bluuebunny/crossref_metadata_2025 huggingface dataset:
A Sartan Derivative with a Very Low Angiotensin II Receptor Affinity Ameliorates Ischemic Cerebral Damage
Authors: Takizawa Shunya , Dan Takashi , Uesugi Tsuyoshi , Nagata Eiichiro , Takagi Shigeharu , van Ypersele de Strihou Charles , Miyata Toshio
Published: June 2009

Angiotensin II receptor blockers (ARBs) have a potent ability to inhibit oxidative stress and advanced glycation, in addition to their protective effects originated from blood pressure lowering and angiotensin II type 1 receptor (AT1)-blockade. To obtain a pharmacological tool to dissect the mechanisms of ARBs’ protective benefits in experimental stroke, we synthesized a novel ARB-derivative, R-147176, which is 6,700 times less potent than olmesartan in AT1-binding inhibition and therefore has a minimal antihypertensive effect, but retains marked inhibitory effects on oxidative stress and advanced glycation. We evaluated the effect of R-147176 (10–30 mg/kg per day), administered orally or intravenously, on brain infarct volume in transient thread occlusion and photothrombotic models in rats. The antioxidative and antiinflammatory properties were also investigated. R-147176 significantly reduced infarct volume, without influence on blood pressure, in both models. R-147176 significantly reduced the numbers of ED-1-positive cells and of TUNEL-positive cells, and protein carbonyl formation in the damaged brain. This ARB derivative, despite its significantly lower AT1 affinity and virtually no antihypertensive effect, ameliorated ischemic cerebral damage through antioxidative and antiinflammatory properties. These findings suggest potential usefulness of R-147176 as a pharmacological tool to investigate the ARBs’ protective effect in experimental stroke and open new therapeutic avenues.
N‐terminal modification increases the stability of the recombinant human endostatinin vitro
Authors: Jiang Li‐Ping , Zou Chang , Yuan Xue , Luo Wei , Wen Yong , Chen Yali
Published: June 2009

Endostar, approved for the treatment of non‐small‐cell lung cancer by the State Food and Drug Administration in China, is a derivative of human endostatin that is modified with an additional metal‐chelating sequence (MGGSHHHHH) at the N‐terminus. This modification contributes to an additional zinc‐binding site in the endostatin sequence. In the present study, zinc‐binding and zinc‐free endostar were compared to further characterize their biochemical and structural properties. Thermally induced denaturation was determined by monitoring changes in fluorescence emission spectra. The data indicated that zinc binding significantly increased the transition temperature of endostar and contributed to a reversible change in protein conformation after recooling. Proteolysis assays demonstrated that the modified protein binding with zinc ions can stabilize the N‐terminus and the C‐terminus of endostar when treated with trypsin, chymotrypsin and carboxypeptidase A and B. Western‐blot analyses using anti‐His6antibody confirmed that the major cleaved fragments of endostar were in the N‐terminus when treated with trypsin and chymotrypsin. In the proliferation assay with human umbilical‐vein endothelial cells, the zinc‐binding and zinc‐free endostar samples with extra zinc‐binding sites displayed similar inhibiting activities.
Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases
Authors: Sexton Daniel J. , Chen Ting , Martik Diana , Kuzmic Petr , Kuang Guannan , Chen Jie , Nixon Andrew E. , Zuraw Bruce L. , Forteza Rosanna M. , Abraham William M. , Wood Clive R.
Published: June 2009

KLK1 (tissue kallikrein 1) is a member of the tissue kallikrein family of serine proteases and is the primary kinin-generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki=0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface-plasmon-resonance biosensor assay when KLK1 was complexed to known active-site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were each tested at a concentration of 1 μM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial epithelial cells grown at the air/liquid interface, DX-2300 blocked oxidative-stress-induced epidermal-growth-factor receptor activation and downstream mucus cell proliferation and hypersecretion, which have been previously shown to be mediated by KLK1. In an allergic sheep model of asthma, DX-2300 inhibited both allergen-induced late-phase bronchoconstriction and airway hyper-responsiveness to carbachol. These studies demonstrate that DX-2300 is a potent and specific inhibitor of KLK1 that is efficacious in in vitro and in vivo models of airway disease.
Coupling oxidative signals to protein phosphorylation via methionine oxidation in Arabidopsis
Authors: Hardin Shane C. , Larue Clayton T. , Oh Man-Ho , Jain Vanita , Huber Steven C.
Published: June 2009

