Hydrogen-Transfer-Mediated α-Functionalization of 1,8-Naphthyridines by a Strategy Overcoming the Over-Hydrogenation Barrier.

A general catalytic hydrogen transfer-mediated α-functionalization of 1,8-naphthyridines is reported for the first time that benefits from a hydrogen transfer-mediated activation mode for non-activated pyridyl cores. The pyridyl α-site selectively couples with the C8-site of various tetrahydroquinolines (THQs) to afford novel α-functionalized tetrahydro 1,8-naphthyridines, a class of synthetically useful building blocks and potential candidates for the discovery of therapeutic and bio-active products. The utilization of THQs as inactive hydrogen donors (HDs) appears to be a key strategy to overcome the over-hydrogenation barrier and address the chemoselectivity issue. The developed chemistry features operational simplicity, readily available catalyst and good functional group tolerance, and offers a significant basis for further development of new protocols to directly transform or functionalize inert N-heterocycles.

[Observation of the changes in ventral prostatic microcirculation in castrated rats].

OBJECTIVE: Castrated rats exhibit significant shrinkage of the ventral prostate and apoptosis of prostatic cells, which can be attributed to the reduced blood supply to the prostate. But what causes the blood decrease in the prostate remains unknown. This study aims to explore the molecular mechanism of the changes in the microcirculation of the ventral prostate of rats following castration. METHODS: We randomized 24 male adult rats into 6 groups of equal number, and collected their ventral prostates at 0, 1/2, 1, 2, 3 and 7 d, respectively, after castration. Then we observed the changes of the microvessels under the transmission electron microscope, detected the apoptosis of endothelial cells by TUNEL, and determined the expressions of VEGF, endostatin, angiostatin and angiopoietin-2 by Western blot. RESULTS: The castrated rats showed dramatic changes in the microvessels of the ventral prostate, obvious apoptosis of the endothelial cells, down-regulated expression of VEGF, and up-regulated expressions of endostatin and angiostatin, while angiopoietin-2 remained unchanged. CONCLUSION: The decreased level of VEGF and increased levels of endostatin and angiostatin might underlie the mechanism of the changes in the microcirculation of the ventral prostate of rats following castration.

Expression of cytochrome P450 arachidonic acid epoxygenase 2J2 in human tumor tissues and cell lines.

BACKGROUND AND OBJECTIVE: Cytochrome P450 arachidonic acid epoxygenase 2J2 (CYP2J2) is a new metabolic pathway of arachidonic acid. However, its biological effects, especially pathophysiologic significance in human beings, remain to be further recognized. This study was to determine the expression of CYP2J2 in human tumor tissues and cell lines. METHODS: The expression of CYP2J2 mRNA and protein in 130 specimens of human carcinoma and related adjacent normal tissues, four specimens of inflammatory pseudotumor tissues, eight human tumor cell lines and two normal cell lines (as control) was detected by reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunohistochemistry. RESULTS: CYP2J2 was highly expressed in 101 (78%) carcinoma tissues, but was not detected in adjacent normal tissues and inflammatory pseudo-tumor tissues. Its mRNA level was obviously correlated to its protein level (r = 0.613, p < 0.01). Immunohistochemistry analysis showed the same results as RT-PCR and western blot. Furthermore, CYP2J2 was only expressed in cancer cells but not in interstitial and inflammatory cells. CYP2J2 was highly expressed in all carcinoma cell lines, but not in two normal cell lines. CONCLUSION: CYP2J2 is highly and selectively expressed in human tumor tissues and cell lines and may be a novel biomarker of human tumors.

Intramuscular delivery of rAAV-mediated kallikrein gene reduces hypertension and prevents cardiovascular injuries in model rats.

