Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures

Mutations in alpha-synuclein, parkin and ubiquitin C-terminal hydrolase L1, and defects in 26/20S proteasomes, cause or are associated with the development of familial and sporadic Parkinson's disease (PD). This suggests that failure of the ubiquitin-proteasome system (UPS) to degrade abnormal proteins may underlie nigral degeneration and Lewy body formation that occur in PD. To explore this concept, we studied the effects of lactacystin-mediated inhibition of 26/20S proteasomal function and ubiquitin aldehyde (UbA)-induced impairment of ubiquitin C-terminal hydrolase (UCH) activity in fetal rat ventral mesencephalic cultures. We demonstrate that both lactacystin and UbA caused concentration-dependent and preferential degeneration of dopaminergic neurons. Inhibition of 26/20S proteasomal function was accompanied by the accumulation of alpha-synuclein and ubiquitin, and the formation of inclusions that were immunoreactive for these proteins, in the cytoplasm of VM neurons. Inhibition of UCH was associated with a loss of ubiquitin immunoreactivity in the cytoplasm of VM neurons, but there was a marked and localized increase in alpha-synuclein staining which may represent the formation of inclusions bodies in VM neurons. These findings provide direct evidence that impaired protein clearance can induce dopaminergic cell death and the formation of proteinaceous inclusion bodies in VM neurons. This study supports the concept that defects in the UPS may underlie nigral pathology in familial and sporadic forms of PD.     

Mechanisms of ligand binding and efficacy at the human D2(short) dopamine receptor

Mechanisms of ligand binding and receptor activation for the human D2(short) dopamine receptor have been probed using two homologous series of monohydroxylated and dihydroxylated agonists (phenylethylamines and 2-dipropylaminotetralins). In ligand binding studies, the majority of compounds exhibited competition curves versus [3H]spiperone that were best fitted using a two site binding model. The compounds had different abilities (potencies and maximal effects) to stimulate [35S]GTPgammaS binding and to inhibit forskolin-stimulated cAMP accumulation. From the data it can be concluded that: (i) the ability of an agonist to stabilize receptor/G protein coupling can be used to predict agonist efficacy for some groups of compounds (2-dipropylaminotetralins) but not for others (phenylethylamines); (ii) the receptor may be activated by unhydroxylated compounds; (iii) single hydroxyl groups or pairs of hydroxyl groups on the agonist may contribute to binding affinity, potency and efficacy; and (iv) for the 2-dipropylaminotetralin series two modes of agonist/receptor interaction have been identified associated with different relative efficacy.     

瓦屋山国家森林公园锄足蟾科6种的繁殖鸣声特性

在地处四川省洪雅县的瓦屋山国家森林公园录取了锄足蟾科 6种的繁殖期求偶鸣叫声。它们分隶 4属 ,即角蟾属 (Megophrys)、齿蟾属 (Oreolalax)、齿突蟾属 (Scutiger)和掌突蟾属 (Leptolalax)。在IBMPC上用“SIGNAL”软件 (EngineeringDesign ,USA)对获取的鸣声资料进行分析 ,分析的频率范围设置为 0～ 10kHz。声学分析结果表明 :峨山掌突蟾 (L oshanensi) ,小角蟾 (M minorr) ,角蟾 1种 (M sp) ,金顶齿突蟾[S (S )chintingensis],峨眉齿蟾 (O omeimontis)和无蹼齿蟾 (O schmidti)的主能峰频率平均值分别是45 2 1 9、 34 5 6 4、 2 2 93 8、 10 76 5、 10 71 0和 1849 4Hz ,每声持续时间的平均值分别是 46 2、 90 8、 99 6、72 2、 78 8和 110 3ms ,声距的平均值分别是 140 4、 2 5 3 0、 6 81 4、 15 17 7、 46 1 3和 6 19 5ms。单因子方差分析结果表明主能峰频率、每声持续时间和各声距在 6个种间差异极显著 (P <0 0 1)。LSD法多重比较的结果指出金顶齿突蟾和峨眉齿蟾间的主能峰频率无显著差异 (P =0 917>0 0 5 ) ;在每声持续时间上 ,只有峨山掌突蟾与小角蟾、角蟾 1种、峨眉齿蟾、无蹼齿蟾间差异极显著 (P <0 0 1) ;在声距上 ,峨山掌突蟾与小角蟾间无显著差异 ,角蟾 1种与无蹼齿蟾之间、峨     

