Interaction of SeqA and Dam methylase on the hemimethylated origin of Escherichia coli chromosomal DNA replication

Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end of oriC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed the in vitro methylation of hemimethylated 13-mers by Dam methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.     

In vitro characterization of protein kinase CKII beta mutants defective in beta-beta dimerization

Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits. The beta subunit mediates tetramer formation through beta-beta homodimerization and alpha-beta heterodimerization. In a previous study R26 and R75, point mutants of CKIIb defective in beta-beta dimerization, were isolated. In the present work we characterized these CKIIbeta mutants in vitro. Purified R26 and R75 bound to CKIIalpha but were defective in binding to CKIIbeta. R75 stimulated the catalytic activity of CKII whereas R26 gave little stimulation, and poly-L-lysine increased the stimulation of catalytic activity by R26 or R75. Circular dichroism and intrinsic fluorescence data pointed to different conformational changes in R26 and R75. Molecular modeling of these mutants provides an explanation of the difference in their ability to interact with CKIIbeta and to activate CKIIalpha.     

（强）热带风暴条件下农田林网对防止棉花倒伏及减产的效应

研究结果表明 ,林带背风面 2 7H范围内 ,林网具有不同程度减轻棉株倒伏及产量损失的功能 ,有效防护范围为 0 .42～ 2 3H ,最大籽棉产量和衣分率效果区为 1 0～ 1 3H .与受灾区相比 ,保护区的籽棉产量增加 45.0 1 % ,皮棉产量增加 52 .69% .整个林网内的籽棉产量增加 2 9 94% ,皮棉产量增加 35 0 6% .     

Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method

AIM:To characterize single-cell-derived mouse clonal mesenchymal stem cells (mcMSCs) established with bone marrow samples from three different mouse strains. METHODS:We established mcMSC lines using subfractionation culturing method from bone marrow samples obtained from long bones.These lines were characterized by measuring cell growth, cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability. Nonclonal MSCs isolated by the conventional gradient centrifugation method were used as controls. RESULTS:All mcMSC lines showed typical nonclonal MSC-like spindle shape morphology. Lines differed inoptimal growth density requirement.Cell surface epitope prof iles of these mcMSC lines were similar to those of nonclonal MSCs. However, some lines exhibited different expression levels in a few epitopes, such as CD44 and CD105. Differentiation assays showed that 90% of the mcMSC lines were capable of differentiating into adipogenic and/or chondrogenic lineages, but only 20% showed osteogenic lineage differentiation. T-cell suppression analysis showed that 75% of the lines exhibited T-cell suppression capability. CONCLUSION:mcMSC lines have similar cell morphology and cell growth rate but exhibit variations in their cell surface epitopes, differentiation potential, lineage-specifi c gene expression and T-cell suppression capability.     

MOSFET-BJT hybrid mode of the gated lateral bipolar junction transistor for C-reactive protein detection

In this study, we propose a novel biosensor based on a gated lateral bipolar junction transistor (BJT) for biomaterial detection. The gated lateral BJT can function as both a BJT and a metal-oxide-semiconductor field-effect transistor (MOSFET) with both the emitter and source, and the collector and drain, coupled. C-reactive protein (CRP), which is an important disease marker in clinical examinations, can be detected using the proposed device. In the MOSFET-BJT hybrid mode, the sensitivity, selectivity, and reproducibility of the gated lateral BJT for biosensors were evaluated in this study. According to the results, in the MOSFET-BJT hybrid mode, the gated lateral BJT shows good selectivity and reproducibility. Changes in the emitter (source) current of the device for CRP antigen detection were approximately 0.65, 0.72, and 0.80 μA/decade at base currents of -50, -30, and -10 μA, respectively. The proposed device has significant application in the detection of certain biomaterials that require a dilution process using a common biosensor, such as a MOSFET-based biosensor.     

Rust secreted protein Ps87 is conserved in diverse fungal pathogens and contains a RXLR-like motif sufficient for translocation into plant cells

Background

Effector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. How obligate fungal pathogen effectors enter host cells is poorly understood. The Ps87 gene of Puccinia striiformis encodes a protein that is conserved in diverse fungal pathogens. Ps87 homologs from a clade containing rust fungi are predicted to be secreted. The aim of this study is to test whether Ps87 may act as an effector during Puccinia striiformis infection.

Methodology/Principal Findings

Yeast signal sequence trap assay showed that the rust protein Ps87 could be secreted from yeast cells, but a homolog from Magnaporthe oryzae that was not predicted to be secreted, could not. Cell re-entry and protein uptake assays showed that a region of Ps87 containing a conserved RXLR-like motif [K/R]RLTG was confirmed to be capable of delivering oomycete effector Avr1b into soybean leaf cells and carrying GFP into soybean root cells. Mutations in the Ps87 motif (KRLTG) abolished the protein translocation ability.

Conclusions/Significance

The results suggest that Ps87 and its secreted homologs could utilize similar protein translocation machinery as those of oomycete and other fungal pathogens. Ps87 did not show direct suppression activity on plant defense responses. These results suggest Ps87 may represent an “emerging effector” that has recently acquired the ability to enter plant cells but has not yet acquired the ability to alter host physiology.