fc⁺ in a manner that could be directly traced to the group electronegativities, χ_R, of the R groups on the β-diketonato ligands, as well as to the values of the free β-diketones. Anodic peak potentials, E_pa,Rh, for the dominant cyclic voltammetry peak associated with rhodium(I) oxidation were between 0.718 (bfcm complex) and 1.022 V (dfcm complex) versus Fc/Fc⁺. Coulometric experiments implicated a second, much less pronounced anodic wave for the apparent two-electron Rh^I oxidation that overlaps with the ferrocenyl anodic wave and that the redox processes associated with these two Rh^I oxidation waves are in slow equilibrium with each other.     

Selective interactions among Rh,ABO, and sex ratio of newborns

Summary The hypothesis that the Rh and ABO blood systems behave like the HLA system in relation to mother-conception tolerance-rejection mechanisms was tested in 25,501 mother-infant pairs. According to this hypothesis, heterozygotes carrying a paternal gene that is not present in their mother should be better tolerated than homozygotes. Significantly more BO infants born to AO mothers. AO infants born to BO mothers, Rh(+) heterozygotes born to Rh(-) mothers, and less significantly AO infants born to OO mothers confirm the hypothesis. Fewer homozygotes occurred in Rh(-) infants born to Rh(+) mothers and in O infants born to non-O mothers. Deviations from the Hardy-Weinberg equilibrium found in the ABO system were modified by the Rh and sex of the infant. These data strongly support the hypothesis that at least two feto-maternal systems influence the denstiny of pregnancies: the classical known incompatibility system which operates late in pregnancy and a new one which is based on the induction of maternal tolerance early in pregnancy: maternal tolerance seems to be better elicited by heterozygous eggs or embryos carrying a gene not present in the mother. The data also support the hypothesis that the sex ratio is influenced by feto-maternal tolerance-rejection mechanisms associated with the ABO and Rh systems.     

The best strategy of blood transfusion in patients with Rhnull syndrome

Rh_null syndrome is a very rare disease. Patients with this syndrome present with negative serological Rh typing of E, e, C, c, and D antigens. Only one study has previously discussed Rh_null syndrome in Chinese individuals. We experienced two patients with Rh_null syndrome in China, Rh genotypes being CcDEe in the first patient and CCDee in the second patient. The first patient was a pregnant woman (gravida 2, para 1) with a negative red blood cell (RBC) antibody screen test. The second patient was a middle-aged man, transfused with ccdee, ccdEe, and ccdee RBC products, the pre-transfusion specimen was negative and post-transfusion specimen was anti-c,e, respectively. The hemoglobin level continued to increase in the second patient after being transfused with ccdEe RBC products. In the first patient, the result of the antibody screen test was still negative after artificial abortion. In patients with Rh_null syndrome, RBC products that have the same Rh genotype as the patient can be safely transfused.     

Kinetics of CO substitution in Co4(CO)12 and Rh4(CO)12

The kinetics of rapid CO substitution by PPh₃ in Co₄(CO)₁₂ and Rh₄(CO)₁₂ have been examined by stopped-flow and low temperature FT-IR methods. In Co₄(CO)₁₂ rapid (k_obs ∼ 1.8 s⁻¹) substitution of CO occurs after a 1–15 s induction period at 28 °C in C₆H₅Cl solvent by a catalytic process. Addition of PPh₃ to Rh₄(CO)₁₂ yields Rh₄(CO)₁₁(PPh₃) according to a predominantly second order rate law k₁[Rh₄- (CO)₁₂] + k₂[Rh₄(CO)₁₂][PPh₃] with k₁ = 25 ± 11 s⁻¹ and k₂ = 2.97 ± 0.27 X 10⁴ M⁻¹ s⁻¹ at 28 °C. Substitution of a second CO ligand also occurs rapidly with k₁ = 0.15 ± 0.09 s⁻¹ and k₂ = 6.54 ± 0.07 X 10² M⁻¹ s⁻¹ at 28 °C. The reactivity of Rh₄(CO)₁₂ toward associative substitution is 10⁴– 10¹¹ faster than for the Co and Ir analogues, In Rh₄(CO)₁₁(PPh₃) the increase in CO substitution rates over Co and Rh analogues is 10²–10⁷. The ordering of associative substitution rates Co << Rh >>> Ir in these clusters exaggerates the trend seen in mononuclear metal complexes.     

Syntheses of linear-type S-bridged trinuclear complexes composed of heavy d metal ions and 2-aminoethanethiolate: comparative characterization by X-ray diffraction, spectroscopy and electrochemistry

The S-bridged trinuclear complexes composed of heavy d⁶ metal ions, [Rh^III{M(aet)₃}₂]³⁺ (M=Ir^III(1), Rh^III(2); aet = 2-aminoethanethiolate), have been prepared by the reactions of fac(S)-[M(aet)₃] with RhCl₃ · 3H₂O. The complexes were separated into meso (1a, 2a) and rac (1b, 2b) isomers by SP-Sephadex C-25 column chromatography. 1b and 2b were optically resolved by the column chromatographic method and characterized by CD spectroscopy. Crystal structures of 1a, 1b and 2a were determined by X-ray diffraction, and it was found that they consist of linear-type trinuclear structures. The central Rh(III) ion in the present complexes has d⁶ electronic configuration with the non-degenerated A-type cubic field term, and showed long Rh?M distances, acute S-M-S angles and obtuse Rh-S-M angles. These are in contrast with the complexes having the degenerated T-type cubic field term such as [M^′{M(aet)₃}₂]ⁿ⁺ (M^′=V^III, Mo^IV and Re^III, M=Ir^III, Rh^III, n=3 or 4). All the isomers have been comparatively characterized and discussed in solid state and the solution for spectrochemical and electrochemical properties.     

