bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the UL25 Protein in Herpes Simplex Virus DNA Encapsidation
Authors: Cockrell Shelley K. , Sanchez Minerva E. , Erazo Angela , Homa Fred L.
Published: October 2008

The herpes simplex virus protein UL25 attaches to the external vertices of herpes simplex virus type 1 capsids and is required for the stable packaging of viral DNA. To define regions of the protein important for viral replication and capsid attachment, the 580-amino-acid UL25 open reading frame was disrupted by transposon mutagenesis. The UL25 mutants were assayed for complementation of a UL25 deletion virus, and in vitro-synthesized protein was tested for binding to UL25-deficient capsids. Of the 11 mutants analyzed, 4 did not complement growth of the UL25 deletion mutant, and analysis of these and additional mutants in the capsid-binding assay demonstrated that UL25 amino acids 1 to 50 were sufficient for capsid binding. Several UL25 mutations were transferred into recombinant viruses to analyze the effect of the mutations on UL25 capsid binding and on DNA cleavage and packaging. Studies of these mutants demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids and that the C terminus is essential for DNA packaging and the production of infectious virus through its interactions with other viral packaging or tegument proteins. Analysis of viral DNA cleavage demonstrated that in the absence of a functional UL25 protein, aberrant cleavage takes place at the unique short end of the viral genome, resulting in truncated viral genomes that are not retained in capsids. Based on these observations, we propose a model where UL25 is required for the formation of DNA-containing capsids by acting to stabilize capsids that contain full-length viral genomes.
The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem
Authors: Gu Haidong , Roizman Bernard
Published: October 2008

ICP0, an α (immediate-early) protein of herpes simplex virus 1, performs at least two key functions. It blocks inhibition of viral-gene expression by interferon, a function dependent on the degradation of the ND10 components PML and SP100 by the ubiquitin ligase expressed by the RING finger (RF), and it blocks silencing of viral DNA mediated by the HDAC1/2-CoREST-REST complex. In the latter case, a mutant CoREST lacking the HDAC1 binding site compensates totally or in part for the absence of ICP0 in a cell-type-dependent manner. Here, we compare the phenotypes of an ICP0 mutant containing disabling amino acid substitutions in the RF with those of a mutant with substitutions in the CoREST binding site (R8507). We report the following: (i) the onset of replication of both mutants was delayed, but the RF mutant yields did not reach wild-type virus levels even as late as 48 h after infection, and (ii) in infected cells, PML is rapidly degraded by wild-type virus, with some delay by the R8507 mutant, and is spared by the RF mutant. The translocation of ICP0 to the cytoplasm is impaired in cells infected with the RF mutant or delayed in cells infected with the R8507 mutant. Finally, in contrast to wild-type viruses, both mutants are inhibited by alpha or gamma interferon. The results indicate that both sets of events, the degradation of PML and the blocking of silencing, are interdependent and in large measure dependent on events in the ND10 nuclear bodies.
Evidence of CD8
+
T-Cell-Mediated Selective Pressure on Human Immunodeficiency Virus Type 1
nef
in HLA-B*57
+
Elite Suppressors
Authors: Bailey Justin R. , Brennan Timothy P. , O'Connell Karen A. , Siliciano Robert F. , Blankson Joel N.
Published: October 2008

Elite suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain viral loads of <50 copies/ml without treatment. The observation that the HLA-B*57 allele is overrepresented in these patients implies that HIV-1-specific CD8
+
T cells play a key role in suppressing viral replication. We have previously shown that while CD8
+
T-cell escape mutations are rarely seen in proviral Gag sequences in resting CD4
+
T cells from peripheral blood, they are present in every clone amplified from the low levels of free virus in the plasma of HLA-B*57
+
ES. In this study, we compared the pattern of mutations in Nef sequences amplified from peripheral blood CD4
+
T cells and from plasma virus. We show that Nef mutations are present in plasma virus but are rare in the cellular sequences and provide evidence that these plasma Nef variants represent novel escape mutants. The results provide further evidence of CD8
+
T-cell-mediated selective pressure on plasma virus in ES and suggest that there must be ongoing HIV-1 replication in spite of the very low viral loads seen for these patients.
Interaction of the Influenza A Virus Nucleocapsid Protein with the Viral RNA Polymerase Potentiates Unprimed Viral RNA Replication
Authors: Newcomb Laura L. , Kuo Rei-Lin , Ye Qiaozhen , Jiang Yunyun , Tao Yizhi Jane , Krug Robert M.
Published: October 2008

The influenza A virus polymerase transcribes and replicates the eight virion RNA (vRNA) segments. Transcription is initiated with capped RNA primers excised from cellular pre-mRNAs by the intrinsic endonuclease of the viral polymerase. Viral RNA replication occurs in two steps: first a full-length copy of vRNA is made, termed cRNA, and then this cRNA is copied to produce vRNA. The synthesis of cRNAs and vRNAs is initiated without a primer, in contrast to the initiation of viral mRNA synthesis, and requires the viral nucleocapsid protein (NP). The mechanism of unprimed viral RNA replication is poorly understood. To elucidate this mechanism, we used purified recombinant influenza virus polymerase complexes and NP to establish an in vitro system that catalyzes the unprimed synthesis of cRNA and vRNA using 50-nucleotide-long RNA templates. The purified viral polymerase and NP are sufficient for catalyzing this RNA synthesis without a primer, suggesting that host cell factors are not required. We used this purified in vitro replication system to demonstrate that the RNA-binding activity of NP is not required for the unprimed synthesis of cRNA and vRNA. This result rules out two models that postulate that the RNA-binding activity of NP mediates the switch from capped RNA-primed transcription to unprimed viral RNA replication. Because we showed that NP lacking RNA-binding activity binds directly to the viral polymerase, it is likely that a direct interaction between NP and the viral polymerase results in a modification of the polymerase in favor of unprimed initiation.
Pollen morphology of North American species of Antennaria (Asteraceae: Inuleae)
Authors: Chmielewski J. G. , Chinnappa C. C. , Warner B. G.
Published: October 2008

