Bioequivalence and Safety Assessment of Two Formulations of Metformin Hydrochloride Sustained-Release Tablets (Yuantang® SR and Glucophage® XR) Under Fed Conditions in Healthy Chinese Adult Subjects: An Open-Label, Two-Way Crossover, Sequence Randomized Phase I Clinical Trial.

OBJECTIVE: The purpose of this single-center, randomized, open, two-period, two-sequence crossover, single-dose administration, bioequivalence research was to evaluate the bioequivalence and safety of the generic formulations of metformin hydrochloride sustained-release (MH-SR) 500 mg tablets (test preparation [T]: Yuantang® SR) and the original formulation (reference preparation [R]: Glucophage® XR) in 36 healthy Chinese volunteers under postprandial conditions. METHODS: Subjects received 500 mg T/R in each period, with a 7-day washout period. Venous blood samples of 4 mL each were collected from each subject 19 times spanning predose (0 h) to 36 h postdose. The metformin concentration in deproteinized plasma was determined by high-performance liquid chromatography-tandem mass spectrometry. Bioequivalence (80.00-125.00%) was assessed by adjusted geometric mean ratios (GMRs) and two-sided 90% confidence intervals (CIs) of the area under the curve (AUC) and maximum concentration (Cmax) for each component. SAS 9.4 software was used for statistical analysis and Phoenix WinNonlin software v7 was used to analyze the pharmacokinetic parameters. RESULTS: Thirty-four volunteers completed the clinical study. The 90% CIs (96.12-105.44% for AUC from time zero to the time of the last measurable concentration [AUCt], 96.22-105.54% for AUC extrapolated from time zero to infinity [AUC∞], and 98.42-105.00% for Cmax) of T/R adjusted GMRs were within the bioequivalence acceptance range of 80.00-125.00%, indicating that they are bioequivalent. No serious adverse events occurred in this study, indicating that the two formulations were effective and well tolerated. CONCLUSIONS: Yuantang® SR was confirmed to be a well tolerated and bioequivalent alternative to Glucophage® XR when taken under postprandial conditions in healthy Chinese volunteers. The Clinical Trials Registry Platform used for this study was http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml . The trial registration numbers (TRNs) and dates of registrations were CTR20180476 (19 April 2018) for this clinical trial and CTR20171595 (11 January 2018) for the pilot trial.

Pharmacokinetic bioequivalence and safety assessment of two metformin hydrochloride tablet formulations using a phase I, randomized, open, two-period, two cross-over, single-dose, fed study in healthy Chinese adult subjects.

OBJECTIVES: To assess the bioequivalence and safety of generic metformin hydrochloride (test preparation) and glucophage (reference preparation) in healthy Chinese subjects. MATERIALS AND METHODS: A bioequivalence and safety assessment of two formulations of metformin (850 mg) using a randomized, open, two-period, two cross-over, single-dose, fed trial in 36 healthy Chinese adult subjects was performed at our center from March 22, 2018, to April 9, 2018. Bioequivalence was determined as two-sided 90% confidence intervals (CI) of the test-to-reference ratio of area under the curve (AUC) and peak concentration (Cmax) for each constituent within 80.00 - 125.00%. SAS 9.4 software was employed for the statistical analysis. RESULTS: One subject was excluded from the trial. The 90% CIs (95.36 - 101.43% for AUC0→t, 95.65 - 101.66% for AUC0→∞; 94.43 - 101.74% for Cmax) of test/reference preparation for these pharmacokinetic parameters were within the range of 80.00 - 125.00%. No severe adverse events were observed during this trial. The two preparations were safe and well-tolerated. CONCLUSION: It was concluded that generic metformin was bioequivalent and as safe as glucophage under fed conditions in healthy Chinese subjects.

Identification and characterisation of cold stress-related proteins in Oryza rufipogon at the seedling stage using label-free quantitative proteomic analysis.

