Demethylenetetrahydroberberine protects dopaminergic neurons in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a multifactorial disorder of the nervous system where a progressive loss of dopaminergic neurons exist. However, the pathogenesis of PD remains undefined, which becomes the main limitation for the development of clinical PD treatment. Demethylenetetrahydroberberine (DMTHB) is a novel derivative of natural product berberine. This study was aimed to explore the neuroprotective effects and pharmacological mechanism of DMTHB on Parkinson's disease using C57BL/6 mice. A PD model of mice was induced by administration of MPTP (20 mg·kg-1) and probenecid (200 mg·kg-1) twice per week for five weeks. The mice were administered with DMTHB daily by gavage at the dose of 5 and 50 mg·kg-1 for one- week prophylactic treatment and five-week theraputic treatment. The therapeutic effects of DMTHB were evaluated by behavior tests (the open field, rotarod and pole tests), immunohistochemical staining of tyrosine hydroxylase (TH), Nissl staining and biochemical assays. The molecular mechanisms of DMTHB on the key biomarkers of PD pathological states were analyzed by Western blot (WB) and qRT-PCR. DMTHB treatment alleviated the behavioral disorder induced by MPTP-probenecid. Nissl staining and TH staining showed that the damage of dopaminergic neurons in the substantia nigra was remarkably suppressed by DMTHB treatment. Western blot results showed that the ratio of Bcl-2/Bax and TH increased, but the level of α-synuclein (α-syn) was remarkably reduced, which indicated that the apoptosis of dopaminergic neurons in mice was significantly reduced. The protein phosphorylation of p-PI3K, p-AKT and p-mTOR also increased about 2-fold, compared with the model group. Furthermore, qRT-PCR results demonstrated that the mRNA levels of pro-inflammatory cytokines, IL-1ß and TNF-α, were reduced, but the level of anti-inflammatory cytokine IL-10 increased after DMTHB treatment. Finally, the cellular assay displayed that DMTHB was also a strong antioxidant to protect neuron cell line PC12 by scavenging ROS. In this study, we demonstrated DMTHB alleviates the behavioral disorder and protects dopaminergic neurons through multiple-target effects includubg anti-apoptotic, anti-inflammatory and antioxidant effects.

[Tradeoffs and synergies of ecosystem services in western mountainous China: A case study of the Bailongjiang watershed in Gansu, China]. / è¥¿éƒ¨å±±åŒºæµåŸŸç”Ÿæ€ç³»ç»ŸæœåŠ¡æƒè¡¡ä¸ŽååŒå ³ç³»­­ä»¥ç”˜è‚ƒç™½é¾™æ±ŸæµåŸŸä¸ºä¾‹.

The Bailongjiang watershed of Gansu is an important water conservation and ecological barrier area in the upper reaches of Yangtze River. It is necessary to reveal the tradeoffs and synergies of ecosystem services (ESs) for the "win-win" of watershed ecological system and social eco-nomy development. Based on the InVEST model, four typical ESs including soil conservation (SC), water conservation (WC), food supply (FS), and habitat quality (HQ) were assessed, and the multi-scale tradeoffs and synergies of ESs and its drivers were analyzed by correlation and root mean square deviation (RMSD). The results showed that there were significant synergies among SC, WC, and HQ, and a significant tradeoff between FS and HQ, SC, WC, respectively. The areas with high tradeoff intensity between the three pairs of synergistic services (SC-WC, SC-HQ, WC-HQ), and between FS and HQ were mainly concentrated in the steep forest area of middle-high mountain in Wenxian, Diebu and Zhouqu. The high intensity of tradeoffs between FS-SC, FS-WC were mainly concentrated in the gentle apricus farming and pastoral areas of middle-low mountain in Tanchang and Wudu. The spatial variation of land use/cover caused by human activities was an important factor affecting the degree of ES tradeoffs and its scale effect.

Effect of BTXA on Inhibiting Hypertrophic Scar Formation in a Rabbit Ear Model.

