The aim of the present study was to investigate whether the physiological features of Ano1 were affected by enhanced green fluorescent protein (EGFP) fusing at Ano1 C-terminal. The eukaryotic expression vectors of Ano1 and EGFP-Ano1 were constructed, and these plasmids were transfected into Fischer rat thyroid follicular epithelial (FRT) cells using liposome. The expression and location of Ano1 were examined by using inverted fluorescence microscope. The ability of Ano1 to transport iodide was detected by kinetics experiment of fluorescence quenching. The results showed that both Ano1 and EGFP-Ano1 were expressed on FRT cell membrane and could be activated by Ca(2+). There was no significant difference of the ability to transport iodide between Ano1 and EGFP-Ano1. These results suggest Ano1 and EGFP-Ano1 have similar physiological feature.
Animals ; Anoctamin-1 ; Cell Membrane ; physiology ; Chloride Channels ; metabolism ; Epithelial Cells ; physiology ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Microscopy, Fluorescence ; Plasmids ; Rats ; Recombinant Fusion Proteins ; metabolism ; Thyroid Gland ; cytology ; Transfection

Objective: To study the chemical constituents from the dried rhizome of Lepidium meyenii (Maca) cultivated in Jilin. Methods: The chemical constituents were isolated and purified with macroreticular resin, silica gel, ODS column chromatography, preparation HPLC, etc. The structures of the compounds were identified by physicochemical properties and various spectroscopic methods. Results: Thirteen compounds were isolated as N-methyl-3-hydroxy-benzeneacetamide (1), N-benzyloctadecanamide (2), benzeneacetamide (3), benzylamine (4), 3-methoxyphenylacetic acid (5), phenylacetic acid (6), 4-hydroxy-3-methoxybenzoic acid (7), nicotinic acid (8), 3, 4-dihydroxy-benzoic acid methyl ester (9), adenosine (10), L-valine (11), daueosterol (12), and β-sitosterol (13). Conclusion: Compound 1 is identifled as a new natural compound, and compounds 3-6 and 9-12 are obtained from L. meyenii for the first time.

OBJECTIVE: To establish the hairy roots culture system of Atractylodes japonica Koidz. ez Kitam. and study the hairy roots growth and analyze the polysaccharide content in hairy roots culturing system.

Objective: To explore the effect of ginsenoside Rgl on the ubiquitin-modified protein aggregation in the cortex after cerebral ischemia reperfusion (I / R) injury in the rats, and to further clarify the therapeutic mechanism of ginsenoside Rgl in the cerebral I/R injury. Methods: The middle cerebral artery occlusion (MCAO) model was set up with suture method for 1. 5 h of embolization. A total of 72 rats were divided into sham operation group, I/R model group, positive drug control (nimodipine) group, low, middle, and high doses 10, 20, and 40 mg ' k g - 1) of ginsenoside Rgl groups. All 12 rats in each group were given intraperitoneal injection. TTC staining and Longa' s score method were used to detect the infarction areas and the neurological deficit scores of the rats in various groups 24 h after modeling. The death of neurons in the cortex and hippocampus after cerebral ischemia of the rats in various groups were observed with HE staining. Immunohistochemistry and Western blotting method were used to detect the expression of ubiquitin-modified protein aggregation in the cortex of the rats in various groups. Results: Compared with I/R group, the percentages of infarction areas of the rats in nimodipine group and ginsenoside Rgl groups were significantly decreased (P < 0 . 05). and the neurological deficit scores were decreased (P < 0 . 05). The HE staining results showed that compared with sham operation group, the neurons in I/R model group were sparse, showing fragmentation and dissolution; compared with I/R model group, the phenomena of cell nucleus fragmentation, dissolution and powder staining in nimodipine group and different doses of ginsenoside Rgl groups were all improved to different degrees. The immunohistochemical results showed that compared with sham operation group, the positive expression level of ubiquitin-modified protein in I/R model group was increased significantly (P < 0 . 05); compared with I/R model group, the positive expression levels of ubiquitin-modified protein in nimodipine group and different doses of ginsenoside Rgl groups were decreased (P < 0 . 05), especially in high dose of ginsenoside Rgl group (P < 0 . 05). The Western blotting results showed that compared with sham operation group, the level of ubiquitin-modified protein aggregates in I/R model group was significantly increased (P < 0 . 0 5); compared with I/R model group, the levels of ubiquitin-modified protein aggregates in nimodipine group and different doses of ginsenoside Rgl were decreased (P < 0 . 05), especially in high dose of ginsenoside Rgl group. Conclusion: Ginsenoside Rgl can inhibit the formation of ubiquitin-modified protein aggregates induced by I/R injury in the cortex, thereby alleviating the I/R injury in the rats.

