bluuebunny/crossref_metadata_2025 huggingface dataset:
ORF73-Null Murine Gammaherpesvirus 68 Reveals Roles for mLANA and p53 in Virus Replication
Authors: Forrest J. Craig , Paden Clinton R. , Allen Robert D. , Collins Julie , Speck Samuel H.
Published: August 2007

Gammaherpesviruses establish lifelong, latent infections in host lymphocytes, during which a limited subset of viral gene products facilitates maintenance of the viral episome. Among the gamma-2-herpesvirus (rhadinovirus) subfamily, this includes expression of the conserved ORF73-encoded LANA proteins. We previously demonstrated by loss-of-function mutagenesis that the murine gammaherpesvirus 68 (MHV68) ORF73 gene product, mLANA, is required for the establishment of latency following intranasal inoculation of mice (N. J. Moorman, D. O. Willer, and S. H. Speck, J. Virol. 77:10295-10303, 2003). mLANA-deficient viruses also exhibited a defect in acute virus replication in the lungs of infected mice. The latter observation led us to examine the role of mLANA in productive viral replication. We assessed the capacity of mLANA-deficient virus (73.Stop) to replicate in cell culture at low multiplicities of infection (MOIs) and found that 73.Stop growth was impaired in murine fibroblasts but not in Vero cells. A recombinant virus expressing an mLANA-green fluorescent protein (GFP) fusion revealed that mLANA is expressed throughout the virus replication cycle. In addition, 73.Stop infection of murine fibroblasts at high MOIs was substantially more cytotoxic than infection with a genetically repaired marker rescue virus (73.MR), a phenotype that correlated with enhanced kinetics of viral gene expression and increased activation of p53. Notably, augmented cell death, viral gene expression, and p53 induction were independent of viral DNA replication. Expression of a mLANA-GFP fusion protein in fibroblasts correlated with both reduced p53 stabilization and reduced cell death following treatment with p53-inducing agonists. In agreement, accentuated cell death associated with 73.Stop infection was reduced in p53-deficient murine embryonic fibroblasts. Additionally, replication of 73.Stop in p53-deficient cells was restored to levels comparable to those of 73.MR. More remarkably, the absence of p53 led to an overall delay in replication for both 73.Stop and 73.MR viruses, which correlated with delayed viral gene expression, indicating a role for p53 in MHV68 replication. Consistent with these findings, the expression of replication-promoting viral genes was positively influenced by p53 overexpression or treatment with the p53 agonist etoposide. Overall, these data demonstrate the importance of mLANA in MHV68 replication and suggest that LANA proteins limit the induction of cellular stress responses to regulate the viral gene expression cascade and limit host cell injury.
Hepatitis B Virus Replication Is Associated with an HBx-Dependent Mitochondrion-Regulated Increase in Cytosolic Calcium Levels
Authors: McClain Stephanie L. , Clippinger Amy J. , Lizzano Rebecca , Bouchard Michael J.
Published: August 2007

The nonstructural hepatitis B virus (HBV) protein HBx has an important role in HBV replication and in HBV-associated liver disease. Many activities have been linked to HBx expression; however, the molecular mechanisms underlying many of these activities are unknown. One proposed HBx function is the regulation of cytosolic calcium. We analyzed calcium levels in HepG2 cells that expressed HBx or replicating HBV, and we demonstrated that HBx, expressed in the absence of other HBV proteins or in the context of HBV replication, elevates cytosolic calcium. We linked this elevation of cytosolic calcium to the association of HBx with the mitochondrial permeability transition pore.
Disease Duration Determines Canine Distemper Virus Neurovirulence
Authors: Bonami François , Rudd Penny A. , von Messling Veronika
Published: August 2007

The
Morbillivirus
hemagglutinin (H) protein mediates attachment to the target cell. To evaluate its contribution to canine distemper virus neurovirulence, we exchanged the H proteins of the wild-type strains 5804P and A75 and assessed the pathogenesis of the chimeric viruses in ferrets. Both strains are lethal to ferrets; however, 5804P causes a 2-week disease without neurological signs, whereas A75 is associated with a longer disease course and neurological involvement. We observed that both H proteins supported neuroinvasion and the subsequent development of clinical neurological signs if given enough time, demonstrating that disease duration is the main neurovirulence determinant.
Role for Amino Acids
212
KLR
214
of Ebola Virus VP40 in Assembly and Budding
Authors: McCarthy Sarah E. , Johnson Reed F. , Zhang Yong-An , Sunyer J. Oriol , Harty Ronald N.
Published: August 2007

Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M- and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids
212
KLR
214
were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i)
212
KLR
214
residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that
12
KLR
214
residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.
Cross-Priming of Cytotoxic T Cells Dictates Antigen Requisites for Modified Vaccinia Virus Ankara Vector Vaccines
Authors: Gasteiger Georg , Kastenmuller Wolfgang , Ljapoci Ronny , Sutter Gerd , Drexler Ingo
Published: August 2007

Recombinant vaccines based on modified vaccinia virus Ankara (MVA) have an excellent record concerning safety and immunogenicity and are currently being evaluated in numerous clinical studies for immunotherapy of infectious diseases and cancer. However, knowledge about the biological properties of target antigens to efficiently induce MVA vaccine-mediated immunity in vivo is sparse. Here, we examined distinct antigen presentation pathways and different antigen formulations contained in MVA vaccines for their capability to induce cytotoxic CD8+T-cell (CTL) responses. Strikingly, we found that CTL responses against MVA-produced antigens were dominated by cross-priming in vivo, despite the ability of the virus to efficiently infect professional antigen-presenting cells such as dendritic cells. Moreover, stable mature protein was preferred to preprocessed antigen as the substrate for cross-priming. Our data are essential for improved MVA vaccine design, as they demonstrate the need for optimal adjustment of the target antigen properties to the intrinsic requirements of the delivering vector system.
Pol-Specific CD8
+
T Cells Recognize Simian Immunodeficiency Virus-Infected Cells Prior to Nef-Mediated Major Histocompatibility Complex Class I Downregulation
Authors: Sacha Jonah B. , Chung Chungwon , Reed Jason , Jonas Anna K. , Bean Alexander T. , Spencer Sean P. , Lee Wonhee , Vojnov Lara , Rudersdorf Richard , Friedrich Thomas C. , Wilson Nancy A. , Lifson Jeffrey D. , Watkins David I.
Published: August 2007

Effective, vaccine-induced CD8
+
T-cell responses should recognize infected cells early enough to prevent production of progeny virions. We have recently shown that Gag-specific CD8
+
T cells recognize simian immunodeficiency virus-infected cells at 2 h postinfection, whereas Env-specific CD8
+
T cells do not recognize infected cells until much later in infection. However, it remains unknown when other proteins present in the viral particle are presented to CD8
+
T cells after infection. To address this issue, we explored CD8
+
T-cell recognition of epitopes derived from two other relatively large virion proteins, Pol and Nef. Surprisingly, infected cells efficiently presented CD8
+
T-cell epitopes from virion-derived Pol proteins within 2 h of infection. In contrast, Nef-specific CD8
+
T cells did not recognize infected cells until 12 h postinfection. Additionally, we show that SIVmac239 Nef downregulated surface major histocompatibility complex class I (MHC-I) molecules beginning at 12 h postinfection, concomitant with presentation of Nef-derived CD8
+
T-cell epitopes. Finally, Pol-specific CD8
+
T cells eliminated infected cells as early as 6 h postinfection, well before MHC-I downregulation, suggesting a previously underappreciated antiviral role for Pol-specific CD8
+
T cells.
Viral Sequence Evolution in Acute Hepatitis C Virus Infection
Authors: Kuntzen Thomas , Timm Joerg , Berical Andrew , Lewis-Ximenez Lia L. , Jones Andrea , Nolan Brian , Schulze zur Wiesch Julian , Li Bin , Schneidewind Arne , Kim Arthur Y. , Chung Raymond T. , Lauer Georg M. , Allen Todd M.
Published: August 2007

CD8
+
-T-cell responses play an important role in the containment and clearance of hepatitis C virus (HCV) infection, and an association between viral persistence and development of viral escape mutations has been postulated. While escape from CD8
+
-T-cell responses has been identified as a major driving force for the evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), a broader characterization of this relationship is needed in HCV infection. To determine the extent, kinetics, and driving forces of HCV sequence evolution, we sequenced the entire HCV genome longitudinally in four subjects monitored for up to 30 months after acute infection. For two subjects the transmission sources were also available. Of 53 total nonenvelope amino acid substitutions detected, a majority represented forward mutations away from the consensus sequence. In contrast to studies in HIV and SIV, however, only 11% of these were associated with detectable CD8
+
T-cell responses. Interestingly, 19% of nonenvelope mutations represented changes toward the consensus sequence, suggesting reversion in the absence of immune pressure upon transmission. Notably, the rate of evolution of forward and reverse mutations correlated with the conservation of each residue, which is indicative of structural constraints influencing the kinetics of viral evolution. Finally, the rate of sequence evolution was observed to decline over the course of infection, possibly reflective of diminishing selection pressure by dysfunctional CD8
+
T cells. Taken together, these data provide insight into the extent to which HCV is capable of evading early CD8
+
T-cell responses and support the hypothesis that dysfunction of CD8
+
T cells may be associated with failure to resolve HCV infections.
The Formation of Viroplasm-Like Structures by the Rotavirus NSP5 Protein Is Calcium Regulated and Directed by a C-Terminal Helical Domain
Authors: Sen Adrish , Sen Nandini , Mackow Erich R.
Published: August 2007

