Site-specific mutations in the N-terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor.

C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C-terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N-terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site-directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (beta-glucuronidase release). Mutants that contained the single residue substitutions Ile-6-->Gly or Tyr-13-->Gly were reduced in potency to 4-30% compared with wild-type rC5a. Other single-site glycine substitutions at positions Leu-2, Ala-10, Lys-4, Lys-5, Glu-7, Glu-8, and Lys-14 showed little effect on C5a potency. The double mutant, Ile-6-->Gly/Tyr-13-->Gly, was reduced in potency to < 0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in alpha-helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N-terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.     

Metal resistance in Acinetobacter and its relation to β-lactamase production

Thirty nine clinical isolates of Acinetobacter belonging to six species were tested for resistance to 20 metal ions and their ability to produce -lactamase. Fifty two percent of the strains produced -lactamase. -Lactamase producers and non-producers were almost equally distributed in the different species. A. baumannii was the predominant biotype and was found to be most resistant to metals. Resistance to mercury was prevalent in -lactamase-producing A. baumannii only. Silver resistant strains of A. baumannii produced -lactamase. Sensitivity and resistance to copper and cadium was equally distributed between -lactamase producers and non-producers. -Lactamase-producer and -non-producer strains were uniformly sensitive to cadmium except Acinetobacter genospecies 1.     

p62蛋白的分子功能及其在疾病中的研究进展

螯合体1(SQSTM1/p62)是一种选择性自噬接头蛋白,在清除待降解蛋白、维持细胞内蛋白质稳态中发挥重要的调控作用。p62蛋白具有多个功能结构域,介导与多种蛋白质发生相互作用进而精确调节特定的信号通路,从而将p62蛋白与氧化防御系统、炎症反应和营养感知等重要生命过程联系起来。研究表明p62的突变或者表达异常与多种疾病的发生发展过程密切相关,包括神经退行性疾病、肿瘤、感染性疾病、遗传性疾病以及慢性疾病等。本文综述了p62蛋白的结构特征、分子功能,并系统介绍其在蛋白质稳态和信号通路调控中的多种功能,总结了p62在疾病发生发展中的复杂性与多面性,以期为p62蛋白的功能与相关疾病研究提供参考。     

基于CRISPR/Cas系统的多重基因编辑与调控技术

规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)及其相关Cas蛋白所构建的CRISPR/Cas系统是古细菌或细菌中特有的一种获得性免疫系统。研究人员将其开发成基因编辑工具之后,凭借其高效、精准和通用性强等优点迅速成为合成生物学领域的热门研究方向,在生命科学、生物工程技术、食品科学及农作物育种等多个领域引发了革命性的影响。目前基于CRISPR/Cas系统单基因编辑与调控技术日益完善,但在多重基因编辑和调控方面仍存在挑战。本文聚焦基于CRISPR/Cas系统的多重基因编辑与调控技术开发及应用,针对单个细胞内实现多位点基因编辑或调控和细胞群体内实现多位点基因编辑或调控技术,依据作用原理对其进行了系统总结和阐述,包括基于CRISPR/Cas系统的双链断裂、单链断裂以及多重基因调控技术等。这些工作丰富了多重基因编辑与调控的工具,为CRISPR/Cas系统在多领域的应用作出了贡献。     

谷氨酸棒杆菌代谢工程高效生产L-缬氨酸北大核心CSCD

L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。     

Atypical lipomatous tumour/well-differentiated liposarcoma found in the buccal mucosa: A rare case report

Oral liposarcomas are uncommon diseases, the most predominant histopathological subtype being atypical lipomatous tumour/well-differentiated liposarcoma. In regard to its clinical aspects in the oral cavity, it is challenging to confirm a diagnosis and develop a treatment plan. In this case report, we present a rare case of atypical lipomatous tumour/well-differentiated liposarcoma in the right cheek of a 77-year-old male patient. Conservative surgery was performed considering the histopathological subtype of the neoplasm. Knowledge of the clinical and histopathological characteristics of this rare disease is essential to maintaining function and aesthetics through conservative treatment in older patients.     

Algae in mosquito breeding sites and the effectiveness of the mosquito larvicide Bacillus thuringiensis H-14

The presence of cyanobacteria generally decreased the effectiveness of Bacillus thuringiensis H-14 (BTI) as a mosquito larvicide. The effect was more pronounced when the mosquito larvae were exposed to BTI in the presence of several cyanobacterial strains. No synergistic or antagonistic effect between the -endotoxin from BTI and the hepatotoxin from cyanobacteria was seen. Neurotoxic cyanobacterial strains caused very fast paralysis in mosquito larvae; the decreases in the effectiveness of BTI when tested in combination with a neurotoxic strain might be due to the effect of this paralytic action on the feeding rate of the mosquito larvae.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

Effects of vitamin D3 on signaling by prostaglandin E2 in osteoblast-like cells

We investigated the effects of vitamin D₃ on the signaling pathways by prostaglandin E₂ (PGE₂) in osteoblast-like MC3T3-E1 cells. The pretreatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), an active form of vitamin D₃, significantly inhibited cAMP accumulation induced by 10 μM PGE₂ in a dose-dependent manner in the range between 1 pM and 1 nM. This effect of 1,25-(OH)₂D₃ was dependent on the time of pretreatment up to 8 h. 1,25-(OH)₂D₃ also inhibited the cAMP accumulation induced by NaF, a GTP-binding protein activator, or forskolin which directly activates adenylate cyclase. On the other hand, 1,25-(OH)₂D₃ significantly inhibited PGE₂-induced IP₃ formation in a dose-dependent manner between 10 pM and 1 nM. However, 1,25-(OH)₂D₃ had little effect on NaF-induced IP₃ formation. The pretreatment with 24,25-dihydroxyvitamin D₃, an inactive form of vitamin D₃, affected neither cAMP accumulation nor IP₃ formation induced by PGE₂. These results strongly suggest that 1,25-(OH)₂D₃ modulates the signaling by PGE₂ in osteoblast-like cells as follows: the inhibitory effect on the cAMP production is exerted at a point downstream from adenylate cyclase and the inhibitory effect on the phosphoinositide hydrolysis is exerted at the point between the PGE₂ receptor and GTP-binding protein, probably G_i2.