[Analysis of flavonoids and antitumor activity of transgenic Saussurea involucrate].

The aim of this paper was to investigate the flavonoids of callus of transgenic and non-transgenic Saussurea involucrate and its antitumor activity on the esophageal cancer CaEs-17 cells. The species and content of mono-phenols were detected by high performance liquid chromatography. The growth of human esophageal cancer CaEs-17 cells was detected using CCK-8 assay, apoptosis morphology observation and flow cytometry. Expression of related apoptotic genes Bax and Bcl-2 were determined by qPCR. The results showed that the content of total flavonoids in the transgenic callus was 2.72 times that of the non-transgenic callus. The cyanidin-galactoside was detected in the transgenic callus, but not in the non-transgenic callus. The inhibitory effect of the extracts from the transgenic callus on CaEs-17 cells was more significant than that of the non-transgenic callus, and the IC50 value had a decreased of 26.4%. Flow cytometry analysis results showed that the apoptosis induction effect of the extracts from the transgenic callus on CaEs-17 cells was significantly better than that of the non-transgenic callus. Fluorescence quantitative PCR analysis results showed that the extracts from the transgenic callus could up-regulate the expression of proapoptotic gene Bax and down-regulate the expression of apoptotic gene Bcl-2, and the regulation effect of the transgenic callus was more significant. Therefore, compared with the non-transgenic callus, the antitumor activity of the extracts from the callus of transgenic S. involucrate on the esophageal cancer CaEs-17 cells was significantly increased, which was closely related to the accumulation of cyanidin-galactoside and its metabolism-related flavonoid compounds in the transgenic callus.

Functional Identification of Allograft Inflammatory Factor 1-Like Gene in Luning Chicken.

Allograft inflammatory factor-1 (AIF-1) is an inflammation-related protein mainly produced by immune cells, such as monocyte/macrophages and activated T lymphocytes. It is essential for the survival and proinflammatory activity of immune cells. However, the function of AIF-1 in chicken still has not been defined. In the present study, AIF-1-like (AIF1L) gene was identified in Luning chicken. Bioinformatics analysis revealed that the molecular weight of the chicken AIF-1 protein was 16290.8 Da. AIF1L contained a Ca2+ binding EF hand and could interact with actin filament. Its transcript was found in all tested tissues including spleen, brain, heart, kidney, liver, thymus, bursa of Fabricius, lung, and a relative low-level expression was detected in leg muscle. Furthermore, AIF1L expression in peripheral blood lymphocyte was depressed in a dose-dependent manner with cadmium exposure and peripheral blood lymphocyte viability decrease displayed a similar pattern with AIF1L expression. The results indicated that newly identified chicken AIF1L might be associated with lymphocyte viability.

The orbicularis oculi muscle resection technique for medial epicanthoplasty: A retrospective review of surgical outcomes in 47 Chinese patients.

BACKGROUND: Epicanthal folds and single eyelids are considered characteristic East Asian traits. This study describes a new approach to medial epicanthoplasty based on an orbicularis oculi muscle resection technique and reports the surgical outcomes in Chinese patients. METHODS: The study presents an uncontrolled retrospective case series review of 47 Chinese patients who underwent medial epicanthoplasty with double eyelid surgery from December 2010 to December 2014 by traditional medial epicanthoplasty (Group I, n = 23) or medial epicanthoplasty using the orbicularis oculi muscle resection technique (Group II, n = 24). Horizontal lid fissure length (HLFL), inner intercanthal distance (IICD), and the HLFL/IICD ratio were measured preoperatively and at 6-month follow-up. Within group and between group comparisons were performed using paired t-test, independent t-test, and Chi-square test. Pre- and postoperative photographic images were compared. RESULTS: In Group I, mean IICD decreased from 38.8 ± 1.06 mm preoperatively to 33.7 ± 0.93 mm postoperatively, and mean HLFL increased from 24.9 ± 1.27 to 29.6 ± 0.63 mm (p < 0.01). The HLFL/IICD ratio increased to 0.237 ± 0.05 (p < 0.0001). In Group II, mean IICD decreased from 38.7 ± 1.30 mm preoperatively to 32.2 ± 1.13 mm postoperatively, and mean HLFL increased from 24.5 ± 1.72 to 30.6 ± 1.08 mm (p < 0.01). The HLFL/IICD ratio increased to 0.315 ± 0.047 (p < 0.0001). The improvement in the HLFL/IICD ratio was significantly higher in Group II than in Group I (p < 0.001). All patients obtained satisfactory results with no complications. CONCLUSIONS: Medial epicanthoplasty using the orbicularis oculi muscle resection technique is safe and effective for epicanthal fold correction in Chinese patients.