The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic α-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 α-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted α-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.
Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth
Authors: Dugan Aisling S. , Gasparovic Megan L. , Tsomaia Natia , Mierke Dale F. , O'Hara Bethany A. , Manley Kate , Atwood Walter J.
Published: August 2007

BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing α(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.
Novel Adeno-Associated Virus Serotypes Efficiently Transduce Murine Photoreceptors
Authors: Allocca Mariacarmela , Mussolino Claudio , Garcia-Hoyos Maria , Sanges Daniela , Iodice Carolina , Petrillo Marco , Vandenberghe Luk H. , Wilson James M. , Marigo Valeria , Surace Enrico M. , Auricchio Alberto
Published: August 2007

Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.
Inhalable β(1→3)glucans as a non-allergenic exposure factor in Dutch bakeries
Authors: Stuurman B , Meijster T , Heederik D , Doekes G
Published: August 2007

Objectives:

To obtain an overview of inhalable β(1→3)glucans levels in Dutch industrial bakeries and explore possible associations with reported respiratory health effects in bakery workers.

Methods:

β(1→3)glucan levels were analysed in 186 personal inhalable dust measurements obtained from a random population of bakery workers. Association between respiratory health effects and exposure to β(1→3)glucan was explored in a population of industrial bakery workers participating in a Health Surveillance System for flour processing sectors. Based on their job, bakery workers were assigned to low or high exposure categories given the average job exposure estimates obtained from the measurement study.

Results:

Bread bakers and dough makers had the highest exposures to β(1→3)glucans (GM 1.48 μg/m3 and 1.37 μg/m3 respectively). Strong correlations were found between airborne levels of inhalable dust and β(1→3)glucans, and between β(1→3)glucans and wheat allergens (Pearson correlation coefficients were 0.74 and 0.68 respectively). No significant associations could be identified between β(1→3)glucan exposure and work-related respiratory symptoms.

Conclusion:

This study has shown that bakery workers are exposed to inhalable β(1→3)glucan levels comparable with exposure levels found in other occupational settings. More refined exposure assessment is necessary to fully understand the role of β(1→3)glucan exposure on respiratory health in bakery workers.
The Evolution of Color Polymorphism: Crypticity, Searching Images, and Apostatic Selection
Authors: Bond Alan B.
Published: August 2007

The development and maintenance of color polymorphism in cryptic prey species is a source of enduring fascination, in part because it appears to result from selective processes operating across multiple levels of analysis, ranging from cognitive psychology to population ecology. Since the 1960s, prey species with diverse phenotypes have been viewed as the evolved reflection of the perceptual and cognitive characteristics of their predators. Because it is harder to search simultaneously for two or more cryptic prey types than to search for only one, visual predators should tend to focus on the most abundant forms and effectively overlook the others. The result should be frequency-dependent, apostatic selection, which will tend to stabilize the prey polymorphism. Validating this elegant hypothesis has been difficult, and many details have been established only relatively recently. This review clarifies the argument for a perceptual selective mechanism and examines the relevant experimental evidence.
Functional ion channels in mouse bone marrow mesenchymal stem cells
Authors: Tao Rong , Lau Chu-Pak , Tse Hung-Fat , Li Gui-Rong
Published: August 2007

Bone marrow mesenchymal stem cells (MSCs) are used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not fully understood. The present study was to investigate the functional ionic channels in undifferentiated mouse bone marrow MSCs using whole cell patch-voltage clamp technique, RT-PCR, and Western immunoblotting analysis. We found that three types of ionic currents were present in mouse MSCs, including a Ca2+-activated K+ current ( IKCa), an inwardly rectifying K+ current ( IKir), and a chloride current ( ICl). IKir was inhibited by Ba2+, and IKCa was activated by the Ca2+ ionophore A-23187 and inhibited by the intermediate-conductance IKCa channel blocker clotrimazole. ICl was activated by hyposmotic (0.8 T) conditions and inhibited by the chloride channel blockers DIDS and NPPB. The corresponding ion channel genes and proteins, KCa3.1 for IKCa, Kir2.1 for IKir, and Clcn3 for ICl, were confirmed by RT-PCR and Western immunoblotting analysis in mouse MSCs. These results demonstrate that three types of functional ion channel currents (i.e., IKir, IKCa, and ICl) are present in mouse bone marrow MSCs.
Aquaporin-2 water channel in developing quail kidney: possible role in programming adult fluid homeostasis
Authors: Nishimura Hiroko , Yang Yimu , Lau Keith , Kuykindoll Rhonda J. , Fan Zheng , Yamaguchi Ken'ichi , Yamamoto Tadashi
Published: August 2007