The mechanisms involved in sensing oxidative signalling molecules, such as H2O2, in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of Met (methionine) to MetSO (Met sulfoxide) can couple oxidative signals to changes in protein phosphorylation. We demonstrate that when a Met residue functions as a hydrophobic recognition element within a phosphorylation motif, its oxidation can strongly inhibit peptide phosphorylation in vitro. This is shown to occur with recombinant soybean CDPKs (calcium-dependent protein kinases) and human AMPK (AMP-dependent protein kinase). To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf NR (nitrate reductase) on Ser534 using modification-specific antibodies. NR was a candidate protein for this mechanism because Met538, located at the P+4 position, serves as a hydrophobic recognition element for phosphorylation of Ser534 and its oxidation substantially inhibits phosphorylation of Ser534in vitro. Two lines of evidence suggest that Met oxidation may inhibit phosphorylation of NR-Ser534in vivo. First, phosphorylation of NR at the Ser534 site was sensitive to exogenous H2O2 and secondly, phosphorylation in normal darkened leaves was increased by overexpression of the cytosolic MetSO-repair enzyme PMSRA3 (peptide MetSO reductase A3). These results are consistent with the notion that oxidation of surface-exposed Met residues in kinase substrate proteins, such as NR, can inhibit the phosphorylation of nearby sites and thereby couple oxidative signals to changes in protein phosphorylation.
Human and rodent pancreatic β-cells express IL-4 receptors and IL-4 protects against β-cell apoptosis by activation of the PI3K and JAK/STAT pathways
Authors: Kaminski Anna , Welters Hannah J. , Kaminski Edward R. , Morgan Noel G.
Published: June 2009

Secretion of pro-inflammatory cytokines is associated with loss of pancreatic β-cell viability and cell death. IL-4 (interleukin-4) has been reported to mediate a protective effect against the loss of pancreatic β-cells, and IL-4 receptors have been found in rat pancreatic β-cells at both the RNA and the protein level. The aim of the present study was to investigate IL-4 receptor expression in human islet cells and to examine the signalling pathways by which IL-4 exerts its effects using the rat β-cell lines, BRIN-BD11 and INS-1E. By means of immunohistochemistry, it was demonstrated that IL-4 receptors are present on human islet cells. Using a flow cytometric method for evaluating cell death, it was confirmed that incubating β-cells with IL-4 attenuated cell death induced by IL-1β and interferon-γ by approx. 65%. This effect was abrogated by the presence of the PI3K (phosphoinositide 3-kinase) inhibitor, wortmannin, suggesting that activation of the PI3K pathway is involved. In support of this, Western blotting revealed that incubation of cells with IL-4 resulted in increased phosphorylation of Akt (also called protein kinase B), a downstream target of PI3K. Increased tyrosine phosphorylation of STAT6 (signal transducer and activator of transcription 6) also occurred in response to IL-4 and a selective JAK3 (Janus kinase 3) inhibitor reduced the cytoprotective response. Both effects were prevented by overexpression of the tyrosine phosphatase, PTP-BL (protein tyrosine phosphatase-BL). We conclude that IL-4 receptors are functionally competent in pancreatic β-cells and that they signal via PI3K and JAK/STAT pathways. These findings may have implications for future therapeutic strategies for the management of diabetes.
DDB2 decides cell fate following DNA damage
Authors: Stoyanova Tanya , Roy Nilotpal , Kopanja Dragana , Bagchi Srilata , Raychaudhuri Pradip
Published: June 2009

The xeroderma pigmentosum complementation group E (XP-E) gene product damaged-DNA binding protein 2 (DDB2) plays important roles in nucleotide excision repair (NER). Previously, we showed that DDB2 participates in NER by regulating the level of p21
Waf1/Cip1
. Here we show that the p21
Waf1/Cip1
-regulatory function of DDB2 plays a central role in defining the response (apoptosis or arrest) to DNA damage. The DDB2-deficient cells are resistant to apoptosis in response to a variety of DNA-damaging agents, despite activation of p53 and the pro-apoptotic genes. Instead, these cells undergo cell cycle arrest. Also, the DDB2-deficient cells are resistant to E2F1-induced apoptosis. The resistance to apoptosis of the DDB2-deficient cells is caused by an increased accumulation of p21
Waf1/Cip1
after DNA damage. We provide evidence that DDB2 targets p21
Waf1/Cip1
for proteolysis. The resistance to apoptosis in DDB2-deficient cells also involves Mdm2 in a manner that is distinct from the p53-regulatory activity of Mdm2. Our results provide evidence for a new regulatory loop involving the NER protein DDB2, Mdm2, and p21
Waf1/Cip1
that is critical in deciding cell fate (apoptosis or arrest) upon DNA damage.
Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state
Authors: Ge Ying , Rybakova Inna N. , Xu Qingge , Moss Richard L.
Published: June 2009

