Characteristic chromanone acids from Calophyllum membranaceum: Determination of C-3 configuration and anti-inflammatory activity.

One undescribed homologous furanochromanone (1) featuring a 6/6/5/3 tetracyclic skeleton and four highly oxidized pyranochromanones (2-5), along with a set of four pyranochromanone stereoisomers [(±)-6a and (±)-6b], were isolated from the leaves of Calophyllum membranaceum Gardn. Et Champ. Their structures were elucidated by using spectroscopic data, Snatzke's method, quantum-chemical calculations, and X-ray crystallographic analysis. The correlation of characteristic Cotton effects and specific chemical shifts with C-3 configuration provided a convenient approach to assign the C-3 configuration of 2,3-dimethylchromanones. The stereochemical assignments of 3-OH substituted pyranochromanones by quantum-based NMR methods following single/double MTPA derivatization were consistent with the ECD/NMR prediction, which verified the feasibility and reliability of the proposed empirical rule. The underlying mechanism was further clarified by conformational and molecular orbital analyses. Moreover, biological evaluation and binding assays demonstrated that compound 3 (KD = 0.45 µM) tightly binds to the TLR4-MD2 target, thereby inhibiting the TLR4/MyD88-dependent and -independent signal pathways. This study provides the first evidence that Calophyllum chromanones are a novel structural type of TLR4 inhibitors, exerting their anti-inflammatory effects by disrupting the binding between TLR4 and MD2.

A new species of Stylicletodes Lang, 1936 (Copepoda: Harpacticoida: Cletodidae) from the East China Sea, including an updated key to species and synopsis of distribution records.

Both sexes of a new species, Stylicletodes wellsi sp. nov. (Harpacticoida: Cletodidae), are described from material collected from sediments in the East China Sea. The new species belongs to a species group whose members are characterized by an anal operculum that has a backwardly directed, median linguiform process and fifth legs that display naked or sparsely pinnate armature elements in both sexes. Within this group, S. wellsi sp. nov. is morphologically closest to S. reductus Wells, 1965 but differs primarily from its European congener in the armature pattern of P4 (both rami) and the female P5. Distribution records of all species are summarized and an updated identification key to the seven valid species in the genus is presented. Taxonomic issues related to the type species S. longicaudatus (Brady, 1880) are briefly discussed.

Patient health questionnaire-15 (PHQ-15) to distinguish bipolar II disorder from major depressive disorder.

A sequential-recruited clinical trial has been conducted to assess capacity of Patient Health Questionnaire-15 (PHQ-15) in distinguishing bipolar II disorder from major depressive disorder. A total of 73 patients (49 BD-II depression patients) filled sociodemographic characteristics, Patient Health Questionnaire-9 (PHQ-9), Generalized Anxiety Disorder-7 Questionnaire (GAD-7), and PHQ-15. Sum score of PHQ-15 showed statistically significant difference in the two groups (t-test, P = 0.027). The area under the curve was 0.663 (P = 0.025), and the specificity was 0.75 at sum score of 13. Patients with BD-II depression has more somatic symptoms than MDD, and PHQ-15 might be used for identification.

[Assemblage composition and distribution of meiofauna in the southern Yellow Sea cold water mass during summer and autumn].

In order to study the impact of the Yellow Sea cold water mass (YSCWM) on meiofauna, the composition, abundance, biomass of meiofauna and their relationships with environmental variables were analyzed through the samples from 8 stations investigated by R/V 'Dongfanghong II' in June and November, 2013. The results showed that the average abundances of meiofauna were 900.8 and 758.4 ind · 10 cm(-2), and biomasses were 886.9 and 615.7 µg · 10 cm(-2) in June and November, 2013, respectively. Results of ANOVA showed that no significant differences of meiofaunal abundance and biomass were detected among the 8 stations in the two study cruises. A total of 17 meiofaunal groups were identified. The most dominant taxonomic group was free-living marine nematodes, with relative dominance of 88.5% in June and 94.0% in November. The following groups were also important, including benthic copepods, polychaetes, kinorhynchs and ostracods. Analysis of meiofaunal vertical distribution indicated that 92.5% and 95.4% of meiofauna distributed in the top 5 cm of the sediment in the two study cruises, while 59.1% of nematodes and 78.2% of copepods were found in the top 2 cm of the sediment. Correlation analysis among meiofaunal abundance and biomass, nematode and copepod abundance and environmental variables showed that meiofaunal abundance and biomass had significant negative correlations with bottom water temperature (BWT) and sediment silt-clay percentage. Copepod abundance also had significant negative correlations with BWT and silt-clay percentage while it had significant positive correlation with sediment median diameter. The results of BIOENV indicated that BWT, bottom water salinity, sediment water content, sediment chlorophyll a and phaeophorbide contents were the most important factors to influence meiofaunal assemblages.