The pollen of 90 individuals representing 28 species and subspecies in the genus Antennaria is described through the use of light and scanning electron microscopy. Two morphological groupings separated on the basis of exine morphology are evident from cluster analysis. One group consists of those species and subspecies in which the exine is perforate and the other group consists of those individuals in which the exine is not perforate (perforate-reticulate or striate-perforate-reticulate). These two pollen morphological groups do not correspond with previously defined groupings based on morphology or cytogenetics. Both large (> 19 μm in diameter) and small pollen grains occur in each of the cluster groups. Similarly, both long- (> 2 μm) and short-spined individuals occur in each of the cluster groups. Used in combination, these characters do however exhibit some sectional affinities. Section Alpinae is characterized by pollen grains with long spines. Section Dioicae is characterized by small pollen grains. Section Plantaginifoliae is characterized by large pollen grains with long spines. Species included in sections other than these are not consistent in their pollen morphology. The pollen groups recognized in this preliminary study form a basis for future studies at the geographic or population level.
Antifungal activity in culture filtrates of the ectomycorrhizal fungus Pisolithus tinctorius
Authors: Kope H. H. , Fortin J. A.
Published: October 2008

The ectomycorrhizal fungus Pisolithus tinctorius secreted a metabolite that lysed hypha and conidia and inhibited the germination of conidia in a range of phytopathogenic fungi. The optimum incubation period for the production of the metabolite by P. tinctorius in liquid culture was 42–56 days. Dilutions of the culture filtrate with fresh medium caused a gradient of inhibitory effects. For hyphal lysis the minimum ratio of culture filtrate to medium for complete growth inhibition was 5:1 for Rhizoctonia praticola and Truncatella hartigii and 3:1 for Sphaerosporella brunnea. At higher dilutions, the hyphae that formed were short celled and highly branched; many hyphae lysed. Conidial germination was completely inhibited at a ratio of 5:1 for both Fusarium solani and T. hartigii, 3:1 for a North American isolate of Brunchorstia pinea (NA), 1:1 for a European isolate of B. pinea (EU), and 1:3 for two strains of Cochliobolus sativus (0910, 0912). Conidial lysis was seen for T. hartigii at a ratio of 5:1 and at a ratio of 1:1 for B. pinea (EU) and C. sativus (0910). Characterization of the cell-free culture filtrate through the separate additions of D-glucose and an adsorbent, activated charcoal, showed that hyphal lysis and conidia germination inhibition did not result from a depletion of carbohydrates from the growth medium but from the presence of some substance adsorbed by charcoal. Key words: Pisolithus tinctorius, allelopathy, antifungal metabolite, hyphal lysis, mycorrhizosphere.
Variability in Fusarium oxysporum f.sp. cubense
Authors: Ploetz R. C.
Published: October 2008

A worldwide collection of 96 isolates of Fusarium oxysporum f.sp. cubense (incitant of fusarial wilt of banana or Panama disease) from 12 countries was used to assess population structure in the pathogen; isolates were diverse for vegetative compatibility (11 vegetative compatibility groups) and race-specific virulence (races 1, 2, and 4). Rates of radial growth on potato dextrose agar differed at temperatures ranging from 8–36 °C for isolates in different VCGs and races (P < 0.05). On a KClO3-amended medium used to generate nitrate-nonutilizing (nit) mutants, variability in chlorate (a toxic analog of nitrate) sensitivity and the time required before nit mutants arose on the medium (mutability) was related primarily to vegetative compatibility group. In addition, cultural morphology on modified Komada's medium and potato dextrose agar was related primarily to vegetative compatibility group, whereas race was not as consistently related to these traits. In studies on the population biology and diversity in F. oxysporum f.sp. cubense, vegetative compatibility was a more useful character than race. On the basis of these results, it is suggested that F. oxysporum f.sp. cubense has had diverse origins.
Active layer slope movement in a continuous permafrost environment, Garry Island, Northwest Territories, Canada
Authors: Mackay J. Ross
Published: October 2008

Field investigations have been carried out at Garry Island, N.W.T. for the 1964–1980 period in order to study downslope active layer movement at sites with two-sided (downward and upward) freezing and active ice-wedge growth. Movements have been determined with reference to semi-flexible plastic tubes inserted vertically into the ground and by deformation of lines of stakes. The results show that the vertical velocity profile on the hillslopes with clayey hummocks is convex downslope; the movement is plug-like and occurs in late summer; the plug-like movement progressively buries the interhummock peat to form a buried organic layer; and most of the plug-like movement can be attributed to frost creep by thaw of an ice-rich layer at the bottom of the active layer. The ice-rich layer forms by upfreezing in winter and the ice content may be augmented by ice lensing in the summer thaw period. In a sedgy drainage swale, the vertical velocity profile is concave downslope. The active layer of ice-wedge polygons shows a net movement outwards from the centres to the troughs. These studies show that active layer movement at sites with two-sided freezing and active ice-wedge polygons may differ substantially from sites with only one-sided freezing and without active ice-wedge polygons.
Fault block rotations in the Southern Province as defined by paleomagnetism of the Nipissing diabase
Authors: Morris W. A.
Published: October 2008