Excited state structures and decay dynamics of 1,3-dimethyluracils in solutions: resonance Raman and quantum mechanical calculation study.

The resonance Raman spectroscopic study of the excited state structural dynamics of 1,3-dimethyluracil (DMU), 5-bromo-1,3-dimethyluracil (5BrDMU), uracil, and thymine in water and acetonitrile were reported. Density functional theory calculations were carried out to help elucidate the ultraviolet electronic transitions associated with the A-, and B-band absorptions and the vibrational assignments of the resonance Raman spectra. The effect of the methylation at N1, N3 and C5 sites of pyrimidine ring on the structural dynamics of uracils in different solvents were explored on the basis of the resonance Raman intensity patterns. The relative resonance Raman intensities of DMU and 5BrDMU are computed at the B3LYP-TD level. Huge discrepancies between the experimental resonance Raman intensities and the B3LYP-TD predicted ones were observed. The underlying mechanism was briefly discussed. The decay channel through the S1((1)nπ*)/S2((1)ππ*) conical intersection and the S1((1)nπ*)/T1((3)ππ*) intersystem crossing were revealed by using the CASSCF(8,7)/6-31G(d) level of theory calculations.

[ShRNA-mediated silencing of MDM2 inhibits growth of HepG2 hepatocellular carcinoma cells xenografted in nude mice].

OBJECTIVE: To construct a short hairpin (sh)RNA targeting the gene encoding the MDM2 oncoprotein in order to investigate its role in human hepatocellular carcinoma (HCC) and its potential for use as a gene therapy strategy to inhibit HCC growth in vivo. METHODS: Small interfering (si)RNAs were designed targeting the MDM2 gene (siMDM2-1 and siMDM2-2) and unrelated sequences (negative control) and cloned into the expression plasmid pGCSilencer-U6-neo-GFP. A HCC mouse model was established by subcutaneous inoculation of HepG2 cells (2 x 10(6) in 0.2 ml) into 20 nude mice. The inoculated mice were divided into four equal groups for tumor-localized injections of saline, negative control siRNA plasmid, siMDM2-1 plasmid, and siMDM2-2 plasmid. Tumor growth was observed daily (by caliper measurement) for one month, when mice were sacrificed by cervical dislocation. The tumor mass was resected for analysis of tumor inhibition rate (% = [(average tumor weight of control group - average tumor weight of treatment group) / average tumor weight of control group x 100]) and effects on MDM2 and p53 mRNA and protein expression (by reverse transcription- PCR and western blotting, both normalized to beta-actin). Significance of between-group differences was assessed by one-way ANOVA or LSD test; pairwise comparisons were made by the Chi-squared test. RESULTS: siMDM2-1 and siMDM2-2 suppressed the xenografted tumor growth remarkably (60.6% and 54.6% inhibition rates, respectively), significantly reduced the expression ofMDM2 gene (62.8% and 61.6%) and protein (60.7% and 59.5%), and significantly increased p53 gene (47.1% and 45.6%) and protein (45.9% and 44.3%) (all, P < 0.05). CONCLUSION: shRNA-mediated silencing of the MDM2 gene effectively inhibits HCC tumorigenesis of subcutaneously xenografted HepG2 cells in nude mice, and the mechanism may involve p53.

Role of AIF-1 in the regulation of inflammatory activation and diverse disease processes.

Allograft Inflammatory Factor-1 (AIF-1) is a 17kDa cytoplasmic, calcium-binding, inflammation-responsive scaffold protein that is mainly expressed in immunocytes. AIF-1 influences the immune system at several key points and thus modulates inflammatory diseases. AIF-1 boosts the expression of inflammatory mediators such as cytokines, chemokines, inducible nitric oxide synthase (iNOS) and promotes inflammatory cell proliferation and migration. Here we provide an overview of the different pathological processes regulated by AIF-1 mainly including allograft rejection, autoimmune diseases, central nervous system (CNS) injury, vasculopathy and cancer et al.

Daintain/AIF-1 (allograft inflammatory factor-1) promotes erythrocyte lysis and heme release probably via binding to hemoglobin.

