Catechol oxidation by peroxidase-positive astrocytes in primary culture: an electron spin resonance study.
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In rodents, chronic estrogenization has been shown to induce degeneration of dendrites and myelin figures in the hypothalamic arcuate nucleus adjacent to peroxidase-positive astrocyte processes. Because in this brain region estradiol is metabolized to 2-hydroxyestradiol (catecholestrogen), we hypothesized that the latter may be oxidized by the astrocytic peroxidase activity to cytotoxic ortho-semiquinones as occurs in peripheral tissues. Cysteamine induces nonenzymatic peroxidase activity in cultured astroglia identical to that observed in vivo. Using electron spin resonance, we demonstrate robust peroxidase-catalyzed oxidation of 2-hydroxyestradiol and dopamine by cysteamine-pretreated astrocyte cultures relative to untreated controls. These results implicate the peroxidase-positive astrocytes in the pathogenesis of estradiol-related hypothalamic damage, parkinsonism, and other free-radical-related neurologic disorders. (+info)
Human PEP-1-ribosomal protein S3 protects against UV-induced skin cell death.
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The consequences of ultraviolet (UV) exposure are implicated in skin aging and cell death. The ribosomal protein S3 (rpS3) is one of the major proteins by which cells counteract the deleterious effects of UV and it plays a role in the repair of damaged DNA. In the present study, we investigated the protective effects of PEP-1-rpS3 fusion protein after UV-induced cell injury. A human rpS3 gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-rpS3 fusion protein. The expressed and purified fusion proteins were efficiently transduced into skin cells in a time- and dose-dependent manner. Once inside the cells, transduced PEP-1-rpS3 fusion protein was stable for 48h. We showed that transduced PEP-1-rpS3 fusion protein increased cell viability and dramatically reduced DNA lesions in the UV exposed skin cells. Immunohistochemical analysis revealed that PEP-1-rpS3 fusion protein efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin. These results suggest that PEP-1-rpS3 fusion protein can be used in protein therapy for various disorders related to UV, including skin aging and cancer. (+info)
Pharmacological studies on lappaconitine: antinociception and inhibition of the spinal action of substance P and somatostatin.
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The pain response of mice to an injection of 0.5% formalin into the dorsal surface of a hindpaw is biphasic, with a first phase lasting for 5 min and a second phase lasting from 10 to 30 min post-injection. Intrathecal (i.t.) injection of [D-Pro2, D-Trp7,9]-substance P inhibited the first phase, and i.t. cysteamine inhibited the second phase. Lappaconitine (LA) and morphine (MOR) inhibited both phases equally in a dose-dependent manner. Diclofenac inhibited both phases, but the second phase was inhibited by lower doses. An i.t. injection of substance P (SP) or somatostatin (SOM) produced a characteristic behavioral response (scratching, biting, and licking). This behavioral response to SP and SOM was inhibited by s.c., intracerebroventricular (i.c.v.), or i.t. injection of MOR. In contrast, LA inhibited the SP- and SOM-induced response when injected s.c. or i.c.v., but had no effect when injected intrathecally. These results indicate that LA may act supraspinally to inhibit the transmission of nociceptive information by SP and/or SOM. (+info)
Pharmacokinetics of cysteamine bitartrate following gastrointestinal infusion.
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AIMS: Although cysteamine was first used in the treatment of cystinosis in 1976 and approved by the FDA as cysteamine bitartrate (Cystagon) in 1994, surprisingly little pharmacological data are available for this compound. Cysteamine and its related drugs are currently being evaluated for the treatment of Huntington's and Parkinson's disease. The aim of te study was to understand the pharmacokinetics of cysteamine bitartrate following gastrointestinal infusion. METHOD: Cysteamine bitartrate was delivered through a naso-enteric catheter into the stomach (n = 8), small intestine (n = 8) and caecum (n = 4) of normal subjects. Plasma cysteamine concentrations were determined using LC-MS/MS. RESULTS: The rate and extent of drug absorption were assessed by comparing AUC(0, infinity), C(max) and t(max), among the gastrointestinal infusion sites. Total cysteamine exposure, expressed as area under the curve (AUC(0, infinity)) was greatest when the drug was infused into the small intestine (4331.3 +/- 1907.6 min x microM) followed by stomach (3901.9 +/- 1591.9 min x microM) and caecum (3141.4 +/- 1627.6 min x microM). Cysteamine infusion into the small intestine resulted in the most rapid rise to maximal plasma concentrations (t(max) = 21 +/- 0.56 min); t(max) was delayed to 50 +/- 26 min and 64 +/- 26 min after gastric and caecal infusion, respectively. The maximum cysteamine plasma concentration (C(max)) was reached after infusion of the drug into the small intestine (51 +/- 21 microM), which was higher than plasma C(max) concentrations after gastric (39 +/- 16 microM) and caecal infusion (23 +/- 15 microM). CONCLUSIONS: The pharmacokinetic data generated help extend our understanding of cysteamine. (+info)
Modification of maturation condition improves oocyte maturation and in vitro development of somatic cell nuclear transfer pig embryos.