Avian kidneys have loopless and looped nephrons; a countercurrent multiplier mechanism operates in the latter by NaCl recycling. We identified an aquaporin-2 (AQP2) homolog in apical/subapical regions of cortical and medullary collecting duct (CD) cells in kidneys of Japanese quail (q), Coturnix japonica. We investigated whether undernutrition during the embryonic/maturation period retards kidney and AQP2 development in quail and programs impaired volume regulation in adults. Protocols included 1) time course and 2) effects of 5–10% egg white withdrawal (EwW) or 48-h post-hatch food deprivation (FD) on nephron growth and qAQP2 mRNA expression, and 3) effects of EwW and FD on qAQP2 mRNA responses to 72-h water deprivation in adults. In metanephric kidneys, qAQP2 mRNA is expressed in medullary CDs at embryonic day 10; distribution and intensity increase during maturation. The number and size of glomeruli continue to increase after birth, whereas nephrogenic zones decrease. In EwW embryos, qAQP2 mRNA expression is initially delayed, then restored; birth weight and hatching rate are lower than in controls. Adults from EwW embryos and FD chicks have fewer ( P < 0.01) glomeruli. Water deprivation reduces body weight more in EwW birds than in controls. The results suggest that qAQP2 evolved in metanephric kidneys and that undernutrition may retard nephrogenesis, leading to impaired adult water homeostasis.
Downregulation of TRPC6 protein expression by high glucose, a possible mechanism for the impaired Ca2+signaling in glomerular mesangial cells in diabetes
Authors: Graham Sarabeth , Ding Min , Sours-Brothers Sherry , Yorio Thomas , Ma Jian-Xing , Ma Rong
Published: August 2007

The present study was performed to investigate whether transient receptor potential (TRPC)6 participated in Ca2+signaling of glomerular mesangial cells (MCs) and expression of this protein was altered in diabetes. Western blots and real-time PCR were used to evaluate the expression level of TRPC6 protein and mRNA, respectively. Cell-attached patch-clamp and fura-2 fluorescence measurements were utilized to assess angiotensin II (ANG II)-stimulated membrane currents and Ca2+responses in MCs. In cultured human MCs, high glucose significantly reduced expression of TRPC6 protein, but there was no effect on either TRPC1 or TRPC3. The high glucose-induced effect on TRPC6 was time and dose dependent with the maximum effect observed on day 7 and at 30 mM glucose, respectively. In glomeruli isolated from streptozotocin-induced diabetic rats, TRPC6, but not TRPC1, was markedly reduced compared with the glomeruli of control rats. Furthermore, TRPC6 mRNA in MCs was also significantly decreased by high glucose as early as 1 day after treatment with maximal reduction on day 4. Patch-clamp experiments showed that ANG II-stimulated membrane currents in MCs were significantly attenuated or enhanced by knockdown or overexpression of TRPC6, respectively. Fura-2 fluorescence measurements revealed that the ANG II-induced Ca2+influxes were markedly inhibited in MCs with TRPC6 knockdown, reminiscent of the impaired Ca2+entry in response to ANG II in high glucose-treated MCs. These results suggest that the TRPC6 protein expression in MCs was downregulated by high glucose and the deficiency of TRPC6 protein might contribute to the impaired Ca2+signaling of MCs seen in diabetes.
Vasopressin-induced membrane trafficking of TRPC3 and AQP2 channels in cells of the rat renal collecting duct
Authors: Goel Monu , Sinkins William G. , Zuo Cheng-Di , Hopfer Ulrich , Schilling William P.
Published: August 2007