A new approach for the treatment of proximal humeral fractures using the TRIGEN proximal humeral nail.

The optimal surgical treatment for displaced proximal humeral fractures continues to be controversial. One of the new treatment options is the minimally invasive intramedullary nail. The purpose of this study was to evaluate the functional outcome of using the TRIGEN proximal humeral nail (PHN) for the treatment of displaced proximal humeral fractures in elderly patients. From January 2004 to December 2008, 64 elderly patients (age > 60 years old) with displaced proximal humeral fractures were treated using TRIGEN PHN. A complete 12-month postoperative follow-up was available for 54 patients. The study cohort included two-part (29 shoulders), three-part (22 shoulders), and four-part (3 shoulders) Neer classification fracture types. The Constant-Murley score was used to assess functional outcome. Radiological outcomes were evaluated, and all complications were recorded. All fractures were united. The Constant-Murley score data indicated that the patients experienced improvement from 6 to 12 months postoperatively. The mean absolute Constant-Murley score on the injured side increased from 71.2 ± 11.2 points at 6 months to 82.4 ± 16.4 points at 12 months (P = 0.01). The mean neck-shaft angle 1 year after surgery was 125° ± 8.1° (95°-140°). Secondary complications were minimal and observed in only 6 of 54 patients. In conclusion, the TRIGEN intramedullary humeral nail is effective for the treatment of proximal humeral fractures.

[Case-control study on cable-pin system in the treatment of olecranon fractures].

OBJECTIVE: To prospectively evaluate the clinical result of Cable-Pin system in the treatment of olecranon fractures and compare with tension band wiring (TBW) method. METHODS: From March 2008 to June 2010,65 patients with olecranon fractures were divided into two groups: 32 patients in Cable-Pin group were treated with Cable-Pin system, including 18 males and 14 females, ranging in age from 21 to 69 years, with an average of (53.69 +/- 13.42) years; 33 patients in TBW group were treated with Kirschner tension bend, including 20 males and 13 females, ranging in age from 20 to 70 years, with an average of (53.18 +/- 13.36) years. The incision length, operation time, the amounts of hemoglobin after operation, fracture healing time, complications and HSS elbow scores were recorded and analyzed statistically. The follow-up period ranged from 12 to 24 months, with an average period of 18.4 months. RESULTS: There were statistical differences (P<0.05) in fracture healing time (t= 2.588, P=0.012), complication rate (chi2=4.534, P=0.033) and HSS elbow joint scores (Z=-2.039, P=0.041) between two groups, which all were superior to TBW in Cable-Pin group. There was no statistical differences (P>0.05) in the length of incision (t= 0.416, P=0.679), operation time (t=0.816, P=0.417) and the postoperative amounts of hemoglobin (t=-0.553, P=0.294) between two groups. CONCLUSION: Cable-Pin system is an easy and reliable method for the treatment of olecranon fractures with less complications and better functions than TBW.

(+)-Medioresinol leads to intracellular ROS accumulation and mitochondria-mediated apoptotic cell death in Candida albicans.