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This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or beta-mercaptoethanol (beta-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 microM CYS or 100 microM beta-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. beta-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos. (+info)
Transduced PEP-1-Grb7 fusion protein suppressed LPS-induced COX-2 expression.
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Although the incidence and severity of atopic dermatitis (AD) is steadily increasing at an alarming rate, its pathogenic mechanisms remain poorly understood yet. Recently, we found that the expression of Grb7 protein was markedly decreased in AD patients using proteomic analysis. In the present study, human Grb7 gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-Grb7 fusion protein. The expressed and purified PEP-1-Grb7 fusion proteins transduced efficiently into skin cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-Grb7 protein was stable for 48 h. In addition, transduced PEP-1-Grb7 fusion protein markedly increased cell viability in macrophage RAW 264.7 cells treated with LPS by inhibition of the COX-2 expression level. These results suggest that the PEP-1-Grb7 fusion protein can be used in protein therapy for inflammatory skin disorders, including AD. (+info)
Cysteamine protects gastric epithelial cell monolayers against drug induced damage: evidence for direct cellular protection by sulphydryl compounds.
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The sulphydryl containing drug cysteamine protects gastric mucosa in vivo against acute injury. It is not known whether this protection includes a direct effect on gastric cells. Using gastric epithelial cell monolayers derived from a well differentiated human cell line, we evaluated whether cysteamine protects against taurocholate or indomethacin induced damage in conditions which completely exclude the influence of vascular, hormonal, and neural factors. The effect of cysteamine on prostaglandin production by monolayer cells in vitro was also assessed. Cysteamine decreased damage brought about by sodium taurocholate and indomethacin by 40% (p less than 0.01) and 50% (p less than 0.01) respectively. The sulphydryl blocker iodoacetamide prevented the protective effect of cysteamine. Pretreatment with indomethacin, which inhibited prostaglandin E2 output by 60%, did not prevent protection by cysteamine; incubation with cysteamine decreased prostaglandin E2 production by cultured cells. We conclude that (i) cysteamine directly protected gastric epithelial cells in vitro (ii) this protection occurred with indomethacin, which interferes with cellular metabolism of prostaglandins, and taurocholate, whose damaging action at neutral pH is unrelated to interference with prostanoid metabolism, (iii) cysteamine protection in vitro is unrelated to endogenous prostaglandins and is probably mediated by endogenous sulphydryl compounds. (+info)
A fall in duodenal PGE2 synthesis precedes histological changes and ulceration in the cysteamine model of duodenal ulceration.
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The temporal association of histological damage and mucosal prostaglandin E2 (PGE2) synthesis was examined in the cysteamine model of duodenal ulceration in the rat. Four groups of 10 animals were used; a control group (which had a light ether anaesthetic, an i.m. injection of vehicle and were killed 18 h later) and three treatment groups (which were similarly treated but injected with cysteamine (0.3 mg/g body weight) and killed at 6, 18 or 24 h). Estimates of duodenal PGE2 synthesis and histological assessment of mucosal damage were performed. There were four early deaths in the 24-h group due to intra-thoracic haemorrhage. The 6-h group appeared normal macroscopically. There were seven established and three early ulcers in the 18-h group and two established and four early ulcers in the survivors of the 24-h group. Microscopically, the 6-h group did not differ from controls, but the 18 and 24-h groups showed evidence of mucosal damage compared to the control group (P less than 0.001). All three treatment groups synthesized less PGE2 than did the control group (P less than 0.001). Thus at 6-h post-injection, a decrease in PGE2 synthesis was evident but mucosal damage occurred later. Histological features correlated negatively with PGE2 synthesis (P less than 0.001). (+info)