BAmSA: Visualising transmembrane regions in protein complexes using biotinylated amphipols and electron microscopy

Membrane protein (MP) complexes play key roles in all living cells. Their structural characterisation is hampered by difficulties in purifying and crystallising them. Recent progress in electron microscopy (EM) have revolutionised the field, not only by providing higher-resolution structures for previously characterised MPs but also by yielding first glimpses into the structure of larger and more challenging complexes, such as bacterial secretion systems. However, the resolution of pioneering EM structures may be difficult and their interpretation requires clues regarding the overall organisation of the complexes. In this context, we present BAmSA, a new method for localising transmembrane (TM) regions in MP complexes, using a general procedure that allows tagging them without resorting to neither genetic nor chemical modification. Labels bound to TM regions can be visualised directly on raw negative-stain EM images, on class averages, or on three-dimensional reconstructions, providing a novel strategy to explore the organisation of MP complexes.     

Methyl B12 protects PC12 cells against cytotoxicity induced by Aβ25−35

Alzheimer's disease (AD) is the most common aging-associated dementia. The population of AD patients is increasing as the world age grows. Currently, there is no cure for AD. Given that methyl vitamin B12 (methylcobalamin) deficiency is related to AD and Aβ-induced oxidative damage and that methylcobalamin can scavenge reactive oxygen species (ROS) by direct or indirect ways, we studied the effect of methylcobalamin on the cytotoxicity of Aβ. PC12 cells were chronically exposed (24 hours) to Aβ_25-35 (25 μM) to establish an AD cell model. The cells were pretreated with or without methylcobalamin (1-100 μM) to investigate the role of methylcobalamin. Cell viability and apoptosis were tested, followed by testing of mitochondrial damage, oxidative stress, and mitochondrial calcium concentration. We observed that methylcobalamin improved the cell viability by decreasing the ratio of apoptosis cells in this AD cell model. Further experiments suggested that methylcobalamin functioned as an antioxidant to scavenge ROS, reducing the endoplasmic reticulum-mitochondria calcium flux through IP3R, preventing mitochondria dysfunction, ultimately protecting cells against apoptosis and cell death. Taken together, our results presented, for the first time, that methyl vitamin B12 can protect cells from Aβ-induced cytotoxicity and the mechanism was mainly relevant to the antioxidative function of methyl B12.     

Roles for osteocalcin in proliferation and differentiation of spermatogonial cells cocultured with somatic cells

Spermatogonial cells (SCs) are key cells for spermatogenesis. These cells are affected by paracrine signals originated from nearby somatic cells, among them Leydig cells have receptors for osteocalcin, a hormone known for exerting positive roles in the promotion of spermatogenesis. The aim of this study was to evaluate roles for osteocalcin on SCs proliferative and differentiation features after coculture with Leydig cells. SCs and Leydig cells were isolated from neonate NMRI offspring mice and adult NMRI mice, respectively. SCs population were then enriched in a differential attachment technique and assessed for morphological features and identity. Then, SCs were cocultured with Leydig cells and incubated with osteocalcin for 4 weeks. Evaluation of proliferation and differentiation-related factors were surveyed using immunocytochemistry (ICC), Western blot, and quantitative real-time polymerase chain reaction (PCR). Finally, the rate of testosterone release to the culture media was measured at the end of 4th week. Morphological and flow cytometry results showed that the SCs were the population of cells able to form colonies and to express ID4, α6-, and β1-integrin markers, respectively. Leydig cells were also able to express Gprc6α as a specific marker for the cells. Incubation of SCs/Leydig coculture with osteocalcin has resulted in an increase in the rate of expressions for differentiation-related markers. Levels of testosterone in the culture media of SCs/Leydig was positively influenced by osteocalcin. It could be concluded that osteocalcin acts as a positive inducer of SCs in coculture with Leydig cells probably through stimulation of testosterone release from Leydig cells and associated signaling.     

非洲猪瘟病毒p35蛋白作为诊断抗原的抗原性比较

为了筛选出酶联免疫吸附测定(Enzyme linked immunosorbent assay,ELISA)反应性最佳的非洲猪瘟病毒(African swine fever virus,ASFV)诊断抗原,通过建立ELISA方法,以杆状病毒昆虫细胞表达系统表达的ASFV p30蛋白诊断抗原为参照,首次探讨原核表达系统表...     

异槲皮苷对Aβ25-35导致的PC12细胞损伤的保护作用及机制研究

探讨异槲皮苷对β-淀粉样蛋白(Aβ25-35)导致的PC12细胞氧化损伤的保护作用.首先通过分子对接技术分析异槲皮苷与AMPK的结合情况.采用Aβ25-35(20 μmol/L)损伤PC12细胞建立细胞氧化损伤模型,采用甲基噻唑蓝(MTT)法检测细胞活力,通过试剂盒检测乳酸脱氢酶(LDH)漏出量、活性氧(ROS)含量、...     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

Enzymatic synthesis of lysophosphatidic acid and phosphatidic acid

Immobilised 1,3-specific lipase from Rhizopus arrhizus was used as catalyst for the esterification of -glycero-3-phosphate and fatty acid or fatty acid vinyl ester in a solvent-free system. With lauric acid vinyl ester as acyl donor, a_w<0.53 favored the synthesis of lysophosphatidic acid (1-acyl-rac-glycero-3-phosphate, LPA1) and the spontaneous acyl migration of the fatty acid on the molecule. Subsequent acylation by the enzyme resulted in high phosphatidic acid (1,2-diacyl-rac-glycero-3-phosphate, PA) formation and high total conversions (>95%). With oleic acid, maximum conversions of 55% were obtained at low water activities. Temperatures below melting point of the product favored precipitation and resulted in high final conversion and high product ratio [LPA/(PA+LPA)]. Thus, LPA was the only product with lauric acid vinyl ester as acyl donor at 25°C. Increased substrate ratio ( -glycero-3-phosphate/fatty acid) from 0.05 to 1 resulted in a higher ratio of LPA to PA formed, but a lower total conversion of -glycero-3-phosphate. Increased amounts of enzyme preparation did not result in higher esterification rates, probably due to high mass-transfer limitations.     

Identification of the site of inhibition of oncogenic ras-p21-induced signal transduction by a peptide from a ras effector domain

We have previously found that a peptide corresponding to residues 35–47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras–p21 peptide corresponding to residues 96–110 that blocks ras–jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35–47, but not the 96–110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35–47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras–p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35–47 and 96–110, act at two different critical downstream sites, the former at raf, the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.