In this study, label-free quantitative proteomics were used to study cold stress-related proteins in Dongxiang wild rice (Oryza rufipogon Griff., DWR) and cold sensitive cultivated rice 'Xieqingzao B'(Oryza sativa L. ssp. indica cv., XB). The results demonstrated the presence of 101 and 216 differentially expressed proteins (DEPs) were detected in DWR and XB, respectively, after cold stress. Bioinformatics analysis showed that DWR and XB differed significantly in their ability to scavenge reactive oxygen species (ROS) and regulate energy metabolism. Of the 101 DEPs of DWR, 46 DEPs related to differential expressed genes were also detected by transcriptome analysis. And 13 out of 101 DEPs were located in previous cold related quantitative trait loci (QTL). Quantitative real-time PCR analysis indicated that protein expression and transcription patterns were not similar in XB and DWR. Protein-protein interaction (PPI) network was constituted using the DEPs of DWR and XB, and the following three centre proteins were identified: Q8H3I3, Q9LDN2, and Q2QXR8. Next, we selected a centre protein and two of the 37 DEPs with high levels of differential expression (fold change ≥ 2) were used for cloning and prokaryotic expression. We found that Q5Z9Q8 could significantly improve the cold tolerance of Escherichia coli.

Fabrication of mechanically resistant superhydrophobic synthetic suede materials.

Functionalization of synthetic suede materials with excellent superhydrophobicity can expand their application ranges. Superhydrophobic synthetic suede was obtained by coating with polydimethylsiloxane (PDMS) and octadecyltrichlorosilane (OTS). Utilizing the synthetic suede effect of the fibrous rough structures in combination with the low surface energy micro-nano rough structure on fibers resulting from PDMS and OTS, the surface was easily turned superhydrophobic with self-cleaning properties. Abrasion tests showed that the superhydrophobic synthetic suede has excellent superhydrophobic performance after more than 2000 severe abrasion tests. This research provides a facile strategy for the preparation of practical superhydrophobic synthetic suede materials.

Unlocking the potential antioxidant and anti-inflammatory activities of Rhododendron molle G. Don.

Rhododendron molle G. Don is an important traditional Chinese medicinal plant, which has been applied to treat some inflammatory diseases. In the present study, ethanol extracts of R. molle flower (RFE) and leaf (RLE) were used for phytochemical, antioxidant and anti-inflammatory analysis. The antioxidant activity was investigated using the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (OH-)-scavenging activity, super oxide anion radical (O2.-)-scavenging activity and iron reducing power (FRAP). Production of nitric oxide (NO) was an indicator to evaluate the anti-inflammatory activity. The results showed that compared with RFE, RLE was more active against DPPH (56.66%), FRAP (51.29%) and hydroxyl radical (OH-) (69.66%) at 100µg/mL. In the same time, RLE and RFE had significant anti-inflammatory activity which could reduce nitrite production from 8.76µM to 5.08µM and 6.01µM, respectively. In addition, GC-MS analysis showed that 43 compounds were identified in R. molle. Among them, 11 compounds had antioxidant and 5 compounds had anti-inflammatory effect. Results showed that ethanol extracts of R. molle have significant antioxidant and anti-inflammatory activity. These results would be helpful for further investigation on the anti-inflammatory mechanism of R. molle.

Transcriptome analysis of phosphorus stress responsiveness in the seedlings of Dongxiang wild rice (Oryza rufipogon Griff.).

BACKGROUND: Low phosphorus availability is a major factor restricting rice growth. Dongxiang wild rice (Oryza rufipogon Griff.) has many useful genes lacking in cultivated rice, including stress resistance to phosphorus deficiency, cold, salt and drought, which is considered to be a precious germplasm resource for rice breeding. However, the molecular mechanism of regulation of phosphorus deficiency tolerance is not clear. RESULTS: In this study, cDNA libraries were constructed from the leaf and root tissues of phosphorus stressed and untreated Dongxiang wild rice seedlings, and transcriptome sequencing was performed with the goal of elucidating the molecular mechanisms involved in phosphorus stress response. The results indicated that 1184 transcripts were differentially expressed in the leaves (323 up-regulated and 861 down-regulated) and 986 transcripts were differentially expressed in the roots (756 up-regulated and 230 down-regulated). 43 genes were up-regulated both in leaves and roots, 38 genes were up-regulated in roots but down-regulated in leaves, and only 2 genes were down-regulated in roots but up-regulated in leaves. Among these differentially expressed genes, the detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in phosphorus deficiency tolerance. Meanwhile, the differentially expressed genes were also annotated with gene ontology terms and key pathways via functional classification and Kyoto Encyclopedia of Gene and Genomes pathway mapping, respectively. A set of the most important candidate genes was then identified by combining the differentially expressed genes found in the present study with previously identified phosphorus deficiency tolerance quantitative trait loci. CONCLUSION: The present work provides abundant genomic information for functional dissection of the phosphorus deficiency resistance of Dongxiang wild rice, which will be help to understand the biological regulatory mechanisms of phosphorus deficiency tolerance in Dongxiang wild rice.

