Comparative Genome Sequence Analysis of Choristoneura occidentalis Freeman and C. rosaceana Harris (Lepidoptera: Tortricidae) Alphabaculoviruses

The complete genome sequences of Choristoneura occidentalis and C. rosaceana nucleopolyhedroviruses (ChocNPV and ChroNPV, respectively) (Baculoviridae: Alphabaculovirus) were determined and compared with each other and with those of other baculoviruses, including the genome of the closely related C. fumiferana NPV (CfMNPV). The ChocNPV genome was 128,446 bp in length (1147 bp smaller than that of CfMNPV), had a G+C content of 50.1%, and contained 148 open reading frames (ORFs). In comparison, the ChroNPV genome was 129,052 bp in length, had a G+C content of 48.6% and contained 149 ORFs. ChocNPV and ChroNPV shared 144 ORFs in common, and had a 77% sequence identity with each other and 96.5% and 77.8% sequence identity, respectively, with CfMNPV. Five homologous regions (hrs), with sequence similarities to those of CfMNPV, were identified in ChocNPV, whereas the ChroNPV genome contained three hrs featuring up to 14 repeats. Both genomes encoded three inhibitors of apoptosis (IAP-1, IAP-2, and IAP-3), as reported for CfMNPV, and the ChocNPV IAP-3 gene represented the most divergent functional region of this genome relative to CfMNPV. Two ORFs were unique to ChocNPV, and four were unique to ChroNPV. ChroNPV ORF chronpv38 is a eukaryotic initiation factor 5 (eIF-5) homolog that has also been identified in the C. occidentalis granulovirus (ChocGV) and is believed to be the product of horizontal gene transfer from the host. Based on levels of sequence identity and phylogenetic analysis, both ChocNPV and ChroNPV fall within group I alphabaculoviruses, where ChocNPV appears to be more closely related to CfMNPV than does ChroNPV. Our analyses suggest that it may be appropriate to consider ChocNPV and CfMNPV as variants of the same virus species.     

Induced osteoblast differentiation by amide derivatives of stilbene: The possible osteogenic agents

A series of amide derivatives of stilbene was synthesized and investigated for osteogenic activity. Out of sixteen, seven compounds viz 19c, 19g, 19i, 24b, 25a, 25c and 26a showed significant osteoblast differentiation within 1 pM–1 µM concentrations. Amongst all, 26a was identified as most active molecule which presented effective mineralization of osteoblasts and expression of mRNA of osteogenic marker gene such as BMP-2, ALP, and Runx-2 at 1 pM. In estrogen-deficient balb/c mice, 26a showed significant osteogenic activity at 5 mg-kg⁻¹ body weight dose. The protein expression study for estrogen receptors α and β (ER-α & ER-β) using mouse calvarial osteoblasts (MCOs) and molecular docking analyses showed preferential expression of ER-β by 26a indicating the possibility of ER-β mediated osteogenic activity of 26a.     

Inhibition by acidic phospholipids of protein degradation by ER-60 protease, a novel cysteine protease, of endoplasmic reticulum.

A protein (ER60) with sequence similarity to phosphoinositide-specific phospholipase C-alpha purified from rat liver endoplasmic reticulum (ER) degraded ER resident proteins and is really a protease [(1992) J. Biol. Chem. 265, 15152-15159]. Therefore, ER60 is called ER-60 protease. We now show that negatively charged phospholipids, phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate and phosphatidylserine inhibit ER protein degradation by ER-60 protease. Phosphatidylcholine and phosphatidylethanolamine show no effect on the activity of ER-60 protease. With the use of protease inhibitors, ER-60 protease is shown to be a novel cysteine protease distinct from those of the cytosol and lysosomes.     