The phytochemical (+)-Medioresinol, a furofuran type lignan identification and isolation on the stem bark of Sambucus williamsii, which is a folk medicinal plant used in traditional medicine. (+)-Medioresinol is known to possess a lesishmanicidal activity and cardiovascular disease risk reduction but its antifungal effects have not yet been identified. In this study, to confirm (+)-Medioresinol's antifungal properties and mode of action, we observed morphological and physiological change in Candida albicans. In cells exposed to (+)-Medioresinol, arrested the cell cycle and intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased. The increase of ROS induced oxidative stress and the mitochondria dysfunction which causes release of pro-apoptotic factors. We investigated a series of characteristic cellular changes of apoptosis by using various apoptosis detection methods. We report here for the first time that (+)-Medioresinol has effects on mitochondria and induced the accumulation of ROS in C. albicans cells. We demonstrated that one of the important features of apoptosis, mitochondrial membrane depolarization is caused by ROS. Substantially, we investigated the release of cytochrome c, which is one of the factors of metacaspase activity. We also show that the effects of (+)-Medioresinol are mediated at an early stage in apoptosis acting on the plasma membrane phosphatidylserine externalization. In addition, (+)-Medioresinol induced apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC). In late stage of confirmation of diagnostic markers in yeast apoptosis include the effects of nucleus morphological change, DNA fragmentation and condensation by influence of oxidative stress. These apoptotic phenomena represent that oxidative stress and mitochondria dysfunctions by inducing the phytochemical (+)-Medioresinol must be an important factors of the apoptotic process in C. albicans. These results support the elucidation of the underlying antifungal mechanisms of (+)-Medioresinol.

Antifungal effect of (+)-pinoresinol isolated from Sambucus williamsii.

In this study, we investigated the antifungal activity and mechanism of action of (+)-pinoresinol, a biphenolic compound isolated from the herb Sambucus williamsii,used in traditional medicine. (+)-Pinoresinol displays potent antifungal properties without hemolytic effects on human erythrocytes. To understand the antifungal mechanism of (+)-pinoresinol, we conducted fluorescence experiments on the human pathogen Candida albicans. Fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated that the (+)-pinoresinol caused damage to the fungal plasma membrane. This result was confirmed by using rhodamine-labeled giant unilamellar vesicle (GUV) experiments. Therefore, the present study indicates that (+)-pinoresinol possesses fungicidal activities and therapeutic potential as an antifungal agent for the treatment of fungal infectious diseases in humans.

[Effects of calcitonin on osteoblast cell proliferation and OPG/RANKL expression: experiment with mouse osteoblasts].

OBJECTIVE: To investigate the effects of calcitonin on the proliferation of osteoblasts and the OPG/RANKL expression therein. METHODS: Osteoblasts were separated by enzyme digestion methods from the cranial bones of 30 newborn mice, cultured, and inoculated in 96-well plate. Calcitonin of the concentrations of 10, 40, and 80 IU/L was administrated into the culture medium for 24, 48, and 72 hours. The proliferation of osteoblasts was detected by MTT method. Flow cytometry was used to analyze the cell cycle. The mRNA expression of osteoprotegerin (OPG) and RANKL and protein expression of OPG were examined by RT-PCR and ELISA respectively. RESULTS: The proliferation rates of the osteoblasts exposed to the calcitonin of the concentrations of 40 and 80 IU/L for 24. 48, and 72 hours cell were all significantly higher than those of the blank control group (P < 0.05, P < 0.01). The index of osteoblasts at the G1 phase was significantly lower and the indexes of the osteoblasts at the S phase and G2-M phase were significantly increased after exposure to calcitonin (all P < 0.01). The OPG mRNA expression of the osteoblasts exposed to 80 IU/L calcitonin was significantly higher and the RANKL mRNA expression was significantly lower compared with the control group (P < 0.05). The OPG protein expression of the osteoblasts exposed to 80 LU/L calcitonin was significantly higher than that of the control group at different time points (P < 0.05, P < 0.01). CONCLUSION: Calcitonin increases the osteoblast proliferation and the OPG mRNA expression in the osteoblasts while RANKL mRNA expression in the osteoblasts is inhibited.

4-Hydroxykobusin inhibits the induction of nitric oxide synthase by inhibiting NF-kappaB and AP-1 activation.

