ATM phosphorylates histone H2AX in response to DNA double-strand breaks

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.     

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

火针层孔菌（桑黄）粗多糖对荷瘤小鼠的免疫调节研究

研究了火针层孔菌不同粗多糖对荷瘤小鼠的免疫调节作用。荷瘤小鼠随机分为四组:胞外粗多糖组、菌丝体粗多糖组、子实体粗多糖组和生理盐水阴性对照组。给药10d后测定荷瘤小鼠脾NK细胞活性和脾淋巴细胞的增殖率。结果显示火针层孔菌粗多糖组与阴性对照组相比能够提高小鼠脾NK细胞活性和脾淋巴细胞的增殖率(P<0.01),表明火针层孔菌液体发酵粗多糖和子实体粗多糖对荷瘤小鼠免疫功能均有调节作用。     

Linking seasonal inorganic nitrogen shift to the dynamics of microbial communities in the Chesapeake Bay

Seasonal shifts of dissolved inorganic nitrogen (DIN) and the dynamics of microbial communities for nitrogen transformation were investigated in the water column of Chesapeake Bay. The relative abundance of nitrogen over phosphorus (N*) showed a strong seasonal and spatial pattern: gradually decreased from upstream to downstream; high in winter and low in summer. Because the phosphorus concentration remained relatively stable, the spatiotemporal pattern of N* implied that a substantial fraction of DIN was removed in the bay, especially in summer. Correlation analyses indicated the functional microbial communities and environmental variables, such as temperature, dissolved oxygen, salinity, played important roles for connecting the seasonal variation of N*. Among them, temperature was the trigger factor. High temperature in the summer induced the growth of functional microbes, which subsequently consumed a large portion of DIN inputted from the tributaries and reduced the N*. The current study provided the relative importance of microbial communities and environmental variables in driving the DIN loss in the bay.     

Protein kinase C delta null mice exhibit structural alterations in articular surface,intra-articular and subchondral compartments

IntroductionStructural alterations in intra-articular and subchondral compartments are hallmarks of osteoarthritis, a degenerative disease that causes pain and disability in the aging population. Protein kinase C delta (PKC-δ) plays versatile functions in cell growth and differentiation, but its role in the articular cartilage and subchondral bone is not known.MethodsHistological analysis including alcian blue, safranin O staining and fluorochrome labeling were used to reveal structural alterations at the articular cartilage surface and bone–cartilage interface in PKC-δ knockout (KO) mice. The morphology and organization of chondrocytes were studied using confocal microscopy. Glycosaminoglycan content was studied by micromass culture of chondrocytes of PKC-δ KO mice.ResultsWe uncovered atypical structural demarcation between articular cartilage and subchondral bone of PKC-δ KO mice. Histology analyses revealed a thickening of the articular cartilage and calcified bone–cartilage interface, and decreased safranin O staining accompanied by an increase in the number of hypertrophic chondrocytes in the articular cartilage of PKC-δ KO mice. Interestingly, loss of demarcation between articular cartilage and bone was concomitant with irregular chondrocyte morphology and arrangement. Consistently, in vivo calcein labeling assay showed an increased intensity of calcein labeling in the interface of the growth plate and metaphysis in PKC-δ KO mice. Furthermore, in vitro culture of chondrocyte micromass showed a decreased alcian blue staining of chondrocyte micromass in the PKC-δ KO mice, indicative of a reduced level of glycosaminoglycan production.ConclusionsOur data imply a role for PKC-δ in the osteochondral plasticity of the interface between articular cartilage and the osteochondral junction.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0720-4) contains supplementary material, which is available to authorized users.     

基因组规模代谢网络模型构建及其应用

微生物制造产业的发展迫切需要进一步提高认识、设计和改造微生物细胞代谢的能力,以推动工业生物技术快速发展。随着微生物全基因组序列等高通量数据的不断积聚和生物信息学策略的持续涌现,使全局性、系统化地解析、设计、调控微生物生理代谢功能成为可能。而基于基因组序列注释和详细生化信息整合的基因组规模代谢网络模型(GSMM)构建为全局理解和理性调控微生物生理代谢功能提供了最佳平台。以下在详述GSMM的应用基础上,描述了如何构建一个高精确度的GSMM,并展望了未来的发展方向。     

High-resolution finite element models with tissue strength asymmetry accurately predict failure of trabecular bone

The ability to predict trabecular failure using microstructure-based computational models would greatly facilitate study of trabecular structure–function relations, multiaxial strength, and tissue remodeling. We hypothesized that high-resolution finite element models of trabecular bone that include cortical-like strength asymmetry at the tissue level, could predict apparent level failure of trabecular bone for multiple loading modes. A bilinear constitutive model with asymmetric tissue yield strains in tension and compression was applied to simulate failure in high-resolution finite element models of seven bovine tibial specimens. Tissue modulus was reduced by 95% when tissue principal strains exceeded the tissue yield strains. Linear models were first calibrated for effective tissue modulus against specimen-specific experimental measures of apparent modulus, producing effective tissue moduli of (mean±S.D.) 18.7±3.4 GPa. Next, a parameter study was performed on a single specimen to estimate the tissue level tensile and compressive yield strains. These values, 0.60% strain in tension and 1.01% strain in compression, were then used in non-linear analyses of all seven specimens to predict failure for apparent tensile, compressive, and shear loading. When compared to apparent yield properties previously measured for the same type of bone, the model predictions of both the stresses and strains at failure were not statistically different for any loading case (p>0.15). Use of symmetric tissue strengths could not match the experimental data. These findings establish that, once effective tissue modulus is calibrated and uniform but asymmetric tissue failure strains are used, the resulting models can capture the apparent strength behavior to an outstanding level of accuracy. As such, these computational models have reached a level of fidelity that qualifies them as surrogates for destructive mechanical testing of real specimens.     

Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis

Small RNAs of 21-25 nucleotides (nt), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), act as guide RNAs to silence target-gene expression in a sequence-specific manner. In addition to a Dicer homolog, DCL1, the biogenesis of miRNAs in Arabidopsis requires another protein, HEN1. miRNAs are reduced in abundance and increased in size in hen1 mutants. We found that HEN1 is a miRNA methyltransferase that adds a methyl group to the 3'-most nucleotide of miRNAs, but the role of miRNA methylation was unknown. Here, we show that siRNAs from sense transgenes, hairpin transgenes, and transposons or repeat sequences, as well as a new class of siRNAs known as trans-acting siRNAs, are also methylated in vivo by HEN1. In addition, we show that the size increase of small RNAs in the hen1-1 mutant is due to the addition of one to five U residues to the 3' ends of the small RNAs. Therefore, a novel uridylation activity targets the 3' ends of unmethylated miRNAs and siRNAs in hen1 mutants. We conclude that 3'-end methylation is a common step in miRNA and siRNA metabolism and likely protects the 3' ends of the small RNAs from the uridylation activity.     

Identification and characterization of the retinoic acid response elements in the human RIG1 gene promoter

The expression of retinoic acid-induced gene 1 (RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all-trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5'-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the -4910/-5509 fragment of the 5'-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between -5048 and -5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5'-TGACCTctattTGCCCT-3' (DR5, -5243/-5259), and an inverted repeat sequence with six nucleotide spacing, 5'-AGGCCAtggtaaTGGCCT-3' (IR6, -5323/-5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the atRA-mediated induction. Taken together, we have identified and characterized the functional atRA response element that is responsible for the atRA-mediated induction of RIG1 gene.