Cardiac myosin binding protein C (cMyBP-C), bound to the sarcomere's myosin thick filament, plays an important role in the regulation of muscle contraction. cMyBP-C is a large multidomain protein that interacts with myosin, titin, and possibly actin. Mutations in cMyBP-C are the most common known cause of heritable hypertrophic cardiomypathies. Phosphorylation of cMyBP-C plays an essential role in the normal cardiac function. cMyBP-C (142 kDa) has 81 serine and 73 threonine residues presenting a major challenge for unequivocal identification of specific phosphorylation sites. Top-down mass spectrometry, which directly analyzes intact proteins, is a powerful technique to universally observe and quantify protein posttranslational modifications without a priori knowledge. Here, we have extended top-down electron capture dissociation mass spectrometry to comprehensively characterize mouse cMyBP-C expressed in baculovirus. We have unambiguously identified all of the phosphorylation sites in the truncated (28–115 kDa) and full-length forms of cMyBP-C (142 kDa) and characterized the sequential phosphorylations, using a combination of top-down and middle-down (limited proteolysis) MS approach, which ensures full sequence coverage. Unit mass resolution and high mass accuracy (<5 ppm) have been achieved for a 115-kDa protein (the largest protein isotopically resolved to date). Remarkably, we discovered that truncations in recombinant proteins, even a seemingly minor one, can dramatically alter its phosphorylation state, which is significant because truncated recombinant proteins are routinely substituted for their full-length forms in crystal structure and functional studies. Our study provides direct evidence of alterations in the posttranslational state between the truncated and full-length recombinant proteins, which can lead to variations in structure and function.
Osmolyte-induced separation of the mechanical folding phases of ubiquitin
Authors: Garcia-Manyes Sergi , Dougan Lorna , Fernández Julio M.
Published: June 2009

Solvent molecules play key roles in the conformational dynamics of proteins. Here we use single molecule force-clamp spectroscopy to probe the role played by the stabilizing osmolyte glycerol on the conformational ensembles visited by a single ubiquitin protein folding after mechanical extension. Using a variety of force-pulse protocols, we find that glycerol stabilizes the native state of ubiquitin, making it more resistant to mechanical unfolding. We also find that although glycerol enhanced the hydrophobic collapse of unfolded and highly extended ubiquitins, it had no effect on the resulting collapsed states that are essential precursors of the folded state. These disparate effects of glycerol may be the result of distinct structural roles played by solvent molecules at the transition state of each folding ensemble. Our results open the way for a detailed analysis of the transition state structures that form along the folding trajectory of a mechanically extended protein.
Diminished sarco/endoplasmic reticulum Ca2+ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma
Authors: Mahn Katharina , Hirst Stuart J. , Ying Sun , Holt Mark R. , Lavender Paul , Ojo Oluwaseun O. , Siew Leonard , Simcock David E. , McVicker Clare G. , Kanabar Varsha , Snetkov Vladimir A. , O'Connor Brian J. , Karner Charlotta , Cousins David J. , Macedo Patricia , Chung K. Fan , Corrigan Christopher J. , Ward Jeremy P. T. , Lee Tak H.
Published: June 2009

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca2+in the cytosol ([Ca2+]i). A rise in [Ca2+]iis normalized by rapid reuptake of Ca2+into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca2+(SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca2+]i, cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca2+]ifollowing cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca2+re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca]iafter stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.
Functional analysis of an Orc6 mutant in
Drosophila
Authors: Balasov Maxim , Huijbregts Richard P. H. , Chesnokov Igor
Published: June 2009

The origin recognition complex (ORC) is a 6-subunit complex required for the initiation of DNA replication in eukaryotic organisms. ORC is also involved in other cell functions. The smallest
Drosophila
ORC subunit, Orc6, is important for both DNA replication and cytokinesis. To study the role of Orc6 in vivo, the
orc6
gene was deleted by imprecise excision of
P
element. Lethal alleles of
orc6
are defective in DNA replication and also show abnormal chromosome condensation and segregation. The analysis of cells containing the
orc6
deletion revealed that they arrest in both the G
1
and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of mutant transgenes of Orc6 with deleted or mutated C-terminal domain results in a release of mutant cells from G
1
arrest and restoration of DNA replication, indicating that the DNA replication function of Orc6 is associated with its N-terminal domain. However, these mutant cells accumulate at mitosis, suggesting that the C-terminal domain of Orc6 is important for the passage through the M phase. In a cross-species complementation experiment, the expression of human Orc6 in
Drosophila
Orc6 mutant cells rescued DNA replication, suggesting that this function of the protein is conserved among metazoans.
Multiple pathways guide oxygen diffusion into flavoenzyme active sites
Authors: Baron Riccardo , Riley Conor , Chenprakhon Pirom , Thotsaporn Kittisak , Winter Remko T. , Alfieri Andrea , Forneris Federico , van Berkel Willem J. H. , Chaiyen Pimchai , Fraaije Marco W. , Mattevi Andrea , McCammon J. Andrew
Published: June 2009

Dioxygen (O
2
) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O
2
uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O
2
diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O
2
diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O
2
from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O
2
reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.
Sporulation in mycobacteria
Authors: Ghosh Jaydip , Larsson Pontus , Singh Bhupender , Pettersson B. M. Fredrik , Islam Nurul M. , Sarkar Sailendra Nath , Dasgupta Santanu , Kirsebom Leif A.
Published: June 2009