Free heme is potentially cytotoxic, particularly in the presence of oxidants or activated phagocytes. Daintain/AIF-1 (allograft inflammatory factor-1) is a macrophage factor that has been implicated in the regulation of inflammation. In the present study, daintain/AIF-1 was found to induce cytolysis of erythrocytes, resulting in heme release in vitro. Furthermore, the interacting protein of daintain/AIF-1 was purified by daintain/AIF-1-6 histidine antigen fusion protein nickel affinity chromatography. MALDI-TOF-MS analysis identified hemoglobin subunit beta-1 as an interacting protein of daintain/AIF-1.These data suggest that daintain/AIF-1 may be involved in heme-associated diseases.

Daintain/AIF-1 (Allograft Inflammatory Factor-1) accelerates type 1 diabetes in NOD mice.

A large body of experimental evidence suggests that cytokines trigger pancreatic ß-cell death in type 1 diabetes mellitus. Daintain/AIF-1 (Allograft Inflammatory Factor-1), a specific marker for activated macrophages, is accumulated in the pancreatic islets of pre-diabetic BB rats. In the present study, we demonstrate that daintain/AIF-1 is released into blood and the levels of daintain/AIF-1 in the blood of type 1 diabetes-prone non-obese diabetic (NOD) mice suffering from insulitis are significantly higher than that in healthy NOD mice. When injected intravenously into NOD mice, daintain/AIF-1 stimulates white blood cell proliferation, increases the concentrations of blood glucose, impairs insulin expression, up-regulates nitric oxide (NO) production in pancreases and accelerates diabetes in NOD mice, while the antibody against daintain/AIF-1 delays or prevents insulitis in NOD mice. These results imply daintain/AIF-1 triggers type 1 diabetes probably via arousing immune cells activation and induction of NO production in pancreas of NOD mice.

[Therapeutic effects of small interfering RNA targeting MDM2 on human osteosarcoma cells].

OBJECTIVE: To investigate the inhibitory effect of the small interfering RNA targeting mdm2 gene on the growth of osteosarcoma cells. METHODS: PGCsilencerTM-mdm2 siRNA was constructed and transfected into the osteosarcoma cell line U2OS cells. The inhibitory effects on mdm2 were determined by RT-PCR and Western blot analysis. The cell growth activity was determined by MTT assay, and the cell apoptosis was examined by flow cytometry. The therapeutic effects of simdm2 was assessed on the nude mouse model of transplanted tumor. RESULTS: The simdm2 plasmid was successfully constructed. After simdm2 being transfected into the U2OS cells, the expressions of mdm2 gene and protein were significantly inhibited. The ability of cell growth activity decreased greatly and cell apoptosis occurred apparently. There was no significant difference between the negative control group and non-transfected group. The growth of xenograft tumor in simdm2 transfected nude mice was inhibited and the expressions of mdm2 gene and protein were down-regulated remarkably. CONCLUSION: siRNA targeting mdm2 gene inhibits the mdm2 expression in osteosarcoma U2OS cells and the growth of osteosarcoma in nude mice.

[Influence of expression of six transmembrane epithelial antigen of the prostate-1 on intracellular reactive oxygen species level and cell growth: an in vitro experiment].

OBJECTIVE: To investigate the STEAP1 gene function of the newly discovered gene six transmembrAne epithelial antigen of the prostate-1 (STEAP1). METHODS: Total RNA was obtained from human prostate cancer tissue and underwent PCR amplification. The full length of STEAP1 gene thus obtained was cloned. Mammalian expression vector pcDNA3. 1-STEAP1 was constructed and stably transfected into the human thyroid epithelial cells of the line FRTAP2320. A growth curve of the transfected cells was drawn. The intracellular reactive oxygen species (ROS) level was determined by flow cytometry (FC) with dichlorodihydrofluorescein diacetate (DCFHDA). RESULTS: The growth curve showed that the STEAP1 transfected cells grew faster than the control cells. FC showed that the fluorescence intensity of he intracellular ROS of the STEAP1 transfected cells was 42.13 +/- 1.13, significantly higher than those of the cell transfected with blank plasmid (10.02 +/- 1.42) and un-transfected cells (13.02 +/- 2.42, both P < 0.01). CONCLUSION: STEAP1 promotes the cell growth through inducing the intracellular ROS level.

[3,3-diindolylmethane enhances the inhibitory effect of idarubicin on the growth of human prostate cancer cells].

