Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Molecular cloning and expression analysis of interferon stimulated gene 15 (ISG15) in turbot, Scophthalmus maximus.

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by double-stranded RNA (polyinosinic: polycytidylic acid, poly I:C) and viral infection. In this study, we described the nucleotide, mRNA tissue distribution and regulation of an ISG15 gene from turbot, Scophthalmus maximus (SmISG15). SmISG15 gene is 862 bp in length, composed of two exons and one intron, and encodes 158 amino acids. The deduced protein exhibits the highest homology (44.7-71.2% identity) with ISG15s from other fishes and possesses two conserved tandem ubiquitin-like (UBL) domains and a C-terminal RLRGG conjugating motif known to be important for the functions of ISG15s in vertebrates. Phylogenetic analysis grouped SmISG15 into fish ISG15. SmISG15 mRNA was constitutively expressed in all tissues examined, with higher levels observed in immune organs. Gene expression analysis was performed for SmISG15 in the spleen, head kidney, gills and muscle of turbots challenged with poly I:C or turbot reddish body iridovirus (TRBIV) over a 7-day time course. The result showed that SmISG15 was upregulated by both stimuli in all four tissues, with induction by poly I:C apparently stronger and initiated more quickly. A two-wave induced expression of SmISG15 was seen in the spleen, head kidney and gills, suggesting an induction of SmISG15 either by IFN-dependent or -independent pathway. These results provide insights into the roles of fish ISG15 in antiviral immunity.

Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus).

Myeloid differentiation factor 88 (MyD88) is an adapter protein involved in the interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR)-mediated activation of nuclear factor-kappaB (NF-κB). In this study, a full length cDNA of MyD88 was cloned from turbot, Scophthalmus maximus. It is 1619 bp in length and contains an 858-bp open reading frame that encodes a peptide of 285 amino acid residues. The putative turbot (Sm)MyD88 protein possesses a N-terminal death domain and a C-terminal Toll/IL-1 receptor (TIR) domain known to be important for the functions of MyD88 in mammals. Phylogenetic analysis grouped SmMyD88 with other fish MyD88s. SmMyD88 mRNA was ubiquitously expressed in all examined tissues of healthy turbots, with higher levels observed in immune-relevant organs. To explore the role of SmMyD88, its gene expression profile in response to stimulation of lipopolysaccharide (LPS), CpG oligodeoxynucleotide (CpG-ODN) or turbot reddish body iridovirus (TRBIV) was studied in the head kidney, spleen, gills and muscle over a 7-day time course. The results showed an up-regulation of SmMyD88 transcript levels by the three immunostimulants in all four examined tissues, with the induction by CpG-ODN strongest and initiated earliest and inducibility in the muscle very weak. Additionally, TRBIV challenge resulted in a quite high level of SmMyD88 expression in the spleen, whereas the two synthetic immunostimulants induced the higher levels in the head kidney. These data provide insights into the roles of SmMyD88 in the TLR/IL-1R signaling pathway of the innate immune system in turbot.

Cloning and expression analysis of a Toll-like receptor 22 (tlr22) gene from turbot, Scophthalmus maximus.

Toll-like receptor 22 (TLR22) exists exclusively in aquatic animals and recognizes double stranded RNA (dsRNA). In the present study, a tlr22 gene and its 5'-flanking sequence were cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (sm)tlr22 gene spans over 5.6 kb with a structure of 4 exon-3 intron and encodes 962 amino acids. The deduced protein shows the highest sequence identity (76.7%) to Japanese flounder Tlr22 and possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 27 LRR motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost Tlr22s. The interferon-stimulated response element (ISRE) and signal transducer and activator of transcription (STAT) binding site important for the basal transcriptional activity of TLR3 were predicted in the 5'-flanking sequence of smtlr22 gene. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of smtlr22 mRNA in all examined tissues with higher levels in the head kidney, kidney and spleen. Further, smtlr22 expression was significantly up-regulated following challenge with polyinosinic: polycytidylic acid (poly I:C), lipopolysaccharide (LPS) or turbot reddish body iridovirus (TRBIV) in the gills, head kidney, spleen and muscle, with maximum increases ranging from 2.56 to 6.24 fold upon different immunostimulants and organs. These findings suggest a possible role of Smtlr22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and Gram-negative bacteria.

Molecular cloning and characterization of interferon regulatory factor 9 (IRF9) in Japanese flounder, Paralichthys olivaceus.