Mycobacteria owe their success as pathogens to their ability to persist for long periods within host cells in asymptomatic, latent forms before they opportunistically switch to the virulent state. The molecular mechanisms underlying the transition into dormancy and emergence from it are not clear. Here we show that old cultures of
Mycobacterium marinum
contained spores that, upon exposure to fresh medium, germinated into vegetative cells and reappeared again in stationary phase via endospore formation. They showed many of the usual characteristics of well-known endospores. Homologues of well-known sporulation genes of
Bacillus subtilis
and
Streptomyces coelicolor
were detected in mycobacteria genomes, some of which were verified to be transcribed during appropriate life-cycle stages. We also provide data indicating that it is likely that old
Mycobacterium bovis
bacillus Calmette–Guérin cultures form spores. Together, our data show sporulation as a lifestyle adapted by mycobacteria under stress and tempt us to suggest this as a possible mechanism for dormancy and/or persistent infection. If so, this might lead to new prophylactic strategies.
NOD2 contributes to cutaneous defense againstStaphylococcus aureusthrough α-toxin-dependent innate immune activation
Authors: Hruz Petr , Zinkernagel Annelies S. , Jenikova Gabriela , Botwin Gregory J. , Hugot Jean-Pierre , Karin Michael , Nizet Victor , Eckmann Lars
Published: June 2009

Staphylococcus aureusis a major cause of community-acquired and nosocomial infections including the life-threatening conditions endocarditis, necrotizing pneumonia, necrotizing fasciitis, and septicemia. Toll-like receptor (TLR)-2, a membrane-bound microbial sensor, detects staphylococcal components, but macrophages lacking TLR2 or both TLR2 and TLR4 remainS. aureusresponsive, suggesting that an alternative microbial recognition receptor might be involved. The cytoplasmic sensor nucleotide-binding oligomerization domain containing (NOD) 2/caspase recruitment domain (CARD) 15 detects muramyl dipeptide from bacterial peptidoglycans and mediates cytokine responses toS. aureusin vitro, but the physiological significance of these observations is not well defined. Here we show that NOD2-deficient mice exhibit a delayed but ultimately exacerbated ulcerative response and impaired bacterial clearance after s.c. infection withS. aureus. NOD2-dependent recognition ofS. aureusand muramyl dipeptide is facilitated by α-toxin (α-hemolysin), a pore-forming toxin and virulence factor of the pathogen. The action of NOD2 is dependent on IL-1β-amplified production of IL-6, which promotes rapid bacterial killing by neutrophils. These results significantly broaden the physiological importance of NOD2 in innate immunity from the recognition of bacteria that primarily enter the cytoplasm to the detection of bacteria that typically reside extracellularly and demonstrate that this microbial sensor contributes to the discrimination between commensal bacteria and bacterial pathogens that elaborate pore-forming toxins.
Low-κ′ dielectric properties of UV-treated bi-axially oriented polypropylene films
Authors: Dervos C T , Tarantili P A , Athanassopoulou M D
Published: June 2009

A 40 µm multilayer bi-axially oriented polypropylene (BOPP) film, was fabricated by the tenter process and its dielectric response was investigated after applying combined action of UV, humidity and heat. Dissipation factor (tan δ) and relative dielectric constant measurements were performed via the capacitance method for frequencies 20Hz–1 GHz. These results show that the relative dielectric constant (κ′) reduces towards ultra low values (1.8) with an increasing number of applied UV–condensation cycles without any subsequent increase in the dielectric loss. Having no added physical porosity and absence of fluorine atoms, the irradiated BOPP structures offer significant advantages over poly(tetrafluoroethylene) PTFE due to reduced polarization effects, lower dielectric constant values and chemical stability to the adjacent copper or aluminium conductors. Possible application fields are dry type high-voltage capacitors and insulation within electronic components.
Novel Roles of Hakai in Cell Proliferation and Oncogenesis
Authors: Figueroa Angélica , Kotani Hirokazu , Toda Yoshinobu , Mazan-Mamczarz Krystyna , Mueller Eva-Christina , Otto Albrecht , Disch Lena , Norman Mark , Ramdasi Rasika Mohan , Keshtgar Mohammed , Gorospe Myriam , Fujita Yasuyuki
Published: June 2009

During tumor development, cells acquire multiple phenotypic changes upon misregulation of oncoproteins and tumor suppressor proteins. Hakai was originally identified as an E3 ubiquitin-ligase for the E-cadherin complex that regulates cell–cell contacts. Here, we present evidence that Hakai plays a crucial role in various cellular processes and tumorigenesis. Overexpression of Hakai affects not only cell–cell contacts but also proliferation in both epithelial and fibroblast cells. Furthermore, the knockdown of Hakai significantly suppresses proliferation of transformed epithelial cells. Expression of Hakai is correlated to the proliferation rate in human tissues and is highly up-regulated in human colon and gastric adenocarcinomas. Moreover, we identify PTB-associated splicing factor (PSF), an RNA-binding protein, as a novel Hakai-interacting protein. By using cDNA arrays, we have determined various specific PSF-associated mRNAs encoding proteins that are involved in several cancer-related processes. Hakai affects the ability of PSF to bind these mRNAs, and expression of PSF short hairpin RNA or a dominant-negative PSF mutant significantly suppresses proliferation of Hakai-overexpressing cells. Collectively, these results suggest that Hakai is an important regulator of cell proliferation and that Hakai may be an oncoprotein and a potential molecular target for cancer treatment.
Evaluation of Influence of Acupuncture and Electro‐Acupuncture for Blood Perfusion of Stomach by Laser Doppler Blood Perfusion Imaging
Authors: Dong Zhang , Shun-Yue Li , Shu-You Wang , Hui-Min Ma
Published: June 2009

