Stoichiometry of tyrosine hydroxylase phosphorylation in the nigrostriatal and mesolimbic systems in vivo: effects of acute haloperidol and related compounds

Electrical stimulation of the medial forebrain bundle increases (32)P incorporation into striatal tyrosine hydroxylase (TH) at Ser (19), Ser(31), and Ser(40). In the present studies, the effects of acute haloperidol and related drugs on sitespecific TH phosphorylation stoichiometry (PS) in the nigrostriatal and mesolimbic systems were determined by quantitative blot immunolabeling using phosphorylation statespecific antibodies. The striatum (Str), substantia nigra (SN), nucleus accumbens (NAc), and ventral tegmental area (VTA) from Sprague-Dawley rats were harvested 30-40 min after a single injection of either vehicle, haloperidol (2 mg/kg), raclopride (2 mg/kg), clozapine (30 mg/kg), or SCH23390 (0.5 mg/kg). In vehicle-injected control rats, Ser(19) PS was 1.5- to 2. 5-fold lower in Str and NAc than in SN and VTA, Ser(31) PS was two-to fourfold higher in Str and NAc than in SN and VTA, and Ser(40) PS was similar between the terminal field and cell body regions. After haloperidol, Ser(40) PS increased twofold in Str and NAc, whereas a smaller increase in SN and VTA was observed. The effects of haloperidol on Ser(19) PS were similar to those on Ser(40) in each region; however, haloperidol treatment increased Ser(31) PS at least 1.6-fold in all regions. The effects of raclopride on TH PS were comparable to those of haloperidol, whereas clozapine treatment increased TH PS at all sites in all regions. By contrast, the effects of SCH23390 on TH PS were relatively small and restricted to the NAc. The stoichiometries of site-specific TH phosphorylation in vivo are presented for the first time. The nigrostriatal and mesolimbic systems have common features of TH PS, distinguished by differences in TH PS between the terminal field and cell body regions and by dissimilar increases in TH PS in the terminal field and cell body regions after acute haloperidol.     

Distribution of Dopamine,its Metabolites,and D1 and D2 Receptors in Heterozygous And Homozygous Weaver Mutant Mice

In weaver mice, besides a postnatal cerebellar developmental anomaly probably caused by alterations of an inwardly rectifying K⁺ channel, there is a progressive loss of mesencephalic dopaminergic neurons. To further evaluate this deficit, endogenous dopamine and its metabolites were measured in 22 brain regions from heterozygous (wv/+) and homozygous (wv/wv) mutants, and compared to wild type (+/+) mice. In both wv/+ and wv/wv mutants there were profound dopamine depletions in all regions; these changes were accompanied by decreases in metabolites but with an increase of turnover indexes. Dopamine D₁ and D₂ receptors were examined by autoradiography, and their distribution was conserved. The results show that the dopaminergic deficit is widespread to all areas of innervation, and is probably compensated for by an increased turnover. Abnormal developmental growth signals, or aberrant cellular responses, may result in defective neurite formation of the midbrain dopaminergic neurons, leading to their postnatal death.     

长引物P CR法构建白念珠菌SCH9-MYC融合菌

目的：构建白念珠菌SCH9?MYC融合菌。方法运用长引物PCR扩增含有MYC标签和ARG4筛选标记的质粒序列,采用醋酸锂转染法将质粒序列同源重组到白念珠菌SN152的SCH9基因开放阅读框的C末端,在SC?Leu?选择性培养基上筛选阳性克隆,抽取阳性克隆基因组进行PCR验证,将验证为阳性的转染子进行生长曲线测定、spot assay、菌丝诱导实验,进一步筛选出表型正常的融合菌。结果通过PCR验证鉴定出3株融合菌构建正确,通过生长曲线测定、spot assay、菌丝诱导实验筛选出两株表型正常的融合菌菌株。结论运用长引物PCR扩增方法同源重组可以正确构建白念珠菌SCH9?MYC融合菌菌株。     

Apoptosis of osteosarcoma cultures by the combination of the cyclin-dependent kinase inhibitor SCH727965 and a heat shock protein 90 inhibitor