The more southerly N1 Nipissing diabase poles show conclusive evidence of fault block rotation of individual segments of the Southern Province subsequent to this intrusive event. The more northerly N2 Nipissing diabase poles do not provide such conclusive evidence. It is presently not known whether this uncertainty in the N2 is because the N2 intrusive event postdates the post N1 faulting event, or whether it is a result of inaccuracies in the paleomagnetic data.
Post-tectonic igneous rocks: north-central Labrador geosyncline
Authors: Dressler B.
Published: October 2008

Post-tectonic albite syenodiorite and cancrinite–arfvedsonite pegmatite are associated with a major fault in the Nachicapau and Marcel Lakes area within the eugeosyncline of the Labrador Trough. These rocks are not considered to be leucocratic associates of country tholeiites but, instead, are tentatively related to an older carbonatite lamprophyre suite of rocks found in the miogeosyncline.
Responses of Benthic Macroinvertebrate Species to Manipulation of Interstitial Detritus in Carnation Creek, British Columbia
Authors: Culp Joseph M. , Davies Ronald W.
Published: October 2008

Experimental manipulations were conducted in Carnation Creek, British Columbia, to determine the response of macroinvertebrate distribution and abundance to differences in detritus source and quantity. Four treatments (no detritus, low hemlock (Tsuga heterophylla), low alder (Alnus rubra), high alder) with a standardized substrate were established in a riffle and left for 28 d. Densities and/or biomasses of 12 of the 20 colonizing macroinvertebrate taxa were significantly different among the detritus treatments. Microbial activity, detritus processing, and macroinvertebrate abundances were highest in substrate patches with alder detritus. Compared with the no detritus and low hemlock treatments, the low alder treatment increased the abundances of nine taxa and decreased three taxa, while the high alder treatment increased the abundances of six taxa and decreased six taxa. Thus, detritus source and quantity strongly influenced macroinvertebrate community composition within the streambed. Although most of the macroinvertebrates in all treatments were either collector–gatherer or shredder detritivores, these macroinvertebrates responded to changes in detritus in a species-specific manner. Therefore, the Carnation Creek experiments emphasize the importance of interstitial detritus in the substrate as a factor that influences the microdistribution of the benthos at the species, rather than trophic feeding, assemblage level.
A REVIEW OF PURIFICATION TECHNIQUES FOR CARBON NANOTUBES
Authors: ISMAIL A. F. , GOH PEI SEAN , TEE JIA CHEE , SANIP SUHAILA M. , AZIZ MADZLAN
Published: October 2008

Purification of carbon nanotubes (CNTs) is a very actively discussed topic in contemporary CNT literature. To a large extent, impurities embedded in CNTs influence the physical and chemical characteristics of the CNTs. Different purification methods yield different CNT characteristics and may be suitable for the production of different types of CNTs. Developments in the purification methods of CNTs are reviewed, and the production methods are briefly discussed and summarized. This is followed by a detailed description of the three major purification methods, viz. chemical, physical, and multi-step purification.
ELECTRON SPIN RESONANCE STUDY OF POLY (ORTHO-ANISIDINE)/SINGLE-WALLED CARBON NANOTUBE COMPOSITE FILMS: SPIN DYNAMICS AND EFFECTS OF PHYSISORPTION PROCESSES
Authors: KONCHITS A. A. , MOTSNYI F. V. , KOLESNIK S. P. , YEFANOV V. S. , TAMBURRI E. , ORLANDUCCI S. , TERRANOVA M. L. , PASSERI D. , ROSSI M.
Published: October 2008

Poly(ortho-anisidine) (POA)/single-walled carbon nanotubes (SWCNTs) composite films have been prepared by means of oxidative potentiostatic electropolymerization of the monomer (ortho-anisidine) in the presence of purified SWCNTs. The magnetic properties of the obtained films have been investigated by electron spin resonance (ESR) (ν ≅ 9.4 GHz ; temperature range 10–300 K) and compared with those of pure POA films in the same oxidation state. The similarity of the ESR signals and the dependence on temperature of the ESR line features suggest a polaron origin of the paramagnetic states. A strong increase of the ESR integral intensity has been detected for composite samples kept for 72 h in hydrogen atmosphere at room temperature. Under the same conditions, a less pronounced increase in the intensity has been detected in the case of pure polymer films. The kinetics of the hydrogen adsorption–desorption processes in the nanocomposite samples has been investigated and interpreted in the frame of an H-physisorption mechanism. The comparison between the H-induced effects in POA and nanocomposite POA/SWCNTs films has been carried out taking into account the structure of the samples and the presence of defects.
THRESHOLD AND SATURATION VOLTAGES MODELING OF CARBON NANOTUBE FIELD EFFECT TRANSISTORS (CNT-FETs)
Authors: MARULANDA JOSE M. , SRIVASTAVA ASHOK , SHARMA ASHWANI K.
Published: October 2008