IFN-gamma inhibition of TRAIL-induced IAP-2 upregulation, a possible mechanism of IFN-gamma-enhanced TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of TNF family and a potent inducer of apoptosis. The anticancer activities of TNF family members are often modulated by interferon (IFN)-gamma. Thus, we investigated whether IFN-gamma enhances TRAIL-induced apoptosis. We exposed HeLa cells to IFN-gamma for 12 h and then treated with recombinant TRAIL protein. No apoptosis was induced in cells pretreated with IFN-gamma, and TRAIL induced 25% cell death after 3 h treatment. In HeLa cells pretreated with IFN-gamma, TRAIL induced cell death to more than 70% at 3 h, indicating that IFN-gamma pretreatment sensitized HeLa cells to TRAIL-induced apoptosis. We investigated molecules that might be regulated by IFN-gamma pretreatment that would affect TRAIL-induced apoptosis. Western blotting analyses demonstrated that TRAIL treatment increased the level of IAP-2 protein and IFN-gamma pretreatment inhibited the upregulation of IAP-2 protein by TRAIL protein. Our data indicate that TRAIL can signal to activate both apoptosis induction and antiapoptotic mechanism, at least, through IAP-2 simultaneously. IFN-gamma or TRAIL treatment alone did not change expression of other pro- or antiapoptotic proteins such as DR4, DR5, FADD, Bax, IAP-1, XIAP, Bcl-2, and Bcl-XL. Our findings suggest that IFN-gamma may sensitize HeLa cells to TRAIL-induced apoptosis by preventing TRAIL-induced IAP-2 upregulation, and IFN-gamma may play a role in anticancer therapy of TRAIL protein through such mechanism.     

Genome sequence of Ensifer medicae strain WSM1369; an effective microsymbiont of the annual legume Medicago sphaerocarpos

Ensifer medicae WSM1369 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1369 was isolated in 1993 from a nodule recovered from the roots of Medicago sphaerocarpos growing at San Pietro di Rudas, near Aggius in Sardinia (Italy). WSM1369 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. sphaerocarpos. Here we describe the features of E. medicae WSM1369, together with genome sequence information and its annotation. The 6,402,557 bp standard draft genome is arranged into 307 scaffolds of 307 contigs containing 6,656 protein-coding genes and 79 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Endoplasmic reticulum and Golgi localization sequences for mammalian target of rapamycin

Mammalian target of rapamycin (mTOR) forms two complexes, mTORC1 and mTORC2, that play central roles in cell growth and functions. Only mTORC1 is directly inhibited by the immunosuppressive drug rapamycin. Despite recent progress in identifying new components and functions of the mTOR pathway, relatively little is known about the spatial arrangement of mTOR signaling and the underlying mechanisms. In a previous study, we showed that a large proportion of mTOR is localized to the endoplasmic reticulum (ER) and Golgi in many common cell lines. Here, we report the identification of an internal mTOR sequence that contains two HEAT (HT) repeats, HT18 and HT19, and two intervening interunit spacers (IUSs), IUS17 and IUS18, which is sufficient to target enhanced green fluorescent protein to the Golgi. Surprisingly, deletion of IUS17 from this Golgi localization sequence (GLS) converts it to an ER localization sequence (ELS). Deletion of HT19, a common element of both GLS and ELS from the full-length mTOR, causes delocalization of mTOR and inhibits the ability of mTOR to promote S6 phosphorylation. Moreover, overexpression of GLS and ELS inhibits both mTOR complexes. Together, our results reveal unusual ER- and Golgi-targeting sequences and suggest that anchoring to these organelles is important for the functions of mTOR complexes.     

A novel mutation in LEPRE1 that eliminates only the KDEL ER- retrieval sequence causes non-lethal osteogenesis imperfecta

Prolyl 3-hydroxylase 1 (P3H1), encoded by the LEPRE1 gene, forms a molecular complex with cartilage-associated protein (CRTAP) and cyclophilin B (encoded by PPIB) in the endoplasmic reticulum (ER). This complex is responsible for one step in collagen post-translational modification, the prolyl 3-hydroxylation of specific proline residues, specifically α1(I) Pro986. P3H1 provides the enzymatic activity of the complex and has a Lys-Asp-Glu-Leu (KDEL) ER-retrieval sequence at the carboxyl terminus. Loss of function mutations in LEPRE1 lead to the Pro986 residue remaining unmodified and lead to slow folding and excessive helical post-translational modification of type I collagen, which is seen in both dominant and recessive osteogenesis imperfecta (OI). Here, we present the case of siblings with non-lethal OI due to novel compound heterozygous mutations in LEPRE1 (c.484delG and c.2155dupC). The results of RNA analysis and real-time PCR suggest that mRNA with c.2155dupC escapes from nonsense-mediated RNA decay. Without the KDEL ER- retrieval sequence, the product of the c.2155dupC variant cannot be retained in the ER. This is the first report of a mutation in LEPRE1 that eliminates only the KDEL ER-retrieval sequence, whereas other functional domains remain intact. Our study shows, for the first time, that the KDEL ER- retrieval sequence is essential for P3H1 functionality and that a defect in KDEL is sufficient for disease onset.     

