Expression,purification and characterization of a cysteine desulfurase,IscS, from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

Iron–sulfur clusters are one of the most common types of redox center in nature. Three proteins of IscS (a cysteine desulfurase), IscU (a scaffold protein) and IscA (an iron chaperon) encoded by the operon iscSUA are involved in the iron–sulfur cluster assembly in Acidithiobacillus ferrooxidans. In this study the gene of IscS from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The molecular mass of recombinant IscS was 46 kDa by SDS-PAGE. The IscS was a pyridoxal phosphate-containing protein, that catalyzed the elimination of S from l-cysteine to yield l-alanine and elemental sulfur or H₂S, depending on whether or not a reducing agent was added to the reaction mixture. Jia Zeng and Yanfei Zhang contributed equally to this work.     

Effects of CXCL4/CXCR3 on the lipopolysaccharide-induced injury in human umbilical vein endothelial cells

Chemokines and inflammatory response of endothelial cells is crucial in the development and progression of inflammatory disease. Lipopolysaccharide (LPS) is a well-known factor to trigger inflammatory response and induce damage of endothelial cells. The present study used lipopolysaccharide (LPS)-treated human vascular endothelial cells (HUVECs) to investigate the function of chemokine CXC chemokine ligand 4 (CXCL4) and its receptor CXC chemokine receptor 3 (CXCR3) in inflammatory-induced endothelial injury. LPS exposure (50, 100, 200 ng/ml) to HUVECs induced a dose- and time-dependent increase in CXCL4 and CXCR3 expression at both mRNA and protein levels. The LPS-induced endothelium hyperpermeability was inhibited by the addition of CXCL4 neutralizing antibody. Moreover, the addition of CXCL4 neutralizing antibody abolished the effects of LPS on tight junction (TJ) protein expression (occludin claudin-4 and Zonula occluden-1[ZO-1]) and p38 phosphorylation, which is supported by the observation of increased TJ protein expression and decreased p38 phosphorylation in LPS-treated HUVECs. SB203580, a p38 inhibitor, protected HUVECs from CXCL4-stimulated damage. In conclusion, CXCL4/CXCR3, which was enhanced by LPS, may be involved in endothelial proliferation, apoptosis, and permeability via the p38 signaling pathway.     

Electrochemical immunosensor for α-fetoprotein detection using ferroferric oxide and horseradish peroxidase as signal amplification labels

An electrochemical immunosensor for quantitative detection of α-fetoprotein (AFP) in human serum was developed using graphene sheets (GS) and thionine (TH) as electrode materials and mesoporous silica nanoparticles (MSNs) loaded with ferroferric oxide (Fe₃O₄) nanoparticles and horseradish peroxidase (HRP) as labels for signal amplification. In this study, the compound of GS and TH (GS–TH) was used as a substrate for promoting electron transfer and immobilization of primary antibody of AFP (Ab₁). MSNs were used as a carrier for immobilization of secondary antibody of AFP (Ab₂), Fe₃O₄, and HRP. The synergistic effect occurred between Fe₃O₄ and HRP and greatly improved the sensitivity of the immunosensor. This method could detect AFP over a wide concentration range from 0.01 to 25 ng ml⁻¹ with a detection limit of 4 pg ml⁻¹. This strategy may find wide potential application in clinical analysis or detection of other tumor markers.     

Transforming Growth Factor (TGF)-β-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and X-linked Protein Kinase (PRKX)

TGF-β-activated kinase 1 (TAK1) is a key kinase in mediating Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling. Although TAK1 activation involves the phosphorylation of Thr-184 and Thr-187 residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1 remains elusive. In this work, we show that the Thr-187 phosphorylation of TAK1 is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1 activation in mediating downstream signaling requires an additional phosphorylation at Ser-412, which is critical for TAK1 response to proinflammatory stimuli, such as TNF-α, LPS, and IL-1β. In vitro kinase and shRNA-based knockdown assays reveal that TAK1 Ser-412 phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit α (PKACα) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser-391 in zebrafish TAK1 plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1 phosphorylation mediated by PKACα and PRKX that contributes to innate immune signaling.     

