1H, 13C, and 15N resonance assignments of SarA monomer from Staphylococcus aureus in complex with DNA.

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarAΔN19) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the 1H, 13C and 15N NMR assignment of SarAΔN19/DNA complex which is the first step towards further structure and function analysis.

Transcriptome analysis of turbot (Scophthalmus maximus) head kidney and liver reveals immune mechanism in response to Vibrio anguillarum infection.

The diseases triggered by Vibrio anguillarum infection have created huge economic losses to the turbot (Scophthalmus maximus) farming industry. However, the immune mechanism of turbot to V. anguillarum infection has not been deeply investigated. To better understand the immune response of turbot to V. anguillarum infection, transcriptome analysis of the head kidney and liver of turbot was performed. A total of 15,948 and 11,494 differentially expressed genes (DEGs) were obtained from the turbot head kidney and liver, respectively. Transcriptome analysis revealed that the head kidney and liver of turbot have some differences in the gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs for the different functions of these two organs. Although there are many uncertain factors in this immune process, such as the occurrence of alternative splicing (AS) events and the differences in the protein structure of the DEGs, the NFκB signalling pathway, MKK-dependent AP-1 activation, JAK-STAT signalling pathway, the signal transmission of MHC Ⅰ and a series of DEGs including HSP90 driving NLRP3 to produce inflammatory factors (IL-1ß, IL-8, TNFα, etc.) were possible important immune response pathways for turbot to V. anguillarum infection. Overall, our research has conducted a preliminary exploration of the immune mechanism of turbot in response to V. anguillarum infection.

Selective Nonmethylated CpG DNA Recognition Mechanism of Cysteine Clamp Domains.

Methylation of DNA at CpG sites is a major mark for epigenetic regulation, but how transcription factors are influenced by CpG methylation is not well understood. Here, we report the molecular mechanisms of how the TCF (T-cell factor) and GEF (glucose transporter 4 enhancer factor) families of proteins selectively target unmethylated DNA sequences with a C-clamp type zinc finger domain. The structure of the C-clamp domain from human GEF family protein HDBP1 (C-clampHDBP1) in complex with DNA was determined using NMR spectroscopy, which adopts a unique zinc finger fold and selectively binds RCCGG (R = A/G) DNA sequences with an "Arg···Trp-Lys-Lys" DNA recognition motif inserted in the major groove. The CpG base pairs are central to the binding due to multiple hydrogen bonds formed with the backbone carbonyl groups of Trp378 and Lys379, as well as the side chain ε-amino groups of Lys379 and Lys380 from C-clampHDBP1. Consequently, methylation of the CpG dinucleotide almost abolishes the binding. Homology modeling reveals that the C-clamp domain from human TCF1E (C-clampTCF1E) binds DNA through essentially the same mechanism, with a similar "Arg···Arg-Lys-Lys" DNA recognition motif. The substitution of tryptophan by arginine makes C-clampHDBP1 prefer RCCGC DNA sequences. The two signature DNA recognition motifs are invariant in the GEF and TCF families of proteins, respectively, from fly to human. The recognition of the CpG dinucleotide through two consecutive backbone carbonyl groups is the same as that of the CXXC type unmethylated CpG DNA binding domains, suggesting a common mechanism shared by unmethylated CpG binding proteins.

GapR binds DNA through dynamic opening of its tetrameric interface.

