Pro-apoptotic actions of exisulind and CP461 in SW480 colon tumor cells involve beta-catenin and cyclin D1 down-regulation

Exisulind and its analogues are inhibitors of cyclic GMP phosphodiesterases (PDEs) that have been shown to activate and induce protein kinase G, resulting in the induction of apoptosis in colon cancer cells. These drugs also reduce beta-catenin protein levels and decrease cyclin D1 mRNA levels in SW480 cells. Herein we report on studies pertaining to exisulind regulation of beta-catenin levels and activity in colon tumor cells. Exisulind and its higher-affinity PDE analogues, (Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)-indenylacetamide hydrochloride (CP461) and (Z)-1H-indene-3-acetamide, 5-fluoro-2-methyl-N-(phenylmethyl)-1-[(3,4,5-trimethoxyphenyl)methylene] (CP248), reduced beta-catenin, including the nuclear beta-catenin in SW480 cells (EC(50) approximately 200 microM, 1 microM, and <1 microM, respectively). The 50% reduction of beta-catenin was seen in 8-14 hr. There was no change in beta-catenin mRNA. Exisulind-induced beta-catenin reduction was blocked by the proteasomal inhibitor MG132 (Z-leu-Leu-Leu-CHO), indicating that the effect of exisulind involved ubiquitin-proteasomal degradation. A consequence of reduced beta-catenin in SW480 cells was that exisulind, CP461, and CP248 caused a concentration- and time-dependent decrease in cyclin D1 levels (EC(50) approximately 300 microM, 1 microM, and <1 microM, respectively) in 4 hr. The effect was via decreased cyclin D1 mRNA levels. Exisulind-induced degradation of beta-catenin was not blocked by the inhibition of caspase-3 activity and/or apoptosis, and some SW480 cells showed a reduction in beta-catenin levels before the appearance of early apoptosis indicators. Expression of the N-terminal 170 amino acid fragment of beta-catenin reduced the effects of beta-catenin degradation, cyclin D1 reduction, and the apoptosis response to exisulind. These results indicate that exisulind-induced beta-catenin degradation precedes the induction of apoptosis and that the down-regulation of inappropriate beta-catenin-activated genes accounts in part for the pro-apoptotic effects of exisulind and CP461 in colon tumor cells.     

Role of preoptic second messenger systems (cAMP and cGMP) in the febrile response

The present study aimed to test the hypothesis that a decrease in preoptic cAMP mediates fever. To this end, body core temperature (T(c)) of unanesthetized, freely moving rats was monitored by biotelemetry before and after pharmacological modulation of the cAMP pathway, and cAMP levels in the anteroventral third ventricular region (AV3V), where the preoptic region (POA) is located, were determined. We observed that intra-POA administration of the cAMP agonist dibutyryl-cAMP (Db-cAMP, 40 microg) reduced T(c). PGE(2) (the proximal mediator of fever, 200 ng) raised T(c) with a concomitant decrease in AV3V cAMP levels from 22.7+/-1.8 to 17.0+/-1.0 fmol/microg protein. Moreover, PGE(2)-induced fever was impaired by the phosphodiesterase inhibitor aminophylline. In order to verify the interaction between the cAMP- and cGMP-dependent pathways in the POA, we then co-injected Db-cAMP and 8-Br-cGMP into the POA. As a result, 8-Br-cGMP augmented the drop in T(c) evoked by Db-cAMP. Lastly, we observed that intra-POA co-microinjection of the protein kinase A inhibitor (Rp-cAMPS, 1 microg) with the protein kinase G inhibitor (Rp-cGMPS, 1 microg), mimicking the effects of reduced production of cAMP and cGMP, respectively, produced a fever-like response. In summary, the present data support that a decrease in the levels of cAMP and cGMP in the POA is associated with the genesis of fever.     