AIM: The overexpression of the human tissue kallikrein (HK) gene can reduce blood pressure and ameliorate the secondary syndromes associated with hypertension in animal models. The current study was designed to investigate hypotensive effect of intramuscular delivery of HK gene. METHODS: We generated an recombinant adeno-associated virus (rAAV) vector expressing human tissue kallikrein under the control of a cytomegalovirus promoter and administered the rAAV-HK vector to a spontaneously hypertensive rat model at a dose of 1 x 10(10) virons/rat through intramuscular injection. RESULTS: A persistent, high-level expression of HK post-gene delivery was confirmed by ELISA. The systolic blood pressure in the rats receiving rAAV-LacZ and saline increased from 171.3 mmHg to 182.3 mmHg 28 weeks' post injection. In contrast, the delivery of the HK gene by AAV vectors attenuated the increase of the systolic blood pressure in the treated group. The systolic blood pressure was only slightly lowered (from a level of 174 mmHg to 170.5 mmHg) post-vector administration. The difference in blood pressure between the treated group and the control groups is statistically significant at 12.6 mmHg. The hypotensive effect of rAAV-HK persisted until the end of the testing period. In addition, a significant amelioration of cardiovascular hypertrophy, renal injury, and collagen depositions in the rAAV-HK-treated animals were also observed. CONCLUSION: All the effects are comparable with those of intravenous delivery. Therefore, the intramuscular administration of rAAV-HK may be used in gene therapy for hypertension.

[Antisense vascular endothelial growth factor suppressed corneal neovascularization in rats].

OBJECTIVE: To explore whether recombinant adeno-associated virus mediated antisense vascular endothelial growth factor (rAAV-aVEGF) gene transfer inhibits the development of corneal neovascularization (CNV) in a rat model. METHODS: rAAV-aVEGF(165) and recombinant adeno-associated virus mediated Lacz (rAAV-Lacz) were constructed by three-plasmid cotransfection methods. Forty-eight Sprague-Dawley (SD) rats were divided into two groups randomly. CNV were induced in vivo by alkaline cauterization of the central cornea. Twenty-four rats were injected with rAAV-aVEGF(165) (10(10) pfu/ml) into conjunctiva; another rats were injected with rAAV-Lacz (10(10) pfu/ml) into conjunctiva as controls. The Lacz gene expression was evaluated by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal) immunohistochemical staining. 1 to 30 days post-cautery, CNV was evaluated by morphometric analysis, and expression of VEGF was evaluated by immunohistochemistry. RESULTS: Two days later, 21.36% +/- 1.07% Lacz gene expression was detected in conjunctival sac in control group. 30 d later, 28.02% +/- 1.16% Lacz gene remained expression. Morphometric analysis and immunohistochemistry demonstrated rAAV-mediated antisense VEGF(165) significantly inhibited CNV. The VEGF(165) protein was decreased post-cautery compared to control group injected with. rAAV-Lacz (F = 1639.22, F = 2187.16, F = 719.17, P < 0.01). CONCLUSIONS: This study suggests that rAAV-aVEGF(165) can sufficiently inhibit the cautery-induced CNV, and the effect is associated with the inhibition of VEGF production.

Long-term modifications of blood pressure in normotensive and spontaneously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase.

Arachidonic acid cytochrome P-450 (CYP) hydroxylase 4A isoforms, including 4A1, 4A2, 4A3 and 4A8 in the rat kidney, catalyze arachidonic acid to produce 19/20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active metabolite, which plays an important role in the regulation of blood pressure. However, controversial results have been reported regarding the exact role of 20-HETE on blood pressure. In the present study, we used recombinant adeno-associated viral vector (rAAV) to deliver CYP 4A1 cDNA and antisense 4A1 cDNA into Sprague-Dawley (SD) rats and spontaneously hypertensive rats (SHR), respectively, to investigate the effects of long-term modifications of blood pressure and the potential for gene therapy of hypertension. The mean systolic pressure increased by 14.2+/-2.5 mm Hg in rAAV.4A1-treated SD rats and decreased by 13.7+/-2.2 mm Hg in rAAV.anti4A1-treated SHR rats 5 weeks after the injection compared with controls and these changes in blood pressure were maintained until the experiments ended at 24 weeks. In 4A1 treated animals CYP4A was overexpressed in various tissues, but preferentially in the kidney at both mRNA and protein levels. In anti-4A1-treated SHR, CYP4A mRNA in various tissues was probed, especially in kidneys, but 4A1 protein expression was almost completely inhibited. These results suggest that arachidonic acid CYP hydroxylases contribute not only to the maintenance of normal blood pressure but also to the development of hypertension. rAAV-mediated anti4A administration strategy has the potential to be used as targeted gene therapy in human hypertension by blocking expression of CYP 4A in kidneys.