The canonical transient receptor potential channels TRPC3 and TRPC6 are abundantly expressed along with the water channel aquaporin-2 (AQP2) in principal cells of the cortical and medullary collecting duct. Although TRPC3 is selectively localized to the apical membrane and TRPC6 is found in both the apical and basolateral domains, immunofluorescence is often observed in the cytoplasm, suggesting that TRPC3 and TRPC6 may exist in intracellular vesicles and may shuttle to and from the membrane in response to receptor stimulation. To test this hypothesis, the effect of arginine-vasopressin (AVP) on the subcellular distribution of TRPC3, TRPC6, and AQP2 was examined in the rat kidney and in cultured cell lines from the cortical (M1) and inner medullary (IMCD-3) collecting duct. Immunofluorescence analysis revealed that TRPC3, but not TRPC6, colocalized with AQP2 in intracellular vesicles. AVP caused the insertion and accumulation of TRPC3 and AQP2 in the apical membrane but had no effect on the subcellular distribution of TRPC6. TRPC3, but not TRPC6, coimmunoprecipitated with AQP2 from both medulla and M1 and IMCD-3 cell lysates. Apical-to-basolateral transepithelial 45Ca2+ flux in polarized IMCD-3 cell monolayers was stimulated by diacylglycerol analogs or by the purinergic receptor agonist ATP but not by thapsigargin. Stimulated 45Ca2+ flux was increased by overexpression of TRPC3 and attenuated by a dominant-negative TRPC3 construct. Furthermore, 45Ca2+ flux was greatly reduced by the pyrazole-derivative BTP2, a known inhibitor of TRPC3 channels. These results demonstrate that 1) TRPC3 and TRPC6 exist in different vesicle populations, 2) TRPC3 physically associates with APQ2 and shuttles to the apical membrane in response to AVP, and 3) TRPC3 is responsible for transepithelial Ca2+ flux in principal cells of the renal collecting duct.
Size and charge selectivity of the glomerular filter in early experimental diabetes in rats
Authors: Rippe Catarina , Rippe Anna , Torffvit Ole , Rippe Bengt
Published: August 2007

Microalbuminuria is an early sign of diabetic nephropathy. The aim of the present study was to investigate whether the changes of the glomerular filtration barrier in early experimental diabetes are due to size- or charge-selective alterations. Wistar rats, made diabetic by streptozotocin (STZ) and having their blood glucose maintained at ∼20 mM for 3 or 9 wk, were compared with age-matched controls. Glomerular clearances of native albumin (Cl-HSA) and neutralized albumin (Cl-nHSA) were assessed using a renal uptake technique. Glomerular filtration rate and renal plasma flow were assessed using51Cr-EDTA and [125I]iodohippurate, respectively. In a separate set of animals, diabetic for 9 wk, and in controls, glomerular sieving coefficients (θ) for neutral FITC-Ficoll (molecular radius: 15–90 Å) were assessed using size exclusion chromatography. At 3 wk of diabetes, Cl-HSA and Cl-nHSA remained unchanged, indicating no alteration in either size or charge selectivity. By contrast, at 9 wk of diabetes, there was a twofold increase of Cl-HSA, whereas Cl-nHSA remained largely unchanged, at first suggesting a glomerular charge defect. However, according to a two-pore model, the number of large pores, assessed from both Ficoll and Cl-HSA, increased twofold. In addition, a small reduction in proximal tubular reabsorption was observed at 3 wk, which was further reduced at 9 wk. In conclusion, no functional changes were observed in the glomerular filtration barrier at 3 wk of STZ-induced diabetes, whereas at 9 wk there was a decrease in size selectivity due to an increased number of large glomerular pores.
Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells
Authors: Wu Ling , Gao Xiaochong , Brown Rachel C. , Heller Stefan , O'Neil Roger G.
Published: August 2007

Gain/loss of function studies were utilized to assess the potential role of the endogenous vanilloid receptor TRPV4 as a sensor of flow and osmolality in M-1 collecting duct cells (CCD). TRPV4 mRNA and protein were detectable in M-1 cells and stably transfected HEK-293 cells, where the protein occurred as a glycosylated doublet on Western blots. Immunofluorescence imaging demonstrated expression of TRPV4 at the cell membranes of TRPV4-transfected HEK and M-1 cells and at the luminal membrane of mouse kidney CCD. By using intracellular calcium imaging techniques, calcium influx was monitored in cells grown on coverslips. Application of known activators of TRPV4, including 4α-PDD and hypotonic medium, induced strong calcium influx in M-1 cells and TRPV4-transfected HEK-293 cells but not in nontransfected cells. Applying increased flow/shear stress in a parallel plate chamber induced calcium influx in both M-1 and TRPV4-transfected HEK cells but not in nontransfected HEK cells. Furthermore, in loss-of-function studies employing small interference (si)RNA knockdown techniques, transfection of both M-1 and TRPV4-transfected HEK cells with siRNA specific for TRPV4, but not an inappropriate siRNA, led to a time-dependent decrease in TRPV4 expression that was accompanied by a loss of stimuli-induced calcium influx to flow and hypotonicity. It is concluded that TRPV4 displays a mechanosensitive nature with activation properties consistent with a molecular sensor of both fluid flow (or shear stress) and osmolality, or a component of a sensor complex, in flow-sensitive renal CCD.
Fibroblast growth factor 23 impairs phosphorus and vitamin D metabolism in vivo and suppresses 25-hydroxyvitamin D-1α-hydroxylase expression in vitro
Authors: Perwad Farzana , Zhang Martin Y. H. , Tenenhouse Harriet S. , Portale Anthony A.
Published: August 2007