Osteosarcoma (OS) is an aggressive bone cancer typically observed in adolescents and young adults. Metastatic relapse accounts primarily for treatment failure, and obstacles to improving cure rates include a lack of efficacious agents. Our studies show apoptosis of OS cells prepared from localized and metastatic tumors by a novel drug combination: SCH727965 (SCH), a cyclin-dependent kinase inhibitor, and NVP-AUY922 (AUY) or other heat shock protein 90 inhibitor. SCH and AUY induced apoptosis when added simultaneously to cells and when AUY was added to and removed from cells before SCH addition. Sequential treatment was most effective when cells received AUY for ∼12 h and when SCH was presented to cells immediately after AUY removal. The apoptotic protein Bax accumulated in mitochondria of cotreated cells but was primarily cytosolic in cells receiving either agent alone. Additional data show that SCH and AUY cooperatively induce the apoptosis of other sarcoma cell types but not of normal osteoblasts or fibroblasts, and that SCH and AUY individually inhibit cell cycle progression throughout the cell cycle. We suggest that the combination of SCH and AUY may be an effective new strategy for treatment of OS.     

Influence of alternating low frequency magnetic fields on reactivity of central dopamine receptors in neonatal 6-hydroxydopamine treated rats

The aim of this study was to evaluate the influence of extremely low frequency magnetic field (ELF MF) on the reactivity of the central dopamine D(1) receptor in rats with dopamine neurons chemically damaged by 6-hydroxydopamine (6-OHDA), an animal model of human's Parkinson's disease. The experiment was carried out on male Wistar rats. On day 3 of postnatal life, a lasting and selective chemical damage of the central dopamine system was induced in the rats by infusion of 6-OHDA HBr (133.4 microg intracerebroventricular, base form) given bilaterally into lateral ventricles of the brain. Control animals received similar treatments injecting only vehicle. At 2 months of age, both 6-OHDA treated and control rats were randomly divided into two groups. Rats from the first group were exposed to 10 Hz sinusoidal, 1.8-3.8 mT magnetic field one hour daily for 14 days. Rats of the second group were sham exposed, with the applicator solenoid turned off. On the day after the final exposure the evaluations were made of the rat's spontaneous irritability, oral activity, and catalepsy. The MF exposed rat with chemically induced dopamine neurons damage exhibited a reduction of irritability and oral activity when stimulated with SKF 38393 (the agonist of central dopamine D(1) receptor) and some increase of catalepsy after administration of SCH 23390(the antagonist of central dopamine D(1) receptor). These results indicate that ELF MF reduce the reactivity of central dopamine D(1) receptors in rats.     

多巴胺受体激动剂对兔动脉cAMP产生系统的影响

实验观察了选择性多巴胺（ＤＡ）ＤＡ１受体激动剂ｆｅｎｏｌｄｏｐａｍ与ＤＡ２受体激动剂ｐｒｏｐｙ１－ｂｕｔｙｌ－ｄｏｐａｍｉｎｅ（ＰＢＤＡ）对兔肾动脉,肺、肠系膜动脉和股动脉环磷酸腺苷（ｃＡＭＰ）产生系统的影响。结果表明：⑴除股动脉外,ｆｅｎｏｌｄｏｐａｍ均可浓度依赖性地增加肺动脉、肾动脉和肠系膜动脉ｃＡＭＰ的生成量。选择性ＤＡ１受体阻断剂ＳＣＨ２３３９０可以显著阻断ｆｅｎｏｌｄｏｐａｍ的效应,而Ｄ     

Pharmacokinetics of the active antifungal enantiomer, SCH 42427 (RR), and evaluation of its chiral inversion in animals following its oral administration and the oral administration of its racemate genaconazole (RR/SS)

Genaconazole (SCH 39304) is a potent triazole antifungal agent that is active both orally and topically. Genaconazole is a racemic mixture which contains 50% of the RR (SCH 42427) and 50% of the SS (SCH 42426) enantiomers. The RR isomer accounts for most of the antifungal activity of genaconazole. Serum concentrations of the RR and SS enantiomers were analyzed by a chiral HPLC method which involved extraction of serum with organic solvent followed by separation on a Cyclobond I column and quantification by UV absorbance at 205 nm. The bioavailability and pharmacokinetic profiles of the two enantiomers after oral administration of the racemate (genaconazole) were very similar in cynomolgus monkeys. In rats following dosing with genaconazole, the RR enantiomer had a lower C(max) and a longer t(1/2) than the SS enantiomer, while the AUC(I) values of the two enantiomers were similar. Based on chiral HPLC analysis, there was no evidence for the inversion of the RR to the SR isomer, or of the SS to the SR isomer, indicating that there was no chiral inversion of the RR or SS enantiomers in either species. Genaconazole at 20 mg/kg and the RR (SCH 42427) enantiomer at 10 mg/kg had very similar serum concentration-time profiles and C(max), AUC(I), and t(1/2) values for the RR enantiomer in both rats and monkeys, indicating that the two treatments were equivalent with respect to the bioavailability of the RR enantiomer.     

