To insight into genetic differences between chloroquine-resistant line (RC) and chloroquine-sensitive strain (N) of Plasmodium berghei. MethodsAfter continous cbloroquine-pressure upon RC line at higher dosage (50mg/kg. d) ,total RNAs from the RC line and the N strain of P. berghei were isolated for simplified differential display reverse transcript polymerase chain reaction (sDDRT-PCR ). The generated differential fragments had been repetitively rescued and identified by PCRs before one pair of suspected differential fragments (N25 and R25 )were cloned and sequenced. Then, their sequences were analyzed through PC-gene program and BLAST search. ResultsThough the identity of two nucleotide sequences of N252 and R251 ,cloned separately from the N25 and R25 fragments, were up to 99.8％ ,their predicted amino acid secondary structures were quite different due to multiple mutations. Compared with the published sequences in GeneBank,EMBL,DDBJ and PDB database ,no similar gene was found ,using BLAST search. However their partial nucleotide sequences (62nt from query 128nt to189nt bore highly homology to part sequence(from 1053nt to1114nt)of rattus norvegicus mRNA for phospholipase B,up to 93.5％ in N251 and 91.9％ in N252 respectively. ConclusionIt is feasible to isolate chloroquine-resistant related genes by using simplified DDRT-PCR combined repetitively rescuing and PCR identifying the interest differential DNAs together with sequence analyses.

Objective: To study the mechanism that erythrocytic stages Plasmodium berghei chloroquine resistant strain (RC strain) were eliminated in their hosts. Methods: Interactions of leukocytes with the parasites in mice livers infected with the RC strain or chloroquine sensitive strain (N strain) of P. berghei were studied by electron transmission microscopy. Results: None of leukocytes proliferated and infiltrated in mice livers infected with the N strain. Whereas in mice livers infected with the RC strain, proliferation and infiltration of monocyte macrophages, lymphocytes and nuetrophils occurred within portal areas and hepatic sinusoids with sequestration of numerous parasitized erythrocytes. The activated monocyte macrophages adhered to the parasitized red blood cells with their surface membranes. A lot of the parasites became crisis form within the parasitized erythrocytes which directly contacted with the phagocytes or not. Phagocytosis of free merozoites by macrophages was rarely revealed, but of the whole parasitized erythrocytes was not found. Conclusion: The crisis form of the parasites induced by the activated monocyte macrophages, rather than the direct phagocytolysis, is mainly responsible for the elimination of the P. berghei RC strain in their hosts. [

To investigate the effects of ceramide pathway on the inhibition of artesunate (Art) to hepatic fibrosis. LX-2 cells were divided into control group, Art treated group with 350 μmol/L, fumonisin B1 (FB1) treated group with 6 μmol/L, and Co-administration group of artesunate 350 μmol/L and fumonisin B1 6 μmol/L. There were 7 compound holes in each group. After 24 hours of treatment, the cells and supernatant were collected and detected. The expressions of homo sapiens longevity assurance homologue 2 (LASS2), peroxisome proliferators-activated receptors-γ (PPAR-γ) and Caspase-3 were evaluated by Western blot, the content of ceramide was evaluated by HPLC-FLD method, MTT assay was adopted to measure the rate of proliferation of LX-2 cells. The content of hydroxyproline was determined by digestive method. Compared with the control group, the expression of ceramide synthase protein and the ceramide content were increased significantly, the proliferation of LX-2 cells was inhibited significantly, the expressions of PPAR-γ and Caspase-3 protein were up-regulated and the secretion of hydroxyproline was inhibited in Art treated group (P＜0.05). In FB1 treated group, the protein expression of ceramide synthase and the ceramide content were decreased significantly, the proliferation of LX-2 cells was increased significantly, the expressions of PPAR-γ and Caspase-3 protein were down-regulated, and the secretion of hydroxyproline was increased (P＜0.05). Compared with the Art alone group, the combination of the two drugs could significantly reduce the effects of Art on the expression of ceramide synthase protein and the increase of ceramide content, and attenuate the effects of Art on the cell proliferation , PPAR-γ, Caspase-3 protein expression and hydroxyproline level of LX-2 cells (P＜0.05). Artesunate could inhibit hepatic fibrosis by increasing the content of ceramide through the ceramide synthase-ceramide pathway.