Many pathological phenomena of male infertility are related to epigenetic changes in male germ cells. Epigenetic regulation during spermatogenesis plays an important role in mitotic/meiotic divisions and spermiogenesis. The histones have various post-translational modifications on different amino acid residues during spermatogenesis. These modifications are crucial to the precise regulation of spermatogenesis. Moreover, the histone-to-protamine transition will occur during spermiogenesis. Many studies have also found that abnormal changes of histone modifications during spermatogenesis may damage the sperm development, leading to male sterility. This article reviews the changes of histone modifications during spermatogenesis, the regulation of the development of male germ cells, and the relationship between histone abnormalities and male sterility.
Epigenesis, Genetic ; Histones ; metabolism ; Humans ; Infertility, Male ; physiopathology ; Male ; Spermatogenesis

The aim of the present study was to establish a cell model of volume-regulated anion channel subunit LRRC8A and investigate the physiological characteristics of LRRC8A. The eukaryotic expression vectors of LRRC8A and YFP-H148Q/I152L were constructed and transfected into Fischer rat thyroid (FRT) cells by Lipofectamine 2000. The FRT cell lines co-expressing LRRC8A and YFP-H148Q/I152L were obtained by antibiotic screening. The expression of LRRC8A and YFP-H148Q/I152L in FRT cells was detected by the inverted fluorescence microscope. The fluorescence quenching kinetic experiment was done to verify the function and effectiveness of the cell model. Then the cell model was utilized to study the physiological characteristics of LRRC8A, such as the characteristics of anion transport, the opening of LRRC8A by osmotic pressure, the effect of anion transport velocity, and the effect of chloride channel inhibitors on LRRC8A anion channel. The results of the inverted fluorescence microscope showed that LRRC8A was expressed on the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm. The results of fluorescence quenching kinetic test showed that under the condition of low osmotic state, LRRC8A could transport some kinds of anions, such as iodine and chloride ions. Osmotic pressure played a key role in the regulation of LRRC8A volume-regulated anion channel opening. Chloride channel inhibitors inhibited ion transport of LRRC8A channel in a dose-dependent manner. It is suggested that LRRC8A has the characteristics of classic volume-regulated anion channels by using the cell model of FRT cells co-expressing LRRC8A and YFP-H148Q/I152L.
Animals ; Anions ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; Ion Transport ; Membrane Proteins ; physiology ; Microscopy, Fluorescence ; Rats ; Rats, Inbred F344 ; Thyroid Gland ; cytology ; Transfection

BACKGROUNDLung cancer is one of the most common cancers worldwide and its mortality rate has increased year after year. Molecular biology has contributed to make people's understanding to the disease at the gene level. A new idea will be given to the early diagnosis and treatment for lung cancer with the method of molecular biology which can be used to find the association between some genes and lung cancer. The aim of this study is to evaluate the relationship between polymorphism of codon 72 (BstU I single nucleotide polymorphism, BstU I SNP) in p53 gene and susceptibility and radiosensitivity of non-small cell lung cancer (NSCLC) in Chinese population.

METHODSThe BstU I single nucleotide polymorphic sites at condon 72 in exon 4 of p53 gene in 50 patients with NSCLC as well as 50 healthy controls were inspected by polymerase chain reaction-restricted fragment length polymorphism assay and the relationship between BstU I SNP and susceptibility and radiosensitivity of NSCLC was analysed by case-control test.

RESULTSThe allelic distribution of the three genotypes (A1/A1, A1/A2, A2/A2) in healthy controls was 32.0%, 42.0% and 26.0% respectively, which differed slightly from that of lung cancer patients, which was 28.0%, 32.0% and 40.0%. These allelic and genotype differences between control and lung cancer groups (A1/A1 OR=0.83, 95% CI 0.36-1.27 and A2/A2 OR=1.90, 95% CI 0.82-4.42) were insignificant. The patients with A1/A1 or A2/A2 genotype were sensitive to radiotherapy and the patients with A1/A2 were not sensitive to radiother-apy (Chi-square=9.2, P < 0.05), the effective rate to radiotherapy were 71.4%, 70.0% and 25.0% respectively.