Transcriptome analysis of phosphorus stress responsiveness in the seedlings of Dongxiang wild rice (Oryza rufipogon Griff.)

Abstract Background: Low phosphorus availability is a major factor restricting rice growth. Dongxiang wild rice (Oryza rufipogon Griff.) has many useful genes lacking in cultivated rice, including stress resistance to phosphorus deficiency, cold, salt and drought, which is considered to be a precious germplasm resource for rice breeding. However, the molecular mechanism of regulation of phosphorus deficiency tolerance is not clear. Results: In this study, cDNA libraries were constructed from the leaf and root tissues of phosphorus stressed and untreated Dongxiang wild rice seedlings, and transcriptome sequencing was performed with the goal of elucidating the molecular mechanisms involved in phosphorus stress response. The results indicated that 1184 transcripts were differentially expressed in the leaves (323 up-regulated and 861 down-regulated) and 986 transcripts were differentially expressed in the roots (756 up-regulated and 230 down-regulated). 43 genes were up-regulated both in leaves and roots, 38 genes were up-regulated in roots but down-regulated in leaves, and only 2 genes were down-regulated in roots but up-regulated in leaves. Among these differentially expressed genes, the detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in phosphorus deficiency tolerance. Meanwhile, the differentially expressed genes were also annotated with gene ontology terms and key pathways via functional classification and Kyoto Encyclopedia of Gene and Genomes pathway mapping, respectively. A set of the most important candidate genes was then identified by combining the differentially expressed genes found in the present study with previously identified phosphorus deficiency tolerance quantitative trait loci. Conclusion: The present work provides abundant genomic information for functional dissection of the phosphorus deficiency resistance of Dongxiang wild rice, which will be help to understand the biological regulatory mechanisms of phosphorus deficiency tolerance in Dongxiang wild rice.

Study on the foot shape characteristics of the elderly in China.

BACKGROUND: With aging, the feet of the elderly above 60 years old in China present degenerative changes, deformities, and diseases, which significantly affect their daily activities. OBJECTIVES: The authors aimed to study the morphological characteristics of the feet and identify the foot type according to size (length and width) and defect characteristics of elderly feet in China. METHODS: A convenient sample of 1000 subjects above 60 years old was recruited mainly in the regions of Shanghai, Shaanxi, Henan, Hebei, and Sichuan in China. Foot images were collected, and 800 (male 398, female 402) valid questionnaires were recovered. A total of 800 elderly subjects as the test group were invited to measure their foot sizes by means of a Footprint Collector (Tong Yuan Tang Health Management Limited, Qingdao in Shandong province). The foot type of the elderly was compared with that of the general adult Chinese population as the control group using the t-test for independent samples. RESULTS: Hallux valgus (46.9%) and flat foot (50.0%) were the most common foot shape deformities. The most frequent foot diseases were foot scaling (91.2%) and calluses (96.3%). The medial width of the first metatarsal-toe joint of the elderly was significantly higher (elderly female, 44.95±4.86mm; elderly male, 48.55±4.94mm) than that of the general adult population (adult female, 40.18±3.43mm; adult male, 43.22±3.20mm) (p<0.01). CONCLUSION: The foot length of the elderly was not significantly different from that of the general adult Chinese population. The width of the first metatarsal-toe joint in the forefoot of the elderly was significantly higher than that of the general adult Chinese population, which was consistent with the result that a high proportion of elderly subjects presented hallux valgus.

Donor-like surface traps on two-dimensional electron gas and current collapse of AlGaN/GaN HEMTs.