SS Bond-activation of diorganyl disulfide by anionic [Mn(CO)5]: crystal structures of [Mn(SC5H4NO)3] and [(CO)3Mn(μ-SR)3Co(μ-SR)3Mn(CO)3] (R=C6H4NHCOPh)

The SS bond-activation of diorganyl disulfide by the anionic metal carbonyl fragment [Mn(CO)₅]⁻ gives rise to an extensive chemistry. Oxidative decarbonylation addition of 2,2′-dithiobis(pyridine-N-oxide) to [Mn(CO)₅]⁻, followed by chelation and metal-center oxidation, led to the formation of [Mn^II(SC₅H₄NO)₃]⁻ (1). The effective magnetic moment in solid state by SQUID magnetometer was 5.88 μ_B for complex 1, which is consistent with the Mn^II having a high-spin d⁵ electronic configuration in an octahedral ligand field. The average Mn(II)S, SC and NO bond lengths of 2.581(1), 1.692(4) and 1.326(4) Å, respectively, indicate that the negative charge of the bidentate 1-oxo-2-thiopyridinato [SC₅H₄NO]⁻ ligand in complex 1 is mainly localized on the oxygen atom. The results are consistent with thiolate-donor [SC₅H₄NO]⁻ stabilization of the lower oxidation state of manganese (Mn(I)), while the O,S-chelating [SC₅H₄NO]⁻ ligand enhances the stability of manganese in the higher oxidation state (Mn(II)). Activation of SS bond as well as OH bond of 2,2′-dithiosalicylic acid by [Mn(CO)₅]⁻ yielded [(CO)₃Mn(μ-SC₆H₄C(O)O)₂Mn(CO)₃]²⁻ (4). Oxidative addition of bis(o-benzamidophenyl) disulfide to [Mn(CO)₅]⁻ resulted in the formation of cis-[Mn(CO)₄(SR)₂]⁻ (R=C₆H₄NHCOPh) which was employed as a chelating metallo ligand to synthesize heterotrinuclear [(CO)₃Mn(μ-SR)₃Co(μ-SR)₃Mn(CO)₃]⁻ (8) possessing a homoleptic hexathiolatocobalt(III) core.     

Reactions of quadruply bonded dimolybdenum(II) and dirhenium(III) complexes with 2,6-bis(dicyclohexylphosphinomethyl)pyridine and the related behavior of the mixed P,S,P ligand bis(diphenylphosphinomethyl)sulfide

The first complexes that contain the 2,6-bis(dicyclohexylphosphinomethyl)pyridine ligand (PNP) have been isolated and characterized. The reactions of K₄Mo₂Cl₈, (n-Bu₄N)₂Re₂Cl₈ and PdBr₂(1,5-COD) afford Mo₂Cl₄(PNP)(HPCy₂) (1), ReCl₃(PNP) (2) and PdBr₂(PNP) (4), respectively, while from the reaction of PNP with cis-Re₂(μ-O₂CCH₃)₂Cl₄(H₂O)₂ the heteromacrocylic dication [Cy₂P{CH₂pyCH₂}₂PCy₂]²⁺ has been isolated as its mixed [Cl]⁻/[ReO₄]⁻ salt (3). The reaction of cis-Re₂(μ-O₂CCH₃)₂Cl₄(H₂O)₂ with bis(diphenylphosphinomethyl)sulfide (PSP) gives the mononuclear Re(V) complex ReO(OEt)Cl₂(PSP) (5) in which the S atom is not coordinated. The structures of 1-5 have been established by X-ray crystallography, that of 5 being the first for a complex of this ligand.     

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.