CONCLUSIONSThere is no significant relationship between the BstUI SNP in p53 and susceptibility in NSCLC .The p53 BstU I SNP is closely associated with radiosensitivity of NSCLC in the Northern Chinese population.

OBJECTIVETo study therapeutic effects of Sky-bone expander for the treatment of osteoporotic vertebral compressed fractures.

METHODSFifteen patients (18 vertebrae) suffering from vertebral compression fractures were treated with Sky bone expander system which expanded and reconstructed the vertebral body with PMMA cement. The clinical effect was evaluated by observing the changing of visual analogue scale (VAS). The preoperative and postoperative mean VAS scores were compared by paired-sample t test. All the patients were followed up by telephone or clinic consulting after being discharged from our hospital.

RESULTSThe procedure was performed successfully in 15 patients. The operation time ranged from 45 to 110 minutes (65 minutes per vertebra on average). The patients were followed up and the duration ranged from 6 to 12 months (8 months on average). The mean VAS score of the patients were improved significantly at the third postoperative day compared with those before the operation (2.5 +/- 1.3, vs 7.7 +/- 1.1, all P < 0.05). The mean VAS score at the end of the follow-up was 2.2 +/- 1.2.

CONCLUSIONSky bone expander system provides significant pain relief effect in the cases of osteoporotic vertebral compression fractures, shortens the duration of lying in bed, and its procedure is convenient with few complications.

Aged ; Female ; Fractures, Compression ; surgery ; Humans ; Male ; Middle Aged ; Osteoporosis ; complications ; Pain Measurement ; Spinal Fractures ; surgery ; Vertebroplasty

OBJECTIVEOxidative stress plays an important role in retinal pigmental epithelium (RPE) death during aging and the development of age-related macular degeneration. Although early reports indicate that reactive oxygen species (ROS) including H₂O₂ can trigger apoptosis at lower concentrations and necrosis at higher concentrations, the exact molecular mechanism of RPE death is still unclear. The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS, especially at higher concentrations.

METHODSCultured ARPE-19 cells were treated with H₂O₂ at different concentrations and cell viability was measured with the MTT assay. Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining. Furthermore, the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG. Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects.

RESULTSH₂O₂ reduced the viability of ARPE-19 cells in a concentration-dependent manner, which was presented as a typical s-shaped curve. Cell death caused by high concentrations of H₂O₂ was confirmed to be programmed necrosis. Morphologically, dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane, positively detected with APOPercentage assay and PI staining. 24-hour treatment with 500 μmol/L H₂O₂ induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells. Moreover, antioxidant treatment using EGCG effectively protected cells from H₂O₂-induced injury, increasing cell viability from 14.17%±2.31% to 85.77%±4.58%. After H₂O₂ treatment, intracellular calcium levels were highly elevated with a maximum concentration of 1200 nM. Significantly, the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway, rescuing the ARPE-19 cell from H₂O₂-induced death.

CONCLUSIONSAt high concentrations, H₂O₂ induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.

Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Cell Culture Techniques ; Cell Line ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Heme Oxygenase-1 ; metabolism ; Humans ; Hydrogen Peroxide ; toxicity ; Necrosis ; drug therapy ; Oxidative Stress ; drug effects ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Retinal Pigment Epithelium ; drug effects ; enzymology ; metabolism ; pathology

OBJECTIVETo investigate the effect of transcriptional regulation of aberrant transcription factor AML1-ETO on p14.

METHODSP14expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. Methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of p14promoter. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and p14promoter in AML1-ETO positive leukemia cell line. And the p14mRNA expression level was detected by qRT-PCR after treatment with 5-Aza.

RESULTSAML1-ETO-expressing cell subclone displayed low level of p14mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, level of p14mRNA was markedly lower when compared with other acute myeloid leukemias lacking this translocation. P14gene promoter was non-methylated in control group and primary leukemia cells of AML patients without t(8;21) and was hyper-methylated in U937-A/E1-4 and primary leukemia cells of AML patients with t(8;21). The enriched regions in transfected cells were located within p14promoter. 5-Aza could increase the expression of p14.

CONCLUSIONP14is a possible target gene of AML1-ETO. The p14silencing induced by hyper-methlylation may be an important factor for occurrence and development of the Msubtype of acute myeloid leukemia.