In order to further investigate the mechanisms of action of berberine (Ber), we assessed the effects of Ber on the mRNA expression of nitric oxide synthases (NOS) in rat corpus cavernosum. After incubation with Ber for 1 or 3 h respectively, the levels of NOS mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that there were iNOS and eNOS mRNA expressions in rat corpus cavernosum. Ber enhanced eNOS mRNA expression in rat penis, but exhibited no effect on the expression of iNOS mRNA (P > 0.05). The present study indicated that the relaxation of Ber involved the NO-cGMP signal transduction pathway. The enhancing effect of Ber on eNOS mRNA expression might associated with its relaxation of corpus cavernosum.
Berberine/*pharmacology ; Connective Tissue/physiopathology ; Nitric Oxide Synthase/*biosynthesis ; Nitric Oxide Synthase/genetics ; Nitric Oxide Synthase Type I/biosynthesis ; Nitric Oxide Synthase Type I/genetics ; Nitric Oxide Synthase Type III/biosynthesis ; Nitric Oxide Synthase Type III/genetics ; Penile Erection/*physiology ; Penis/*metabolism ; Penis/physiology ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics

The different effects of capsaicin on I(A) and I(K) currents in pain-conduct neurons of trigeminal ganglia (TG) were investigated. In cultured TG neurons of rats, whole-cell patch clamp techniques were used to record the I(A) and I(K) before and after capsaicin perfused. Results revealed that 1 micromol/L capsaicin could inhibit the amplitude of I(A) by 48.2% (n = 10, P < 0.05), but had no inhibitory effect on I(K) (n = 7, P > 0.05). Ten micromol/L capsaicin could significantly inhibit the amplitude of I(A) by 93.2% (n = 8, P < 0.01), but only slightly inhibit the amplitude of I(K) by 13.2% (n = 7, P < 0.05). Neither 1 micromol/L nor 10 micromol/L capsaicin had effects on the active curve of I(A) and I(K). It was concluded that capsaicin could selectively inhibit the I(A) current, and this effect might involve in the analgesic mechanisms of capsaicin.

To further investigate the mechanisms of action of icariin (ICA), we assessed the effects of ICA on the in vitro formation of cGMP and cAMP in isolated rabbit corpus cavernosum. Isolated segments of rabbit corpus cavernosum were exposed to increasing concentrations of ICA and the dose-dependent accumulation of cGMP and cAMP was determined in the tissues samples by means of 125I radioimmunoassay. Responses of the isolated tissues preparations to ICA were compared with those obtained with the reference compounds sildenafil (Sild). Furthermore, the effects of ICA on the mRNA expression of specific cGMP-binding phosphodiesterase type V (PDE5) in rat penis were also observed. After incubation with ICA for 6 h or 14 h respectively, the levels of PDE5 mRNA were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that ICA increased cGMP concentrations directly (P < 0.05), but there was no significant effect on cAMP concentrations (P > 0.05). In the presence of sodium nitroprusside (SNP), a stimulatory agent of cGMP, both ICA and Sild increased cGMP concentrations with increasing dose (P < 0.01). Their EC50 was 4.62 (ICA) and 0.42 (Sild) micromol/L respectively. Under the same condition, ICA and Sild unaltered cAMP level significantly (P > 0.05). There were PDE5A1 and PDE5A2 mRNA expressions in rat corpus cavernosum with PDE5A2 being the dominant isoform. ICA could obviously inhibit these two isoforms mRNA expression in rat penis, and decrease PDE5A1 more pronouncedly (P < 0.01). The present study indicated that the aphrodisiac mechanisms of icariin involved the NO-cGMP signal transduction pathway, with increasing cGMP levels in the corpus cavernosum smooth muscle. The inhibitory effect of icariin on PDE5 mRNA expression, especially on PDE5A1, might account for its molecular mechanisms for its long-term activity.