GapR is a nucleoid-associated protein that is an essential regulator of chromosome replication in the cell cycle model Caulobacter crescentus. Here, we demonstrate that free GapR is a homotetramer, but not a dimer as previously reported (Guo et al., Cell 175: 583-597, 2018). We have determined the crystal structure of GapR in complex with a 10-bp A-tract DNA, which has an open tetrameric conformation, different from the closed clamp conformation in the previously reported crystal structure of GapR/DNA complex. The free GapR adopts multiple conformations in dynamic exchange equilibrium, with the major conformation resembling the closed tetrameric conformation, while the open tetrameric conformation is a representative of minor conformers. As it is impossible for the circular genomic DNA to get into the central DNA binding tunnel of the major conformation, we propose that GapR initially binds DNA through the open conformation, and then undergoes structural rearrangement to form the closed conformation which fully encircles the DNA. GapR prefers to bind DNA with 10-bp consecutive A/T base pairs nonselectively (Kd ∼12 nM), while it can also bind GC-rich DNA sequence with a reasonable affinity of about 120 nM. Besides, our results suggest that GapR binding results in widening the minor groove of AT-rich DNA, instead of overtwisting DNA.

Detection of Cytomegalovirus Antibodies Using a Biosensor Based on Imaging Ellipsometry.

BACKGROUND: Cytomegalovirus (CMV) is the most common infectious cause of mental disability in newborns in developed countries. There is an urgent need to establish an early detection and high-throughput screening method for CMV infection using portable detection devices. METHODS: An antibody analysis method is reported for the detection and identification of CMV antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (BIE). CMV antigen (CMV-3A) was immobilized on silicon wafers and used to capture CMV antibodies in serum. An antibody against human immunoglobulin G (anti-IgG) was used to confirm the IgG antibody against CMV captured by the CMV-3A. RESULTS: Our results show that this assay is rapid and specific for the identification of IgG antibody against CMV. Further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. The CMV antibody detection sensitivity of BIE reached 0.01 IU/mL. CONCLUSIONS: This novel biosensor may be a valuable diagnostic tool for analysis of IgG antibody against CMV during CMV infection screening.

Structural and expression studies of interferon regulatory factor 8 in Japanese flounder, Paralichthys olivaceus.

Interferon regulatory factor 8 (IRF8) plays a role in both innate and adaptive systems in mammals. In this study, the gene and promoter sequences of Japanese flounder, Paralichthys olivaceus, (Po) IRF8 were cloned, and its expression in response to polyinosinic:polycytidylic acid (poly I:C) and lymphocystis disease virus (LCDV) challenges was studied in vivo. The PoIRF8 gene spans over 3.3 kb with a structure of 9 exon-8 intron and encodes 420 amino acids. The putative protein shows the highest sequence identity (69.5-89.0%) to fish IRF8 and possesses a DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS) of vertebrate IRF8. Phylogenetic analysis classified PoIRF8 into the cluster of fish IRF8 within vertebrate IRF8 group of IRF4 subfamily. A number of transcription factor binding sites were identified in the 2348-bp 5' flanking region of PoIRF8 gene, including those of transcription factors for type Ⅰ and type Ⅱ interferon (IFN) inducible genes and genes regulating the development and function of lymphomyeloid cells in mammals. The PoIRF8 transcripts were expressed in all examined tissues of healthy flounders, with higher levels observed in the immune relevant tissues. They were up-regulated by both poly I:C and LCDV treatments in the spleen, head kidney, gills and muscle in an early phase of immune responses, with initiation and peak time points of induction prior to type Ⅰ IFN and Mx. Relative to LCDV, the induction by poly I:C was quicker in all four tissues. These results indicate an involvement of PoIRF8 in the host's antiviral responses and a functional conservation of IRF8 between fish and mammals.

Molecular cloning and expression analysis of interferon regulatory factor 8 (IRF8) in turbot, Scophthalmus maximus.