We recently isolated a novel lignan, 4-hydroxykobusin from Geranium thunbergii (Liu et al., Arch. Pharm. Res., 29, 1109-1113, 2006). Here, we studied its effect on the expression of inducible nitric oxide synthase (iNOS) gene in RAW264.7 cells. 4-Hydroxykobusin inhibited nitric oxide (NO) production in a concentration-dependent manner and blocked the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS). To identify the mechanistic basis for its inhibition of iNOS induction, we examined the effect of 4-hydroxykobusin on the transactivation of iNOS gene by luciferase reporter activity using -1.59 kb flanking region. The lignan suppressed the reporter gene activity and the LPS-induced reporter activations of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) were also significantly blocked by 4-hydroxykobusin. These findings suggest that the inhibition of LPS-induced NO formation by 4-hydroxykobusin is due to its inhibition of NF-kappaB and AP-1 activation.

7,7'-Dihydroxy bursehernin inhibits the expression of inducible nitric oxide synthase through NF-kappaB DNA binding suppression.

This study isolated a lignan, 7,7'-dihydroxy bursehernin, from Geranium thunbergii and investigated whether or not the lignan affects the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). The gel shift analysis and luciferase reporter gene assays using the iNOS promoter and nuclear factor-kappaB (NF-kappaB) minimal promoter showed that a treatment with 7,7'-dihydroxy bursehernin reduced the reporter activities and binding of NF-kappaB to the NF-kappaB consensus sequence, while it had no effect on the nuclear translocation of p65 and the phosphorylation/degradation of I-kappaBalpha. It was reported that a few natural compounds directly suppressed the binding activity of the NF-kappaB components to DNA. The NF-kappaB binding activity was not reversed by the in vitro exposure of the nuclear extracts to 7,7'-dihydroxy bursehernin, which suggest that a metabolite(s) of 7,7'-dihydroxy bursehernin might target the binding of the NF-kappaB complex to the DNA binding domain region in the promoter region of the iNOS gene. After incubation of RAW264.7 cells with 7,7-dihydroxy bursehernin for 18h, the levels of parent compound were negligible; while a main metabolite, 4-[4-(n-hydroxy-phenyl)-2,3-dimethyl-buta-1,3-dienyl]-benzene-1,2-diol was detected in cell lysates and culture medium.

[Effects of carbon monoxide on polymorphonuclear leukocyte apoptosis stimulated by limb ischemia-reperfusion patient serum and the role of nuclear factor-kappaB].

OBJECTIVE: To explore the role of exogenous carbon monoxide (CO) in apoptosis of polymorphonuclear leukocyte (PMN) stimulated by ischemia-reperfusion (IR). METHODS: PMNs isolated from the venous blood of a healthy volunteer, incubated, put in 24-well plates, and randomly divided into 4 groups of 8 wells: control group (exposed to 5% CO2), control + CO group (exposed to 0.025% CO and 5% CO2 for I hour and then serum of healthy person was used to replace the culture fluid), IR group (exposed to 5% CO2 and then IR serum was used to replace the culture fluid), and IR + CO group (exposed to 0.025% CO and 5% CO2 for I hour and then serum of healthy person was used to replace the culture fluid). The IR serum was obtained from 8 male patients with osteoarthritis of knee undergoing knee replacement. After 24-hour incubation the PMNs underwent flow cytometry and electrophoresis to examine the apoptosis of PMNs. Electrophoretic mobility shift assay (EMSA) was used to detect the NF-kappaB binding activity. RESULTS: The PMN apoptotic rate of the IR + CO group was 9.38% +/- 1.58%, significantly higher than that of the control group (4.18% +/- 1.02%, P < 0.05). The PMN apoptotic rate of the IR group was 2.15% +/- 1.02%, significantly lower than that of the control group (P < 0.05). However, the PMN apoptotic rate of the control + CO group was 4.16% +/- 1.12%, not significantly different from that of the control group (P > 0.05). Electrophoresis showed that PMN apoptosis DNA ladder was seen in the control, control + CO, and IR + CO groups, but not in the IR group. EMSA showed that after co-incubation of PMN nuclear extract and isotope- labeled NF-kappaB probe in term of the strength of radiation self-development band the result the IR group was significantly greater than that of the control group, and the result of the IR + CO group was significantly lower than that of the IR group, however, there was no significant difference between the control and control + CO groups. CONCLUSION: Exogenous CO improves the inhibitory effect of IR blood on the PMN apoptosis with a mechanism of suppressing the NF-kappaB binding activity.