The objective of this study is to observe effects of acupuncture and electro‐acupuncture (EA) on blood perfusion in the stomach, and probe into the application of laser Doppler blood perfusion imaging technique in the study of the effect of acupuncture and moxibustion on the entrails. In the acupuncture group of 20 rats, acupuncture was given at “Zusanli” (ST 36) and in EA group of 18 rats, EA was applied at “Zusanli” (ST 36), with 18 rats without acupuncture used as control group. Changes of blood perfusion and microcirculation distribution in the stomach were investigated with laser Doppler blood perfusion imager (LDPI). The laser Doppler blood perfusion image could clearly display changes of blood flow distribution in the stomach before and after acupuncture. After acupuncture or EA was given at “Zusanli” (ST 36), the blood perfusion in the stomach increased significantly, the blood perfusion in the blood vessels and microcirculation of other parts significantly increased, and the maximum increase of the blood perfusion was found at 10 min after acupuncture or EA, with increases of 0.50 _ 0.11 (PU) and 0.66 _ 0.16 (PU), respectively, and the blood perfusion still kept at a higher degree within 10 min after ceasing of the acupuncture or EA. While the blood perfusion in the stomach in the rat of the control group tended to gradual decrease. It has been concluded that both acupuncture and EA can increase blood perfusion in the stomach, the EA having stronger action, and LDPI can display the regulative action of acupuncture on the blood vessel of the stomach by using an image.
Adenovirus serotype 5 infects human dendritic cells via a coxsackievirus–adenovirus receptor-independent receptor pathway mediated by lactoferrin and DC-SIGN
Authors: Adams William C. , Bond Emily , Havenga Menzo J. E. , Holterman Lennart , Goudsmit Jaap , Karlsson Hedestam Gunilla B. , Koup Richard A. , Loré Karin
Published: June 2009

The coxsackievirus–adenovirus receptor (CAR) is the described primary receptor for adenovirus serotype 5 (Ad5), a common human pathogen that has been exploited as a viral vector for gene therapy and vaccination. This study showed that monocytes and dendritic cells (DCs), such as freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, are susceptible to recombinant Ad5 (rAd5) infection despite their lack of CAR expression. Langerhans cells and dermal DCs from skin expressed CAR, but blocking CAR only partly decreased rAd5 infection, together suggesting that other receptor pathways mediate viral entry of these cells. Lactoferrin (Lf), an abundant protein in many bodily fluids known for its antiviral and antibacterial properties, promoted rAd5 infection in all cell populations except plasmacytoid DCs using a CAR-independent process. Lf caused phenotypic differentiation of the DCs, but cell activation played only a minor role in the increase in infection frequencies. The C-type lectin receptor DC-SIGN facilitated viral entry of rAd5–Lf complexes and this was dependent on high-mannose-typeN-linked glycans on Lf. These results suggest that Lf present at high levels at mucosal sites can facilitate rAd5 attachment and enhance infection of DCs. A better understanding of the tropism and receptor mechanisms of Ad5 may help explain Ad5 pathogenesis and guide the engineering of improved rAd vectors.
Inhibition of hepatitis C virus replication and internal ribosome entry site-dependent translation by an RNA molecule
Authors: Romero-López Cristina , Díaz-González Raquel , Barroso-delJesus Alicia , Berzal-Herranz Alfredo
Published: June 2009

Hepatitis C virus (HCV) protein synthesis is mediated by a highly conserved internal ribosome entry site (IRES), mostly located at the 5′ untranslatable region (UTR) of the viral genome. The translation mechanism is different from that used by cellular cap-mRNAs, making IRESs an attractive target site for new antiviral drugs. The present work characterizes a chimeric RNA molecule (HH363-50) composed of two inhibitors: a hammerhead ribozyme targeting position 363 of the HCV genome and an aptamer directed towards the essential stem–loop structure in domain IV of the IRES region (which contains the translation start codon). The inhibitor RNA interferes with the formation of a translationally active complex, stalling its progression at the level of 80S particle formation. This action is likely related to the effective and specific blocking of HCV IRES-dependent translation achieved in Huh-7 cells. The inhibitor HH363-50 also reduces HCV RNA levels in a subgenomic replicon system. The present findings suggest that HH363-50 could be an effective anti-HCV compound and highlight the possibilities of antiviral agents based on RNA molecules.
Genetic and temporal dynamics of human immunodeficiency virus type 1 CRF07_BC in Xinjiang, China
Authors: Xin Ruolei , He Xiang , Xing Hui , Sun Feng , Ni Mingjian , Zhang Yuanzhi , Meng Zhefeng , Feng Yi , Liu Shengya , Wei Jing , Shao Yiming
Published: June 2009