Interferon regulatory factor 8 (IRF8) in mammals is known to be involved in antiviral response. In this study, the gene of IRF8 was cloned from the turbot (Scophthalmus maximus) fish and its expression in response to polyinosinic:polycytidylic acid (poly I:C) and turbot reddish body irrdovirus (TRBIV) challenges was studied. Turbot (Sm)IRF8 gene is 4363bp long, comprises nine exons and eight introns and encodes a putative 420 amino acid (aa) protein. The predicted protein sequence possesses a DNA binding domain (DBD), an IRF association domain (IAD) and a nuclear localization signal (NLS). Constitutive expression of SmIRF8 was detectable in all tested organs, with higher levels observed in the spleen, kidney and head kidney. SmIRF8 transcript levels were up-regulated by both poly I:C and TRBIV treatments in the spleen, head kidney, gills and muscle in an early phase of a 7-day time course and the poly I:C was a quicker inducer. In both challenge cases, the highest and earliest inductions were detected in the spleen, while the induction in the muscle was quite faint. These results provide insights into the role of SmIRF8 in antiviral response.

Characteristics of the interferon regulatory factor 5 (IRF5) and its expression in response to LCDV and poly I:C challenges in Japanese flounder, Paralichthys olivaceus.

Interferon regulatory factor 5 (IRF5) has been identified as a key transcriptional mediator regulating expression of both type I interferons (IFNs) and proinflammatory cytokines. In this study, the cDNA and genomic sequences of IRF5 were isolated from Japanese flounder, Paralichthys olivaceus. The gene of Japanese flounder (Jf)IRF5 is 7326 bp long, contains 9 exons and 8 introns and encodes a putative protein of 472 amino acids. The predicted protein sequence shares 61.1-81.9% identity to fish IRF5 and possesses a DNA-binding domain (DBD), a middle region (MR), an IRF association domain (IAD), a virus activated domain (VAD) and two nuclear localization signals (NLSs) conserved in all known IRF5s. Phylogenetic analysis clustered it into the teleost IRF5 subgroup within vertebrate IRF5 group. JfIRF5 mRNA was constitutively expressed in all tissues examined, with higher levels observed in the gills and head kidney. Gene expression of JfIRF5 was analyzed over a 7-day time course in the gills, head kidney, spleen and muscle of Japanese flounders challenged with lymphocystis disease virus (LCDV) and polyinosinic:polycytidylic acid (poly I:C). The data showed that JfIRF5 expression was slightly up-regulated by LCDV, but its induction time was clearly moved up; in contrast, the induction upon poly I:C challenge started not earlier than day 2 post-injection and was stronger and more persistent with a later peak time in all four organs. The late and long-lasting inductive expression of JfIRF5 following poly I:C challenge suggests that it might be an interferon stimulated gene (ISG), the induction of which is driven by poly I:C-induced type I IFNs.

Molecular characterization and expression analysis of interferon regulatory factor 5 (IRF-5) in turbot, Scophthalmus maximus.

Interferon regulatory factor 5 (IRF-5) plays a role both in the antiviral and inflammatory responses. In this study, we described the structure, mRNA tissue distribution and regulation of an IRF-5 gene from turbot, Scophthalmus maximus (SmIRF-5). The gene sequence of SmIRF-5 is 4275 bp long, composed of 9 exons and 8 introns similar to known IRF-5 genes of vertebrates, and encodes a peptide of 487 amino acids. The deduced protein sequence shares the highest identity of ∼60-70% with fish IRF-5 and possesses a DNA-binding domain (DBD), a middle region (MR), an IRF association domain (IAD) and a virus activated domain (VAD) known to be important for the functions of IRF-5 in mammals. Phylogenetic analysis grouped SmIRF-5 with other IRF-5s of vertebrates. SmIRF-5 transcripts were detectable in a wide range of tissue types of healthy fish with higher levels observed in the head kidney, kidney and spleen. The SmIRF-5 was transcriptionally up-regulated by turbot reddish body iridovirus (TRBIV) but not by polyinosinic:polycytidylic acid (poly I:C) in the gills, head kidney, spleen and muscle. Both the highest inducibility and earliest induction of SmIRF-5 expression were observed in the spleen where it reached a maximum level at day 1 after infection, prior to that of turbot Mx. These findings may help to better understand the roles of SmIRF-5 in antiviral response.

