Identification of an amino acid residue important for binding of methiothepin and sumatriptan to the human 5-HT(1B) receptor.

Site-directed mutagenesis of the human 5-HT_1B receptor was performed to investigate the role of the amino acid residues cysteine 326 and tryptophan 327 in transmembrane region VI and aspartic acid 352 in transmembrane region VII in ligand binding. Binding studies were performed with the antagonist radioligand [³H]GR125743 on mutant and wild-type receptors stably expressed in Chinese hamster ovary cells (CHO)-K1 cells. Substitution of tryptophan 327 by alanine resulted in decreased affinities of all ligands tested. The most prominent changes in affinity were observed for the antagonist methiothepin and the antimigraine drug sumatriptan, which were reduced approximately 300- and 60-fold, respectively. Nevertheless, the affinity of 5-HT remained the same. Replacement of the aspartic acid 352 by alanine reduced high-affinity binding of 5-HT. Substitution of cysteine 326 by alanine had minor effects on ligand binding. Some of these results agree with the results from mutagenesis studies of the corresponding amino acids in other receptors. However, some notable differences also emerge showing that functional roles of individual amino acid residues must be tested experimentally in each receptor subtype.     

L-701,324, a selective antagonist at the glycine site of the NMDA receptor, counteracts haloperidol-induced muscle rigidity in rats

  Rationale: It has recently been suggested that the overactivity of glutamatergic neurotransmission may contribute to the pathophysiology of Parkinson’s disease. Therefore, a search for new compounds which block glutamatergic receptors and show antiparkinsonian properties in animal models of this disease seems to be justified. Objective: The aim of this study was to determine whether L-701,324 [7-chloro-4-hydroxy-3(3-phenoxy) phenylquinoline-2-(H)-one], a selective and full antagonist at the glycine site of the NMDA receptor, counteracts parkinsonian-like muscle rigidity and catalepsy induced by haloperidol in rats. Methods: The muscle tone was measured as the resistance developed to passive flexion and extension of the hind limb. Electromyographic (EMG) activity was additionally recorded in the gastrocnemius and tibialis anterior muscles. Results: L-701,324 (2.5–40 mg/kg IP) dose-dependently decreased the muscle tone enhanced by haloperidol (1–5 mg/kg IP). Likewise, the haloperidol-enhanced resting EMG activity and the EMG reflex response to passive movements were diminished by lower and almost abolished by higher doses of L-701,324. However, up to a dose of 20 mg/kg IP, L-701,324 did not influence haloperidol (0.5 mg/kg IP)-induced catalepsy. Moreover, L-701,324 (1.25–5 mg/kg IP) given alone or together with haloperidol (0.5–1 mg/kg IP) disturbed rotarod performance. Gross observation of behaviour indicated that rats injected with L-701,324 in doses equal to or higher than 5 mg/kg, alone or in combination with haloperidol, were markedly ataxic, i.e. rats showed signs of disturbed balance and loss of control over their hind limbs. Conclusions: The present study suggests that L-701,324 exhibits a beneficial action in the animal model of parkinsonian rigidity, but not that of parkinsonian akinesia. Nonetheless, this compound is not devoid of motor side-effects. Received: 1 February 1998 / Final version: 20 October 1998     

Prodrugs of Peptides. 9. Bioreversible N-α-Hydroxyalkylation of the Peptide Bond to Effect Protection Against Carboxypeptidase or Other Proteolytic Enzymes

Various N--hydroxyalkyl derivatives of N-acyl amino acids and di- and tripeptides were prepared by hydrolysis or aminolysis of N-acyl 5-oxazolidinones. The stability of these derivatives was studied in aqueous solution as a function of pH. The compounds were all degraded quantitatively to their parent N-acylated amino acid or peptide and aldehyde but with vastly different rates. At pH 7.4 and 37°C the half-lives of decomposition ranged from 4 min to 1500 hr. The structural factors influencing the stability included both steric and polar effects within the acyl and N--hydroxyalkyl moieties as well as within the amino acid attached to the N--hydroxyalkylated N-acyl amino acid. Whereas the N-benzyloxycarbonyl (Z) derivatives of the dipeptides Gly-L-Leu and Gly-L-Ala were readily hydrolyzed by carboxypeptidase A, the N-hydroxymethylated compounds, i.e., Z-Gly(CH₂OH)-Leu and Z-Gly(CH₂OH)-Ala, were resistant to cleavage by the enzyme as revealed by their similar rates of decomposition in the presence or absence of the enzyme at pH 7.4 and 37°C. The results suggest that N--hydroxyalkylation of a peptide bond protects not only this bond but also an adjacent peptide bond against proteolytic cleavage. Since the N--hydroxyalkyl derivatives are readily bioreversible, undergoing spontaneous hydrolysis at physiological pH, this prodrug approach promises to overcome the enzymatic barrier to absorption of various peptides.     