The effect of donor-like surface traps on two-dimensional electron gas (2DEG) and drain current collapse of AlGaN/GaN high electron mobility transistors (HEMTs) has been investigated in detail. The depletion of 2DEG by the donor-like surface states is shown. The drain current collapse is found to be more sensitive to the addition of positive surface charges. Surface trap states with higher energy levels result in weaker current collapse and faster collapse process. By adopting an optimized backside doping scheme, the electron density of 2DEG has been improved greatly and the current collapse has been greatly eliminated. These results give reference to the improvement in device performance of AlGaN/GaN HEMTs.

[Influence of histatin 1 on the proliferation and migration of HaCaT cells].

OBJECTIVE: To study the influence of histatin 1 (Hst1) on the proliferation and migration of human epidermal cell line HaCaT. METHODS: (1) HaCaT cells were routinely cultured and divided into control group, 100, 30, and 3 µg/mL Hst1 groups, 10 ng/mL recombinant human epidermal growth factor (rhEGF) group, and 30 µg/mL Hst1 + 10 ng/mL rhEGF group, according to the random number table (the same dividing method used for following grouping), with 27 samples in each group. NO stimulating factor was added in control group, while Hst1 and(or) rhEGF in corresponding concentration(s) was (were) added in the latter 5 groups. Cell proliferation was assayed by cell counting method at post culture hour (PCH) 24, 48, and 72. (2) HaCaT cells were divided into control group and 100, 30, and 3 µg/mL Hst1 groups, with 27 samples in each group. NO stimulating factor was added in control group, while Hst1 in corresponding concentration was added in the latter 3 groups. Cell cycle was assayed with flow cytometry at PCH 24, 48, and 72, and PI was calculated. (3) HaCaT cells were divided into control group, 30 µg/mL Hst1 group, 10 ng/mL rhEGF group, 30 µg/mL Hst1 + 10 ng/mL rhEGF group, 15 µg/mL Hst1 + 5 ng/mL rhEGF group, and 15 µg/mL Hst1 + 10 ng/mL rhEGF group, with 10 samples in each group. NO stimulating factor was added in control group, while Hst1 and(or) rhEGF in corresponding concentration(s) was (were) added in the latter 5 groups. Cells in each group were divided into two portions: cells in one portion were treated by mitomycin C for 2 hours, while cells in the other portion were not. Scratching assay was conducted in both portions of cells. Cell migration was measured at post scratching hour (PSH) 0, 16, and 24, and the wound-area healing rate was calculated. Data were processed with analysis of variance, and LSD- t test or Dunnett t test was applied in paired comparison among groups. RESULTS: (1) At PCH 24, the cell numbers in 10 ng/mL rhEGF group and 30 µg/mL Hst1 + 10 ng/mL rhEGF group were significantly higher than that in control group (with t values respectively 3.813, 5.410, P < 0.05 or P < 0.01). Except for cell numbers in 30 µg/mL Hst1 group and 3 µg/mL Hst1 group at PCH 48, cell numbers in the other groups as treated by Hst1 and (or) rhEGF were significantly higher than those in control group at PCH 48 and 72 (with t values from 7.754 to 24.979, P values all below 0.01). At PCH 72, the cell number was obviously higher in 100 µg/mL Hst1 group [(19.21 ± 0.59)×104] than in 30 µg/mL Hst1 group [(16.19 ± 0.53)×104)] and 3 µg/mL Hst1 group [(15.38 ± 0.13)×104], with t values respectively 11.391, 19.017, P values all below 0.01. The cell number was higher in 30 µg/mL Hst1 + 10 ng/mL rhEGF group than in 30 µg/mL Hst1 group, 3 µg/mL Hst1 group, and 10 ng/mL rhEGF group (with t values from 4.579 to 34.884, P < 0.05 or P < 0.01). Cell numbers in all groups increased with prolongation of time. (2) Compared with those in control group at PCH 24 and 48, the percentage of cells in G0/G1 phase was decreased, the percentage of cells in S phase was increased (except for cell percentage of 30 µg/mL Hst1 group at PCH 24), and PI value was significantly increased in 100 µg/mL Hst1 group and 30 µg/mL Hst1 group (with t values from 4.752 to 16.104, P values all below 0.01). The PI value in 3 µg/mL Hst1 group was obviously higher than that in control group only at PCH 48 (t = 4.609, P < 0.01). At PCH 72, only the PI value in 100 µg/mL Hst1 group was higher than that in control group (t = 8.005, P < 0.01). Compared among the groups treated by Hst1, the percentage of cells in G0/G1 phase showed an elevating trend, and the percentage of cells in S phase and the PI value showed a declining trend along with the decrease in Hst1 concentration at each time point. Compared within each group treated by Hst1, the percentage of cells in G0/G1 phase declined first and then elevated, while the percentage of cells in S phase and the PI value elevated first and then declined along with prolongation of time. (3) Without treatment of mitomycin C, the wound-area healing rate in 30 µg/mL Hst1 group (75.9 ± 3.9)% at PSH 16 was significantly higher than those in control group and 10 ng/mL rhEGF group [(53.0 ± 3.5)%, (61.7 ± 2.5)%, with t values respectively 12.241, 7.598, P values all below 0.01], but lower than those in 30 µg/mL Hst1 + 10 ng/mL rhEGF group, 15 µg/mL Hst1 + 5 ng/mL rhEGF group, and 15 µg/mL Hst1 + 10 ng/mL rhEGF group [(95.0 ± 4.1)%, (97.0 ± 3.7)%, (80.5 ± 5.9)%, with t values from -11.324 to -2.502, P < 0.05 or P < 0.01]. After being treated by mitomycin C, the wound-area healing rate in 30 µg/mL Hst1 group at PSH 16 [(54.1 ± 4.5)%] was higher than that in control group [(35.8 ± 5.7)%, t = 7.790, P < 0.01], but lower than that in the same Hst1 concentration but without mitomycin C treatment group (t = -10.863, P < 0.01). There was no statistically significant difference in the wound-area healing rate between 30 µg/mL Hst1 group and other groups treated by Hst1 and rhEGF at PSH 16 (with t values from 0.061 to 2.030, P values all above 0.05). Compared within each group with or without treatment of mitomycin C, the wound-area healing rate at PSH 16 was not significantly different from that at PSH 24 (with F values from 0.856 to 3.062, P values all above 0.05). CONCLUSIONS: Hst1 can promote the proliferation and migration of HaCaT cells. It has synergic effect with rhEGF on the promotion of cell proliferation, but their synergic effect on cell migration is not obvious.