Interferon regulatory factor 3 (IRF-3) in Japanese flounder, Paralichthys olivaceus: sequencing, limited tissue distribution, inducible expression and induction of fish type I interferon promoter.

Two cDNAs with different 3'-untranslated region (UTR) encoding an interferon regulatory factor 3 (IRF-3) were cloned from head kidney of Japanese flounder, Paralichthys olivaceus, by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Sequence analysis reveals that they were generated by alternative polyadenylation. The predicted protein consists of 467 amino acid residues which shares the highest identity of 50.7-57.6% to fish IRF-3 and possesses a DNA-binding domain (DBD), an IRF association domain (IAD) and a serine-rich domain (SRD) of vertebrate IRF-3. The presence of these domains along with phylogenetic analysis places it into the IRF-3 group of the IRF-3 subfamily. RT-PCR analysis revealed that flounder IRF-3 was expressed constitutively in limited tissue types including head kidney, spleen, kidney, heart, gill, intestine and liver. A quantitative real time PCR assay was employed to monitor expression of IRF-3, type I interferon (IFN) and Mx in flounder head kidney and gill. All three genes were up-regulated by polyinosinic:polycytidylic acid (polyI:C) and lymphocystis disease virus (LCDV) with an earlier but slight and less persistent increase in transcription levels seen for the IRF-3. Finally, flounder IRF-3 was proved to induce fish type I IFN promoter in FG9307 cells, a flounder gill cell line, by a luciferase assay. These results provide insights into the roles of fish IRF-3 in the antiviral immunity.

Cloning and expression analysis of interferon regulatory factor 7 (IRF-7) in turbot, Scophthalmus maximus.

Interferon regulatory factor (IRF) 7 is known as the master regulator of type I interferon (IFN)-dependent immune responses in mammals. In this study, the cDNA and genomic sequences of turbot (Scophthalmus maximus) IRF-7 (SmIRF-7) were cloned and found to encode a putative protein of 439 amino acids. The gene is composed of 10 exons and 9 introns similar to known IRF-7 genes of fish. The SmIRF-7 shows the highest amino acid identity of 49.0-80.3% to fish IRF-7 and possesses a DNA-binding domain (DBD), an IRF association domain (IAD) and a serine-rich domain (SRD) of vertebrate IRF-7. In addition, the tryptophan cluster of SmIRF-7 DBD consists of only four tryptophans, which is a characteristic unique to all fish IRF-7 members. The SmIRF-7 transcripts were expressed constitutively in all analyzed tissues of healthy turbot, with higher levels observed in immune relevant tissues. Gene expressions of SmIRF-7 and Mx were monitored over a 7-day time course by quantitative real time PCR in head kidney and muscle of turbot challenged with turbot reddish body iridovirus (TRBIV), which is a prevalent viral pathogens in farmed turbot in China. Both genes were up-regulated by TRBIV although their inducibility was much weaker in the muscle. The peak levels of SmIRF-7 transcripts were detected at day 2 post-infection in the two organs with a 12- and 4.5-fold increase, respectively. Further, the Mx showed two waves of induced expression and the maximum expression of SmIRF-7 arose earlier than the second wave of the Mx expression in both organs. These findings contribute to an understanding of functions of SmIRF-7 in antiviral response.

Molecular cloning and characterization of interferon regulatory factor 7 (IRF-7) in Japanese flounder, Paralichthys olivaceus.

