Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus)

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus–oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.     

Activation of the Ghrelin Receptor is Described by a Privileged Collective Motion: A Model for Constitutive and Agonist-induced Activation of a Sub-class A G-Protein Coupled Receptor (GPCR)

Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser _[octa]-Phe-NH ₂ and Gly-Ser-Ser _[octa]-Phe-Leu-NH ₂ allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.     

Optical biosensor provides insights for bradykinin B(2) receptor signaling in A431 cells

The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B(2) receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dose-dependent inhibition by a B(2) specific antagonist HOE140, suggested that the optical signature is a direct result of B(2) receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-beta-cyclodextrin argued that B(2) receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B(2) signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B(2) receptor activation results in signaling via at least dual pathways - G(s)- and G(q)-mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B(2) signaling. The roles of endocytosis and cytoskeleton modulation in B(2) signaling were also demonstrated.     

The Effect of Hydrophilic Substitutions and Anionic Lipids upon the Transverse Positioning of the Transmembrane Helix of the ErbB2 (neu) Protein Incorporated into Model Membrane Vesicles

The sequence of the transmembrane (TM) helix of ErbB2, a member of the epidermal growth factor receptor (ErbB) family, can influence its activity. In this report, the sequence and lipid dependence of the transverse position of a model-membrane-inserted peptides containing the ErbB2 TM helix and some of the juxtamembrane (JM) residues were studied. For the ErbB2 TM helix inserted into phosphatidylcholine vesicles, the activating V664E mutation was found to induce a transverse shift involving the movement of the E residue toward the membrane surface. This shortened the effective length of the TM-spanning portion of the sequence. The transverse shift was observed with the E664 residue in both the uncharged and charged states, but the extent of the shift was larger when the E residue was charged. When a series of hydrophilic residues was substituted for V664, the resulting transverse shifts at pH 7.0 decreased in the order D,H > E > Q > K > G > V. Except for His, this order is strongly correlated to that reported for the degree to which these substitutions induce cellular transformation when introduced into full-length ErbB2. To examine the effect of lipid on transverse shift, we studied the uncharged V664Q mutation. The presence of 20% of the anionic lipid DOPS (dioleoylphosphatidylserine) in the model membrane vesicles, which introduces a physiologically relevant level of anionic lipid, did not affect the degree of transverse shift. However, in the case of a peptide containing a V674Q substitution, in which the Q is closer to the C-terminus of the ErbB2 TM helix than the N-terminus, transverse shift was suppressed in vesicles containing 20% DOPS. This suggests that the interaction of the cationic JM residues flanking the C-terminus of the ErbB2 TM helix interact with anionic lipids to anchor the C-terminal end of the TM helix. This anchoring site may act as a pivot that amplifies transverse movements of the ErbB2 TM segment to induce a large swinging-type motion in the extracellular domain of the protein, affecting ErbB2 activity. Interactions interrupting C-terminal JM residue association with anionic lipid might partly impact ErbB2 activity by disrupting this pivoting.     

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.     

The mitochondrial genomes of Amphiascoides atopus and Schizopera knabeni (Harpacticoida: Miraciidae) reveal similarities between the copepod orders Harpacticoida and Poecilostomatoida

Members of subclass Copepoda are abundant, diverse, and—as a result of their variety of ecological roles in marine and freshwater environments—important, but their phylogenetic interrelationships are unclear. Recent studies of arthropods have used gene arrangements in the mitochondrial (mt) genome to infer phylogenies, but for copepods, only seven complete mt genomes have been published. These data revealed several within-order and few among-order similarities. To increase the data available for comparisons, we sequenced the complete mt genome (13,831 base pairs) of Amphiascoides atopus and 10,649 base pairs of the mt genome of Schizopera knabeni (both in the family Miraciidae of the order Harpacticoida). Comparison of our data to those for Tigriopus japonicus (family Harpacticidae, order Harpacticoida) revealed similarities in gene arrangement among these three species that were consistent with those found within and among families of other copepod orders. Comparison of the mt genomes of our species with those known from other copepod orders revealed the arrangement of mt genes of our Harpacticoida species to be more similar to that of Sinergasilus polycolpus (order Poecilostomatoida) than to that of T. japonicus. The similarities between S. polycolpus and our species are the first to be noted across the boundaries of copepod orders and support the possibility that mt-gene arrangement might be used to infer copepod phylogenies. We also found that our two species had extremely truncated transfer RNAs and that gene overlaps occurred much more frequently than has been reported for other copepod mt genomes.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

Molecular cloning and tissue distribution of the phosphotyrosine interaction domain containing 1 (PID1) gene in Tianfu goat

Phosphotyrosine interaction domain containing 1 (PID1) is an important mediator in the development of obesity-related insulin resistance in humans and animals. For a better understanding of the structure and function of the PID1 gene and to study its effect in caprine, the cDNA of the PID1 gene from the abdominal muscle of Tianfu goat was cloned and sequenced. The structure of PID1 was analyzed using bioinformatics tools. The results showed that the full sequence of the caprine PID1 cDNA was 896 bp long and contained a 654 bp long coding region that encoded a 217 amino acid sequence. Fifteen phosphorylation sites were predicted in the translated PID1 protein. The protein had a phosphotyrosine-binding domain between Arg⁵³ and Ile¹⁹⁹. A phylogenic tree based on the PID1 proteins from other species revealed that the caprine protein was closely related to cattle PID1. Fluorescence quantitative PCR analyses revealed that PID1 was expressed in the heart, liver, spleen, lung, kidney, leg muscle, abdominal muscle and longissimus dorsi muscle of goats. In particular, high expression levels of PID1 were detected in liver and abdominal muscle, and low expression levels were seen in lung. Furthermore, the PID1 mRNA expression levels in the longissimus dorsi muscles increased gradually with the age of the goats (P < 0.05). Western blotting results detected the PID1 protein in six of the tissues in which PID1 was shown to be expressed; the two exceptions were liver and spleen.     