Fibroblast growth factor-23 (FGF-23) is critical to the pathogenesis of a distinct group of renal phosphate wasting disorders: tumor-induced osteomalacia, X-linked hypophosphatemia, and autosomal dominant and autosomal recessive hypophosphatemic rickets. Excess circulating FGF-23 is responsible for their major phenotypic features which include hypophosphatemia due to renal phosphate wasting and inappropriately low serum 1,25(OH)2D concentrations. To characterize the effects of FGF-23 on renal sodium-phosphate (Na/Pi) cotransport and vitamin D metabolism, we administered FGF-23(R176Q) to normal mice. A single injection (0.33 μg/g body wt) induced significant hypophosphatemia, 20 and 29% decreases ( P < 0.001) in brush-border membrane (BBM) Na/Picotransport at 5 and 17 h after injection, respectively, and comparable decreases in the abundance of type IIa Na/Picotransporter protein in BBM. Multiple injections (6, 12, and 24 μg/day for 4 days) induced dose-dependent decreases (38, 63, and 75%, respectively) in renal abundance of 1α-hydroxylase mRNA ( P < 0.05). To determine whether FGF-23(R176Q) exerts a direct action on 1α-hydroxylase gene expression, we examined its effects in cultured human (HKC-8) and mouse (MCT) renal proximal tubule cells. FGF-23(R176Q) (1 to 10 ng/ml) induced a dose-dependent decrease in 1α-hydroxylase mRNA with a maximum suppression of 37% ( P < 0.05). Suppression was detectable after 6 h of exposure and maximal after 21 h. In MCT cells, FGF-23(R176Q) suppressed 1α-hydroxylase mRNA and activated the ERK1/2 signaling pathway. The MAPK inhibitor PD98059 effectively abolished FGF-23-induced suppression of 1α-hydroxylase mRNA by blocking signal transduction via ERK1/2. These novel findings provide evidence that FGF-23 directly regulates renal 1α-hydroxylase gene expression via activation of the ERK1/2 signaling pathway.
Retention of intravenously infused [13C]bicarbonate is transiently increased during recovery from hard exercise
Authors: Henderson Gregory C. , Fattor Jill A. , Horning Michael A. , Faghihnia Nastaran , Luke-Zeitoun Mona , Brooks George A.
Published: August 2007

The effects of exercise on energy substrate metabolism persist into the postexercise recovery period. We sought to derive bicarbonate retention factors ( k) to correct for carbon tracer oxidized, but retained from pulmonary excretion before, during, and after exercise. Ten men and nine women received a primed-continuous infusion of [13C]bicarbonate (sodium salt) under three different conditions: 1) before, during, and 3 h after 90 min of exercise at 45% peak oxygen consumption (V̇o2peak); 2) before, during, and 3 h after 60 min of exercise at 65% V̇o2peak; and 3) during a time-matched resting control trial, with breath samples collected for determination of 13CO2 excretion rates. Throughout the resting control trial, k was stable and averaged 0.83 in men and women. During exercise, average k in men was 0.93 at 45% V̇o2peak and 0.94 at 65% V̇o2peak, and in women k was 0.91 at 45% V̇o2peak and 0.92 at 65% V̇o2peak, with no significant differences between intensities or sexes. After exercise at 45% V̇o2peak, k returned rapidly to control values in men and women, but following exercise at 65% V̇o2peak, k was significantly less than control at 30 and 60 min postexercise in men (0.74 and 0.72, respectively, P < 0.05) and women (0.75 and 0.76, respectively, P < 0.05) with no significant postexercise differences between men and women. We conclude that bicarbonate/CO2 retention is transiently increased in men and women for the first hour of postexercise recovery following endurance exercise bouts of hard but not moderate intensity.
Lack of Spike-Count and Spike-Time Correlations in the Substantia Nigra Reticulata Despite Overlap of Neural Responses
Authors: Nevet Alon , Morris Genela , Saban Guy , Arkadir David , Bergman Hagai
Published: August 2007