To explore the temporal genetic variation of human immunodeficiency virus type 1 CRF07_BC and reconstruct its epidemic in Xinjiang, China, we studied 216 C2–V4 fragments of env genes sampled from 1996 to 2008. Phylogenetic analysis indicates that the viruses prevailing in Xinjiang form a large monophyletic cluster and may have originated from a common ancestor. The epidemic in Xinjiang was probably established around 1995 (95 % confidence interval, 1994–1996). We noted an increased diversity of CRF07_BC over time, with a rapid evolutionary rate we estimated to be 8.3×10−3 substitutions per site per year in the env gene. After 5–6 years of the epidemic (1997–2002), the transmission rate of CRF07_BC in Xinjiang slowed down, although CRF07_BC infection remained at a high prevalence.
Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation
Authors: Malik Tahir H. , Wolbert Candie , Nerret Laura , Sauder Christian , Rubin Steven
Published: June 2009

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.
Vertical transmission of Prunus necrotic ringspot virus: hitch-hiking from gametes to seedling
Authors: Amari Khalid , Burgos Lorenzo , Pallás Vicente , Sánchez-Pina Maria Amelia
Published: June 2009

The aim of this work was to follow Prunus necrotic ringspot virus (PNRSV) infection in apricot reproductive tissues and transmission of the virus to the next generation. For this, an analysis of viral distribution in apricot reproductive organs was carried out at different developmental stages. PNRSV was detected in reproductive tissues during gametogenesis. The virus was always present in the nucellus and, in some cases, in the embryo sac. Studies within infected seeds at the embryo globular stage revealed that PNRSV infects all parts of the seed, including embryo, endosperm and testa. In the torpedo and bent cotyledon developmental stages, high concentrations of the virus were detected in the testa and endosperm. At seed maturity, PNRSV accumulated slightly more in the embryo than in the cotyledons. In situ hybridization showed the presence of PNRSV RNA in embryos obtained following hand-pollination of virus-free pistils with infected pollen. Interestingly, tissue-printing from fruits obtained from these pistils showed viral RNA in the periphery of the fruits, whereas crosses between infected pistils and infected pollen resulted in a total invasion of the fruits. Taken together, these results shed light on the vertical transmission of PNRSV from gametes to seedlings.
Comparison of vesicular stomatitis virus pseudotyped with the S proteins from a porcine and a human coronavirus
Authors: Schwegmann-Weßels Christel , Glende Jörg , Ren Xiaofeng , Qu Xiuxia , Deng Hongkui , Enjuanes Luis , Herrler Georg
Published: June 2009

The surface proteins S of severe acute respiratory syndrome coronavirus (SARS-CoV) and transmissible gastroenteritis virus (TGEV) were compared for their ability to mediate infection of viral pseudotypes based on vesicular stomatitis virus (VSV). The cell tropism of the respective pseudotypes corresponded to the tropism of the viruses from which the S protein was derived. Higher infectivity values were obtained with the SARS-CoV S protein than with the TGEV S protein. Differences were observed with respect to the importance of the cytoplasmic tail and the membrane anchor of the S proteins. In the case of the SARS-CoV S protein, truncation of the cytoplasmic tail resulted in increased infectivity. For the TGEV S protein, the inactivation of an intracellular retention signal in the cytoplasmic tail was required. Exchange of the membrane anchor of the S proteins led to a low infection efficiency. Our results indicate that related glycoproteins may show substantial differences in their ability to mediate pseudotype infection.
Determinants of in vitro expansion of different human virus-specific FoxP3+ regulatory CD8+ T cells in chronic hepatitis C virus infection
Authors: Billerbeck Eva , Nakamoto Nobuhiro , Seigel Bianca , Blum Hubert E. , Chang Kyong-Mi , Thimme Robert
Published: June 2009

It has been shown previously that suppressive virus-specific FoxP3+ regulatory CD8+ T cells can be expanded from human peripheral blood mononuclear cells after in vitro antigen-specific stimulation. This study extended this finding by analysing the mechanisms of virus-specific FoxP3+ regulatory CD8+ T-cell generation during peptide-specific expansion in vitro. It was shown that hepatitis C virus (HCV)-, influenza virus (FLU)-, Epstein–Barr virus (EBV)- and cytomegalovirus (HCMV)-specific FoxP3+ regulatory CD8+ T cells could be expanded differentially from the blood of chronically HCV-infected patients following in vitro peptide-specific stimulation. The different ability of virus-specific CD8+ T-cell populations to express FoxP3 after continuous antigen stimulation in vitro correlated significantly with the ex vivo differentiation status. Indeed, CD27+ CD28+ CD57− HCV-, FLU- and EBV-specific CD8+ T cells displayed a significantly higher ability to give rise to FoxP3+ regulatory CD8+ T cells compared with CD27− CD28− CD57+ HCMV-specific CD8+ T cells. Similar T-cell receptor expression patterns of FoxP3+ versus FoxP3− CD8+ T cells of the same antigen specificity indicated that both cell populations were probably expanded from the same virus-specific CD8+ T-cell precursor. In addition, no specific antigen-presenting cell populations were required for the generation of FoxP3+ CD8+ T cells, as CD8+-selected virus-specific FoxP3+ CD8+ T cells could be expanded by peptide presentation in the absence of antigen-presenting cells. Taken together, these results suggest that the ability to expand FoxP3+ regulatory CD8+ T cells from virus-specific CD8+ T cells differs among distinct virus-specific CD8+ T-cell populations depending on the differentiation status.
Oral pravastatin prolongs survival time of scrapie-infected mice
Authors: Vetrugno Vito , Di Bari Michele Angelo , Nonno Romolo , Puopolo Maria , D'Agostino Claudia , Pirisinu Laura , Pocchiari Maurizio , Agrimi Umberto
Published: June 2009