Effect of ER-27856, a novel squalene synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-coa reductase inhibitors

Squalene synthase (SQS; EC 2.5.1.21) plays an important role in the cholesterol biosynthetic pathway. We discovered ER-28448, 5-(N-[2-butenyl-3-(2-methoxyphenyl)]-N-methylamino)-1, 1-penthylidenebis(phosphonic acid) trisodium salt, as a potent and selective inhibitor of SQS. ER-28448 inhibited SQS in rat liver microsome with an IC(50) value of 3.6 nm. We also prepared ER-27856, the tripivaloyloxymethyl ester prodrug of ER-28448. Although less active than ER-28448 in a liver microsome assay, ER-27856 more potently inhibited cholesterol biosynthesis in rat hepatocytes; and ER-27856 orally inhibited de novo cholesterol biosynthesis in Sprague-Dawley rats, with an ED(50) value of 1.6 mg/kg. In HepG2 cells, ER-27856 upregulated low density lipoprotein receptor activity to 2.1 times that of control. A time-course study indicated that the inhibitory effect of ER-27856 on cholesterol biosynthesis in rats continued for up to 8 h. In a study of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HMGRIs), atorvastatin actively suppressed cholesterol biosynthesis for 8 h, whereas the effect of pravastatin and simvastatin diminished at 4 and 8 h, respectively. In rhesus monkeys, 4 days of oral administration of ER-27856 lowered plasma total cholesterol (TCHO) more potently than did these HMGRIs. Whereas atorvastatin significantly elevated plasma alanine aminotransferase, a marker of hepatotoxicity, to 3.7 times at 100 mg/kg, ER-27856 increased the level only 1.4 times at 10 mg/kg, at which doses the hypocholesterolemic effect was equivalent. During 28 days of administration, ER-27856 reduced TCHO and non-high density lipoprotein (non-HDL) cholesterol by 72 and 95%, respectively.These results demonstrate that ER-27856 had more potent hypocholesterolemic activity and less hepatotoxic effect than HMGRIs. ER-27856 may contribute to the treatment of hypercholesterolemic patients.     

Thermodynamic investigation of monoclonal antibody alkylated nucleoside interaction as a model for epitope recognition on nucleic acids

The objective of this investigation is examination of the dominant forces that govern complex formation between a series of monoclonal antibodies directed against O6-ethyl-2'-deoxyguanosine. These monoclonal antibodies (coded as ER-6, ER-3, and EM-1) provide the basis for a thermodynamic comparative evaluation of the potentially different forces that stabilize the various monoclonal antibody (mAb) alkylated nucleoside complexes. The binding affinities of ER-6, ER-3, and EM-1 are measured in terms of specific (O6-ethyl-2'-deoxyguanosine, or O6-EtdGuo) and nonspecific (O6-methyl-2'-deoxyguanosine, or O6-MedGuo) antigens, under a variety of experimental conditions, including pH, sodium chloride addition, 1-propanol addition, and temperature, via a nitrocellulose affinity filter assay. The binding isotherms were analyzed via a least-squares routine fit to a two independent binding sites model. The temperature dependence of the van't Hoff enthalpies for the specific O6-EtdGuo interaction ranges from -15.18 to -18.60 kcal mol-1, while for O6-MedGuo the range was extended from -2.72 to -20.66 kcal mol-1. The standard and unitary entropies were negative for those mAb interactions with O6-EtdGuo as well as for ER-6/O6-MedGuo complex formation. However, it was found that the interactions between ER-3 and EM-1 with O6-MedGuo led to decidedly positive entropic values. These results indicate two different dominant forces at work in complex stabilization. The interaction of the three mAb's with their specific antigen, as well as ER-6/O6-MedGuo interaction (nonspecific), may well be controlled by van der Waals type forces, while ER-3 and EM-1 interactions with nonspecific antigen imply formal charge neutralization electrostatics as the dominant force.     

马传染性贫血病毒驴白细胞弱毒疫苗gag基因的序列测定及分析

以马传染性贫血病毒（ＥＬＡＶ）驴白细胞弱毒疫苗株（ＤＬＡ）病毒基因组ＲＮＡ为材料,用ＲＴ－ＰＣＲ方法扩增出ＥＩＡＶ ｇａｇ基因,以平端针其克隆到质粒载体ｐＵＣ１９中,由于疫苗不是克隆株,因此通过５次单独克隆与测序,推导出ＥＩＡＶ－ＤＬＡ ｇａｇ基因的优势序列。ｇａｇ基因全长１４５８个碱基,编码一４８６个氨基酸残基的前体蛋白。与美国ＥＩＡＶ Ｗｙｏｍｉｎｇ１３６９株比较,核苷酸同源性为８０％,氨基酸     

Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity

ATP-sensitive K⁺ (K_ATP) channels play an important role in insulin secretion. K_ATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue–residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms.     