BACKGROUND: Hypertrophic scar (HS) is a refractory skin disease caused by major physical damage or other inflammation. Some reports found that botulinum toxin type A (BTXA) could be an alternative treatment of the HS. Therefore, the authors studied the effects of BTXA on the treatment of HS and the dose response of BTXA. METHODS: Hypertrophic scars were harvested from the ears of 18 young adult New Zealand big-eared rabbits and treated with BTXA or triamcinolone acetonide (TAC) in vivo experiment. The hypertrophic index (HI) was measured by histological examination. Collagen fibrils were checked by sirius red straining, and the cell nucleuses of fibroblasts were checked by Ki67. RESULTS: The HI of hypertrophic scars with BTXA treatment was lower than that with phosphate-buffered saline treatment (P < 0.05). Compared with the TAC treatment group, the efficacy of treatment with the middle dose of BTXA (1.0, 1.5 IU) had no significant difference, as shown by sirius red staining and immunohistochemistry Ki67. CONCLUSION: These results demonstrated that BTXA effectively improved the appearance of hypertrophic scars and inhibited the formation of collagen fibrils and fibroblasts in vivo. Treatment with the middle dose of BTXA achieved similar efficacy as TAC treatment, indicating that BTXA might be useful for inhibiting hypertrophic scars and worth investigating further. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

De novo design of VEGFR-2 tyrosine kinase inhibitors based on a linked-fragment approach.

Vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors have been demonstrated to possess substantial antitumor activity. VEGFR-2 tyrosine kinase inhibitors are crucial for development of antitumor drugs. Based on the crystal structure of VEGFR-2 tyrosine kinase, a linked-fragment strategy was employed to design novel VEGFR-2 tyrosine kinase inhibitors, and 1000 compounds were generated in this process. Absorption, distribution, metabolism, excretion and toxicity (ADMET) were used to screen the 1000 compounds, and 59 compounds were acceptable. Scaffold hopping was then used for further screening, and only four compounds were obtained in this way. Then, the binding energy of the four molecules to VEGFR-2 tyrosine kinase was calculated using molecular docking, and their values were found to be lower than that of Sorafenib. Finally, molecular dynamics simulations were performed on the complex of the compound with the lowest binding energy with VEGFR-2 tyrosine kinase, and the binding model was analyzed. At the end, four chemical entities with novel structures were obtained, and were suggested for experimental testing in future studies.

QSAR and molecular docking studies on oxindole derivatives as VEGFR-2 tyrosine kinase inhibitors.

The three-dimensional quantitative structure-activity relationships (3D-QSAR) were established for 30 oxindole derivatives as vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors by using comparative molecular field analysis (CoMFA) and comparative similarity indices analysis comparative molecular similarity indices analysis (CoMSIA) techniques. With the CoMFA model, the cross-validated value (q(2)) was 0.777, the non-cross-validated value (R(2)) was 0.987, and the external cross-validated value ([Formula: see text]) was 0.72. And with the CoMSIA model, the corresponding q(2), R(2) and [Formula: see text] values were 0.710, 0.988 and 0.78, respectively. Docking studies were employed to bind the inhibitors into the active site to determine the probable binding conformation. The binding mode obtained by molecular docking was in good agreement with the 3D-QSAR results. Based on the QSAR models and the docking binding mode, a set of new VEGFR-2 tyrosine kinase inhibitors were designed, which showed excellent predicting inhibiting potencies. The result revealed that both QSAR models have good predictive capability to guide the design and structural modification of homologic compounds. It is also helpful for further research and development of new VEGFR-2 tyrosine kinase inhibitors.

Production of herbicide-resistant medicinal plant Salvia miltiorrhiza transformed with the bar gene.

In this study, we successfully performed Agrobacterium-mediated genetic transformation of Salvia miltiorrhiza and produced herbicide-resistant transformants. Leaf discs of S. miltiorrhiza were infected with Agrobacterium tumefaciens EHA105 harboring pCAMBIA 3301. The pCAMBIA 3301 includes an intron-containing gus reporter and a bar selection marker. To increase stable transformation efficiency, a two-step selection was employed which consists of herbicide resistance and gus expression. Here, we put more attention to the screening step of herbicide resistance. The current study provides an efficient screening system for the transformed plant of S. miltiorrhiza harboring bar gene. To determine the most suitable phosphinothricin concentration for plant selection, non-transformed leaf discs were grown on selection media containing six different phosphinothricin concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/l). Based on the above results of non-transformed calluses, the sensitivity of phosphinothricin (0, 0.4, 0.8, 1.2, 1.6 mg/l) was tested in the screening of transgenic S. miltiorrhiza. We identified that 0.6 mg/l phosphinothricin should be suitable for selecting putatively transformed callus because non-transformed callus growth was effectively inhibited under this concentrations. When sprayed with Basta, the transgenic S. miltiorrhiza plants were tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to S. miltiorrhiza.