[Drought resistance evaluation system for backcross lines of Dongxiang common wild rice (Oryza rufipogon Griff.)].

A total of 79 rice materials containing Dongxiang wild rice (Oryza rufipogon Griff. ) backcross lines (Dwr)/Xie-qingzao B (Xqz B)//Xqz B and their parents were chosen as the test objects to study the relationships between the drought resistance of these materials and the 31 drought resistance indices at germinating stage, seedling stage, booting stage, and mature stage. The results showed that the drought resistance index or the drought resistance coefficient of these materials were significantly correlated to the relative germination energy (RGE) under 15% PEG-6000 drought stress, the germination drought resistance index (GDRI) and relative germination energy (RGE) under 20% PEG-6000 drought stress, and the relative value of maximum root length (MRL), seeding height (SH), fresh root mass (FRM), dry root mass (DRM), root relative water content (RRWC), wilting rate (WR), leaf soluble sugar content (LSSC), leaf proline content (LPC), leaf MDA content (LMDAC), leaf relative water content (LRWC), level of rolling leaf (RL), plant height (PH), tiller number per plant (TNP), productive tiller number per plant (PTNP), filled spikelets per panicle (FSP), panicle density (PD), seed setting rate (SR), and 1000-grain mass (TGM) under water stress. Through stepwise regression analysis, nine drought resistance indices including the RGE under 20% PEG-6000 drought stress and the relative values of DRM, RRWC, LSSC, LPC, LMDAC, ETNP, SR, and TGM under water stress were selected. Base on these indices and their partial correlation coefficients, the drought resistance evaluation equation (D value) and evaluation system were established, which could well assess the drought resistance of the Dongxiang common wild rice backcross lines at different growth stages.