Aims To study the effects of clenbuterol on anoxia/reoxygenation( A/R) injury in neonatal Wistar rat cardiomyocytes and to explore whether its mecha-nism is related to reperfusion injury salvage kinase ( RISK) or not. Methods The cultured primary neo-natal cardiomyocytes were randomly divided into eight groups: ①normal culture group; ②anoxia/reoxygen-ation( A/R) group;③ clenbuterol ( 1 μmol · L-1 ) +A/R;④ICI118,551(10 μmol·L-1) + clenbuterol ( 1 μmol · L-1 ) + A/R; ⑤Metoprolol ( 10μmol · L-1 ) + clenbuterol(1μmol·L-1 ) + A/R group;⑥Metoprolol ( 10 μmol · L-1 ) + A/R group; ⑦PD98059 ( 20 μmol · L-1 ) + clenbuterol ( 1 μmol · L-1 ) + A/R group;⑧ LY294002(10 μmol·L-1 ) +clenbuterol(1 μmol · L-1 ) + A/R group. Cell via-bility was determined by the conventional MTT reduc-tion assay. The content of LDH in cultured medium was measured with colorimetry. Cardiomyocyte apopto-sis was determined by Hoechst33342 . Intracellular re-active species( ROS) were monitored by the fluorescent DCFH-DA. Total ERK2 and phosphorylated ERK were detected by western blot. Results Compared with A/R group, clenbuterol significantly increased vaibility of cells, reduced LDH release, lowered the rate of apop-tosis and ROS production. When addedβ2 receptor an-tagonist ICI118 , 551 , PI3 K inhibitor LY294002 and ERK inhibitor PD98059 , the effects of clenbuterol a-bove were inhibited; but β1 receptor antagonist Meto-prolol protected the cardiomyocytes from A/R injury, as evidenced by decreased LDH release and increased cell viability. There were no synergistic effects in the combined use of clenbuterol and Metoprolol. Conclu-sion clenbuterol exerts cardioprotective effects against A/R injury by inhibiting oxidative stress and apopto-sis. The protection of clenbuterol is inhibited by ICI118 , 551 , LY294002 and PD98059 . clenbuterol protects cardiomyocytes against A/R injury via RISK pathway by activation of β2 receptor.

In order to further investigate the mechanisms of action of berberine (Ber), we assessed the effects of Ber on the mRNA expression of nitric oxide synthases (NOS) in rat corpus cavernosum. After incubation with Ber for 1 or 3 h respectively, the levels of NOS mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that there were iNOS and eNOS mRNA expressions in rat corpus cavernosum. Ber enhanced eNOS mRNA expression in rat penis, but exhibited no effect on the expression of iNOS mRNA (P＞0.05). The present study indicated that the relaxation of Ber involved the NO-cGMP signal thansduction pathway. The enhancing effect of Ber on eNOS mRNA expression might associated with its relaxation of corpus cavernosum.

OBJECTIVE: To investigate the expression and clinical significance of BUB1 protein in laryngeal squa mous cell carcinoma (LSCC) tissues. METHOD: The expression of BUB1 protein was measured by immunohistochemistry (EliVision two steps) in 55 cases of LSCC tissues,30 cases of adjacent normal laryngeal mucosa tissues, the relationship of BUB1 expression with clinical pathologic factors in LSCC was also analyzed. RESULT: The positive rate of BUB1 protein in LSCC tissues (50.9%, 28/55) was significantly lower than that in adjacent normal laryngeal mucosa tissues (83.3%, 25/30), P < 0.01. The positive rate of BUB1 protein was correlated with differentiation grading and lymph node metastasis of LSCC (P < 0.05, respectively), but not with patient's sex, age, smoking, tumor site, T-stage and clinical stage(P > 0.05, respectively). CONCLUSION The low expresssion of BUB1 protein is closely related to the tumorigenesis and development of LSCC, and can be a molecular marker for prognosticating metastasis and prognosis of LSCC.
Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Laryngeal Mucosa ; metabolism ; Laryngeal Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Prognosis ; Protein-Serine-Threonine Kinases ; metabolism

Objective To measure the pressure-volume changes and uroflow rate,and assess the function of upper urinary tract in sigmoidorcctal pouch patients.Methods Between 2007 and 2011,a to-tal of 10 patients who had a radical cystectomy because of muscle-invasive bladder tumor underwent a Mainz pouch Ⅱ procedure were evaluated.The ureters were implanted into the post walls of the detubularized sigmoid segment at least 30 cm in length.In all cases,sigmoidoscopy was done and the anal pressure was measured preoperatively,and video urodynamic study of sigmoidorectal pouch as well as the intravenous pyelography was conducted after 3 to 6 months postoperatively.Results Video urodynamic study of sigmoidorectal pouch revealed that no reflux up to an average volume of 360 ml (270-532 ml) of the descending colon.The sigmoidal colon pressure was 26 cm H2O during the 3 to 6 months post-operative follow-up.The anal sphincter pressure was 90 cm H2O on average preoperatively and did not change postoperatively.The renal function and upper urinary tract were preserved well.The Qmax was 30 ml/s and the average uroflow rate was 8 ml/s under abdominal strain.All the patients suffered slight incontinence in the first two months and became continent since the 3 month postoperatively with nocturnal voiding one to four times.Conclusions The sigmoidorectal pouch provides a reservoir with a higher capacity,lower pressure without reflux to the upper urinary tract and descending colon and lower metabolic acidosis problem.It is also a good alternative diversion procedure that would preserve upper urinary tract and good quality of life.