The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

Many studies have shown that the ubiq-uitin-proteasome pathway (UPP) for the degradation of short-lived proteins plays a key role in regulating cell cycle progression[1—3]. At least two distinct prote-olytic pathways are required for cell cycle process. The first pathway promotes transition from G1 to S phase, and the second initiates the onset of anaphase and exit from mitosis. The inhibition of UPP will re-sult in the blockage of cell cycle process. The knowl-edge of the role of UPP in…     

大肠杆菌中高效表达rhBMP-4的探讨及初步活性测定

目的 在大肠杆菌中表达具有生物活性的rhBMP-4。方法 在不改变氨基酸序列的前提下,以全基因合成的方式对人BMP-4成熟肽基因全长进行定点突变,将之重组入pET-3c表达载体并转化至大肠杆菌BL21(DE)plysS。IPTG诱导和包涵体复性后,利用C2C12细胞横向成骨细胞分化实验以及小鼠肌袋异位骨形成实验检测其活性。 结果 获得0.348 kb的BMP-4 DNA序列,表达的目的蛋白主要以包涵体的形式存在。经纯化及复性后,体内与体外的活性检测表明rhBMP-4有良好的诱骨生成活性。结论 该方案能够实现rhBMP-4在大肠杆菌中的高效表达。     

Effect of 2-mercaptoethanol and cysteine supplementation during in vitro maturation on the developmental competence of oocytes from hormone-stimulated lambs

The objective was to determine the effect of 2-mercaptoethanol and cysteine on in vitro developmental competence of oocytes from lambs (4-8-week old) stimulated with eCG and pFSH. Oocytes were matured in medium (TCM199) with no supplement (Control group) or with 100muM 2-mercaptoethanol and 600muM cysteine (GSH group). Oocytes from adult sheep were also included (Adult group). The addition of 2-mercaptoethanol and cysteine did not improve nuclear maturation or microtubule configuration 12, 15, 18, or 24h after placement in maturation medium. Sperm head decondensation and male pronucleus formation were evaluated at 6, 12, and 18h after commencement of IVF; sperm decondensation appeared earlier in the GSH group (6h after the start of IVF). There were differences (P<0.05) between the Control group and the GSH and Adult groups for: fertilization rate at both 12h (55.4, 77.0, and 80.6%, respectively) and 18h (67.9, 86.9, and 88.7%); parthenogenesis rate at both 12h (25.0, 10.8, and 5.6%) and 18h (28.3, 9.8, and 4.5%); and polyspermy rate at 18h (26.4, 4.9, and 5.7%). Blastocyst rate at 7d was higher in the GSH group than the Control group (23.9% vs. 14.9%, P<0.05), but both were lower (P<0.05) than the Adult group (38.3%). The addition of 2-mercaptoethanol and cysteine improved sperm decondensation and rates of fertilization and the blastocyst development to 7d, with no effect on blastocyst rate at 9d.     

Distinct functional sites for human immunodeficiency virus type 1 and stromal cell-derived factor 1alpha on CXCR4 transmembrane helical domains

The entry of human immunodeficiency virus type 1 (HIV-1) into the cell is initiated by the interaction of the viral surface envelope protein with two cell surface components of the target cell, CD4 and a chemokine coreceptor, usually CXCR4 or CCR5. The natural ligand of CXCR4 is stromal cell-derived factor 1alpha (SDF-1alpha). Whereas the overlap between HIV-1 and SDF-1alpha functional sites on the extracellular domains of CXCR4 has been well documented, it has yet to be determined whether there are sites in the transmembrane (TM) helices of CXCR4 important for HIV-1 and/or SDF-1alpha functions, and if such sites do exist, whether they are overlapping or distinctive for the separate functions of CXCR4. For this study, by employing alanine-scanning mutagenesis, (125)I-SDF-1alpha competition binding, Ca(2+) mobilization, and cell-cell fusion assays, we found that the mutation of many CXCR4 TM residues, including Tyr(45), His(79), Asp(97), Pro(163), Trp(252), Tyr(255), Asp(262), Glu(288), His(294), and Asn(298), could selectively decrease HIV-1-mediated cell fusion but not the binding activity of SDF-1alpha. Phe(87) and Phe(292), which were involved in SDF-1alpha binding, did not play a significant role in the coreceptor activity of CXCR4, further demonstrating the disconnection between physiological and pathological activities of CXCR4 TM domains. Our data also show that four mutations of the second extracellular loop, D182A, D187A, F189A, and P191A, could reduce HIV-1 entry without impairing either ligand binding or signaling. Taken together, our first detailed characterization of the different functional roles of CXCR4 TM domains may suggest a mechanistic basis for the discovery of new selective anti-HIV agents.     