Adrenomedullin promotes human endothelial cell proliferation via HIF-1α.

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.

[Characterisation of a rice dwarf and twist leaf 1 (dtl1) mutant and fine mapping of DTL1 gene].

Plant height is one of the most important agronomic traits, which determines grain yield. By a largescale screening of our mutant population, we identified a dwarf with twisty leaf mutant (dwarf and twist leaf 1, dtl1). Besides dwarf with twisty leaf, dtl1 also showed reduced tiller number and sterile phenotypes. Based on the internode length of dtl1, this mutant belongs to the nl type of dwarfing phenotype. Physiological assay with two phytohormones, gibberellin (GA), and brassinosteroid (BR), suggested that dtl1 was neither deficient nor insensitive to GA and BR. Genetic analysis showed that the phenotype of dtl1 was controlled by a single recessive gene. Using F2 population derived from a cross between dtl1 and an indica cultivar Taichung Native 1, the DTL1 gene was narrowed down to a 70.4 kb between two SSR markers, RM25923 and RM6673, on the long arm of chromosome 10, and co-segregated with InDel marker Z10-29, where thirteen open reading frames were predicted without known gene involved in controlling plant height. Thus, the DTL1 gene might be a novel gene which is related to plant height in rice.

[Effect of substrate stiffness on biological behavior of fibroblasts].

OBJECTIVE: To study the effect of substrate stiffness on proliferation, migration of fibroblast and integrin ß(1) expression in fibroblast. METHODS: Fibroblasts were inoculated on silicon substrate with stiffness of (16.2 ± 0.5), (19.8 ± 1.1), and (200.1 ± 2.6) kPa. After being cultured for 5 days or 6 days, cells were counted and cell proliferative activities (recorded as absorbance value) were assessed with methyl thiazolyl blue (MTT). After being cultured for 3 days, cell cycle was detected and proliferation index (PI) was calculated. The cell scratch test was used for determination of cell migration rate on post scratch day (PSD) 0 (the day of scratch), 1, 2, and 3. After being cultured for 2 days, the expression of integrin ß(1) was determined by flow cytometry with fluorescence. Data were processed with one-way analysis of variance. RESULTS: (1) The proliferative speed and proliferative activity of fibroblasts were all increased along with the increase in substrate stiffness. PI of fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5), (19.8 ± 1.1), and (200.1 ± 2.6) kPa was respectively 24.8%, 27.4%, 32.4%. On PSD 2, migration rate of fibroblasts inoculated on silicon substrate with stiffness of (19.8 ± 1.1) and (200.1 ± 2.6) kPa was respectively (91.4 ± 5.1)%, (100.0 ± 1.3)%, which were higher than that of fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5) kPa [(55.8 ± 6.8)%, with F value respectively 3.5, 4.0, P values all below 0.01]. (3) The expression rate of integrin ß(1) in fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5) kPa was the lowest (43.22%), and that in fibroblast inoculated on silicon substrate with stiffness of (200.1 ± 2.6) kPa was the highest (81.26%). CONCLUSIONS: Substrate stiffness may have a great effect on proliferation and migration of fibroblast during the process of wound healing and scar formation, which can be related to regulation of integrin ß(1) expression.

[Effect of calcium on the activity and expression of integrin beta1 promoter in HaCaT cells and cell migration].