OBJECTIVE: To study the effects of idarubicin (IDA) combined with 3, 3-diindolylmethane (DIM) on the growth inhibition of human prostate cancer cells. METHODS: Human prostate cancer cells of the line PC-3M were cultured and then divided into the following groups: control group with solvent added into the culture fluid; IDA groups, with IDA of the terminal concentrations of 0.5, 1 or 5 mg/L added into the culture fluid; DIM groups, with DIM of the terminal concentrations of 30, 60 or 100 micromol/L added into the culture fluid; and DIM + IDA groups, with 0. 5 mg/L IDA and DIM 30, 60 or 100 micromol/L added into the culture fluid. 48 h later the cell growth inhibition rate was detected by MTT assay. Flow cytometry and acridine orange staining were used to detect the cell cycle and apoptosis. RT-PCR and Western blotting were used to detect the mRNA and protein expression of caspase 9, an apoptosis gene. RESULTS: Both IDA and DIM dose-dependently inhibited the growth of the PC-3M cells. The growth inhibition rate of the 60 micromol/L DIM + 0.5 mg/L IDA group was 69.9%, almost 10 times as that of the 0.5 mg/L IDA group. The apoptosis rate of the 60 micromol/L DIM + 0. 5 mg/L IDA group was 47.0%, significantly higher than that of the 0.5 mg/L IDA group (3.2%, P < 0.05). RT-PCR and Western blotting showed that the combination of DIM and IDA significantly enhanced the mRNA and protein expression of caspase 9. CONCLUSION: DIM enhances the growth inhibition effect of IDA on human prostate cancer cells by the mechanism of induction of apoptosis.

Daintain/AIF-1 promotes breast cancer proliferation via activation of the NF-kappaB/cyclin D1 pathway and facilitates tumor growth.

Recent research indicates that inflammatory factors play important roles in the initiation and progression of cancers, including breast cancer. Daintain/allograft inflammatory factor-1 (AIF-1) is a crucial mediator in the inflammatory response, but it has not yet been reported whether daintain/AIF-1 is involved in the development of breast cancers. In this study, immunohistochemical analysis found strong positive expression of daintain/AIF-1 in breast ductal tumor epithelia, but only weakly positive or negative expression in the adjacent histologically normal ductal epithelia. Then, the effect of daintian/AIF-1 on the proliferation of the breast cancer cell line MDA-MB-231 was explored via transduction of the daintian/AIF-1 gene into the cells, and via inhibition of the expression of daintain/AIF-1 through short interference RNA. The results demonstrated that up-regulation and down-regulation of daintain/AIF-1 expressions promoted and inhibited the proliferation of MDA-MB-231, respectively. More interestingly, daintain/AIF-1 overexpression facilitated tumor growth in female nude mice. Furthermore, we found that daintain/AIF-1 overexpression up-regulated the expression of cyclin D1 and enhanced the transcriptional activity of nuclear factor-kappa B (NF-kappaB), a regulator of cyclin D1 expression. In contrast, the down-regulation of daintain/AIF-1 expression decreased cyclin D1 expression and inhibited the transcriptional activity of NF-kappaB. These results strongly suggest that daintain/AIF-1 can promote the growth of breast tumors via activating NF-kappaB signaling, which consequently up-regulates the expression of cyclin D1, implying that daintain/AIF-1 may be a novel target molecule for the prognosis and therapy of breast cancer.

Preparation and identification of monoclonal antibodies against recombinant human thioredoxins.

Thioredoxin (Trx) is a member of the thioredoxin protein family and has a conserved catalytic domain (-Trp-Cys-Gly-Pro-Cys-Lys-) with reduction/oxidation (redox) activity. There are two main members in this family, Trx-1, a cytosolic and nuclear form, and Trx -2, a mitochondrial form. Trx-1 is a 104 amino acid multifunctional protein that has been extensively studied. Here we report the preparation of monoclonal antibodies (MAb) against recombinant human Trx-1(hTrx). To enhance its immunogenicity, Trx-1 was coupled to carrier protein bovine serum albumin by a two-step glutaraldehyde method. Using conventional procedures, we prepared three stable hybridoma cell lines that can produce and secret anti-Trx MAbs. We further analyzed their isotypes, titer, and affinity and found that those MAbs belong to the G1 subclass with kappa light chains, respectively. The MAbs were capable of recognizing hTrx-1, as determined by Western blotting.

Preparation of monoclonal antibodies against insulin and their applications.

Monoclonal antibodies (MAbs) against insulin are useful for insulin assays because of their specificity and plentiful supply. We have produced four monoclonal cell strains stably secreting the monoclonal antibodies against insulin; further established subpopulation, titer, affinity; and identified the antibodies as being of subclass IgG(2b)(kappa), one strain, or IgG(1)(kappa), three strains. The smallest detectable level of human insulin by ELISA using this IgG(1) was 1.25 microg/L. The monoclonal antibodies have been used for analyzing insulin content from the sera of type 2 diabetes patients and normal subjects.