Interferon regulatory factor 9 (IRF9) in mammals is known to be involved in antiviral response. In this study, we studied the structure, mRNA tissue distribution and regulation of IRF9 from Japanese flounder, Paralichthys olivaceus. The cDNA sequence of IRF9 is 3305 bp long, containing an open reading frame (ORF) of 1308 bp that encodes a peptide of 435 amino acids. The predicted protein sequence shares 33.7-72.0% identity to other fish IRF9s. Japanese flounder IRF9 possesses a DNA-binding domain (DBD), an IRF association domain (IAD), two nuclear localization signals (NLSs) and a proline-rich domain (PRD). The IRF9 transcripts were detectable in all examined tissues of healthy Japanese flounders, with higher levels in the head kidney, kidney, liver and spleen. The IRF9 mRNA levels were up-regulated in the gills, head kidney, spleen and muscle when challenged with polyinosinic:polycytidylic acid (poly I:C) or lymphocystis disease virus (LCDV). The up-regulations were stronger and arose earlier in the case of poly I:C treatment in most tested organs in a 7-day time course, with maximum increases ranging from 1.37- to 8.59-fold and peak time points from 3 h to 3 d post injection depending on different organs, relative to those in the case of LCDV treatment which ranged from 1.32- to 3.21-fold and from 18 h to 3 d post injection, respectively. The highest and earliest inductions were detected in the spleen in both challenge cases, while the inductions by LCDV in the muscle were quite faint. These results demonstrate a role of Japanese flounder IRF9 in the host's antiviral responses.

Whole cell cryo-electron tomography suggests mitochondria divide by budding.

Eukaryotes rely on mitochondrial division to guarantee that each new generation of cells acquires an adequate number of mitochondria. Mitochondrial division has long been thought to occur by binary fission and, more recently, evidence has supported the idea that binary fission is mediated by dynamin-related protein (Drp1) and the endoplasmic reticulum. However, studies to date have depended on fluorescence microscopy and conventional electron microscopy. Here, we utilize whole cell cryo-electron tomography to visualize mitochondrial division in frozen hydrated intact HeLa cells. We observe a large number of relatively small mitochondria protruding from and connected to large mitochondria or mitochondrial networks. Therefore, this study provides evidence that mitochondria divide by budding.

[Effects of nitrogen application rate on faba bean fusarium wilt and rhizospheric microbial metabolic functional diversity].

A field plot experiment was conducted to study the effects of different nitrogen (N) application rates on the microbial functional diversity in faba bean rhizosphere and the relationships between the microbial functional diversity and the occurrence of faba bean fusarium wilt. Four nitrogen application rates were installed, i. e. , N0(0 kg hm-2 , N1 (56. 25 kg hm-2) , N2(112. 5 kg hm-2), and N3 (168.75 kg hm-2), and Biolog microbial analysis system was applied to study the damage of faba bean fusarium wilt and the rhizospheric microbial metabolic functional diversity. Applying N (N1 N2, and N3) decreased the disease index of faba bean fusarium wilt and the quantity of Fusarium oxysporum significantly, and increased the quantities of bacteria and actinomyces and the ratios of bacteria/fungi and actinomyces/fungi significantly, with the peak values of bacteria and actinomyces, bacteria/fungi, and actinomyces/fungi, and the lowest disease index and F. oxysporum density in N2. As compared with N0, applying N increased the AWCD value significantly, but the effects of different N application rates on the ability of rhizospheric microbes in utilizing six types of carbon sources had definite differences. Under the application of N, the utilization rates of carbohydrates, carboxylic acids, and amino acids by the rhizospheric microbes were higher. Principal component analysis demonstrated that applying N changed the rhizospheric microbial community composition obviously, and the carbohydrates, carboxylic acids, and amino acids were the sensitive carbon sources differentiating the changes of the microbial community induced by N application. Applying N inhibited the utilization of carbohydrates and carboxylic acids but improved the utilization of amino acids and phenolic acids by the rhizospheric microbes, which could be one of the main reasons of applying N being able to reduce the harm of faba bean fusarium wilt. It was suggested that rationally applying N could increase the quantities of rhizospheric bacteria and actinomyces, alter the microbial metabolic function, and decrease F. oxysporum density, being an effective measure to control the occurrence of faba bean fusarium wilt.

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.

An IRF-3 homolog that is up-regulated by DNA virus and poly I:C in turbot, Scophthalmus maximus.

In this study, we described the structure, mRNA tissue distribution and regulation of an IRF-3 gene from turbot, Scophthalmus maximus (SmIRF-3). The gene sequence of SmIRF-3 is 6077 bp long, composed of 11 exons and 10 introns similar to known IRF-3 genes of fish, and encodes a peptide of 466 amino acids. The deduced protein sequence shares the highest identity of 56.0-81.2% with fish IRF-3 and possesses a DNA-binding domain (DBD), an IRF association domain (IAD) and a serine-rich domain (SRD) known to be important for the functions of IRF-3 in vertebrates. Phylogenetic analysis grouped SmIRF-3 with other IRF3s of vertebrates. SmIRF-3 transcripts were detectable in limited tissue types of healthy fish, with higher expression observed in head, kidney, spleen and kidney,. The SmIRF-3 was transcriptionally up-regulated by turbot reddish body iridovirus (TRBIV) and polyinosinic:polycytidylic acid (poly I:C) in the head kidney, spleen and gills, with showing a two wave induced expression during a 7-day time course in all cases. The highest inducibility and the likely earliest increase of SmIRF-3 expression were observed in the spleen, and poly I:C was a stronger inducer. In addition, the maximal expression level of SmIRF-3 arose prior to that of the Mx in all the cases.

Localization of diversified relaxin gene transcripts in the brain of eels.