Previous studies of single neurons in the substantia nigra reticulata (SNr) have shown that many of them respond to similar events. These results, as well as anatomical studies, suggest that SNr neurons share inputs and thus may have correlated activity. Different types of correlation can exist between pairs of neurons. These are traditionally classified as either spike-count (“signal” and “noise”) or spike-timing (spike-to-spike and joint peristimulus time histograms) correlations. These measures of neuronal correlation are partially independent and have different implications. Our purpose was to probe the computational characteristics of the basal ganglia output nuclei through an analysis of these different types of correlation in the SNr. We carried out simultaneous multiple-electrode single-unit recordings in the SNr of two monkeys performing a probabilistic delayed visuomotor response task. A total of 113 neurons (yielding 355 simultaneously recorded pairs) were studied. Most SNr neurons responded to one or more task-related events, with instruction cue (69%) and reward (63%) predominating. Response-match analysis, comparing peristimulus time histograms, revealed a significant overlap between response vectors. However, no measure of average correlation differed significantly from zero. The lack of significant SNr spike-count population correlations appears to be an exceptional phenomenon in the brain, perhaps indicating unique event-related processing by basal ganglia output neurons to achieve better information transfer. The lack of spike-timing correlations suggests that the basal high-frequency discharge of SNr neurons is not driven by the common inputs and is probably intrinsic.
TMS Pulses on the Frontal Eye Fields Break Coupling Between Visuospatial Attention and Eye Movements
Authors: Neggers S.F.W. , Huijbers W. , Vrijlandt C. M. , Vlaskamp B.N.S. , Schutter D.J.L.G. , Kenemans J. L.
Published: August 2007

While preparing a saccadic eye movement, visual processing of the saccade goal is prioritized. Here, we provide evidence that the frontal eye fields (FEFs) are responsible for this coupling between eye movements and shifts of visuospatial attention. Functional magnetic resonance imaging (fMRI)–guided transcranial magnetic stimulation (TMS) was applied to the FEFs 30 ms before a discrimination target was presented at or next to the target of a saccade in preparation. Results showed that the well-known enhancement of discrimination performance on locations to which eye movements are being prepared was diminished by TMS contralateral to eye movement direction. Based on the present and other reports, we propose that saccade preparatory processes in the FEF affect selective visual processing within the visual cortex through feedback projections, in that way coupling saccade preparation and visuospatial attention.
BDNF Induces Calcium Elevations Associated WithIBDNF, a Nonselective Cationic Current Mediated by TRPC Channels
Authors: Amaral Michelle D. , Pozzo-Miller Lucas
Published: August 2007

Brain-derived neurotrophic factor (BDNF) has potent actions on hippocampal neurons, but the mechanisms that initiate its effects are poorly understood. We report here that localized BDNF application to apical dendrites of CA1 pyramidal neurons evoked transient elevations in intracellular Ca2+concentration, which are independent of membrane depolarization and activation of N-methyl-d-aspartate receptors (NMDAR). These Ca2+signals were always associated with IBDNF, a slow and sustained nonselective cationic current mediated by transient receptor potential canonical (TRPC3) channels. BDNF-induced Ca2+elevations required functional Trk and inositol-tris-phosphate (IP3) receptors, full intracellular Ca2+stores as well as extracellular Ca2+, suggesting the involvement of TRPC channels. Indeed, the TRPC channel inhibitor SKF-96365 prevented BDNF-induced Ca2+elevations and the associated IBDNF. Thus TRPC channels emerge as novel mediators of BDNF-induced intracellular Ca2+elevations associated with sustained cationic membrane currents in hippocampal pyramidal neurons.
Withdrawal From Intermittent Ethanol Exposure Increases Probability of Burst Firing in VTA Neurons In Vitro
Authors: Hopf F. Woodward , Martin Miquel , Chen Billy T. , Bowers M. Scott , Mohamedi Maysha M. , Bonci Antonello
Published: August 2007

Changing the activity of ventral tegmental area (VTA) dopamine neurons from pacemaker to burst firing is hypothesized to increase the salience of stimuli, such as an unexpected reward, and likely contributes to withdrawal-associated drug-seeking behavior. Accordingly, pharmacological, behavioral, and electrophysiological data suggest an important role of the VTA in mediating alcohol-dependent behaviors. However, the effects of repeated ethanol exposure on VTA dopamine neuron ion channel function are poorly understood. Here, we repeatedly exposed rats to ethanol (2 g/kg ethanol, ip, twice per day for 5 days), then examined the firing patterns of VTA dopamine neurons in vitro after 7 days withdrawal. Compared with saline-treated animals, the function of the small conductance calcium-dependent potassium channel (SK) was reduced in ethanol-treated animals. Consistent with a role for SK in regulation of burst firing, NMDA applied during firing facilitated the transition to bursting in ethanol-treated but not saline-treated animals; NMDA consistently induced bursting only in saline-treated animals when SK was inhibited. Also, enhanced bursting in ethanol-treated animals was not a result of differences in NMDA-induced depolarization. Further, Ihwas also reduced in ethanol-treated animals, which delayed recovery from hyperpolarization, but did not account for the increased NMDA-induced bursting in ethanol-treated animals. Finally, repeated ethanol exposure and withdrawal also enhanced the acute locomotor-activating effect of cocaine (15 mg/kg, ip). Thus withdrawal after repeated ethanol exposure produced several alterations in the physiological properties of VTA dopamine neurons, which could ultimately increase the ability of VTA neurons to produce burst firing and thus might contribute to addiction-related behaviors.
What Controls the Strength of Snow-Albedo Feedback?
Authors: Qu Xin , Hall Alex
Published: August 2007

