Effects of Apelin-13/putative receptor protein related to AT1 homodimer on behaviors of human umbilical vein endothelial cells / 中国药理学通报

Aim To explore the effects of putative receptor protein related to ATI (APJ) homodimer on the behaviors-the proliferation, migration and tube formation of human umbilical vein endothelial cells (HU-VECs). Methods HUVECs at logarithmic growth stage were randomly divided into PBS, Apelin-13 + TM1 (APJ monomer group) and Apelin-13 + PBS group (APJ homodimer group). Western blot and Matrix-Assisted Laser Desorption/Ionization Time of Fligh Mass Spectrometry (MALDI-TOF MS) were used to detect the expression of APJ and APJ homodimer in HUVECs, respectively. Real-Time Cell Analyzers (RT-CA) was used to detect the concentration of the maximum effect of Apelin-13. Cell viability was detected by CCK-8. The cell migration ability was detected by scratch test, and the number of tubes formed on matri-gel that made artificial basement membrane was counted. Results Western blot and MALDI-TOF MS showed that APJ and APJ homodimer were expressed in HUVECs. The EC50 of Apelin-13 was 2.26 x 10

The treatment effect of novel hGHRH homodimer to male infertility hamster

Extra-hypothalamic growth hormone-releasing hormone (GHRH) plays an important role in reproduction. To study the treatment effect of Grin (a novel hGHRH homodimer), the infertility models of 85 male Chinese hamsters were established by intraperitoneally injecting 20 mg/kg of cyclophosphamide once in a week for 5 weeks and the treatment with Grin or human menopausal gonadotropin (hMG) as positive control was evaluated by performing a 3-week mating experiment. 2–8 mg/kg of Grin and 200 U/kg of hMG showed similar effect and different pathological characteristics. Compared to the single cyclophosphamide group (0%), the pregnancy rates (H-, M-, L-Grin 26.7, 30.8, 31.3%, and hMG 31.3%) showed significant difference, but there was no difference between the hMG and Grin groups. The single cyclophosphamide group presented loose tubules with pathologic vacuoles and significant TUNEL positive cells. Grin induced less weight of body or testis, compactly aligned tubules with little intra-lumens, whereas hMG caused more weight of body or testis, enlarging tubules with annular clearance. Grin presented a dose-dependent manner or cell differentiation-dependentincrease in testicular GHRH receptor, and did not impact the levels of blood and testicular GH, testosterone. Grin promotes fertility by proliferating and differentiating primitive cells through up-regulating testicular GHRH receptor without triggering GH secretion, which might solve the etiology of oligoasthenozoospermia.

Structural studies on MRG701 chromodomain reveal a novel dimerization interface of MRG proteins in green plants

MRG proteins are conserved during evolution in fungi, flies, mammals and plants, and they can exhibit diversified functions. The animal MRGs were found to form various complexes to activate gene expression. Plant MRG1/2 and MRG702 were reported to be involved in the regulation of flowering time via binding to H3K36me3-marked flowering genes. Herein, we determined the crystal structure of MRG701 chromodomain (MRG701). MRG701 forms a novel dimerization fold both in crystal and in solution. Moreover, we found that the dimerization of MRG chromodomains is conserved in green plants. Our findings may provide new insights into the mechanism of MRGs in regulation of gene expression in green plants.

Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection

Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95 percent ± 3.56, p < 0.0001, for 24 hours and mean of 37.67 percent ± 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.

Characterization of the homodimer and antigenicity of ORF2 polypeptides of genotype 4 hepatitis Evirus / 中华微生物学和免疫学杂志

Objective To characterize the dimerization and the antigenicity of the ORF2 polypep-tide of hepatitis E virus (HEV, genotype 4). Methods HEV ORF2 gene was cloned from the serum of a patient with hepatitis E. The genotype was determined by sequencing. Three ORF2 polypeptides differing in size and other polypeptides with point mutations were produced in E. coli. The recombinant polypeptides were purified and analyzed by SDS-PAGE and Western blot. Results The ORF2 polypeptide containing 459-607 amino acid formed homedimer even in 8 mol/L urea. The truncated polypeptides containing amino acid 472-607 or 459-594 formed monomer only. The mutations at amino acid 562 or 595 disrupted the ho-modimer, whereas the mutations at amino acid 476 or 580 did not. Anti-HEV from hepatitis E patients only reacted with the homodimer form of the polypeptide 459-607 and did not react with monomer or tnmcated pol-ypeptides. Conclusion The amino acid 459-607 of HEV ORF2 is essential for dimerization of the ORF2 polypeptide. Residues at amino acid 562 and 595 are critical for the dimerization. The antigenicity of the polypeptide 459-607 mainly depends on its homodimer form.

An Improved Measurement of Fluorescence Resonance Energy Transfer to Analyse Protein-protein Interaction in Protein Homodimer / 中国生物化学与分子生物学报

Fluorescence resonance energy transfer(FRET)is increasingly used to study inter-and intramolecular interactions in living cells.Since being proportional to the concentration of the donor-acceptor complex.FRET value must be normalized to exclude the influence of the ratio and the concentration of donor and acceptor for comparison.Different from the intra.molecular FRET which is simplified by the fact that the COncentration of the donor is equal to that of the acceptor,the inter-molecular FRET is usually too complicated for most existing measurements to quantify exactly.We deduced the exact proportion of the donor-acceptor complex based on a unique characteristic of homodimer,a special kind of the intermolecular interaction,developed an exact quantification measurement of the FRET.We proved the novel method Can generate more reliable estimation of FRET value by comparison with other methods using a homodimer,estrogen receptor alpha(ERa),as a FRET pair.