Relaxin 3 (RLN3) is a newly-discovered member of the insulin superfamily. We isolated three RLN3-like cDNAs from the brain of the Japanese eel (Anguilla japonica). The deduced amino acid sequences of the RLN3-like cDNAs contained the two-chain structure common to relaxin including a RXXXRXXI/V motif in the B-chain. Phylogenetic analysis assigned the two prepropeptides into teleost/mammalian RLN3 group, which are a pair of duplicates generated by the teleost-specific third-round whole genome duplication, and the other one into teleost RLN group. Therefore, they have been named eel rln3a, rln3b and rln. rln3a transcripts were abundant in the middle-posterior region of the brain and detected at lower levels in the gills, head kidney and kidney. rln3b transcripts were also detected in the middle-posterior region of the brain, but the expression levels were lower than those of rln3a. Low levels of rln transcripts were detected in all brain areas, pituitary, digestive tract and gonad. Quantitative PCR analysis did not detect differences in expression of any rln3 or rln gene between freshwater- and seawater-acclimated eels. In situ hybridization showed that rln3a was expressed in neurons of the lateral lemniscus of the midbrain and of the griseum centrale (GC) of the hindbrain, while low amounts of rln transcripts were found in neurons of the periventricular nucleus of the posterior tuberculum of the diencephalon and the GC. These results suggest that the multiple RLN3-like peptides may play regulatory roles in the brain of euryhaline fish.

Toroidal surface complexes of bacteriophage ϕ12 are responsible for host-cell attachment.

Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage ϕ12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of ϕ12 demonstrates that the gene for the hexameric spike is located in ϕ12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimates derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity.

WITHDRAWN: Molecular cloning and expression analysis of relaxin 3c in Japanese eel, Anguilla japonica.

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A novel immortalization vector for the establishment of penaeid shrimp cell lines.

Cell immortalization technology based on gene transfer has been successfully used to generate cell lines from a wide variety of cell types. The inability to stably introduce and express foreign genes has hampered application of this strategy in shrimp cells. We report here the use of replication-defective pantropic retrovirus to achieve a novel immortalization vector in which simian virus 40 large T antigen (SV40T) gene is expressed from Moloney murine leukemia virus (MoMLV) promoter. Data confirmed the presence of transferred SV40T gene and its stable mRNA expression in transduced lymphoid cells of Penaeus chinensis. The transduced cells showed a higher growth rate and a longer replication life-span compared with their untransduced counterparts. These results indicate the pantropic retrovirus-based immortalization-inducing gene delivery system is a potential tool for establishing cell lines from shrimp.

Electron cryo-tomographic structure of cystovirus phi 12.

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.

Three-dimensional structure of the KdpFABC complex of Escherichia coli by electron tomography of two-dimensional crystals.

The KdpFABC complex (Kdp) functions as a K+ pump in Escherichia coli and is a member of the family of P-type ATPases. Unlike other family members, Kdp has a unique oligomeric composition and is notable for segregating K+ transport and ATP hydrolysis onto separate subunits (KdpA and KdpB, respectively). We have produced two-dimensional crystals of the KdpFABC complex within reconstituted lipid bilayers and determined its three-dimensional structure from negatively stained samples using a combination of electron tomography and real-space averaging. The resulting map is at a resolution of 2.4 nm and reveals a dimer of Kdp molecules as the asymmetric unit; however, only the cytoplasmic domains are visible due to the lack of stain penetration within the lipid bilayer. The sizes of these cytoplasmic domains are consistent with Kdp and, using a pseudo-atomic model, we have described the subunit interactions that stabilize the Kdp dimer within the larger crystallographic array. These results illustrate the utility of electron tomography in structure determination of ordered assemblies, especially when disorder is severe enough to hamper conventional crystallographic analysis.

Apoptosis-inducing factor and apoptosis.

Apoptosis-inducing factor (AIF) is a phylogenetically conserved mitochondrial intermembrane flavoprotein which has the ability to induce apoptosis via a caspase-independent pathway. AIF plays an important role in inducing nuclear chromatin condensation as well as large-scale DNA fragmentation (approximately 50 kb), and is essential for programmed cell death during cavitation of embryoid bodies. Two homologous proteins, AIF-homologous mitochondrion-associated inducer of death (AMID) and p53-responsive gene 3 (PRG3), also have apoptosis-inducing effects. Recent studies on mechanisms of AIF-mediated apoptotic DNA degradation in Caenorhabditis elegans reveal that WAH-1(an AIF homolog in C. elegans) induced apoptosis is CED-3-dependent. AIF also interacts with cytochrome c and caspases during mammalian apoptosis processes, indicating that different apoptotic pathways may be mutually cross-regulated to activate an apoptotic program.

[Differences in gene expression between Taxus chinensis cells during Taxol-synthesis phase and those during non-Taxol-synthesis phase].

In plant, evocation of secondary metabolism is associated with complex biochemical and molecular events that are regulated by developmental and environmental factors. In order to get more information about Taxol biosynthesis, comparison of mRNA populations from Taxus chinensis cells during Taxol-synthesis phase and those during non-Taxol-synthesis phase were performed by mRNA differential display. The results suggested that genes specifically expressed in the Taxol-synthesis phase might be involved in Taxol biosynthesis.