We present analytical model equations for threshold voltage (Vth) and saturation voltage (Vds,sat) characterizing CNT-FETs. These model equations have been obtained from the charge and potential distributions between the gate and substrate in a CNT-FET. It is shown that both Vth and Vds,sat are strongly dependent on chiral vectors of CNTs. The results show close agreement between theoretical and graphical modeling techniques. It is also shown that the calculated Vth of a CNT-FET with chiral vector (3, 1) is in close agreement with the corresponding published work.
FACTORS CONTROLLING THE STRONGEST SIZES IN THE INVERSE HALL–PETCH RELATIONSHIP
Authors: PAN YONG , ZHOU ZHAO FENG , FU SHAO-YUN , NIE YANGUANG , SUN CHANG Q.
Published: October 2008

Incorporating the bond–order–length–strength correlation mechanism [C. Q. Sun, Prog. Solid State Chem.35, 1 (2007)] and Born's criterion for melting [J. Chem. Phys.7, 591 (1939)] into the conventional Hall–Petch relationship has turned out an analytical expression for the size and temperature dependence of the mechanical strength of nanograins, known as the inverse Hall–Petch relationship (IHPR). Reproduction of the measured IHPR of Ni , NiP , and TiO 2 nanocrystals revealed that: (i) the competition between the size-induced energy–density gain and atomic cohesive energy loss in the surface skins of nanograins originate from the IHPR; (ii) the competition between the activation and inhibition of atomic dislocations motion activate the entire IHPR behavior; (iii) the bond nature involved and the T/Tm ratio between the temperature of operating and the temperature of melting dictate the measured strongest sizes of a given specimen; (iv) a quasimolten phase present before melting determines the size-induced softening and the superplasticity of nanostructures.
SELF-ASSEMBLY OF Si NANOSTRUCTURES IN SF6/O2 PLASMA
Authors: YANG CHENG , RYU SEUNG-HEON , LIM YEONG-DAE , YOO WON JONG
Published: October 2008

Self-assembled Si nanostructure arrays were formed in an inductively coupled plasma (ICP) reactor using SF 6/ O 2 plasma at noncryogenic temperature. It was possible to form nanopillar arrays of a mean diameter of ~100 nm and a mean height up to 4.77 μm over areas >100 cm2. The self-assembly of the nanostructures was studied as a function of time, bias RF-power, and O 2/ SF 6 ratio. It was found that the nanostructure arrays could be formed only when O 2/ SF 6 was in the range of 0.8 to 2.5. Two types of the self-assembled nanostructure arrays were formed at the different bias RF-power ranges: one was nanohole arrays and the other was nanopillar arrays. The hole-type nanostructure was formed when the bias power was low at ~10 W, while the pillar-type nanostructure was formed when the bias power increased to 30 W. It was also found that, the height of the nanostructure arrays increased with the onset of an etching time of 40 s, but it decreased after excessively long etching time as the nanostructure arrays could no longer sustain themselves. The correlation between the formation of nanostructures and plasma properties was investigated using OES, XPS, AFM, and SEM analyses. According to the analyses, sidewall passivation layers which were formed by the reaction of Si with F and O radicals generated from the SF 6/ O 2 plasma were responsible for giving rise to various nanostructure arrays.
Primary Myelofibrosis: Update on Definition, Pathogenesis, and Treatment
Authors: Abdel-Wahab Omar I. , Levine Ross L.
Published: October 2008

Primary myelofibrosis (PMF) is a clonal stem cell disorder that manifests clinically as anemia, splenomegaly due to extramedullary hematopoiesis, leukoerythroblastosis, and constitutional symptoms, which are the clinical hallmarks of PMF. Within the past three years it has been determined that a single, recurrent, somatic mutation in the gene encoding the cytoplasmic tyrosine kinase Janus kinase 2 (JAK2) occurs in the majority of patients with PMF, and more recently, activating mutations in the gene encoding the thrombopoietin receptor MPL have also been identified in a subset of PMF patients. These discoveries have yielded important insights into the pathogenesis of PMF and have brought about the first opportunity for rationally targeted therapy for this disorder. Here we present an updated review of the pathogenesis, definition, and treatment of PMF in light of the discovery of JAK2 and MPL mutations, as well as other recent work in the myeloproliferative neoplasm field.
Emerging Concepts in the Immunopathogenesis of AIDS
Authors: Douek Daniel C. , Roederer Mario , Koup Richard A.
Published: October 2008

There is an intense interplay between HIV and the immune system, and the literature is replete with studies describing various immunological phenomena associated with HIV infection. Many of these phenomena seem too broad in scope to be attributable either to HIV-infected cells or to the HIV-specific immune response. Recently, a more fundamental understanding of how HIV affects various T cells and T cell compartments has emerged. This review covers the role of immune activation in HIV immunopathogenesis, how that activation could be mediated directly by HIV replicating within and damaging the gut mucosal barrier, how HIV affects multiple T cell functions and phenotypes, and how chronic HIV replication induces immune modulatory pathways to negatively regulate certain functions in HIV-specific T cells.
Biomarkers for Prostate Cancer
Authors: Makarov Danil V. , Loeb Stacy , Getzenberg Robert H. , Partin Alan W.
Published: October 2008