Statins are potent inhibitors of HMG–CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase in the cholesterol-biosynthesis pathway. They are either lipophilic (e.g. simvastatin) or hydrophilic [e.g. pravastatin (PRV)] compounds, considered mainly for long-term treatment of hypercholesterolaemic individuals. Beneficial effects of statins are not related exclusively to their lipid-lowering action; they also possess cholesterol-independent, pleiotropic effects (e.g. anti-inflammatory and antioxidant). Recent studies revealed that simvastatin treatment increased survival significantly in scrapie-infected mice. Although PRV treatment results in measurable drug levels in the mouse brain, the anti-prion effect of this compound has not been investigated. Therefore, we aimed to test the potential therapeutic action of PRV in a murine scrapie model. Our study showed that high-dose and long-term oral PRV treatment prolonged survival times of strain 139A scrapie-infected mice significantly (194 versus 177 days) in the absence of any obvious toxicity, suggesting that protective effects of statins may be independent of absolute solvent or water solubility of the drug.
Phenotypic similarities and differences between UL37-deleted pseudorabies virus and herpes simplex virus type 1
Authors: Leege Tobias , Granzow Harald , Fuchs Walter , Klupp Barbara G. , Mettenleiter Thomas C.
Published: June 2009

In the absence of the tegument protein pUL37, virion formation of pseudorabies virus (PrV) and herpes simplex virus type 1 (HSV-1) is severely impaired. Non-enveloped nucleocapsids accumulate in clusters in the cytoplasm, whereas only a few enveloped particles can be detected. Although a contribution of pUL37 to nuclear egress of HSV-1 has been suggested, the nuclear stages of morphogenesis are not impaired in PrV-ΔUL37-infected cells. Moreover, HSV-1 pUL37 has been described as essential for replication, whereas PrV is able to replicate productively without pUL37, although to lower titres than wild-type virus. Thus, there may be functional differences between the respective pUL37 proteins. This study compared the phenotypes of UL37-deleted PrV and HSV-1 in parallel assays, using a novel pUL37 deletion mutant of HSV-1 strain KOS, HSV-1ΔUL37[86–1035]. Aggregates of seemingly ‘naked’ nucleocapsids were present in the cytoplasm of African green monkey (Vero) or rabbit kidney (RK13) cells infected with HSV-1ΔUL37[86–1035] or PrV-ΔUL37. Nuclear retention of nucleocapsids was not observed in either virus. However, in contrast to PrV-ΔUL37, HSV-1ΔUL37[86–1035] was unable to replicate productively in, and to form plaques on, either Vero or RK13 cells. Trans-complementation of respective deletion mutants with the heterologous pUL37 did not ensue. These data demonstrate that the conserved pUL37 in HSV-1 and PrV have similar but distinct functions.
Epstein–Barr virus protein kinase BGLF4 interacts with viral transactivator BZLF1 and regulates its transactivation activity
Authors: Asai Risa , Kato Ai , Kawaguchi Yasushi
Published: June 2009

BGLF4 is a serine/threonine protein kinase encoded by Epstein–Barr virus. One of the physiological substrates of BGLF4 is viral transactivator BZLF1. In the present study, it was demonstrated that alanine substitution of the serine residue at position 209 (S209A) in BZLF1 eliminated phosphorylation of the protein by BGLF4 in vitro. The S209A mutation in BZLF1, as well as a K102I mutation in BGLF4, which inactivated catalytic activity of the viral kinase, also inhibited formation of a stable BGLF4–BZLF1 complex and downregulation of BZLF1 autotransactivation activity mediated by BGLF4. These results indicate that formation of a stable complex of BGLF4–BZLF1 enables downregulation of BZLF1 autoregulation activity and it appears that BGLF4 phosphorylation of BZLF1 may be involved in these processes.
Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants
Authors: Hemmes Hans , Kaaij Lucas , Lohuis Dick , Prins Marcel , Goldbach Rob , Schnettler Esther
Published: June 2009

The NS3 protein of rice hoja blanca virus represents a viral suppressor of RNA interference (RNAi) that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, NS3 was altered by alanine point mutations and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at positions 173–175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta. This indicates that siRNA binding of NS3 is indeed essential for the suppressor function of NS3 and that residues at positions 173–175 are involved in the siRNA binding and suppressor activities.
Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells
Authors: Bungyoku Yasuaki , Shoji Ikuo , Makine Tatsuhiko , Adachi Tetsuya , Hayashida Kazumi , Nagano-Fujii Motoko , Ide Yoshi-Hiro , Deng Lin , Hotta Hak
Published: June 2009