Interferon regulatory factor (IRF) 7 in mammals is known to be a key player in regulating the type I interferon (IFN) response to viral infection as a transcription activator of IFNs and IFN-stimulated genes (ISGs). In this study, a full-length cDNA of Japanese flounder, Paralichthys olivaceus, (Po)IRF-7 was cloned and characterized. PoIRF-7 is 2032 bp in length, with an open reading frame (ORF) of 1293 bp that encodes 430 amino acid residues. The putative amino acid sequence shows the highest homology to fish IRF-7 with 51.5-76.3% identity and possesses a DNA-binding domain (DBD), an IRF association domain (IAD) and a serine-rich domain of vertebrate IRF-7. In addition, the tryptophan cluster of PoIRF-7 DBD consists of only four tryptophans, which is a characteristic unique to all fish IRF-7 members. The PoIRF-7 was expressed constitutively in all tested tissues of healthy flounders, with high levels in head kidney, spleen, gill, intestine and skin, and moderately expressed in FG9307 cells, a flounder gill epithelial cell line. Using a luciferase assay, PoIRF-7 was proved to be capable of activating fish type I IFN promoter in FG9307 cells. A quantitative real time PCR assay was employed to monitor the gene expression of PoIRF-7 and Mx in FG9307 cells and flounder head kidney and gill. Both genes were up-regulated by polyinosinic:polycytidylic acid (poly I:C) and lymphocystis disease virus (LCDV) though to a much lesser extent in FG9307 cells. Further, their transcription kinetics were similar in fish organs but different in FG9307 cells. These data provide insights into the functions of PoIRF-7 and imply a difference in PoIRF-7-related signaling pathways in antiviral response between cultured cells and live fish.

[Heterogenous expression of antimicrobial peptides].

Antimicrobial peptides (AMPs), a class of short proteins with a broad spectrum of antibacterial activities, are isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. They are a key component of the innate immune response in most multicellular organisms. Owing to their potent, broad-spectrum antibacterial activities and uneasy developing of drug resistance, these peptides are of great clinical significance. However, preparation of AMPs at a large scale is a severe challenge to the development of the commercial products. Undoubtedly, construction of high-level biological expression systems for the production of AMPs is the key in its clinical application process. Herein, we summarize the progress in researches on heterogenous expression of AMPs in prokaryotic expression systems and eukaryotic expression systems.

Nitric oxide mediates stretch-induced Ca2+ release via activation of phosphatidylinositol 3-kinase-Akt pathway in smooth muscle.

BACKGROUND: Hollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca(2+) release occurs in the form of Ca(2+) sparks and Ca(2+) waves in urinary bladder myocytes. While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined the transduction mechanism linking cell stretch to Ca(2+) release. The probability and frequency of Ca(2+) sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO) and the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca(2+) sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204+/-28.6% and 258+/-36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level. CONCLUSIONS/SIGNIFICANCE: Taking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle.

Localized Ca2+ uncaging induces Ca2+ release through IP3R in smooth muscle.

AIM: Our previous study indicated that there are two types of Ca2+ release events seen in intact mouse bladder tissue. In this study our aim is to investigate the mechanism that underlies the phenomena of Ca2+ release in smooth muscle. METHODS: Single cells were isolated and tissue segments were prepared by cutting the detrusor into 0.1 cm x 0.5 cm strips running along the axis from the neck to the fundus. Single cells and intact tissue strips were co-loaded with the Ca2+ indicator and caged Ca2+ by incubation with 10 micromol/L Fluo-4 AM and DMNP-EDTA-AM. Fluo-4 AM fluorescence was detected by laser scanning confocal microscopy, and local uncaging of DMNP-EGTA was achieved by brief exposure to the output of a diode-pumped, Ti:sapphire laser tuned to 730 nm. RESULTS: Local uncaging of caged Ca2+ was able to trigger Ca2+ release events in both single cells and tissue strips from mouse bladder. The Ca2+ release events could not be blocked by ryanodine alone, but the property of the Ca2+ release was markedly altered. Surprisingly, in the presence of ryanodine, Xestospongin C completely inhibited the Ca2+ release events both in single cell and tissue experiments. CONCLUSION: (1) Two photon flash photolysis (TPFP) triggers Ca2+ induced Ca2+ release. This process involves release through type 2 ryanodine receptor channels; (2) TPFP results in the release of Ca2+ through inositol 1,4,5-trisphosphate receptors in the absence of phospholipase C activation.