The development of biomarkers for prostate cancer screening, detection, and prognostication has revolutionized the management of this disease. Prostate-specific antigen (PSA) is a useful, though not specific, biomarker for detecting prostate cancer. We review the literature on prostate cancer biomarkers, including serum markers (PAP, tPSA, fPSA, proPSA, PSAD, PSAV, PSADT, EPCA, and EPCA-2), tissue markers (AMACR, methylated GSTP1, and the TMPRSS2-ETS gene rearrangement), and a urine marker (DD3PCA3/UPM-3). Future research should focus on validation of already existing biomarkers and the discovery of new markers to identify men with aggressive prostate cancer.
Polycystic Kidney Disease
Authors: Harris Peter C. , Torres Vicente E.
Published: October 2008

A number of inherited disorders result in renal cyst development. The most common form, autosomal dominant polycystic kidney disease (ADPKD), is a disorder most often diagnosed in adults and caused by mutation in PKD1 or PKD2. The PKD1 protein, polycystin-1, is a large receptor-like protein, whereas polycystin-2 is a transient receptor potential channel. The polycystin complex localizes to primary cilia and may act as a mechanosensor essential for maintaining the differentiated state of epithelia lining tubules in the kidney and biliary tract. Elucidation of defective cellular processes has highlighted potential therapies, some of which are now being tested in clinical trials. ARPKD is the neonatal form of PKD and is associated with enlarged kidneys and biliary dysgenesis. The disease phenotype is highly variable, ranging from neonatal death to later presentation with minimal kidney disease. ARPKD is caused by mutation in PKHD1, and two truncating mutations are associated with neonatal lethality. The ARPKD protein, fibrocystin, is localized to cilia/basal body and complexes with polycystin-2. Rare, syndromic forms of PKD also include defects of the eye, central nervous system, digits, and/or neural tube and highlight the role of cilia and pathways such as Wnt and Hh in their pathogenesis.
Genetic Testing in Clinical Practice
Authors: Lamberts Steven W.J. , Uitterlinden André G.
Published: October 2008

In the practice of internal medicine, the value of genetic testing in common (mono)genetic diseases such as familial hemochromatosis, hypercholesterolemia, Mediterranean fever, and thrombophilia is limited. The genotype insufficiently predicts the phenotype because of the powerful effects of other modifying genes, environmental influences, and lifestyle factors. Many common diseases, including diabetes mellitus, osteoporosis, and cardiovascular disease, have strong genetic influences but are called complex genetic traits. The underlying genetic factors are currently investigated using new molecular tools such as genome-wide association studies, analyzing up to 500,000 markers in huge numbers of patients. Many new (often unexpected) markers have been identified, and in many instances their functional significance is unknown. Genomic profiles play a rapidly growing role in the field of pharmacogenomics. A number of recently identified pharmacogenomic biomarkers are helpful to predict drug-related toxic effects.
High glucose promotes retinal endothelial cell migration through activation of Src, PI3K/Akt1/eNOS, and ERKs
Authors: Huang Qiong , Sheibani Nader
Published: October 2008

Hyperglycemia impacts retinal vascular function and promotes the development and progression of diabetic retinopathy, which ultimately results in growth of new blood vessels and loss of vision. How high glucose affects retinal endothelial cell (EC) properties requires further investigation. Here we determined the impact of high glucose on mouse retinal EC function in vitro. High glucose significantly enhanced the migration of retinal EC without impacting their proliferation, apoptosis, adhesion, and capillary morphogenesis. The enhanced migration of retinal EC under high glucose was reversed in the presence of the antioxidant N-acetylcysteine, suggesting increased oxidative stress under high-glucose conditions. Retinal EC under high-glucose conditions also expressed increased levels of fibronectin, osteopontin, and αvβ3-integrin, and reduced levels of thrombospondin-1. These changes were concomitant with sustained activation of the downstream prosurvival and promigratory signaling pathways, including Src kinase, phosphatidylinositol 3-kinase/Akt1/endothelial nitric oxide synthase, and ERKs. The sustained activation of these signaling pathways was essential for enhanced migration of retinal EC under high-glucose conditions. Together, our results indicate the exposure of retinal EC to high glucose promotes a promigratory phenotype. Thus alterations in the proangiogenic properties of retinal EC during diabetes may contribute to the development and pathogenesis of diabetic retinopathy.
Slow myosin heavy chain expression in the absence of muscle activity
Authors: Agbulut O. , Vignaud A. , Hourde C. , Mouisel E. , Fougerousse F. , Butler-Browne G. S. , Ferry A.
Published: October 2008

Innervation has been generally accepted to be a major factor involved in both triggering and maintaining the expression of slow myosin heavy chain (MHC-1) in skeletal muscle. However, previous findings from our laboratory have suggested that, in the mouse, this is not always the case ( 30 ). Based on these results, we hypothesized that neurotomy would not markedly reduced the expression of MHC-1 protein in the mouse soleus muscles. In addition, other cellular, biochemical, and functional parameters were also studied in these denervated soleus muscles to complete our study. Our results show that denervation reduced neither the relative amount of MHC-1 protein, nor the percentage of muscle fibers expressing MHC-1 protein ( P > 0.05). The fact that MHC-1 protein did not respond to muscle inactivity was confirmed in three different mouse strains (129/SV, C57BL/6, and CD1). In contrast, all of the other histological, biochemical, and functional muscle parameters were markedly altered by denervation. Cross-sectional area (CSA) of muscle fibers, maximal tetanic isometric force, maximal velocity of shortening, maximal power, and citrate synthase activity were all reduced in denervated muscles compared with innervated muscles ( P < 0.05). Contraction and one-half relaxation times of the twitch were also increased by denervation ( P < 0.05). Addition of tenotomy to denervation had no further effect on the relative expression of MHC-1 protein ( P > 0.05), despite a greater reduction in CSA and citrate synthase activity ( P < 0.05). In conclusion, a deficit in neural input leads to marked atrophy and reduction in performance in mouse soleus muscles. However, the maintenance of the relative expression of slow MHC protein is independent of neuromuscular activity in mice.
A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP
Authors: Qiu Feng , Dahl Gerhard
Published: October 2008