Cytochrome P450 2J2 promotes the neoplastic phenotype of carcinoma cells and is up-regulated in human tumors.

Cytochrome P450 (CYP) arachidonic acid epoxygenase 2J2 converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids, which exert diverse biological activities in cardiovascular system and endothelial cells. However, it is unknown whether this enzyme highly expresses and plays any role in cancer. In this study, we found that very strong and selective CYP2J2 expression was detected in human carcinoma tissues in 101 of 130 patients (77%) as well as eight human carcinoma cell lines but undetectable in adjacent normal tissues and nontumoric human cell lines by Western, reverse transcription-PCR, and immunohistochemical staining. In addition, forced overexpression of CYP2J2, and CYP BM3F87V or addition of epoxyeicosatrienoic acids (EET) in cultured carcinoma cell lines in vitro markedly accelerated proliferation by analyses of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell accounts, and cell cycle analysis, and protected carcinoma cells from apoptosis induced by tumor necrosis factor alpha (TNF-alpha) in cultures. In contrast, antisense 2J2 transfection or addition of epoxygenase inhibitors 17-ODYA inhibited proliferation and accelerated cell apoptosis induced by TNF-alpha. Examination of signaling pathways on the effects of CYP2J2 and EETs revealed activation of mitogen-activated protein kinases and PI3 kinase-AKT systems and elevation of epithelial growth factor receptor phosphorylation level. These results strongly suggest that CYP epoxygenase 2J2 plays a previously unknown role in promotion of the neoplastic cellular phenotype and in the pathogenesis of a variety of human cancers.

Expression of cyclooxygenase-2 in human esophageal squamous cell carcinomas.

AIM: To determine whether cyclooxygenase-2 (COX-2) was expressed in human esophageal squamous cell carcinoma. METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, immunohistoc-hemistry and immunofluorescence were used to assess the expression level of COX-2 in esophageal tissue. RESULTS: COX-2 mRNA levels were increased by >80-fold in esophageal squamous cell carcinoma when compared to adjacent noncancerous tissue. COX-2 protein was present in 21 of 30 cases of esophageal squamous cell carcinoma tissues, but was undetectable in noncancerous tissue. Immunohistochemistry was performed to directly show expression of COX-2 in tumor tissue. CONCLUSION: These results suggest that COX-2 may be an important factor for esophageal cancer and inhibition of COX-2 may be helpful for prevention and possibly treatment of this cancer.

[Adeno-associated virus vector carrying human minidystrophin gene SMCKA3999 effectively ameliorates dystrophic pathology in mdx model mice].

OBJECTIVE: Duchenne muscular dystrophy (DMD) is the most common and letal genetic skeletal muscle disorder, caused by recessive mutations in the dystrophin gene and no treatment is available. The present paper is aimed to study if recombinant adeno-associated virus vector (rAAV) mediated dystrophin minigene SMCKA3999 could effectively ameliorates dystrophic pathology in mdx model mice. METHODS: We used dystrophin minigene SMCKA3999 to construct rAAVSMCKA3999. When injected into the skeletal muscle of mdx mice (DMD model), we adopted methods of immunofluorescent (IF) staining, Evans Blue and electromicroscopy to observe if rAAVSMCKA3999 could effectively ameliorates dystrophic pathology in mdx model mice. RESULTS: rAAVSMCK3999 resulted in efficient and stable expression and restoration of the missing dystrophin onto the plasma membrane. rAAVSMCKA3999 can also protect myofiber membrane integrity. For the first time we prove that rAAVSMCKA3999 can improve ultrastructure changes of DMD. CONCLUSION: We have demonstrated the effectiveness of rAAVSMCKA3999 in correcting pathology changes of skeletal muscle, which offer a promising avenue for DMD gene therapy.