The unbinding studies of vascular endothelial growth factor receptor-2 protein tyrosine kinase type II inhibitors.

Vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase has two conformations, active and inactive conformations. Type II inhibitors bind to inactive conformation. It has two possible binding/unbinding paths. To explore the unbinding path of inhibitor 01-435 that was generated by fragment build in the binding pocket of VEGFR-2, molecular dynamics (MD) simulation was performed on the crystal structure of VEGFR-2 in complex with 01-435, then steered molecular dynamics (SMD) simulation was executed on the crystal structure of VEGFR-2 in complex with 01-435. Pull force, van der Waals and electrostatic interaction along the two paths were calculated by using SMD simulation. The SMD simulation results indicate that the more favorable path for inhibitor dissociation is along with the traditional ATP-channel rather than the allosteric-pocket-channel, which is mainly due to the less electrostatic interaction that the ligand suffers during dissociation process along the traditional ATP-channel.

3-[(E)-2-Chloro-3,3,3-trifluoro-prop-1-en-1-yl]-N-(2-fluoro-phen-yl)-2,2-dimethyl-cyclo-propane-1-carboxamide.

The phenyl ring in the title compound, C(15)H(14)ClF(4)NO, makes a dihedral angle of 80.3 (3)° with the cyclo-propane ring. In the crystal, mol-ecules are linked by N-H⋯O hydrogen bonds into chains running along the a axis.

3-[(E)-2-Chloro-3,3,3-trifluoro-prop-1-en-1-yl]-N-(2-chloro-phen-yl)-2,2-dimethyl-cyclo-propane-1-carboxamide.

In the title compound, C(15)H(14)Cl(2)F(3)NO, synthesized by the reaction of 3-[(E)-2-chloro-3,3,3-trifluoro-prop-1-en-yl]-2,2-dimethyl-cyclo-propane-carb-oxy-lic acid and 2-chloro-aniline, the aromatic ring makes a dihedral angle of 76.7 (3)° with the plane of the cyclo-propane ring. In the crystal, inter-molecular N-H⋯O hydrogen bonds link the mol-ecules into chains running along the b axis.

(E)-2-(2-Chloro-3,3,3-trifluoro-prop-1-en-yl)-N-(2,4-dimethyl-phen-yl)-3,3-dimethyl-cyclo-propane-1-carboxamide.

The title compound, C(17)H(19)ClF(3)NO, crystallizes with three mol-ecules in the asymmetric unit. The aromatic ring makes dihedral angles of 38.69 (13), 46.68 (12) and 50.52 (11)° with the plane of the cyclo-propane ring in the three mol-ecules. The crystal packing is stabilized by inter-molecular N-H⋯O hydrogen bonds.

[Visfatin promotes production of monocyte chemotactic protein-1 and interleukin-6 in human endothelial cells via insulin receptor].

OBJECTIVE: Visfatin is a new novel proinflammatory adipocytokine affecting insulin resistance by binding to insulin receptor. To investigate whether visfatin stimulates monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) production in human umbilical vein endothelial cells (HUVEC) and mediates insulin receptor (IR) is involved in are not known. METHODS: Cultured HUVEC was treated with different doses and durations of visfatin. Furthermore, HUVEC was pretreated with hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester (HNMPA-(AM)3), a specific inhibitor of IR followed by visfatin (100 ng/ml) treatment. Enzyme-linked immunosorbent assay (ELISA) were used to measure MCP-1 and IL-6 production in HUVEC. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) was used for determining MCP-1 and IL-6 mRNA expression. RESULTS: Visfatin significantly dose- and time- dependently up-regulated protein production of MCP-1 and IL-6 in HUVEC. We therefore found visfatin- induced MCP-1 and IL-6 production and gene expression in HUVEC were inhibited by HNMPA-(AM)3. CONCLUSION: Visfatin induces endothelial MCP-1 and IL-6 production in HUVEC in a dose and time-dependently manner. This action appears to be mediated via insulin receptor pathway.

(E)-3-(2-Chloro-3,3,3-trifluoro-prop-1-en-yl)-2,2-dimethyl-N-p-tolyl-cyclo-propane-carboxamide.

