Nap1 and Chz1 have Separate Htz1 Nuclear Import and Assembly Functions

We analyzed the nuclear import and regulation of the yeast histone variant Htz1 (H2A.Z), and the role of histone chaperones Nap1 and Chz1 in this process. Copurification suggested that Htz1 and H2B dimerized in the cytoplasm prior to import. Like H2B, Htz1 contained a nuclear localization signal (NLS) in its N‐terminus that is recognized by multiple karyopherins (also called importins), indicating multiple transport pathways into the nucleus. However, Kap114 and Kap123 appeared to play the major role in Htz1 import. We also identified a role for Nap1 in the import of Htz1/H2B heterodimers, and Nap1 formed a RanGTP‐insensitive import complex with Htz1/H2B and Kap114. Nap1 was necessary for maintaining a soluble pool of Htz1, indicating that its chaperone function may be important for the dynamic exchange of histones within nucleosomes. In contrast, Chz1 was imported by a distinct import pathway, and Chz1 did not appear to interact with Htz1 in the cytoplasm. Genetic analysis indicated that NAP1 has a function in the absence of HTZ1 that is not shared with CHZ1. This provides further evidence that the histone chaperones Nap1 and Chz1 have separate Htz1‐dependent and ‐independent functions.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Enhanced understanding of predator–prey relationships using molecular methods to identify predator species,individual and sex

Predator species identification is an important step in understanding predator‐prey interactions, but predator identifications using kill site observations are often unreliable. We used molecular tools to analyse predator saliva, scat and hair from caribou calf kills in Newfoundland, Canada to identify the predator species, individual and sex. We sampled DNA from 32 carcasses using cotton swabs to collect predator saliva. We used fragment length analysis and sequencing of mitochondrial DNA to distinguish between coyote, black bear, Canada lynx and red fox and used nuclear DNA microsatellite analysis to identify individuals. We compared predator species detected using molecular tools to those assigned via field observations at each kill. We identified a predator species at 94% of carcasses using molecular methods, while observational methods assigned a predator species to 62.5% of kills. Molecular methods attributed 66.7% of kills to coyote and 33.3% to black bear, while observations assigned 40%, 45%, 10% and 5% to coyote, bear, lynx and fox, respectively. Individual identification was successful at 70% of kills where a predator species was identified. Only one individual was identified at each kill, but some individuals were found at multiple kills. Predator sex was predominantly male. We demonstrate the first large‐scale evaluation of predator species, individual and sex identification using molecular techniques to extract DNA from swabs of wild prey carcasses. Our results indicate that kill site swabs (i) can be highly successful in identifying the predator species and individual responsible; and (ii) serve to inform and complement traditional methods.     

Molecular evidence of natural hybridization between abies veitchii and A. homolepis (Pinaceae) revealed by chloroplast, mitochondrial and nuclear DNA markers

Sub-alpine Abies veitchii and A. homolepis are distributed in the central part of Honshu Island, Japan, and their habitats are segregated vertically. These species sometimes form a mixed forest in the overlapping area of the two species, that is, in the upper limit of the A. homolepis habitat and the lower limit of A. veitchii. These species have been considered to be distantly related because they were classified into different sections by most conventional classifications. No natural hybridization has been reported between the two species. The aim of this study was to demonstrate, through the use of molecular markers, whether natural hybridization takes place between these two species at two experimental sites on Mt. Fuji, where the species occur naturally. DNA markers from paternally inherited chloroplast DNA (cpDNA), maternally inherited mitochondrial DNA (mtDNA) and biparentally inherited nuclear DNA (nDNA), were used for this study. As organelle DNA markers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) markers were developed to determine the maternal and paternal species for each individual. Two of 334 individuals possessed a cpDNA haplotype derived from A. homolepis and a mtDNA haplotype from A. veitchii. Furthermore, the nDNA of these two individuals was analysed using the random amplified polymorphic DNA (RAPD) assay to investigate their genomic composition. RAPD analysis indicated that the nuclear genomes of the two individuals were derived from both species. We conclude that A. veitchii and A. homolepis produce natural hybrids, and that their systematic relationship should be re-evaluated.     