A new furofuran lignan from Geranium thunbergii Sieb. et Zucc.

A new furofuran lignan, 4-hydroxykobusin (3), together with known lignans, kobusin (1), and 7,7'-dihydroxybursherenin (2), were isolated from the whole plant of Geranium thunbergii Sieb. et Zucc (Geraniaceae). The structures were determined based on the spectral data and a comparison with the published data. This is the first report of the presence of furofuran lignan in Geranium species.

[Effect of exogenous carbon monoxide on sequestration of polymorphonuclear neutrophils in the lung following limb ischemia-reperfusion: an experimental study].

OBJECTIVE: To investigate the effect of exogenous carbon monoxide (CO) in inhibiting the sequestration of polymorphonuclear neutrophils (PMNs) in the lung following limb ischemia-reperfusion (IR) and the mechanism thereof. METHODS: PMNs of peripheral blood were isolated from the venous blood of a healthy volunteer. Serum was collected from a patient undergoing bilateral knee joint replacement as IR serum. Human pulmonary microvascular endothelial cells (PMVECs) were cultured and divided into 4 groups: control group (cultured under the condition of room air containing 5% CO2 for 5 h and cultured in normal human serum instead of medium during the last 4 hours of experiment), IR group (cultured under the condition of air containing 5% CO2 for 5h and cultured in the serum of IR patient during the last 4 hours), IR + CO group (cultured under the condition of air containing 0.025% CO and 5% CO2 for 5 hours and cultured in IR serum during the last 4 hours), and control + CO group (cultured under the condition of air containing and 0.025% CO and 5% CO2 for 5 hours and cultured in normal human serum during the last 4 hours). Immunofluorescence flow cytometry was used to detect the expression of intercellular adhesion molecule (ICAM)-1 and integrin CD11b in the PMVECs. Human PMVECs were put into the wells of a 96-well plate and added with PMNs to calculate the PMVEC-PMN adhesion rate. Tourniquettes were bound at the bilateral hind thighs of 32 healthy male SD rats for 4 hours so as to establish a rate IR model. The rats were randomly divided into 4 equal groups: control group (undergoing the same operation without causing limb ischemia and exposed to room air), IR group (undergoing bilateral hind limb ischemia for 4 h and reperfusion for 4 h and exposed to room air), IR + CO group (exposed to the containing 0.025% CO one hour before reperfusion till 4 hours after reperfusion), and control + CO group (exposed to air containing 0.025% CO at the corresponding time point as that of the IR + CO group). Then the rats were killed and their middle pulmonary lobes were taken out for microscopy and calculation of the number of PMNs in alveolar septum. Western blotting was used to examine the ICAM-1 protein expression in the lung. RESULTS: The ICAM-1 expression and integrin CD11b expression of the IR group PMVECs were significantly stronger than those of the IR + CO group PMVECs (both P < 0.05) and there were no significant differences in the ICAM-1 expression and CD11b expression between the control + CO and control groups (both P > 0.05). The PMN-PMVEC adhesion rate of the IR group PMVECs was 30 +/- 2.9%, significantly higher than those of the IR + CO group and control group PMVECs (19.8 +/- 1.5% and 13.4 +/- 1.1% respectively, both P < 0.05) and there was no significant difference in the PMN-PMVEC adhesion rate between the CO + control group and control group (P > 0.05). The lung tissues of the IR group rats showed edema and hemorrhage. The number of PMNs in the alveolar septum was 60.6 +/- 1.7/10 high power fields, significantly higher than those of the IR + CO group and control group (36.4 +/- 1.6 and 22.5 +/- 1.6 respectively, both P < 0.05) and there was no significant difference between the latter 2 groups (P > 0.05). The ICAM-1 protein expression in the lung of the IR group was the strongest, followed by the IR + CO group, control + CO group, and control group. CONCLUSION: Exogenous CO inhibits the limb/IR-induced PMN sequestration in the lung, probably by the mechanism of down-regulation of the expression of adhesion molecules and suppression of the PMN\PMVEC adhesion following IR.