Robust production of infectious hepatitis C virus (HCV) in cell culture was realized by using the JFH1 strain and the homologous chimeric J6/JFH1 strain in Huh-7.5 cells, a highly HCV-permissive subclone of Huh-7 cells. In this study, we aimed to establish a more efficient HCV-production system and to gain some insight into the adaptation mechanisms of efficient HCV production. By serial passaging of J6/JFH1-infected Huh-7.5 cells, we obtained culture-adapted J6/JFH1 variants, designated P-27, P-38 and P-47. Sequence analyses revealed that the adaptive mutant viruses P-27, P-38 and P-47 possessed eight mutations [four in E2, two in NS2, one in NS5A and one in NS5B), 10 mutations [two additional mutations in the 5′-untranslated region (5′-UTR) and core] and 11 mutations (three additional mutations in 5′-UTR, core and NS5B), respectively. We introduced amino acid substitutions into the wild-type J6/JFH1 clone, generated recombinant viruses with adaptive mutations and analysed their infectivity and ability to produce infectious viruses. The viruses with the adaptive mutations exhibited higher expression of HCV proteins than did the wild type in Huh-7.5 cells. Moreover, we provide evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level. We thus demonstrate that an efficient HCV-production system could be obtained by introducing adaptive mutations into the J6/JFH1 genome. The J6/JFH1-derived mutant viruses presented here would be a good tool for producing HCV particles with enhanced infectivity and for studying the molecular mechanism of HCV entry.
A Pivotal Role of the Basic Leucine Zipper Transcription Factor bZIP53 in the Regulation ofArabidopsisSeed Maturation Gene Expression Based on Heterodimerization and Protein Complex Formation
Authors: Alonso Rosario , Oñate-Sánchez Luis , Weltmeier Fridtjof , Ehlert Andrea , Diaz Isabel , Dietrich Katrin , Vicente-Carbajosa Jesús , Dröge-Laser Wolfgang
Published: June 2009

Transcription of Arabidopsis thaliana seed maturation (MAT) genes is controlled by members of several transcription factor families, such as basic leucine zippers (bZIPs), B3s, MYBs, and DOFs. In this work, we identify Arabidopsis bZIP53 as a novel transcriptional regulator of MAT genes. bZIP53 expression in developing seeds precedes and overlaps that of its target genes. Gain- and loss-of-function approaches indicate a correlation between the amount of bZIP53 protein and MAT gene expression. Specific in vivo and in vitro binding of bZIP53 protein to a G-box element in the albumin 2S2 promoter is demonstrated. Importantly, heterodimerization with bZIP10 or bZIP25, previously described bZIP regulators of MAT gene expression, significantly enhances DNA binding activity and produces a synergistic increase in target gene activation. Full-level target gene activation is strongly correlated with the ratio of the correspondent bZIP heterodimerization partners. Whereas bZIP53 does not interact with ABI3, a crucial transcriptional regulator in Arabidopsis seeds, ternary complex formation between the bZIP heterodimers and ABI3 increases the expression of MAT genes in planta. We therefore propose that heterodimers containing bZIP53 participate in enhanceosome formation to produce a dramatic increase in MAT gene transcription.
Biochemical Evidence for Translational Repression by Arabidopsis MicroRNAs
Authors: Lanet Elodie , Delannoy Etienne , Sormani Rodnay , Floris Maïna , Brodersen Peter , Crété Patrice , Voinnet Olivier , Robaglia Christophe
Published: June 2009

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally through RNA silencing, a mechanism conserved in eukaryotes. Prevailing models entail most animal miRNAs affecting gene expression by blocking mRNA translation and most plant miRNAs, triggering mRNA cleavage. Here, using polysome fractionation in Arabidopsis thaliana, we found that a portion of mature miRNAs and ARGONAUTE1 (AGO1) is associated with polysomes, likely through their mRNA target. We observed enhanced accumulation of several distinct miRNA targets at both the mRNA and protein levels in an ago1 hypomorphic mutant. By contrast, translational repression, but not cleavage, persisted in transgenic plants expressing the slicing-inhibitor 2b protein from Cucumber mosaic virus. In agreement, we found that the polysome association of miR168 was lost in ago1 but maintained in 2b plants, indicating that translational repression is correlated with the presence of miRNAs and AGO1 in polysomes. This work provides direct biochemical evidence for a translational component in the plant miRNA pathway.
13C and 1H NMR Study of Lysophosphatidylcholine (LPC) Isolated from the Surface of Wheat Starch Granules
Authors: Jimeno M.L. , Valverde S. , Błaszczak W. , Fornal J. , Amarowicz R.
Published: June 2009

Lysophosphatidylcholine (LPC) has been isolated from the surface of starch granules by extraction and subsequent chromatographic purification. The purified extract was examined by mono and bidimensional 1H and 13C NMR and identified as LPC. The NMR information also allowed the assignment of the length and nature of the acyl chains (18—20 carbon atoms, saturated).