Pannexin 1 forms a large membrane channel that, based on its biophysical properties and its expression pattern, is a prime candidate to represent an ATP release channel. Pannexin 1 channel activity is potentially deleterious for cells as indicated by its involvement in the P2X7 death complex. Here we describe a negative feedback loop controlling pannexin 1 channel activity. ATP, permeant to pannexin 1 channels, was found to inhibit its permeation pathway when applied extracellularly to oocytes expressing pannexin 1 exogenously. ATP analogues, including benzoylbenzoyl-ATP, suramin, and brilliant blue G were even more effective inhibitors of pannexin 1 currents than ATP. These compounds also attenuated the uptake of dyes by erythrocytes, which express pannexin 1. The rank order of the compounds in attenuation of pannexin 1 currents was similar to their binding affinities to the P2X7 receptor, except that receptor agonists and antagonists both were inhibitory to the channel. Mutational analysis identified R75 in pannexin 1 to be critical for ATP inhibition of pannexin 1 currents.
Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart
Authors: O'Driscoll Kate E. , Hatton William J. , Burkin Heather R , Leblanc Normand , Britton Fiona C.
Published: October 2008

Bestrophins are a novel family of proteins that encode calcium-activated chloride channels. In this study we establish that Bestrophin transcripts are expressed in the mouse and human heart. Native mBest3 protein expression and localization in heart was demonstrated by using a specific polyclonal mBest3 antibody. Immunostaining of isolated cardiac myocytes indicates that mBest3 is present at the membrane. Using the patch-clamp technique, we characterized the biophysical and pharmacological properties of mBest3 cloned from heart. Whole cell chloride currents were evoked in both HEK293 and COS-7 cells expressing mBest3 by elevation of intracellular calcium. mBest3 currents displayed a KDfor Ca2+of ∼175 nM. The calcium-activated chloride current was found to be time and voltage independent and displayed slight outward rectification. The anion permeability sequence of the channel was SCN−>I−>Cl−, and the current was inhibited by niflumic acid and DIDS in the micromolar range. In addition, we generated a site-specific mutation (F80L) in the putative pore region of mBest3 that significantly altered the ion conduction and pharmacology of this channel. Our functional and mutational studies examining the biophysical properties of mBest3 indicate that it functions as a pore-forming chloride channel that is activated by physiological levels of calcium. This study reports novel findings regarding the molecular expression, tissue localization, and functional properties of mBest3 cloned from heart.
High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction
Authors: Mohan Sumathy , Konopinski Ryszard , Yan Bo , Centonze Victoria E. , Natarajan Mohan
Published: October 2008

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor κB kinases (IKK)α, β, and γ] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 ± 0.65-fold) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.45 ± 0.4-fold). Both IKKβ and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 μM) or Radicicol (20 μM) mitigated (0.45 ± 0.04-fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.42-fold). Blocking of IKKβ expression by IKK inhibitor II (15 μM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.
LPS-binding protein mediates LPS-induced liver injury and mortality in the setting of biliary obstruction
Authors: Minter Rebecca M. , Bi Xiaoming , Ben-Josef Gal , Wang Tianyi , Hu Bin , Arbabi Saman , Hemmila Mark R. , Wang Stewart C. , Remick Daniel G. , Su Grace L.
Published: October 2008

It is generally accepted that low levels of lipopolysaccharide (LPS)-binding protein (LBP) augment the cell's response to LPS, whereas high levels of LBP have been shown to inhibit cell responses to LPS. Clinical studies and in vitro work by our group have demonstrated that, in the setting of liver disease, increased or acute-phase levels of LBP may actually potentiate rather than inhibit an overwhelming proinflammatory response. Therefore, in the present studies we sought to determine the role of acute-phase LBP in mediating morbidity and mortality in animals challenged with LPS in the setting of biliary obstruction. Using LBP-deficient mice and LBP blockade in wild-type mice, we demonstrate that high levels of LBP are deleterious in the setting of cholestasis. Following biliary obstruction and intraperitoneal LPS challenge, hepatic injury, hepatic neutrophil infiltration, and mortality were significantly increased in animals with an intact LBP acute-phase response. Kupffer cell responses from these animals demonstrated a significant increase in several inflammatory mediators, and Kupffer cell-associated LBP appears to be responsible for these differences, at least in part. Our results indicate that the role of LBP signaling in inflammatory conditions is complex and heterogeneous, and elevated levels of LBP are not always protective. Increased LBP production in the setting of cholestatic liver disease appears to be deleterious and may represent a potential therapeutic target for preventing overwhelming inflammatory responses to LPS in this setting.
Elevated production of 20-HETE in the cerebral vasculature contributes to severity of ischemic stroke and oxidative stress in spontaneously hypertensive rats
Authors: Dunn Kathryn M. , Renic Marija , Flasch Averia K. , Harder David R. , Falck John , Roman Richard J.
Published: October 2008