一种基于配体肽的CRP荧光检测探针的研制

C反应蛋白（C-reaction protein,CRP）是反映机体炎症的有效标志物,早期检测是判断炎症相关疾病的关键,因此,研制CRP新型检测制剂具有重要意义。利用噬菌体表面展示技术对CRP特异性亲和配体进行了筛选,采用固相肽合成技术对目标配体进行了合成,并经生物标记、高效液相色谱法（high performance liquid chromatography,HPLC）分析及质谱（mass spectrometry,MS）鉴定,成功制得检测CRP的荧光探针。经3轮筛选、ELISA检测、重组噬菌体测序及序列比对后得到1个目标配体肽：S-P-H-N-R-S-N-L-V-Q-E-L;经肽合成及生物标记获得1种CRP荧光探针：FITC-(Acp)-S-P-H-N-R-SN-L-V-Q-E-L。研究结果为CRP的有效检测提供了一种新型制剂。     

Silencing Nrf2 attenuates chronic suppurative otitis media by inhibiting pro-inflammatory cytokine secretion through up-regulating TLR4

Compromised TLR-mediated chronic inflammation contributes to bacterial infection-caused chronic suppurative otitis media, but the mechanisms are unclear. The present study examined the expression status of nuclear erythroid 2-related factor 2 (Nrf2) and TLRs in human middle-ear mucosae tissues collected from patients with chronic suppurative otitis media, chronic otitis media and non-otitis media, and found that Nrf2 was high-expressed, whereas TLR4, instead of other TLRs, was low expressed in chronic suppurative otitis media compared to chronic otitis media and non-chronic otitis media groups. Consistently, inflammatory cytokines were significantly up-regulated in the chronic suppurative otitis media group, instead of the chronic otitis media and non-chronic otitis media groups. Next, LPS-induced acute otitis media and chronic suppurative otitis media models in mice were established, and high levels of inflammatory cytokines were sustained in the mucosae tissues of chronic suppurative otitis media mice compared to the non-otitis media and acute otitis media groups. Interestingly, continuous low-dose LPS stimulation promoted Nrf2 expression, but decreased TLR4 levels in chronic suppurative otitis media mice mucosae. In addition, knock-down of Nrf2 increased TLR4 expression levels in chronic suppurative otitis media mice, and both Nrf2 ablation and TLR4 overexpression inhibited the pro-inflammatory cytokine expression in chronic suppurative otitis media. Finally, we found that both Nrf2 overexpression and TLR4 deficiency promoted chronic inflammation in LPS-induced acute otitis media mice models. Taken together, knock-down of Nrf2 reversed chronic inflammation to attenuate chronic suppurative otitis media by up-regulating TLR4.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Polydatin promotes the neuronal differentiation of bone marrow mesenchymal stem cells in vitro and in vivo: Involvement of Nrf2 signalling pathway

Bone marrow mesenchymal stem cell (BMSC) transplantation represents a promising repair strategy following spinal cord injury (SCI), although the therapeutic effects are minimal due to their limited neural differentiation potential. Polydatin (PD), a key component of the Chinese herb Polygonum cuspidatum, exerts significant neuroprotective effects in various central nervous system disorders and protects BMSCs against oxidative injury. However, the effect of PD on the neuronal differentiation of BMSCs, and the underlying mechanisms remain inadequately understood. In this study, we induced neuronal differentiation of BMSCs in the presence of PD, and analysed the Nrf2 signalling and neuronal differentiation markers using routine molecular assays. We also established an in vivo model of SCI and assessed the locomotor function of the mice through hindlimb movements and electrophysiological measurements. Finally, tissue regeneration was evaluated by H&E staining, Nissl staining and transmission electron microscopy. PD (30 μmol/L) markedly facilitated BMSC differentiation into neuron‐like cells by activating the Nrf2 pathway and increased the expression of neuronal markers in the transplanted BMSCs at the injured spinal cord sites. Furthermore, compared with either monotherapy, the combination of PD and BMSC transplantation promoted axonal rehabilitation, attenuated glial scar formation and promoted axonal generation across the glial scar, thereby enhancing recovery of hindlimb locomotor function. Taken together, PD augments the neuronal differentiation of BMSCs via Nrf2 activation and improves functional recovery, indicating a promising new therapeutic approach against SCI.