OBJECTIVE: To study the effect of calcium on the activity and protein expression of integrin beta1 promoter in human immortal keratinocyte colony HaCaT cell and cell migration. METHODS: (1) HaCaT cells were cultured in vitro (12-slot plate) and divided into 5 groups according to the random number table, with 18 slots in each group: reporter plasmid pGL3 promoter (positive control group, PC), pGL3 empty vector (negative control group, NC), pGL3-1756 bp (total length promoter group, TL), pGL3-1442 bp (distal promoter group, D), and pGL3-261 bp (proximal promoter group, P) was respectively used to transfect HaCaT cells in non-serum RPMI 1640 culture medium with 0.00, 0.03, 0.09, 0.30, 0.80, or 1.20 mmol/L calcium (3 slots in each group with each concentration). Luciferase activity was detected with dual-luciferase reporter assay system 24 hours after transfection. (2) HaCaT cells steadily transfected with small interfering RNA-integrin beta1 vector (steadily transfected in brief) constructed in our laboratory were normally cultured and divided into 6 parts according to the random number table. And then they were treated with former 6 different concentrations of calcium, with 3 samples for each concentration. Expression level of integrin beta1 protein was determined with Western blot. (3) Normal and steadily transfected HaCaT cells were cultured in 6-slot plate, 18 slots for each kind of cells. They were cultured with former 6 kinds of calcium culture media (divided according to the random number table, with 3 slots of cells for each concentration) for 12 hours after scratch test. Cell migration rate was observed and determined. (4) Data were processed with one-way analysis of variance and independent samples t test. RESULTS: (1) The luciferase activity of cells in TL group increased from 0.16+/-0.09 to 0.39+/-0.09 and 0.35+/-0.05 (with t value respectively 3.143, 3.140, P values all below 0.05) as calcium concentration increasing from 0.00 mmol/L to 0.09 and 0.30 mmol/L, and it decreased as calcium concentration increased to 0.80 and 1.20 mmol/L. The change pattern of luciferase activity of cells along with calcium concentration in D group was similar to that in TL group, but its activity (0.56+/-0.32, 0.64+/-0.06) at the concentration of 0.09, 0.30 mmol/L was respectively higher than that in TL group (with t value respectively 0.887, 6.122, P values all below 0.05). There was no obvious influence of calcium in either concentration on the luciferase activity of cells in P group. (2) The expression amount of integrin beta1 of steadily transfected HaCaT cells cultured with 0.03, 0.09, 0.30, 0.80, 1.20 mmol/L calcium (0.58+/-0.09, 1.40+/-0.29, 1.41+/-0.09, 0.99+/-0.10, 1.16+/-0.15) were all increased as compared with that cultured with 0.00 mmol/L calcium (0.53+/-0.10, with t value respectively 0.687, 4.880, 11.210, 5.578, 6.199, P values all below 0.05). (3) Migration speed of normal HaCaT cells cultured with 0.09, 0.30 mmol/L calcium increased obviously as compared with that cultured with 0.00 mmol/L calcium, and it slowed down when cultured with 0.80, 1.20 mmol/L calcium. There was no obvious difference of migration rate among steadily transfected HaCaT cells treated with different concentration of calcium. CONCLUSIONS: Distal promoter region of integrin beta1 plays a vital role in regulating integrin beta1 transcription in human epidermal cells. And calcium regulates activity, protein expression of integrin beta1 promoter and cell migration.

[Subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 protein].

OBJECTIVE: To study the subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) protein in endothelial cells. METHODS: Human umbilical vein endothelial cell strain ECV304 were cultured in vitro. The fusion protein of enhanced green fluorescent protein (EGFP)-EOLA1 expressing plasmid was constructed. Empty plasmid with EGFP at N side (pEGFP-N2) and fusion protein expressing plasmid EGFP-EOLA1 was respectively transfected into ECV304 cells with liposome. After being cultured for 48 hours, the expression levels of EGFP and fusion protein EGFP-EOLA1 in cells were detected with Western blot. The subcellular localization of EOLA1 protein was detected by laser scanning confocal microscope and immunoelectron microscopy. RESULTS: The EGFP-EOLA1 coexpression plasmid was verified to be successfully constructed by enzyme cutting and gene sequencing. The fusion protein of EGFP-EOLA1 was observed to express in transfected cells through Western blot. Green fluorescence scattered all over the ECV304 cells transfected with empty plasmid and cells transfected with fusion protein expressing plasmid, and it gathered obviously in the nuclei in the latter cells. Immune deposits were observed in the matrix of cells transfected with fusion protein expressing plasmid but not in the cells transfected with empty plasmid. CONCLUSIONS: EOLA1 protein is localized in the nucleus and the matrix of ECV304 cell, and it plays its role as a signal transduction factor.

Significance and relationship between Yes-associated protein and survivin expression in gastric carcinoma and precancerous lesions.