Crystal structure of the novel complex formed between zinc alpha2-glycoprotein (ZAG) and prolactin-inducible protein (PIP) from human seminal plasma

This is the first report on the formation of a complex between zinc α2-glycoprotein (ZAG) and prolactin-inducible protein (PIP). The complex was purified from human seminal plasma and crystallized using 20% polyethylene glycol 9000 and 5% hexaethylene glycol. The structure of the complex has been determined using X-ray crystallographic method and refined to an R_cryst of 0.199 (R_free = 0.239). The structure of ZAG is broadly similar to the structure of serum ZAG. The scaffolding of PIP consists of seven β-strands that are organized in the form of two antiparallel β-pleated sheets, resulting in the formation of a sandwiched β-sheet. The amino acid sequence of PIP contains one potential N-glycosylation site at Asn77, and the same is found glycosylated with four sugar residues. The structure of the complex shows that the β-structure of PIP is ideally aligned with the β-structure of domain α3 of ZAG to form a long interface between two proteins. The proximal β-strands at the long interface are arranged in an antiparallel manner. There are 12 hydrogen bonds and three salt bridges between ZAG and PIP. At the two ends of vertical interface, two salt bridges are formed between pairs of Lys41-Asp233 and Lys68-Glu229. On the perpendicular interface involving α1-α2 domains of ZAG and a loop of PIP, another salt bridge is formed. The internal space at the corner of the L-shaped structure is filled with solvent molecules including a carbonate ion. The overall buried area in the complex is approximately 914 Å², which is considerably higher than the 660 Å² reported for the class I major histocompatibility complex structures.     

Vascular targeting to the SST2 receptor improves the therapeutic response to near-IR two-photon activated PDT for deep-tissue cancer treatment

Background

Broader clinical acceptance of photodynamic therapy is currently hindered by (a) poor depth efficacy, and (b) predisposition towards establishment of an angiogenic environment during the treatment. Improved depth efficacy is being sought by exploiting the NIR tissue transparency window and by photo-activation using two-photon absorption (2PA). Here, we use two-photon activation of PDT sensitizers, untargeted and targeted to SST2 receptors or EGF receptors, to achieve deep tissue treatment.

Methods

Human tumor lines, positive or negative for SST2r expression were used, as well as murine 3LL cells and bovine aortic endothelial cells. Expression of SST2 receptors on cancer cells and tumor vasculature was evaluated in vitro and frozen xenograft sections. PDT effects on tumor blood flow were followed using in vivo scanning after intravenous injection of FITC conjugated dextran 150 K. Dependence of the PDT efficacy on the laser pulse duration was evaluated. Effectiveness of targeting to vascular SST2 receptors was compared to that of EGF receptors, or no targeting.

Results

Tumor vasculature stained for SST2 receptors even in tumors from SST2 receptor negative cell lines, and SST2r targeted PDT led to tumor vascular shutdown. Stretching the pulse from ~ 120 fs to ~ 3 ps led to loss of the PDT efficacy especially at greater depth. PDT targeted to SST2 receptors was much more effective than untargeted PDT or PDT targeted to EGF receptors.

General significance

The use of octreotate to target SST2 receptors expressed on tumor vessels is an excellent approach to PDT with few recurrences and some long term cures.     

Short- and medium-chain fatty acids enhance the cell surface expression and transport capacity of the bile salt export pump (BSEP/ABCB11)

The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We previously reported that 4-phenylbutyrate (4PBA), an approved drug for urea cycle disorders, is a promising agent for intrahepatic cholestasis because it increases both the cell surface expression and the transport capacity of BSEP. In the present study, we searched for effective compounds other than 4PBA by focusing on short- and medium-chain fatty acids, which have similar characteristics to 4PBA such as their low-molecular-weight and a carboxyl group. In transcellular transport studies using Madin–Darby canine kidney (MDCK) II cells, all short- and medium-chain fatty acids tested except for formate, acetate, and hexanoic acid showed more potent effects on wild type (WT) BSEP-mediated [³H]taurocholate transport than did 4PBA. The increase in WT BSEP transport with butyrate and octanoic acid treatment correlated with an increase in its expression at the cell surface. Two PFIC2-type variants, E297G and D482G BSEP, were similarly affected with both compounds treatment. The prolonged half-life of cell surface-resident WT BSEP was responsible for this increased octanoic acid-stimulated transport, but not for that of butyrate. In conclusion, short- and medium-chain fatty acids have potent effects on the increase in WT and PFIC2-type BSEP-mediated transport in MDCK II cells. Although both short- and medium-chain fatty acids enhance the transport capacity of WT and PFIC2-type BSEP by inducing those expressions at the cell surface, the underlying mechanism seems to differ between fatty acids.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.     

Molecular characterization and transcriptional analysis of non-mammalian type Toll like receptor (TLR21) from rock bream (Oplegnathus fasciatus)

Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919 bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112 kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.