制药企业GMP改造中有关操作平台的设计问题

根据在对制药企业GMP改造中的实践，建议洁净区操作平台采用经抛光的304矩形钢管制作，既可达到GMP的要求且经济合理，并列表选出常用规格的承载数据供读者选用。     

Fetal ontogeny and role of metanephric bradykinin B2 receptors

Previous studies in rats have shown that blockade of bradykinin B₂ receptors (B₂R) in combination with a high-salt intake during gestation result in poor postnatal survival and long-term hypertension in the offspring. In this study, we examined the fetal ontogeny of B₂R and determined the consequences of gestational B₂R blockade and high salt on kidney development. B₂R gene expression is induced on embryonic day (E16) of fetal metanephrogenesis and remains sustained until term. The earliest expression of the B₂R protein is observed on apical membranes of ureteric bud branches and in capillary loop stage glomeruli. By the end of gestation, B₂R becomes restricted to more-differentiated tubules in the deep cortex and medulla. Pairs of rats on normal (0.12 mmol/g) or high (0.84 mmol/g) salt diets were mated at 14 weeks of age. The B₂R antagonist, Icatibant (previously known as Hoe-140) (300 nmol/kg per day) or saline (vehicle) was infused intraperitoneally during gestation via osmotic minipumps. Fetuses were examined on E20 (n=27–36 per group). No significant differences in litter size or body weight were observed among the groups. Combined high-salt and Icatibant treatment caused aberrant fetal renal development characterized by tubular dysgenesis, widened stromal mesenchyme, and glomerular cysts. The dysgenetic tubules stained positively for the distal nephron lectin, Dolichos biflorus, and exhibited enhanced Bax expression and apoptosis. Renal microvascular development, the number of mature glomeruli, and percentage of proliferating glomerular cells were not affected. Gestational Icatibant or high salt alone had no deleterious effects on fetal nephrogenesis. We conclude that gestational blockade of the kallikrein-kinin system impairs fetal nephrogenesis if combined with an intrauterine stressor such as high-salt intake. B₂R may play a protective role during segmental nephron differentiation. Received: 7 January 1999 / Revised: 25 February 1999 / Accepted: 26 February 1999     

Physiological significance of nitrergic transmission in human penile erection

The corpora cavemosa (CC) muscles of the human penis and their structural arrangements are essential for the physiology of erection. Contraction of this muscle causes detumescence, and relaxation, tumescence. The motor excitatory neurotransmission is adrenergic, acting through the alpha adrenoceptors. Continuous adrenergic transmitter (noradrenaline) release is necessary for the maintenance of non-erectile (contractile) state of the penis. The inhibitory neurotransmitter that relaxes CC muscle to produce erection is nitrergic i.e., the chemical messenger being nitric oxide (NO). The latter can also be released from cavernous endothelium. Presence of NO increases intracellular cGMP through activation of the enzyme guanylate cyclase. This causes relaxation of CC muscle. Phosphodiesterase type 5 (PDE5) is responsible for the degradation of cGMP and regulation of CC muscle tone. Specific PDE inhibitors such as sildenafil enhance the intracellular cGMP to improve erection. Increase in intracellular cAMP can also bring about pharmacological erection in man (eg. PGE1, papaverine and histamine). Inhibition of excessive adrenergic tone with appropriate alpha - adrenergic blocking agents (eg. phentolamine) can also contribute to the onset of pharmacological erection. ( Asian J Androl 2000 ; 2 : 51 - 56 )     

中药材加工与中药现代化

实现中药现代化，促进中药更广泛地为全人类做贡献是整个中药业应负的责任，尽管中药材加工业是被人们忽视的一部分，在整个中药业所占比重不大，但是，没有中药材加工业（包括中药饮片加工业）的现代化发展，不实施GMP管理也就谈不上实现中药现代化。     

AWD 140-190: a potent anticonvulsant in the amygdala-kindling model of partial epilepsy

PURPOSE: Evaluation of the effect of the new anticonvulsant drug, AWD 140-190 [4-(p-bromophenyl)-3-morpholino-1H-pyrrole-2-carboxylic acid methyl ester] on focally induced seizures and on epileptogenesis in the kindling model. METHODS: Effects of AWD 140-190 were studied in amygdala kindled rats after oral and intraperitoneal administration. In addition, the effect on kindling development was evaluated. In all experiments, behavioral changes in the rats in response to AWD 140-190 were monitored closely. RESULTS: AWD 140-190 exerted potent anticonvulsant activity against focal seizures. After intraperitoneal and oral administration in fully kindled rats, the substance dose-dependently increased the threshold for induction of afterdischarges starting at 15 mg/kg. AWD 140-190 only weakly influenced the seizure severity of the animals after stimulation at the elevated afterdischarge threshold current. No adverse effects were observed up to 30 mg/kg after intraperitoneal and oral administration in the open field and in the rotarod test. No differences were found between kindled and nonkindled rats when comparing neurotoxicity of AWD 140-190. Prolonged treatment with AWD 140-190 during kindling acquisition did not prevent kindling, but significantly retarded the development of fully kindled seizures during the treatment. CONCLUSIONS: This study demonstrates that AWD 140-190 has anticonvulsant effects in the amygdala kindling model in rats, suggesting that the substance is particularly effective against partial seizures. AWD 140-190 is orally active and devoid of neurotoxic effects in anticonvulsant doses, thus indicating that this compound has potential for antiepileptic therapy. AWD 140-190 retards the kindling development during the treatment. This effect could be explained by the acute anticonvulsant effect of the substance.     

Nitrite,nitrate and cGMP in the cerebrospinal fluid in degenerative neurologic diseases

Summary To investigate whether nitric oxide (NO) plays a role in degenerative neurologic disease (DND), we measured nitrite, nitrate and cyclic GMP in cerebrospinal fluid (CSF) samples from patients with Parkinson's disease (PD), spinocerebellar ataxia (SCA) and amyotrophic lateral sclerosis (ALS). We found no significant change in CSF nitrite, nitrate or cyclic GMP in patients with any DND compared with control values. These results suggest that NO production is preserved in PD, SCA and ALS.