Involvement of Upregulated SYF2 in Schwann Cell Differentiation and Migration After Sciatic Nerve Crush

SYF2 is a putative homolog of human p29 in Saccharomyces cerevisiae. It seems to be involved in pre-mRNA splicing and cell cycle progression. Disruption of SYF2 leads to reduced α-tubulin expression and delayed nerve system development in zebrafish. Due to the potential of SYF2 in modulating microtubule dynamics in nervous system, we investigated the spatiotemporal expression of SYF2 in a rat sciatic nerve crush (SNC) model. We found that SNC resulted in a significant upregulation of SYF2 from 3 days to 1 week and subsequently returned to the normal level at 4 weeks. At its peak expression, SYF2 distributed predominantly in Schwann cells. In addition, upregulation of SYF2 was approximately in parallel with Oct-6, and numerous Schwann cells expressing SYF2 were Oct-6 positive. In vitro, we observed enhanced expression of SYF2 during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation. SYF2-specific siRNA-transfected Schwann cells did not show significant morphological change in the process of Schwann cell differentiation. Also, we found shorter and disorganized microtubule structure and a decreased migration in SYF2-specific siRNA-transfected Schwann cells. Together, these findings indicated that the upregulation of SYF2 was associated with Schwann cell differentiation and migration following sciatic nerve crush.     

Molecular evolution of the HD-ZIP I gene family in legume genomes

Homeodomain leucine zipper I (HD-ZIP I) genes were used to increase the plasticity of plants by mediating external signals and regulating growth in response to environmental conditions. The way genomic histories drove the evolution of the HD-ZIP I family in legume species was described; HD-ZIP I genes were searched in Lotus japonicus, Medicago truncatula, Cajanus cajan and Phaseolus vulgaris, and then divided into five clades through phylogenetic analysis. Microsynteny analysis was made based on genomic segments containing the HD-ZIP I genes. Some pairs turned out to conform with syntenic genome regions, while others corresponded to those that were inverted, expanded, or contracted after the divergence of legumes. Besides, we dated their duplications by Ks analysis and demonstrated that all the blocks were formed after the monocot–dicot split; we observed Ka/Ks ratios representing strong purifying selections in the four legume species which might have been followed by gene loss and rearrangement.     

Candesartan antagonizes pressure overload-evoked cardiac remodeling through Smad7 gene-dependent MMP-9 suppression

The present study was designed to investigate the underlying molecular mechanism for Angiotensin II type 1 receptor blockers (ARBs) mediated cardio-protection against pressure overload-induced cardiac remodeling with a focus on Smad7. ROCK-1, Smad3 and fibronectin expressions were increased in male C57BL/6 mice underwent transverse aortic constriction (TAC) for 2weeks. Treatment with Candesartan (2mg/kg per day) could effectively downregulate Smad3 and fibronectin accompanied by upregulating of Smad7. Further data showed that Candesartan inhibited TGF-β1 signal-induced epithelial-to-mesenchymal transition (EMT) through attenuating matrix metalloproteinases (MMP-9), such effect was abolished by knocking-down Smad7. Moreover, TAC for 2weeks caused increased collagen deposition, thickness of left ventricular anterior and posterior wall at end-diastole (LVAWD and LVPWD) and LVEF% reduction, which were reversed by treatment with Candesartan, but failed after knocking-down Smad7. In addition, LV dP/dt(max) and dP/dt(min) were increased by TAC for 2weeks, and treatment with Candesartan or Nifedipine effectively depressed the high levels of dP/dt(min) induced by TAC. However, only Candesartan-mediated protective role in improving cardiac function was suppressed by tail-vein injection of Smad7 siRNA. This study uncovered a novel role for ARBs in preventing pressure overload-induced cardiac remodeling via Smad7 upregulation, which suppressed MMP-9 expression and TGF-β1 signal-mediated EMT progress.     

Characterization of the complete genome segments from BmCPV-SZ, a novel Bombyx mori cypovirus 1 isolate

A novel Bombyx mori cypovirus 1 isolated from infected silkworm larvae and tentatively assigned as Bombyx mori cypovirus 1 isolate Suzhou (BmCPV-SZ). The complete nucleotide sequences of genomic segments S1-S10 from BmCPV-SZ were determined. All segments possessed a single open reading frame; however, bioinformatic evidence suggested a short overlapping coding sequence in S1. Each BmCPV-SZ segment possessed the conserved terminal sequences AGUAA and GUUAGCC at the 5' and 3' ends, respectively. The conserved A/G at the -3 position in relation to the AUG codon could be found in the BmCPV-SZ genome, and it was postulated that this conserved A/G may be the most important nucleotide for efficient translation initiation in cypoviruses (CPVs). Examination of the putative amino acid sequences encoded by BmCPV-SZ revealed some characteristic motifs. Homology searches showed that viral structural proteins VP1, VP3, and VP4 had localized homologies with proteins of Rice ragged stunt virus , a member of the genus Oryzavirus within the family Reoviridae. A phylogenetic tree based on RNA-dependent RNA polymerase sequences demonstrated that CPV is more closely related to Rice ragged stunt virus and Aedes pseudoscutellaris reovirus than to other members of Reoviridae, suggesting that they may have originated from common ancestors.