The strength of snow-albedo feedback (SAF) in transient climate change simulations of the Fourth Assessment of the Intergovernmental Panel on Climate Change is generally determined by the surface-albedo decrease associated with a loss of snow cover rather than the reduction in snow albedo due to snow metamorphosis in a warming climate. The large intermodel spread in SAF strength is likewise attributable mostly to the snow cover component. The spread in the strength of this component is in turn mostly attributable to a correspondingly large spread in mean effective snow albedo. Models with large effective snow albedos have a large surface-albedo contrast between snow-covered and snow-free regions and exhibit a correspondingly large surface-albedo decrease when snow cover decreases. Models without explicit treatment of the vegetation canopy in their surface-albedo calculations typically have high effective snow albedos and strong SAF, often stronger than observed. In models with explicit canopy treatment, completely snow-covered surfaces typically have lower albedos and the simulations have weaker SAF, generally weaker than observed. The authors speculate that in these models either snow albedos or canopy albedos when snow is present are too low, or vegetation shields snow-covered surfaces excessively. Detailed observations of surface albedo in a representative sampling of snow-covered surfaces would therefore be extremely useful in constraining these parameterizations and reducing SAF spread in the next generation of models.
Simulation of the Stable Water Isotopes in Precipitation over South America: Comparing Regional to Global Circulation Models
Authors: Sturm Christophe , Hoffmann Georg , Langmann Bärbel
Published: August 2007

A simulation of the stable water isotope cycle over South America by the regional circulation model REMOiso is discussed. The performance of the regional model, with a resolution of 0.5° (∼55 km), is compared to simulations by the global circulation model ECHAMiso at two coarser resolutions and evaluated against observations of precipitation and δ18O.

Here REMOiso is demonstrated to reproduce reasonably well climatic and isotopic features across South America. This paper explores further insights of δ18O as a climate proxy, based on REMOiso’s improvements as compared to ECHAMiso. In particular, the authors focus on the seasonal variation of the amount effect (δ18O decrease with precipitation amounts) and the anomalous δ18O continental gradient across the Amazon basin, as inferred from the REMOiso, ECHAMiso, and GNIP datasets. The finer resolution of topography in REMOiso enables a detailed analysis of the altitude effect: not only the first, but also the second derivative of δ18O with altitude is considered. It appears that high-altitude grid cells show an isotopic signature similar to Rayleigh distillation, in accordance with experimental studies. Finally, a Lagrangian reference frame is adopted to describe the evolution of δ18O in precipitation along its trajectory, in order to relate the simulation analysis to the fractionation mechanisms. This confirms that the amount effect, via Rayleigh distillation processes, is dominant during the wet season. During the dry season, the δ18O in precipitation is controlled by isotopic reequilibration of rain droplets with surrounding vapor, reflecting the impact of nonfractionating transpiration by the vegetation.
Comparison of Spectrally Resolved Outgoing Longwave Radiation over the Tropical Pacific between 1970 and 2003 Using IRIS, IMG, and AIRS
Authors: Griggs J. A. , Harries J. E.
Published: August 2007

The observation of changes in the earth’s spectrally resolved outgoing longwave radiation (OLR) provides a direct method of determining changes in the radiative forcing of the climate system. An earlier study showed that satellite-observed changes in the clear-sky outgoing longwave spectrum between 1997 and 1970 from the Infrared Interferometer Spectrometer (IRIS) and Interferometic Monitor of Greenhouse Gases (IMG) instruments could be related to changes in greenhouse gas composition. The authors present a new study that extends this to 2003, through the first use of a new, independent source of global atmospheric infrared spectra, from the Atmospheric Infrared Sounder (AIRS) experiment. AIRS is a dispersion grating spectrometer, while the other two were Fourier transform spectrometers, and this is taken into account in the analysis. The observed difference spectrum between the years 2003 and 1970 generally shows the signatures of greenhouse gas forcing, and also shows the sensitivity of the signatures to interannual variations in temperature. The new 2003 data support the conclusions found in the earlier work, though, interestingly, the methane (CH4) Q branch centered at 1304 cm−1 exhibits more complex behavior, showing a decrease in intensity in the difference spectrum between 1997 and 2003. Sensitivity analysis indicates that this is due to changes in temperature structure, superposed on an underlying increase in CH4. Radiative transfer calculations based on reanalysis data are used to simulate the changes in the OLR spectrum; limitations in such data and possible variations that could account for several observed effects are discussed.