Hypertension is a major risk factor for stroke, but the factors that contribute to the increased incidence and severity of ischemic stroke in hypertension remain to be determined. 20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to be a potent constrictor of cerebral arteries, and inhibitors of 20-HETE formation reduce infarct size following cerebral ischemia. The present study examined whether elevated production of 20-HETE in the cerebral vasculature could contribute to the larger infarct size previously reported after transient middle cerebral artery occlusion (MCAO) in hypertensive strains of rat [spontaneously hypertensive rat (SHR) and spontaneously hypertensive stroke-prone rat (SHRSP)]. The synthesis of 20-HETE in the cerebral vasculature of SHRSP measured by liquid chromatography-tandem mass spectrometry was about twice that seen in Wistar-Kyoto (WKY) rats. This was associated with the elevated expression of cytochrome P-450 (CYP)4A protein and CYP4A1 and CYP4A8 mRNA. Infarct volume after transient MCAO was greater in SHRSP (36 ± 4% of hemisphere volume) than in SHR (19 ± 5%) or WKY rats (5 ± 2%). This was associated with a significantly greater reduction in regional cerebral blood flow (rCBF) in SHR and SHRSP than in WKY rats during the ischemic period (78% vs. 62%). In WKY rats, rCBF returned to 75% of control following reperfusion. In contrast, SHR and SHRSP exhibited a large (166 ± 18% of baseline) and sustained (1 h) postischemic hyperperfusion. Acute blockade of the synthesis of 20-HETE with N-hydroxy- N'-(4-butyl-2-methylphenyl)-formamidine (HET0016; 1 mg/kg) reduced infarct size by 59% in SHR and 87% in SHRSP. HET0016 had no effect on the fall in rCBF during MCAO but eliminated the hyperemic response. HET0016 also attenuated vascular O2•− formation and restored endothelium-dependent dilation in cerebral arteries of SHRSP. These results indicate the production of 20-HETE is elevated in the cerebral vasculature of SHRSP and contributes to oxidative stress, endothelial dysfunction, and the enhanced sensitivity to ischemic stroke in this hypertensive model.
Increased renal adrenomedullin expression in rats with ureteral obstruction
Authors: Nørregaard Rikke , Bødker Tina , Jensen Boye L. , Stødkilde Lene , Nielsen Søren , Frøkiær Jørgen
Published: October 2008

Ureteral obstruction is characterized by decreased renal blood flow that is associated with hypoxia within the kidney. Adrenomedullin (AM) is a peptide hormone with tissue-protective capacity that is stimulated through hypoxia. We tested the hypothesis that ureteral obstruction stimulates expression of AM and hypoxia-inducible factor-1 (HIF-1α) in kidneys. Rats were exposed to bilateral ureteral obstruction (BUO) for 2, 6, 12, and 24 h or sham operation and compared with unilateral obstruction (UUO). AM mRNA expression was measured by quantitative PCR in cortex and outer medulla (C+OM) and inner medulla (IM). AM and HIF-1α protein abundance and localization were determined in rats subjected to 24-h BUO. AM mRNA expression in C+OM increased significantly after 12-h BUO and further increased after 24 h. In IM, AM mRNA expression increased significantly in response to BUO for 6 h and further increased after 24 h. AM peptide abundance was enhanced in C+OM and IM after 24-h BUO. Immunohistochemical labeling of kidneys showed a wider distribution and more intense AM signal in 24-h BUO compared with Sham. In UUO rats, AM mRNA expression increased significantly in IM of the obstructed kidney compared with nonobstructed and Sham kidney whereas AM peptide increased in IM compared with Sham. HIF-1α protein abundance increased significantly in IM after 24-h BUO compared with Sham and HIF-1α immunoreactive protein colocalized with AM. In summary, AM and HIF-1α expression increases in response to ureteral obstruction in agreement with expected oxygen gradients. Hypoxia acting through HIF-1α accumulation may be an important pathway for the renal response to ureteral obstruction.
Role of the hypothalamic PVN in the reflex reduction in mesenteric blood flow elicited by hyperthermia
Authors: Chen Feng , Dworak Melissa , Wang Yuliang , Cham Joo Lee , Badoer Emilio
Published: October 2008

The hypothalamic paraventricular nucleus (PVN) is an important integrative center in the brain. In the present study, we investigated whether the PVN is a key region in the mesenteric vasoconstriction that normally accompanies an increase in core body temperature. Anesthetized rats were monitored for blood pressure, heart rate, mesenteric blood flow, and vascular conductance. In control rats, elevation of core body temperature to 41°C had no significant effect on blood pressure, increased heart rate, and reduced mesenteric blood flow by 21%. In a separate group of rats, muscimol was microinjected bilaterally (1 nmol/side) into the PVN. Compared with the control group, there was no significant difference in the blood pressure and heart rate responses elicited by the increase in core body temperature. In contrast to control animals, however, mesenteric blood flow did not fall in the muscimol-treated rats in response to the elevation in core body temperature. In a separate group, in which muscimol was microinjected into regions outside the PVN, elevating core body temperature elicited the normal reduction in mesenteric blood flow. The results suggest that the PVN may play a key role in the reflex decrease in mesenteric blood flow elicited by hyperthermia.
Avian renal proximal tubule epithelium urate secretion is mediated by Mrp4
Authors: Bataille Amy M. , Goldmeyer James , Renfro J. Larry
Published: October 2008

