Oxidative stress response of Deinococcus geothermalis via a cystine importer.

A cystine-dependent anti-oxidative stress response is characterized in Deinococcus geothermalis for the first time. Nevertheless, the same transcriptional directed Δdgeo_1985F mutant strain was revealed to have an identical phenotype to the wild-type strain, while the reverse transcriptional directed Δdgeo_1985R mutant strain was more resistant to oxidative stress at a certain concentration of H2O2 than the wild-type strain. The wild-type and mutant strains expressed equal levels of superoxide dismutase and catalase under H2O2-induced stress. Although the expression levels of the general DNA-damage response-related genes recA, pprA, ddrA, and ddrB were up-regulated by more than five-fold in the wild-type strain relative to the Δdgeo_1985R mutant strain, the mutant strain had a higher survival rate than the wild-type under H2O2 stress. The Δdgeo_1985R mutant strain highly expressed a cystine-transporter gene (dgeo_1986), at levels 150-fold higher than the wild-type strain, leading to the conclusion that this cystine transporter might be involved in the defensive response to H2O2 stress. In this study, the cystine transporter was identified and characterized through membrane protein expression analysis, a cystine-binding assay, and assays of intracellular H2O2, cysteine, and thiol levels. The genedisrupted mutant strain of the cystine importer revealed high sensitivity to H2O2 and less absorbed cystine, resulting in low concentrations of total thiol. Thus, the absorbed cystine via this cystine-specific importer may be converted into cysteine, which acts as a primitive defense substrate that non-enzymatically scavenges oxidative stress agents in D. geothermalis.

Wohlfahrtiimonas larvae sp. nov., isolated from the larval gut of Hermetia illucens (Diptera: Stratiomyidae).

A novel, Gram-negative, facultative anaerobic, motile and short rod-shaped bacterium, strain KBL006(T) was isolated from the larval gut of Hermetia illucens, Black soldier fly. The 16S rRNA gene sequence of strain KBL006(T) showed 96.4 % similarity to that of Wohlfahrtiimonas chitiniclastica S5(T). Strain KBL006(T) grew optimally at 30 °C, at pH 8.0 and in the presence of 1-2 % (w/v) NaCl. Oxidase activity and catalase activity were positive. The major fatty acids were C18:1 ω7c, C14:0, and C16:0. The major respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol, and two phospholipids. The G+C content of the genomic DNA was 45.2 mol%. Based on these polyphasic data, strain KBL006(T) is considered to represent a novel species in the genus Wohlfahrtiimonas, for which the name Wohlfahrtiimonas larvae sp. nov. is proposed. The type strain is KBL006(T) (= KACC 16839(T) = JCM 18424(T)).

Intermolecular cross-talk between NTR1 and NTR2 neurotensin receptor promotes intracellular sequestration and functional inhibition of NTR1 receptors.

G-protein-coupled receptors (GPCR) are now regarded as being able to acquire heterodimer conformations affecting their pharmacology, signaling and trafficking. In co-immunoprecipitation studies using differentially epitope-tagged receptors, we herein provide direct evidence for heterodimerization of human neurotensin type 1 receptor (hNTR1) and type 2 receptor (hNTR2). Using chimeric constructs, we also identified the hNTR2 transmembrane 2 (TM2) to TM4 region as crucial for the formation of the dimerization interface. At the functional level, we demonstrated that the co-expression of hNTR2 suppressed hNTR1-mediated adenylate cyclase/cAMP and phospholipase C activation. Finally, confocal microscopy revealed that whereas tagged hNTR1 expressed alone were localized to the plasma membrane, co-expression of hNTR2 caused the retention of hNTR1 in sub-cellular compartments, indicating that heterodimerization with hNTR2 interferes with the proper recruitment of hNTR1 to the plasma membrane. Overall, this study proposes a novel function of NTR2 in the regulation of NTR1 activity.

Hepatocyte growth factor family negatively regulates hepatic gluconeogenesis via induction of orphan nuclear receptor small heterodimer partner in primary hepatocytes.

Hepatic gluconeogenesis is tightly balanced by opposing stimulatory (glucagon) and inhibitory (insulin) signaling pathways. Hepatocyte growth factor (HGF) is a pleiotropic growth factor that mediates diverse biological processes. In this study, we investigated the effect of HGF and its family member, macrophage-stimulating factor (MSP), on hepatic gluconeogenesis in primary hepatocytes. HGF and MSP significantly repressed expression of the key hepatic gluconeogenic enzyme genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (Glc-6-Pase) and reduced glucose production. HGF and MSP activated small heterodimer partner (SHP) gene promoter and induced SHP mRNA and protein levels, and the effect of HGF and MSP on SHP gene expression was demonstrated to be mediated via activation of the AMP-activated protein kinase (AMPK) signaling pathway. We demonstrated that upstream stimulatory factor-1 (USF-1) specifically mediated HGF effect on SHP gene expression, and inhibition of USF-1 by dominant negative USF-1 significantly abrogated HGF-mediated activation of the SHP promoter. Elucidation of the mechanism showed that USF-1 bound to E-box-1 in the SHP promoter, and HGF increased USF-1 DNA binding on the SHP promoter via AMPK and DNA-dependent protein kinase-mediated pathways. Adenoviral overexpression of USF-1 significantly repressed PEPCK and Glc-6-Pase gene expression and reduced glucose production. Knockdown of endogenous SHP expression significantly reversed this effect. Finally, knockdown of SHP or inhibition of AMPK signaling reversed the ability of HGF to suppress hepatocyte nuclear factor 4alpha-mediated up-regulation of PEPCK and Glc-6-Pase gene expression along with the HGF- and MSP-mediated suppression of gluconeogenesis. Overall, our results suggest a novel signaling pathway through HGF/AMPK/USF-1/SHP to inhibit hepatic gluconeogenesis.

Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein.

The human papillomavirus (HPV) DNA replication origin (ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.

Structure and function of the atypical orphan nuclear receptor small heterodimer partner.

The small heterodimer partner (SHP; NROB2) is a member of the nuclear receptor superfamily and is classified as an "orphan" subgroup, as its ligand has not yet been identified. SHP lacks the classical DNA-binding domain found in most nuclear receptors and functions as a transcriptional coregulator by directly interacting with nuclear receptors and other transcription factors. SHP regulates the transcription of a variety of target genes and controls a variety of physiological functions. For the past 10 years, great progress has been made in our understanding of the mechanism of action of SHP and the regulation of SHP gene expression. Many of the results imply that SHP has a variety of roles in the regulation of metabolic homeostasis. In this review, we discuss the current state of understanding of the structure, expression, and function of the orphan nuclear receptor, SHP.

Whitefly transmission of sweet potato viruses.

Three genera of plant viruses, Begomovirus (Geminiviridae), Crinivirus (Closteroviridae) and Ipomovirus (Potyviridae), contain members that infect sweet potato (Ipomoea batatas) and are transmitted by whiteflies. The begomoviruses, Sweet potato leaf curl virus (SPLCV) and Ipomoea leaf curl virus (ILCV), and the ipomovirus Sweet potato mild mottle virus are transmitted by Bemisia tabaci, the sweet potato whitefly. The crinivirus, Sweet potato chlorotic stunt virus (SPCSV), is transmitted by B. tabaci and Trialeurodes abutilonea, the bandedwinged whitefly. Transmission experiments were done with three of these viruses using laboratory-reared whiteflies and 2-day acquisition and transmission feeding periods. SPLCV and ILCV were transmitted from single and double infections by B. tabaci at rates of 5-10%. Transmission rates for SPLCV by B. tabaci were 15-20%. T. abutilonea transmitted SPCSV at a rate of ca. 3% but did not transmit ILCV or SPLCV.

Purification and electron microscopic visualization of functional human spliceosomes.

Pre-mRNA splicing takes place in a large and highly dynamic complex known as the spliceosome. Here we report the optimization of a maltose-binding protein (MBP) affinity-purification method to isolate functional spliceosomes for electron microscopic analysis. Visualization of the spliceosome preparations revealed distinct 40-60 nm particles. Immunogold-conjugated antibodies to spliceosome components specifically label these particles, which are eliminated by treatment with either RNase or protease. Moreover, spliceosomes assembled on two different pre-mRNAs are indistinguishable. This first visualization of purified functional spliceosomes assembled in vitro reveals striking structural features, including one or more central cavities and multiple elongate lobes.

Occurrence of a Strain of Texas pepper virus in Tabasco and Habanero Pepper in Costa Rica.

A viral disease causing severe leaf malformation and yellow mottle on Tabasco (Capsicum frutescens) and Habanero (C. chinense) pepper plants was observed in 1997 on farms in southwestern Costa Rica. Whiteflies (Bemisia tabaci) were present on affected farms and transmitted the putative virus. Total DNA was extracted from a whitefly-transmitted isolate, and polymerase chain reaction (PCR) was performed using degenerate primers. The expected PCR product (550 bp) was obtained, suggesting the presence of a geminivirus. This was confirmed by Southern analysis using a geminivirus-specific probe. The virus was mechanically transmitted from pepper to pepper. Electron microscopy of ultrathin sections from infected Tabasco pepper plants revealed fibrillar rings and viruslike particles in the nucleus of the vascular parenchyma cells. The sequence of DNA A was obtained from three overlapping PCR fragments amplified using three pairs of degenerate primers; PAL1v1978/PAR1c496, PCRc1/AV494, and PCRv181/ AC1048. The complete sequence of DNA A of this begomovirus consisted of 2,619 bp (GenBank accession number: AF149227) containing four open reading frames (ORF). The nucleotide sequence of the virus was 92.3% identical to DNA A of the Tamaulipas strain of Texas pepper virus (TPV-TAM). Phylogenetic analyses using AC1 and AV1 nucleotide sequences also indicated a close relationship between this virus and TPV. Based on the biological characteristics, the high percentage of nucleotide and derived amino acid sequence identities, and phyloge-netic analyses, we concluded that this virus is a distinct strain of TPV, and designated it as the Costa Rica strain. This is the first report of TPV in Costa Rica.