Functional alteration by NMDA antagonist: effects of L-Dopa,neuroleptics drug and postnatal administration

Summary.  Antiakinsic effects of the uncompetitive NMDA antagonists, memantine, amantadine and MK-801, and competitive antagonists, CGP 40116, alone or in co-administration with acute subthreshold dose of L-Dopa (5 mg/kg) in MPTP-treated mice, functional alterations induced by acute MK-801 in combinations with neuroleptic compounds or behavioural deficits following postnatal administration of MK-801 were investigated. Memantine and amantadine injected 60 min before the subthreshold dose of L-Dopa (5 mg/kg), induced antiakinesic actions in hypokinesic MPTP-treated mice. Concurrently, higher doses of memantine and MK-801 caused dyskinesic changes, reducing further rearing (10 and 30 mg/kg) and locomotor (30 mg/kg) behaviour of the MPTP mice; MK-801 elevated locomotion (0.1 mg/kg) but reduced rearing (0.3 mg/kg). In control, saline-treated mice, memantine (3, 10 and 30 mg/kg) and MK-801 (0.1 and 0.3 mg/kg) increased locomotor behaviour but decreased rearing behaviour. In rats, MK-801 induced marked increases in locomotor activity and disruptions of circular swim maze acquisition that were to greater or lesser extents blocked or potentiated by neuroleptic compounds: SCH 23390 (0.005 and 0.05 mg/kg) and clozapine (5.0 and 10.0 mg/kg) dose-dependently antagonised MK-801 (0.3 mg/kg) induced locomotor activity whereas raclopride (0.1 mg/kg) and haloperidol (0.1 mg/kg) attenuated it dose-specifically. Amperozide (0.5 mg/kg) attenuated the MK-801 effect but potentiated it at the 2.0 mg/kg dose. In the circular swim maze, raclopride (0.01 mg/kg) and SCH 23390 (0.05 mg/kg) improved the acquisitive performance of rats administered MK-801 (0.03 mg/kg) acutely whereas clozapine (10.0 mg/kg) and amperozide (2.0 mg/kg) deteriorated the performance of MK-801-treated rats. Postnatal administration of MK-801 (0.05 mg/kg, day 11 after birth) induced severe functional alterations in adult mice. At 70 days of age, MK-801 mice showed an initial hypoactivity followed by marked hyperactivity in the motor activity test chambers. These mice showed deficits in habituation, a nonassociative form of learning. Their hyperactivity in the test chambers was reversed by a low dose of d-amphetamine (0.25 mg/kg). Taken together, these findings display a wide range of acute/long-term functional alterations induced by NMDA antagonists, particularly MK-801, associated with animal models of brain disorders. Received July 9, 2001 Accepted August 6, 2001 Published online June 17, 2002     

Comparison of 125I-SCH 23982 and [3H]SCH 23390 as Ligands for the D-1 Dopamine Receptor

125I-SCH 23982, an antagonist with high affinity and selectivity for the D-1 subtype of dopamine receptors, has recently been synthesized. Densities of D-1 receptors in rat brain obtained from autoradiographic studies using this iodinated ligand are 5- to 10-fold less than densities reported with tritiated analogues such as [3H]SCH 23390. A direct comparison of these two ligands using striatal homogenates confirmed this discrepancy. One explanation for this difference is that 125I-SCH 23982 labels a subset of the sites labeled by [3H]SCH 23390. However, the distributions of sites labeled by the ligands in autoradiograms of horizontal sections of rat brain were virtually identical. Furthermore, 127I-SCH 23982 displaced 100% of the specifically bound [3H]SCH 23390 in striatal homogenates with a Hill coefficient of approximately 1. These results are not consistent with the existence of a subset of receptors recognized by 125I-SCH 23982 and suggest that both ligands label the same population of receptors. An alternative explanation for the discrepancy in Bmax values is that an unlabeled inhibitor is present in commercial preparations of 125I-SCH 23982. When all of the solvent (including any volatile inhibitors) was removed from commercial preparations of 125I-SCH 23982 prior to use in radioligand binding experiments, the discrepancy in Bmax values was eliminated.