Human brain tubulin purification: Decrease in soluble tubulin with age

The soluble tubulin of human cerebral cortex, as assessed by [³H]colchicine binding of the 100,000g supernatant fraction, decreases drastically with age, 75 percent from age 0 to age 90. There is also a considerably lower concentration of high molecular weight proteins in the soluble fraction of postmortem human cerebral cortex than in that of nonhuman species. Human brain tubulin can be polymerized into microtubules with DEAE-dextran. The DEAE-dextran induced microtubules are stable to cold temperature (4°) and calcium. However, in the presence of 1 M glutamate, the microtubules become cold labile and depolymerize at 4°. Thus we have developed a novel method for purifying polymerization competent tubulin from fresh or frozen human cerebral cortex. Human brain tubulin purified by our novel method is very similar to tubulin from the brains of other mammals in molecular weight, amino acid composition, polymerization-depolymerization parameters, and structural dimensions of the microtubules formed.Some aspects of this work have been published as an abstract in 1981. Fed. Proc. 40:1548.     

Way out of Africa: Early Pleistocene paleoenvironments inhabited by Homo erectus in Sangiran, Java

A sequence of paleosols in the Solo Basin, Central Java, Indonesia, documents the local and regional environments present when Homo erectus spread through Southeast Asia during the early Pleistocene. The earliest human immigrants encountered a low-relief lake-margin landscape dominated by moist grasslands with open woodlands in the driest landscape positions. By 1.5 Ma, large streams filled the lake and the landscape became more riverine in nature, with riparian forests, savanna, and open woodland. Paleosol morphology and carbon isotope values of soil organic matter and pedogenic carbonates indicate a long-term shift toward regional drying or increased duration of the annual dry season through the early Pleistocene. This suggests that an annual dry season associated with monsoon conditions was an important aspect of the paleoclimate in which early humans spread from Africa to Southeast Asia.     

Arabidopsis ADC1 functions as an N~δ-acetylornithine decarboxylase

Polyamines are small aliphatic amines found in almost all organisms, ranging from bacteria to plants and animals. In most plants, putrescine, the metabolic precursor for longer polyamines, such as spermidine and spermine, is produced from arginine, with either agmatine or ornithine as intermediates. Here we show that Arabidopsis thaliana(Arabidopsis) arginine decarboxylase 1(ADC1), one of the two known arginine decarboxylases in Arabidopsis, not only synthesizes agmatine from arginine, but also converts N~δ-acetylornithine to N-acetylputrescine. Phylogenetic analyses indicate that duplication and neofunctionalization of ADC1 and NATA1, the enzymes that synthesize N~δ-acetylornithine in Arabidopsis, co-occur in a small number of related species in the Brassicaceae. Unlike ADC2, which is localized in the chloroplasts, ADC1 is in the endoplasmic reticulum together with NATA1, an indication that these two enzymes have access to the same substrate pool. Together, these results are consistent with a model whereby NATA1 and ADC1 together provide a pathway for the synthesis of N-acetylputrescine in Arabidopsis.     

The Brassicaceae‐specific secreted peptides,STMPs, function in plant growth and pathogen defense

Low molecular weight secreted peptides have recently been shown to affect multiple aspects of plant growth, development, and defense responses.Here, we performed stepwise BLAST filtering to identify unannotated peptides from the Arabidopsis thaliana protein database and uncovered a novel secreted peptide family, secreted transmembrane peptides(STMPs). These low molecular weight peptides, which consist of an N-terminal signal peptide and a transmembrane domain, were primarily localized to extracellular compartments but were also detected in the endomembrane system of the secretory pathway, including the endoplasmic reticulum and Golgi. Comprehensive bioinformatics analysis identified 10 STMP family members that are specific to the Brassicaceae family. Brassicaceae plants showed dramatically inhibited root growth uponexposure to chemically synthesized STMP1 and STMP2.Arabidopsis overexpressing STMP1, 2, 4, 6, or 10 exhibited severely arrested growth, suggesting that STMPs are involved in regulating plant growth and development. In addition, in vitro bioassays demonstrated that STMP1,STMP2, and STMP10 have antibacterial effects against Pseudomonas syringae pv. tomato DC3000, Ralstonia solanacearum, Bacillus subtilis, and Agrobacterium tumefaciens, demonstrating that STMPs are antimicrobial peptides. These findings suggest that STMP family members play important roles in various developmental events and pathogen defense responses in Brassicaceae plants.     

Split Nano luciferase complementation for probing protein‐protein interactions in plant cells

Deciphering protein‐protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual‐use system enabling the detection of PPIs through the Nluc‐based SLC and co‐immunoprecipitation assays. This was realized by coexpression of two proteins under investigation in fusion with the HA‐ or FLAG‐tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium‐transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a coreceptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc‐based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells.     

Tumour‐derived exosomal miR‐3473b promotes lung tumour cell intrapulmonary colonization by activating the nuclear factor‐κB of local fibroblasts

Tumour‐derived exosomes have been shown to induce pre‐metastatic niche formation, favoring metastatic colonization of tumour cells, but the underlying molecular mechanism is still not fully understood. In this study, we showed that exosomes derived from the LLC cells could indeed significantly enhance their intrapulmonary colonization. Circulating LLC‐derived exosomes were mainly engulfed by lung fibroblasts and led to the NF‐κB signalling activation. Further studies indicated that the exosomal miR‐3473b was responsible for that by hindering the NFKB inhibitor delta's (NFKBID) function. Blocking miR‐3473b could reverse the exosome‐mediated NF‐κB activation of fibroblasts and decrease intrapulmonary colonization of lung tumour cells. Together, this study demonstrated that the miR‐3473b in exosomes could mediate the interaction of lung tumour cells and local fibroblasts in metastatic sites and, therefore, enhance the metastasis of lung tumour cells.     