Activity of the plant peptide aglycin in mammalian systems.

A 37 residue peptide, aglycin, has been purified from porcine intestine. The sequence is identical to that of residues 27-63 of plant albumin 1 B precursor (PA1B, chain b) from pea seeds. Aglycin resists in vitro proteolysis by pepsin, trypsin and Glu-C protease, compatible with its intestinal occurrence and an exogenous origin from plant food. When subcutaneously injected into mice (at 10 microg.g(-1) body weight), aglycin has a hyperglycemic effect resulting in a doubling of the blood glucose level within 60 min. Using surface plasmon resonance biosensor technology, an aglycin binding protein with an apparent molecular mass of 34 kDa was detected in membrane protein extracts from porcine and mice pancreas. The polypeptide was purified by affinity chromatography and identified through peptide mass fingerprinting as the voltage-dependent anion-selective channel protein 1. The results indicate that aglycin has the potential to interfere with mammalian physiology.

[Purification and characterization of weak-acid antibacterial peptide MD7095 from Musca domestica larvae].

Musca domestica,which belongs to insecta, diptera, cyclorrhapha, muscidae, is the most common muscae and the richest resource. It is very significant and valuable to isolate antibacterial peptides from Musca domestica and to develop these peptides into antibacterial medicine. Due to purify a pure peptide from the natural materials (animal, plant and microorganism tissue) is very difficult and complex, few research is going on. It had been reported that the most antibacterial peptides from Musca domestica were alkaline, no weak-acid antibacterial peptides had been reported so far. Based on a high sensitivity detection method, using dilute acetic acid extraction, alginic acid absorption, NaCl salting-out, Sephadex G-25 gel filtration, CMC23 ion-exchange chromatography, electrophoresis, a group of weak-acid antibacterial peptides had been purified from Musca domestica larvae and partial characterized. The peptides had characters of broad antibacterial spectrum and low minimum bactericidal concentration against Gram-positive bacterium such as Bacillus thuringiensis, Bacillus subtilis, Staphylococcus aureus and Gram-negative bacterium such as Pseudomonas aeruginosa, Escherichia coli. The peptides were very stable to keep the antibacterial activity even kept in 95 degrees C for 120 min and frozen-thawed for 10 times. A weak-acid antibacterial peptide MD7095 had been purified in high degree of purity by electro-elution,and was determined Mr 7095Da with MALDI-TOF-MS and pl 5.59 with IEF-PAGE. Peptide mass fingerprinting (PMF) analysis showed MD7095 was a novel bioactive peptide. Few peptides with antibacterial activity against Bacillus thuringiensis had been reported. Observation by scanning electron microscopy (SEM), it was suggested that the bioactivity mechanism of antibacterial peptides from Musca domestica larvae against Bacillus thuringiensis was to perforate cell membrane and lead to bacterium lysis and die. It is hopeful to develop the antibacterial peptides from Musca domestica to candidate medicine.

Preparation and identification of monoclonal antibodies against daintain.

Daintain is a 17-kDa polypeptide originally purified from porcine intestine. This polypeptide is associated with insulin secretion and inflammatory responses. Daintain is highly similar in amino acid sequence to allograft inflammatory factor-1 (AIF-1). Here we report the preparation and identification of monoclonal antibodies (MAbs) against daintain. To enhance its immunogenicity, daintain was coupled to carrier protein bovine serum albumin (BSA) by a two-step glutaraldehyde method. Using conventional procedures, we obtained four stable hybridoma cell lines that can produce and secret anti-daintain MAbs. We further analyzed their isotypes, titer, and affinity, and found that those MAbs belong to the G1 subclass with kappa light chains. The MAbs were capable of recognizing daintain as determined by Western blotting. The produced MAbs will be a useful tool for further investigation of daintain functions in organisms.

Preparation and identification of monoclonal antibodies against pea albumin 1b (PA 1b).

PA 1b (pea albumin 1b), extracted from pea seeds, is thermostable and is multifunctional. It has an attractive peros toxicity, and is also involved in the regulation of callus growth and cell proliferation. Here we report the preparation of monoclonal antibodies (MAbs) against this peptide for further investigation of peptide distribution and functions. PA 1b was coupled to carrier protein using the two-step glutaraldehyde method as an immunal antigen. Five stable cell lines producing anti-PA 1b MAbs were obtained. We analyzed their isotypes, titer, and affinity and found that those MAbs belong to the G(1) and G(2b) subclasses with kappa light chain, respectively. Using these antibodies, a competitive inhibition ELISA was developed, and approximately 15 nmol/L of antigen was detected.