There are two mol-ecules in the asymmetric unit of the title compound, C(16)H(17)ClF(3)NO. The benzene ring in each mol-ecule makes a dihedral angle of 66.6 (3)° [116.3 (4)° in the second mol-ecule] with the plane of the cyclo-propane ring. The F atoms of the CF(3) groups are disordered equally over two positions. The amide hydrogen is linked with the amide oxygen in another mol-ecule by an inter-molecular N-H⋯O hydrogen bond. The packing can be described as a dimeric arrangement of mol-ecules linked through N-H⋯O hydrogen bonds.

(E)-3-(2-Chloro-3,3,3-trifluoro-prop-1-en-yl)-2,2-dimethyl-N-(2-naphth-yl)cyclo-propane-carboxamide.

The title compound, C(19)H(17)ClF(3)NO, was synthesized from 3-[(E)-2-chloro-3,3,3-trifluoro-prop-1-en-yl]-2,2-dimethyl-cyclopropane-carboxylic acid and 2-aminona-phthalene. There are two molecules in the asymmetric unit. The dihedral angle between the naphthalene and cyclo-propane units is 111.6 (5)°. Molecules are connected into chains by intermol-ecular N-H⋯O hydrogen bonds. One of the Cl atoms is disordered over two positions with occupancies 0.653 (15) and 0.347 (15).

AOP-1 interacts with cardiac-specific protein kinase TNNI3K and down-regulates its kinase activity.

In the present study, a yeast two-hybrid screening system was used to identify the interaction partners of cardiac troponin I-interacting kinase (TNNI3K) that might serve as regulators or targets, and thus in turn to gain some insights on the roles of TNNI3K. After screening the adult heart cDNA library with a bait construct encoding the ANK motif of TNNI3K, antioxidant protein 1 (AOP-1) was isolated. The interaction between TNNI3K and AOP-1 was confirmed by the in vitro binding assay and coexpression experiments in vivo. The colocalization of TNNI3K and AOP-1 was clarified by confocal immunofluorescence. Moreover, coexpression of AOP-1 inhibited TNNI3K kinase activity in the in vitro kinase assay.

(E)-3-(2-Chloro-3,3,3-trifluoro-prop-1-en-yl)-N-(3-methoxy-phen-yl)-2,2-dimethyl-cyclo-propane-carboxamide.

The title compound, C(16)H(17)ClF(3)NO(2), was synthesized from 3-[(E)-2-chloro-3,3,3-trifluoro-prop-1-en-yl]-2,2-dimethyl-cyclo-propane-carboxylic acid and 3-methoxy-benzenamine. The propenyl and carboxamide substituents lie on the same side of the cyclo-propane ring plane, with the two methyl substituents on either side of the plane. The benzene ring makes a dihedral angle of 76.4 (3)° with the plane of the cyclo-propane ring. The crystal structure involves intermolecular N-H⋯O hydrogen bonds.

(E)-3-(2-Chloro-3,3,3-trifluoro-prop-1-en-yl)-2,2-dimethyl-N,N-diphenyl-cyclo-propane-carboxamide.

The title compound, C(21)H(19)ClF(3)NO, was synthesized from 3-[(E)-2-chloro-3,3,3-trifluoro-prop-1-en-yl]-2,2-dimethyl-cyclo-propane-carboxylic acid and diphenyl-amine. The propenyl and carboxamide substituents lie on the same side of the cyclo-propane ring plane, with the two methyl substituents on either side of the plane. The phenyl rings of the carboxamide are inclined at an angle of 84.6 (3)° to one another. The F atoms are disordered over two positions; the site occupancy factors are ca 0.6 and 0.4.

[Analysis of 216 cases of total laparoscopic hysterectomy].

OBJECTIVE: To investigate the effectiveness and safety of total laparoscopic hysterectomy (TLH). METHODS: A retrospective study of laparoscopic hysterectomy was conducted in this setting. From March 2002 through March 2004, 216 women were subjected to TLH. The average age of the patients was 45.5 years (38-60 years). Out of the 216 patients, 24 had dysfunctional uterine bleeding, 5 atypical endometrial hyperplasia, 139 uterine fibroid, 46 adenomyosis, 2 cervical carcinoma in situ and 36 had a previous lower abdominal surgery. The TLH was carried out using ultrasonic scalpel and the amputated uterus was removed transvaginally. The vagina and peritoneum were closed under laparoscopy. RESULTS: Of the 216 cases who underwent TLH, 23 had bilateral adnexectomy, 36 had ovarian cystectomy, and 54 had adhesiolysis simultaneously. No case was converted to laparotomy. The mean operating time was (103 +/- 35) min. The average amount of blood loss was 83 +/- 45 ml (60-320 ml) during operation. The average hospital stay after operation was (5.3 +/- 1.9) days. There were 4 patients with urinary tract injury in this study population. One bladder perforation was found during operation and repaired under laparoscopy. Two patients had vesicovaginal fistula formation. One ureteral-vaginal fistula was found after operation. The fistula was all closed spontaneously with a prolonged catheter drainage. CONCLUSIONS: TLH appears a safe, effective and reproducible procedure. It is an alternative method for those women who need hysterotomy.

Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty. Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E. coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively. We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing. To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells. METHODS: Adenoviral vectors, including Ad-EF1alpha-CD-cytomegalovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1alpha-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA + E. coli cells. Integration and expression of CD and/or TK gene were identified by PCR and Western blot. Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV. Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays. Flow cytometry analysis was used to detect cell death. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis. RESULTS: Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed. Both single and double suicide genes could be integrated into adenoviral genome and expressed. Cytotoxic effects of Ad-EF1alpha-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1alpha-CD single gene groups. The rate of cell survival was only (9 +/- 3)% in the Ad-EF1alpha-CD-CMV-TK group, but (37 +/- 3)% in the Ad-CMV-TK and (46 +/- 4)% in the Ad-EF1alpha-CD groups (P < 0.05). Flow cytometry analysis indicated that the killing mechanisms of the groups were different. Necrosis and apoptosis were involved in the mechanism of the double gene group. Based on the DNA stainability with Hoechst 33342, the apoptotic rates of VSMCs in the Ad-EF1alpha-CD-CMV-TK [(11.0 +/- 2.1)%] and Ad-CMV-TK [(12.0 +/- 2.2)%] groups were higher than those in Ad-CMV-LacZ [(1.2 +/- 0.11)%] and Ad-EF1alpha-CD [(5.0 +/- 1.8)%] groups (P < 0.05, respectively). DNA smear could be observed in both Ad-CMV-TK and Ad-EF1alpha-CD-CMV-TK groups after administration of prodrugs. CONCLUSIONS: The killing effect on rat VSMCs mediated by adenoviral CD/TK double suicide genes is superior to that of single suicide gene. The killing mechanism of recombinant adenovirus coexpressing CD/TK double suicide genes is mainly through cytotoxic effect and apoptosis.

[Cloning and characterization of a novel cardiac-specific kinase gene p93 related to sarcomere].

Myocyte's contraction is regulated by signal transduction pathway composed with many protein factors, but the definite mechanism is still uncertain. A novel cardiac-specific kinase gene which participates in regulation of signal transduction, p93 named, was cloned from adult heart cDNA library. p93, coding a family member of MAPKKK, localized on 1p31.1 based on bioinformatics analyses. Northern blot and 76-tissue array analyses determined that p93 was merely expressed in heart, but was undetectable in other tissues. Immunohistochemical study showed that p93 predominantly localized in the nucleus of cardiac myocytes. In vitro kinase assay indicated that p93 was a functional kinase capable of autophosphorylation. p93 could directly interact with cardiac troponin I by yeast two-hybrid system assessed utilizing bait plasmid containing p93 C-terminus (733 835 aa) and results were further confirmed by co-immunoprecipitation in vivo. Our data suggest that p93 is a cardiac-specific kinase and may play important role in regulation of sarcomeric contraction protein with signal transduction pathway similar ILK.

Cloning and characterization of a novel cardiac-specific kinase that interacts specifically with cardiac troponin I.

Cardiac-restricted genes play important roles in cardiovascular system. In an effort to identify such novel genes we identified a novel cardiac-specific kinase gene TNNI3K localized on 1p31.1 based on bioinformatics analyses. Sequence analysis suggested that TNNI3K is a distant family member of integrin-linked kinase. Northern blot and 76-tissue array analyses showed that TNNI3K is highly expressed in heart, but is undetectable in other tissues. Immunohistochemical analysis predominantly localized TNNI3K in the nucleus of cardiac myocytes. In vitro kinase assay showed that TNNI3K is a functional kinase. The yeast two-hybrid system showed that TNNI3K could directly interact with cardiac troponin I, results that were further confirmed by coimmunoprecipitation in vivo. Our data suggest that TNNI3K is a cardiac-specific kinase and play important roles in cardiac system.