Effect of Enhanced UV-B Radiation on Polyamine Content and Membrane Permeability in Cucumber Leaves

Cucumber plants (Cucumis sativus L., cv. Jingchun 3) were grown in a greenhouse under PAR illumination of 400–600 mol/(m² s) at 30/15°C (day/night) temperature. Two enhanced biologically effective UV-B radiation levels per day were applied: 8.82 kJ/m² (T₁) and 12.6 kJ/m² (T₂). Cucumber seedlings were irradiated 7 h per day for 25 days under T₁ and T₂. A comparative study of growth, membrane permeability, and polyamine content in cucumber leaves under T₁ and T₂ treatments was conducted. UV-B radiation resulted in the dose-dependent decrease in leaf area, dry weight of foliage, and plant height. The T₁ and T₂ treatments caused an increase in the contents of putrescine, spermine, and spermidine. However, the total polyamine content declined slightly when electrolyte leakage increased dramatically on the 18th day of treatment, especially after T₂ treatment. It can be concluded that polyamine accumulation in the cucumber leaves is an adaptive mechanism to the stress caused by UV-B radiation.     

Optimization of cryopreservation of Artemisia annua L. callus

Cryopreservation of callus tissue of Artimisia annua L. was optimized. Two lines of calli were precultured on MS medium with 5% (v/v) dimethyl sulfoxide, and protected by a cryoprotectant containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, 30% (v/v) glycerol and 13.6% (w/v) sucrose. The highest survival rate of callus A201 reached 87% after it was pretreated at 25°C, cryopreserved by liquid nitrogen, recovered in water bath at 25°C and reloaded at 25°C with 34% (w/v) sucrose solution, and that of callus A202 reached 78% after it was treated as callus A201, except pretreated at 35°C, recovered at 35°C and reloaded with 47.8% (w/v) sucrose solution.     

Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.     

Synthesis and anti-tumor evaluation of new trisulfide derivatives

New bis-aromatic and heterocyclic trisulfide derivatives 5, 7-10 were synthesized by optimizing lead dibenzyl trisulfide natural product (4) to evaluate their anti-tumor activities. Five compounds 5-7, 9, and 10 exhibited potent anti-tumor activities against eight different tumor cell lines with low cytotoxicity against HepG2. Initial SAR was discussed, and MOA of these anti-microtubule agents was suggested based on cell kinetic response patterns observed on RT-CES system.     

Increased efflux of glutathione conjugate in acutely diabetic cardiomyocytes

In diabetes, cell death and resultant cardiomyopathy have been linked to oxidative stress and depletion of antioxidants like glutathione (GSH). Although the de novo synthesis and recycling of GSH have been extensively studied in the chronically diabetic heart, their contribution in modulating cardiac oxidative stress in acute diabetes has been largely ignored. Additionally, the possible contribution of cellular efflux in regulating GSH levels during diabetes is unknown. We used streptozotocin to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac GSH. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. The effect on GSH was not associated with any change in either synthesis or recycling, as both gamma-glutamylcysteine synthetase gene expression (responsible for bio syn thesis) and glutathione reductase activity (involved with GSH recycling) remained unchanged. However, gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was elevated. GSH conjugate efflux mediated by multidrug resistance protein 1 also increased in diabetic cardiomyocytes, an effect that was blocked using MK-571, a specific inhibitor of this transporter. As MK-571 also decreased oxidative stress in diabetic cardiomyocytes, an important role can be proposed for this transporter in GSH and reactive oxygen species homeostasis in the acutely diabetic heart.     

Dynamics of NK,CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID‐19

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8⁺ T, CD4⁺ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8⁺ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3⁺ Tfh‐like cell and CD226⁺ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS⁺ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8⁺ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4⁺ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.