AIM: To analyze the differences and relevance of Yes-associated protein (YAP) and survivin, and to explore the correlation and significance of their expression in gastric carcinoma and precancerous lesions. METHODS: The PV9000 immunohistochemical method was used to detect the expression of YAP and survivin in 98 cases of normal gastric mucosa, 58 intestinal metaplasia (IM), 32 dysplasia and 98 gastric carcinoma. RESULTS: The positive rates of YAP in dysplasia (37.5%) and gastric carcinoma (48.0%) were significantly higher than that in normal gastric mucosa (13.3%), P < 0.01. The positive rates of survivin in IM (53.4%), dysplasia (59.4%) and gastric carcinoma (65.3%) were significantly higher than in normal gastric mucosa (11.2%), P < 0.01. Survivin expression gradually increased from 41.7% in well differentiated adenocarcinoma through 58.3% in moderately differentiated adenocarcinoma to 75.6% in poorly differentiated adenocarcinoma, with significant Rank correlation, r(k) = 0.279, P < 0.01. The positive rate of survivin in gastric carcinoma of diffused type (74.6%) was significantly higher than that in intestinal type (51.3%), P < 0.05. In gastric carcinoma with lymph node metastasis (76.9%), the positive rate of survivin was significantly higher than that in the group without lymph node metastasis (41.2%), P < 0.01. In 98 cases of gastric carcinoma, the expression of YAP and of survivin were positively correlated, r(k) = 0.246, P < 0.01. CONCLUSION: YAP may play an important role as a carcinogenic factor and may induce survivin expression. Detecting both markers together may help in early diagnosis of gastric carcinoma.

Analysis of the early adaptive response of endothelial cells to hypoxia via a long serial analysis of gene expression.

Activation of endothelial cells in humans is an early event in the response to hypoxia that may contribute to the endothelium's endogenous capacity to reduce tissue injury. To better understand the mechanism underlying this process, we utilized Long Serial Analysis of Gene Expression to study the transcriptome of human vein umbilical endothelial cells (EA.hy926) shortly after the induction of hypoxia. Of over 13,000 genes detected in each pool, 112 showed obvious differences in expression. Metabolic processes such as protein biosynthesis and proteolysis, aminoglycan metabolism, ribonucleotide biosynthesis, adenosine salvage, and lipid metabolism were reinforced. Pro-proliferation and pro-apoptotic states suggest the co-existence of pro- and anti-injury forces in endothelium shortly after the induction of hypoxia. Other adaptive responses include reinforced angiogenesis and vasodilation. Additionally, gene transcription in the endothelium shortly after the induction of hypoxia was regulated independently of HIF-1alpha. Our efforts to elucidate the adaptive response at an early post-hypoxia stage should contribute to further investigation of the protective processes that occur in the endothelium and has potential clinical implications.

Clinicopathological significance of B-cell-specific Moloney murine leukemia virus insertion site 1 expression in gastric carcinoma and its precancerous lesion.

AIM: To explore the relation between B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) expression and the clinicopathological features of gastric carcinoma (GC). METHODS: Immunohistochemistry was used to detect the expression of Bmi-1 and ki-67. Double-labeling staining was used to display the distribution of Bcl-2(+)/ki-67(-) cells in 162 cases of GC and its matched normal mucosa and precancerous lesion. RESULTS: The positive rate of Bmi-1 expression in GC (52.5%) was significantly higher than that in normal gastric mucosa (21.6%, chi(2) = 33.088, P < 0.05). The Bmi-1 expression in GC was closely related with the Lauren's and Borrmann's classification and clinical stage (chi(2) = 4.400, 6.122 and 11.190, respectively, P < 0.05). The expression of ki-67 was related to the Borrmann's classification (chi(2) = 13.380, P < 0.05). Bcl-2 expression was correlated with the Lauren's classification (chi(2) = 4.725, P < 0.05), and the Bmi-1 expression both in GC (r(k) = 0.157, P < 0.05) and in intestinal metaplasia (r(k) = 0.270, P < 0.05). CONCLUSION: Abnormal Bmi-1 expression in GC may be involved in cell proliferation, apoptosis and cancerization. This marker can objectively indicate the clinicopathological characteristics of GC.