Exosome derived from CD137‐modified endothelial cells regulates the Th17 responses in atherosclerosis

The role of exosomes derived from endothelial cells (ECs) in the progression of atherosclerosis (AS) and inflammation remains largely unexplored. We aimed to investigate whether exosome derived from CD137‐modified ECs (CD137‐Exo) played a major role in AS and to elucidate the potential mechanism underlying the inflammatory effect. Exosomes derived from mouse brain microvascular ECs treated with agonist anti‐CD137 antibody were used to explore the effect of CD137 signalling in AS and inflammation in vitro and vivo. CD137‐Exo efficiently induced the progression of AS in ApoE^?/? mice. CD137‐Exo increased the proportion of Th17 cells both in vitro and vivo. The IL‐6 contained in CD137‐Exo which is regulated by Akt and NF‐КB pathway was verified to activate Th17 cell differentiation. IL‐17 increased apoptosis, inhibited cell viability and improved lactate dehydrogenase (LDH) release in ECs subjected to inflammation induced by lipopolysaccharide (LPS). The expression of soluble intercellular adhesion molecule1 (sICAM‐1), monocyte chemoattractant protein‐1 (MCP‐1) and E‐selectin in the supernatants of ECs after IL‐17 treatment was dramatically increased. CD137‐Exo promoted the progression of AS and Th17 cell differentiation via NF‐КB pathway mediated IL‐6 expression. This finding provided a potential method to prevent local and peripheral inflammation in AS.     

Allopurinol reduces oxidative stress and activates Nrf2/p62 to attenuate diabetic cardiomyopathy in rats

Allopurinol (ALP) attenuates oxidative stress and diabetic cardiomyopathy (DCM), but the mechanism is unclear. Activation of nuclear factor erythroid 2‐related factor 2 (Nrf2) following the disassociation with its repressor Keap1 under oxidative stress can maintain inner redox homeostasis and attenuate DCM with concomitant attenuation of autophagy. We postulated that ALP treatment may activate Nrf2 to mitigate autophagy over‐activation and consequently attenuate DCM. Streptozotocin‐induced type 1 diabetic rats were untreated or treated with ALP (100 mg/kg/d) for 4 weeks and terminated after heart function measurements by echocardiography and pressure‐volume conductance system. Cardiomyocyte H9C2 cells infected with Nrf2 siRNA or not were incubated with high glucose (HG, 25 mmol/L) concomitantly with ALP treatment. Cell viability, lactate dehydrogenase, 15‐F2t‐Isoprostane and superoxide dismutase (SOD) were measured with colorimetric enzyme‐linked immunosorbent assays. ROS, apoptosis, was assessed by dihydroethidium staining and TUNEL, respectively. The Western blot and qRT‐PCR were used to assess protein and mRNA variations. Diabetic rats showed significant reductions in heart rate (HR), left ventricular eject fraction (LVEF), stroke work (SW) and cardiac output (CO), left ventricular end‐systolic volume (LVVs) as compared to non‐diabetic control and ALP improved or normalized HR, LVEF, SW, CO and LVVs in diabetic rats (all P < .05). Hearts of diabetic rats displayed excessive oxidative stress manifested as increased levels of 15‐F2t‐Isoprostane and superoxide anion production, increased apoptotic cell death and cardiomyocytes autophagy that were concomitant with reduced expressions of Nrf2, heme oxygenase‐1 (HO‐1) and Keap1. ALP reverted all the above‐mentioned diabetes‐induced biochemical changes except that it did not affect the levels of Keap1. In vitro, ALP increased Nrf2 and reduced the hyperglycaemia‐induced increases of H9C2 cardiomyocyte hypertrophy, oxidative stress, apoptosis and autophagy, and enhanced cellular viability. Nrf2 gene silence cancelled these protective effects of ALP in H9C2 cells. Activation of Nrf2 subsequent to the suppression of Keap1 and the mitigation of autophagy over‐activation may represent major mechanisms whereby ALP attenuates DCM.     

Integrin-associated molecules and signalling cross talking in osteoclast cytoskeleton regulation

In the ageing skeleton, the balance of bone reconstruction could commonly be broken by the increasing of bone resorption and decreasing of bone formation. Consequently, the bone resorption gradually occupies a dominant status. During this imbalance process, osteoclast is unique cell linage act the bone resorptive biological activity, which is a highly differentiated ultimate cell derived from monocyte/macrophage. The erosive function of osteoclasts is that they have to adhere the bone matrix and migrate along it, in which adhesive cytoskeleton recombination of osteoclast is essential. In that, the podosome is a membrane binding microdomain organelle, based on dynamic actin, which forms a cytoskeleton superstructure connected with the plasma membrane. Otherwise, as the main adhesive protein, integrin regulates the formation of podosome and cytoskeleton, which collaborates with the various molecules including: c-Cbl, p130^Cas, c-Src and Pyk2, through several signalling cascades cross talking, including: M-CSF and RANKL. In our current study, we discuss the role of integrin and associated molecules in osteoclastogenesis cytoskeletal, especially podosomes, regulation and relevant signalling cascades cross talking.