Protooncogenes and growth factors associated with normal and abnormal liver growth

Hepatocyte replication during liver regeneration depends on extrinsic (circulating) and intrinsic (intrahepatic) factors. Two important growth factors produced in the regenerating liver are discussed, TGF, an autocrine, stimulatory growth factor, and TGF, a paracrine inhibitory factor. The balance between the activities of these factors is likely to play an important role in regulating hepatocyte proliferation. The experession of some protooncogenes occurs sequentially during the first few hours after partial hepatectomy and is a marker for the entry of hepatocytes into the cell cycle (proliferative competence). As hepatocytes become competent to proliferate, they respond to TGF and other growth factors and enter a proliferative phase. It is possible that TGF1 serves as a stop signal for liver regeneration but the mechanisms by which TGF inhibits hepatocyte DNA synthesis are still unknown.Presented at the Proceedings of the International Meeting on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989.     

Cyclosporin A induced internalization of the bile salt export pump in isolated rat hepatocyte couplets.

Isolated rat hepatocyte couplets were used to perform the comparative study of two widely used immunosuppressors, cyclosporin A (CsA) and tacrolimus (FK506) on hepatocanalicular function. We assessed canalicular function by counting the percentage of couplets that were able to accumulate the fluorescent cholephile, cholyl-lysyl-fluorescein (CLF), into the canalicular vacuole between the two cells, i.e., canalicular vacuole accumulation (CVA) of CLF. Compared to controls (DMSO-treated cells), CsA, in the approximate range of concentrations used therapeutically, caused inhibition of CVA of CLF, disorganization of the bile salt export pump (Bsep) localization at canalicular level resulting in its relocation into the cell, and disruption of the pericanalicular F-actin cytoskeleton. In contrast, FK506, at both approximately therapeutic and supratherapeutic concentrations, had no deleterious effect upon CVA of CLF, upon the localization of the bile salt transporter at the canalicular membrane, or on the organization of the pericanalicular F-actin cytoskeleton. These results point to transporter and cytoskeletal disorganization as contributors or determinants of CsA-induced cholestasis at canalicular level, whereas FK506 does not appear to produce these cholestasis-determining responses even at supratherapeutic concentrations.     

Comparative study of bioartificial liver support and plasma exchange for treatment of pigs with fulminant hepatic failure

Recently, bioartificial liver (BAL) treatment was reported to provide beneficial effects for patients with fulminant hepatic failure (FHF). Some success in experimental or clinical trials has been reported; however, the evaluation of BAL efficacy remains unclear, especially in comparison with other treatments for FHF. The purpose of this study was to compare the efficacy between BAL and plasma exchange (PE) in experimentally induced FHF in pigs. Pigs undergoing hepatic devascularization (HD) were placed into the following groups: no treatment (control; n = 6), BAL treatment (BAL; n = 5), and plasma exchange (PE; n = 5). Each treatment was initiated 6 h after HD and lasted for 4 h. BAL treatment significantly improved liver functions in FHF pigs. The decrease in cerebral perfusion pressure was also significantly suppressed in the pigs with BAL, and their survival time was prolonged compared with the results in pigs with PE. The effects of BAL outperform those of PE in the treatment of experimental FHF model.     

肝细胞生长因子及其受体c-met在卵巢上皮性肿瘤组织中的表达及意义

目的:研究肝细胞生长因子(hepatocytegrowthfactor,HGF)和cmet蛋白在卵巢上皮性肿瘤组织中的表达以及与临床病理特征的关系。方法:应用免疫组织化学SP法对45例卵巢上皮性恶性肿瘤及10例卵巢上皮性良性肿瘤组织中HGF和cmet蛋白的表达进行定位分析;应用蛋白印迹法检测HGF、cmet蛋白在卵巢上皮性肿瘤中的表达。结果:在卵巢癌组织中,HGF在上皮细胞的表达强于间质细胞,cmet在上皮细胞强表达,间质细胞无表达。HGF和cmet蛋白在卵巢癌组织中阳性率分别为60.0%(27/45)和73.0%(33/45),较良性肿瘤组织的阳性率20.0%(2/10)和50.0%(5/10)显著升高,P值分别为0.000和0.000。手术病理分期Ⅲ～Ⅳ期者HGF和cmet蛋白表达的阳性率为70.4%(19/27)和88.9%(24/27),较Ⅰ～Ⅱ期者的38.9%(7/18)和50.0%(9/18)显著升高,P值分别为0.048和0.000。淋巴结转移者阳性率分别为78.6%(11/14)和92.8%(13/14),较无淋巴结转移者阳性率51.6%(16/31)和64.5%(21/31)升高显著,P值分别为0.025和0.000。HGF和cmet蛋白表达阳性率与年龄和病理分级无关,P值分别为0.436、0.549、0.465和0.124。HGF和cmet蛋白相对表达强度在卵巢癌组织中为1.29±1.25和1.57±1.25,在良性肿瘤组织中为0.17±0.34和0.44±0.46,两组比较差异有统计学意义,P值分别为0.000和0.007。在卵巢癌组织中HGF和cmet蛋白的表达呈正相关性,P=0.009。结论:卵巢上皮性肿瘤组织中存在HGF和cmet蛋白的表达,HGF和cmet与卵巢癌的发生、侵袭和转移密切相关。