Birds are uricotelic and, like humans, maintain high plasma urate concentrations (∼300 μM). The majority of their urate waste, as in humans, is eliminated by renal proximal tubular secretion; however, the mechanism of urate transport across the brush-border membrane of the intact proximal tubule epithelium during secretion is uncertain. The dominance of secretory urate transport in the bird provides a convenient model for examining this process. The present study shows that short hairpin RNA interference (shRNAi) effectively knocked down gene expression of multidrug resistance protein 4 (Mrp4; 25% of control) in primary monolayer cultures of isolated chicken proximal tubule epithelial cells (cPTCs). Control and Mrp4-shRNAi-treated cPTCs were mounted in Ussing chambers and unidirectional transepithelial fluxes of urate were measured. To detect nonspecific effects, transepithelial electrical resistance (TER) and sodium-dependent glucose transport (Iglu) were monitored throughout experiments. Knocking down Mrp4 expression resulted in a reduction of transepithelial urate secretion to 35% of control with no effects on TER or Iglu. Although electrical gradient-driven urate transport in isolated brush-border membrane vesicles was confirmed, potassium-induced depolarization of the plasma membrane in intact cPTCs failed to inhibit active transepithelial urate secretion. However, electrical gradient-dependent vesicular urate transport was inhibited by the MRP4 inhibitor MK-571 also known to inhibit active transepithelial urate transport by cPTCs. Based on these data, direct measure of active transepithelial urate secretion in functional avian proximal tubule epithelium indicates that Mrp4 is the dominant apical membrane exit pathway from cell to lumen.
Afferent renal denervation impairs baroreflex control of efferent renal sympathetic nerve activity
Authors: Kopp Ulla C. , Jones Susan Y. , DiBona Gerald F.
Published: October 2008

Increasing efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA), which decreases ERSNA to prevent sodium retention. High-sodium diet enhances ARNA, suggesting an important role for ARNA in suppressing ERSNA during excess sodium intake. Mean arterial pressure (MAP) is elevated in afferent renal denervated by dorsal rhizotomy (DRX) rats fed high-sodium diet. We examined whether the increased MAP in DRX is due to impaired arterial baroreflex function. In DRX and sham DRX rats fed high-sodium diet, arterial baroreflex function was determined in conscious rats by intravenous nitroprusside and phenylephrine or calculation of transfer function gain from arterial pressure to ERSNA (spontaneous baroreflex sensitivity). Increasing MAP did not suppress ERSNA to the same extent in DRX as in sham DRX, −60 ± 4 vs. −77 ± 6%. Maximum gain, −4.22 ± 0.45 vs. −6.04 ± 0.90% ΔERSNA/mmHg, and the maximum value of instantaneous gain, −4.19 ± 0.45 vs. −6.04 ± 0.81% ΔERSNA/mmHg, were less in DRX than in sham DRX. Likewise, transfer function gain was lower in DRX than in sham DRX, 3.9 ± 0.2 vs. 6.1 ± 0.5 NU/mmHg. Air jet stress produced greater increases in ERSNA in DRX than in sham DRX, 35,000 ± 4,900 vs. 20,900 ± 3,410%·s (area under the curve). Likewise, the ERSNA responses to thermal cutaneous stimulation were greater in DRX than in sham DRX. These studies suggest impaired arterial baroreflex suppression of ERSNA in DRX fed high-sodium diet. There were no differences in arterial baroreflex function in DRX and sham DRX fed normal-sodium diet. Impaired arterial baroreflex function contributes to increased ERSNA, which would eventually lead to sodium retention and increased MAP in DRX rats fed high-sodium diet.
ACE2 expression and activity are enhanced during pregnancy
Authors: Levy Anat , Yagil Yoram , Bursztyn Michael , Barkalifa Ronit , Scharf Shimon , Yagil Chana
Published: October 2008

In the current study, we investigated the expression and activity of ACE2 during pregnancy in normotensive and hypertensive rats, focusing on the relative contribution of the uterus and the placentas, the kidney serving as a reference. We used the Sabra rat model of salt-sensitive hypertension. We confirmed a systemic vasodilatory state during the third trimester of pregnancy, as evidenced by a reduction in blood pressure, both in normotensive and hypertensive rats. At the time that blood pressure was reduced, ACE2 was expressed abundantly in the reproductive organs. The relative levels of ACE2 mRNA in the pregnant animal were placenta > kidneys ≥ uterus and of ACE2 activity kidney > placenta > uterus. In the uterus and the placenta, ACE2 expression was unaffected by strain, salt-loading, or the level of blood pressure. ACE2 activity in the uterus of the nonpregnant rat was not affected by any of these variables either, but during pregnancy increased in salt-loaded animals. When estimating the total contribution of the uterus to ACE2 mRNA and activity during pregnancy, we found that the amount of ACE2 mRNA increased in both strains irrespective of diet, but that ACE2 activity increased only in salt-loaded animals. We further estimated the relative total contribution of the uterus, placentas, and kidneys to ACE2 expression and activity during pregnancy by adjusting for mass and number of organs and found that the placentas were the major contributors, followed by the kidney and the uterus. We conclude that during pregnancy, the placentas, in particular, but also the uterus, constitute important sources of ACE2, in addition to its normal production in the kidney, leading to an estimated twofold increase in total ACE2 activity. These data are consistent the hypothesis that transient ACE2 overexpression and increased activity during pregnancy may be important in modulating systemic, as well as local hemodynamics in the uteroplacental unit.