Dopamine Receptors Modulate Cytotoxicity of Natural Killer Cells via cAMP-PKA-CREB Signaling Pathway

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 were blocked by {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.     

Ocular Hypertension after Pediatric Cataract Surgery: Baseline Characteristics and First-Year Report

Monitoring intraocular pressure (IOP) is essential for pediatric cataract treatment but always difficult due to lack of cooperation in young children. We present the baseline characteristics and the first-year results of a long-term prospective cohort study, which are aimed to determine the relationship of the incidence of ocular hypertension (OH) in children after cataract surgery during the first-year period and the risk of developing late-onset glaucoma. Children were included with the following criteria: they were≤10 years old and scheduled to undergo cataract surgery with/without intraocular lens implantation; they were compliant with our follow-up protocol, which included monitoring IOP using a Tono-Pen under sedation or anesthesia. Incidence of OH, peak OH value, OH onset time and OH duration within a 12-month period following surgery were measured. In brief, 206 patients (379 eyes) were included and OH developed in 66 of 379 (17.4%) eyes. The mean follow-up period was 14.0±3.2 months (median, 12 months; range, 10–16 months). Moreover, 33 of 196 (16.8%) aphakic eyes and 33 of 183 (18.0%) IOL eyes were diagnosed with OH. The peak OH onset times were at 1-week (34/66, 51.5%) and 1-month (14/66, 21.2%) appointments postsurgery. The peak IOP value in the OH eyes was 29.9±7.5 mmHg (median, 29 mmHg; range, 21–48 mmHg). The duration of OH was 30.9±31.2 days (median, 30 days; range, 3–150 days). OH recurred in 13 eyes with a history of OH diagnosed within 1 month postsurgery (13/54, 24.1%), which needed temporary or long term use of antiglaucoma medications. In conclusion, the incidence of OH in children after cataract surgery was 17.4% during the first-year period. Children who have suffered elevated IOP in the first year after cataract surgery should be followed closely to determine if there is an increased risk of developing late-onset glaucoma.     

Single,Double and Quadruple Alanine Substitutions at Oligomeric Interfaces Identify Hydrophobicity as the Key Determinant of Human Neutrophil Alpha Defensin HNP1 Function

HNP1 is a human alpha defensin that forms dimers and multimers governed by hydrophobic residues, including Tyr¹⁶, Ile²⁰, Leu²⁵, and Phe²⁸. Previously, alanine scanning mutagenesis identified each of these residues and other hydrophobic residues as important for function. Here we report further structural and functional studies of residues shown to interact with one another across oligomeric interfaces: I20A-HNP1 and L25A-HNP1, plus the double alanine mutants I20A/L25A-HNP1 and Y16A/F28A-HNP1, and the quadruple alanine mutant Y16A/I20A/L25A/F28A-HNP1. We tested binding to HIV-1 gp120 and HNP1 by surface plasmon resonance, binding to HIV-1 gp41 and HNP1 by fluorescence polarization, inhibition of anthrax lethal factor, and antibacterial activity using the virtual colony count assay. Similar to the previously described single mutant W26A-HNP1, the quadruple mutant displayed the least activity in all functional assays, followed by the double mutant Y16A/F28A-HNP1. The effects of the L25A and I20A single mutations were milder than the double mutant I20A/L25A-HNP1. Crystallographic studies confirmed the correct folding and disulfide pairing, and depicted an array of dimeric and tetrameric structures. These results indicate that side chain hydrophobicity is the critical factor that determines activity at these positions.     

细胞色素P450酶的结构、功能与应用研究进展

细胞色素P450 (cytochrome P450,CYP)酶是广泛存在于微生物、动植物及人体中与膜结合的血红蛋白类酶,具有氧化、环氧化、羟化、去甲基化等多种生物催化活性。CYP酶在药物、类固醇、脂溶性维生素和许多其他类型化学物质的代谢中具有重要作用,其在异源物质的解毒、药物相互作用和内分泌功能等领域的研究是热点问题。本综述对CYP的结构、功能、临床应用与开发前景进行了概述,并对其最新的研究现状和发展前景进行探讨。     

Baicalin suppresses the cell cycle progression and proliferation of prostate cancer cells through the CDK6/FOXM1 axis

Molecular and Cellular Biochemistry - Population data have consistently demonstrated a correlation between circulating branched-chain amino acids (BCAA) and insulin resistance. Most recently valine...     

蚯蚓提取物对家蚕中肠氧化损伤、总抗氧化能力及抗氧化酶含量的影响

【目的】研究蚯蚓提取物对家蚕Bombyx mori中肠组织氧化应激及抗氧化酶含量的影响,探究蚯蚓提取物的抗氧化功能。【方法】分别给家蚕5龄幼虫饲喂蚯蚓(赤子爱胜蚓Eisenia foetida)提取液原液的50, 100和200倍稀释液处理后的桑叶,测定处理6 d后家蚕中肠组织中氧化损伤产物丙二醛(MDA)和乳酸脱氢酶(LDH)的含量;利用qRT-PCR检测家蚕中肠组织中抗氧化酶关键基因(Cat,Sod和GSH-Px)的mRNA表达水平;同时采用ELISA法检测中肠组织中抗氧化酶[过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)]含量及总抗氧化能力(T-AOC)。【结果】与对照组相比,50, 100和200倍稀释组家蚕中肠组织中MDA, CAT, SOD和GSH-Px含量及T-AOC均差异不显著,200倍稀释组LDH含量显著降低了9.85%,100倍稀释组中肠中Cat和GSH-Px的表达量均显著高于对照组,3个稀释组中Sod的表达量均显著高于对照组。【结论】添食蚯蚓提取物可通过降低家蚕中肠LDH含量、提高抗氧化酶基因的表达水平进而提高家蚕的抗氧化能力。     

Long noncoding RNA LINC01234 promoted cell proliferation and invasion via miR-1284/TRAF6 axis in colorectal cancer

Colorectal cancer is one of the most common and leading malignancies globally. Long noncoding RNAs (lncRNAs) function as potentially critical regulator in colorectal cancer. LINC01234, a novel lncRNA in tumor biology, regulates the progression of various tumors. However, the tumorigenic mechanism of LINC01234 in colorectal cancer is still unclear. This study was performed with the aim to prospectively investigate clinical significance, effect, and mechanism of lncRNA LINC01234 in colorectal cancer. First, we found that LINC01234, localized in the cytoplasm, was increased in both colorectal cancer cell lines and tissues. Subsequent functional assays suggested LINC01234 knockdown suppressed cell proliferation, migration, and invasion of colorectal cancer cells, while blocked cell cycle and induced cell apoptosis. Moreover, we identified that miR-1284 was target of LINC01234, we further demonstrated a negative correlation with LINC01234 in colorectal cancer tissues and cells. Furthermore, miR-1284 targeted and suppressed tumor necrosis factor receptor–associated factor 6 (TRAF6). Loss-of-function assay revealed that LINC01234 silencing suppressed colorectal cancer progression through inhibition of miR-1284. In vivo subcutaneous xenotransplanted tumor model indicated LINC01234 knockdown inhibited in vivo tumorigenic ability of colorectal cancer via downregulation of TRAF6. Collectively, this study clarified the biological significance of LINC01234/miR-1284/TRAF6 axis in colorectal cancer progression, providing insights into LINC01234 as novel potential therapeutic target for colorectal cancer therapeutic from bench to clinic.     

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.