The major opsin in bees (Insecta: Hymenoptera): A promising nuclear gene for higher level phylogenetics.

We report the phylogenetic utility of the nuclear gene encoding the long-wavelength opsin (LW Rh) for tribes of bees. Aligned nucleotide sequences were examined in multiple taxa from the four tribes comprising the corbiculate bees within the subfamily Apinae. Phylogenetic analyses of sequence variation in a 502-bp fragment (approx 40% of the coding region) strongly supported the monophyly of each of the four tribes, which are well established from previous studies of morphology and DNA. Trees estimated from parsimony and maximum likelihood analyses of LW Rh sequences show a strongly supported relationship between the tribes Meliponini and Bombini, a relationship that has been found uniformly in studies of other genes (28S, 16S, and cytochrome b). All of the tribal clades as well as relationships among the tribes are supported by high bootstrap values, suggesting the utility of LW Rh in estimating tribal and subfamily rank for these bees. The sequences exhibit minimal base composition bias. Both 1st + 2nd and 3rd position sites provide information for estimating a reliable tree topology. These results suggest that LW Rh, which has not been reported previously in studies of organismal phylogenetics, could provide important new data from the nuclear genome for phylogeny reconstruction.     

Studies on reaction of dirhodium(II) aquo cation with dioxygen

The reactions of dirhodium(II) aquo cation {Rh_2(aq)⁴⁺} with dioxygen were examined. It has been found that the nature of the oxidation product depends upon the concentration of dioxygen in the solution. The dimeric or polymeric Rh(III)_(aq) cationic species with a charge of greater than +3 is formed when air oxygen slowly diffuses into a solution containing Rh_2(aq)⁴⁺. The paramagnetic cation of proposed formula [(H₂O)₄Rh(O₂⁻)(OH)₂Rh(H₂O)₄]³⁺ is formed when molecular oxygen is bubbled through a 2–3 M HClO₄ solution of Rh_2(aq)⁴⁺. This species has been isolated and characterized in solution.     

Water gas shift reaction and nitrobenzene reduction catalysis by tetracarbonyldi-μ-chlorodirhodium(I) complex immobilized on poly(4-vinylpyridine)

The preparation, characterization and catalytic properties of Rh₂(CO)₄Cl₂ immobilized on poly(4-vinylpyridine) are reported. The polymer-immobilized Rh catalyst in contact with 80% aqueous 2-ethoxyethanol is active for the water gas shift reaction and nitrobenzene reduction to azoxybenzene under carbon monoxide at 100 °C. The polymer-immobilized Rh complex was found to be reusable. The Rh complex was coordinatively bonded to the pyridine groups of the organic polymer as suggested by UV-Vis/diffuse reflectance (UV-Vis/DR), Fourier transform infrared (FT-IR), and electron paramagnetic resonance (EPR) spectroscopies. The morphology and the elemental analysis of the immobilized catalyst were studied by scanning electron microscopy-energy dispersive X-ray (SEM-EDX) technique.     

Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor

A novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. RhEst1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The K_m values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (S)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (ee) of 97.5%, indicating an extremely high enantioselectivity (E = 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals.     

A 31P1H NMR study of the reactions of [Rh2(μ-Cl)2(cod)2] with unsymmetrical,bidentate ligands and hydrogen

[Rh₂(μ-Cl)₂(cod)₂] reacts with Ph₂PCH₂CH₂OMe (PC₂O), Ph₂P(CH₂)₃NMe₂ (PC₃N), PBuⁿPh₂ or PPh₃ to give [Rh(cod)L₂]Cl (L = PC₂O, PC₃N, PBuⁿPh₂, PPh₃). In the presence of hydrogen, [Rh(cod)L₂]Cl is converted to [RhClH₂L₃]. In contrast, [Rh(cod)(PC₂O)₂]BPh₄ reacts with H₂ to give [RhH₂(PC₂O)₂S₂]BPh₄ (S = solvent). With Ph₂PCH₂CH₂NMe₂ (PC₂N) or Ph₂PCH₂CH₂SMe (PC₂S), [Rh₂(μ-Cl)₂(cod)₂] reacts to form the chelate complexes cis- [Rh(PC₂N)₂]⁺ or cis-[Rh(PC₂S)₂]⁺, neither of which reacts with hydrogen under ambient conditions. The products of the reactions are characterized in situ by ³¹P¹H NMR spectroscopy.