Candidate gene approach: potentional association of caspase-1, inhibitor of apoptosis protein-1, and prosaposin gene polymorphisms with response to Salmonella enteritidis challenge or vaccination in young chicks

Salmonella enteritidis (SE) contamination of poultry products is a major cause of foodborne disease worldwide. Caspase-1 and inhibitor of apoptosis protein-1 (IAP-1) were selected as candidate genes for chicken response to SE because their proteins play critical roles in the apoptotic pathway when intracellular bacteria interact with host cells. Prosaposin (PSAP) was selected as a positional candidate gene based on a previous quantitative trait loci (QTL) linkage study using the same population. The F1 offspring of outbred sires crossed with three diverse, highly inbred dam lines (two major histocompatibility complex-congenic Leghorn lines named G-B1 and G-B2, and one Fayoumi line) were used to define the phenotypes. The F1 birds were involved in either pathogenic SE challenge, in which spleen and cecum content bacterial load were quantified, or SE vaccination, in which plasma antibody level to SE vaccine was evaluated. A polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay was developed to identify single-nucleotide polymorphism (SNP) in the three genes. The F1 offspring of heterozygous sires for each gene were genotyped. The sire caspase-1 gene was significantly associated with cecum content bacterial load (P = 0.04) in the three combined dam line crosses, and with spleen bacterial load in the G-B1 cross (P=0.02). The sire caspase-1 gene was also significantly associated with antibody level to SE vaccine (P=0.03) in F1 males in the three combined dam line crosses. The sire IAP-1 gene was significantly associated with spleen bacterial load (P=0.04) in the three combined dam-line crosses, and interacted with dam-line genetics (P = 0.01) for cecum content bacterial load. The sire PSAP gene significantly interacted with sex for spleen bacterial load (P = 0.004). This study is the first to demonstrate the association of SNPs for caspase-1, IAP-1, and PSAP genes with SE vaccine and with pathogen challenge response in chickens.     

Epidermal growth factor receptors in breast cancer: association with early relapse and death, poor response to hormones and interactions with neu

Epidermal growth factor receptors (EGFRs) were measured in 221 primary breast cancers by ligand binding with 125I-labelled EGF, and high-affinity sites were quantitated. There was a highly significant inverse relationship between oestrogen receptor (ER) and EGFR (15 EGFR-positive [EGFR+]ER+ and 92 EGFR-negative [EGFR-]ER+: 54 EGFR- ER- and 60 EGFR+ ER-). The relapse-free survival and overall survival were significantly shorter for EGFR+ vs EGFR- tumours (P less than 0.001) by about 2 yr in the case of relapse-free survival. When ER- tumours were substratified by EGFR status, the EGFR- ER- tumours had a prognosis almost as good as the ER+ tumours. In 31 of 184 cases, high expression of neu, correlating with amplification, was found. Expression of neu conferred similar poor prognosis to EGFR expression in all prognostic subgroups. Coexpression of neu and EGFR had an additive adverse effect. Epidermal growth factor receptors (EGFR) and oestrogen receptors (ER) were analysed in 221 patients with primary operable breast cancer by means of radioligand assays. After median follow-up of 24 months (range 3-60 months), there had been recurrences in 99 patients, of whom 72 (median age 56 yr, range 32-77 yr) received tamoxifen alone as first-line treatment for recurrence. 14 patients (19%) showed a response to this therapy and 58 (81%) did not. Of 32 ER+ tumours, 12 (37.5%) showed an objective response to tamoxifen compared with only 2 of 40 (5%) ER- tumours (P less than 0.005). Of 35 EGFR+ tumours, 3 (8.5%) achieved an objective response compared with 11 of 36 (30%) EGFR tumours (P less than 0.05). Only 1 of 28 EGFR+, ER- tumours achieved an objective response. Including patients whose disease remained stable for more than 6 months with the responders, however, EGFR status was a better predictor of response to tamoxifen; 15 of 37 EGFR- patients and 5 of 35 EGFR+ patients responded (P less than 0.01).