bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of 24-Hour Posaconazole and Voriconazole MIC Readings versus the CLSI 48-Hour Broth Microdilution Reference Method: Application of Epidemiological Cutoff Values to Results from a Global Candida Antifungal Surveillance Program
Authors: Pfaller M. A. , Boyken L. B. , Hollis R. J. , Kroeger J. , Messer S. A. , Tendolkar S. , Diekema D. J.
Published: February 2011

We performed 24- and 48-h MIC determinations of posaconazole and voriconazole against more than 16,000 clinical isolates of
Candida
species. By using the 24- and 48-h epidemiological cutoff values (ECVs), the categorical agreement between the 24-h and reference 48-h broth microdilution results ranged from 97.1% (
C. parapsilosis
and voriconazole) to 99.8% (
C. krusei
and voriconazole), with 0.0 to 2.9% very major discrepancies (VMD). The essential agreement (within 2 log
2
dilutions) between the 24- and 48-h results was 99.6% for both posaconazole and voriconazole. The MIC results obtained for both posaconazole and voriconazole after only 24 h of incubation may be used to determine the susceptibilities of
Candida
spp. to these important antifungal agents. The applications of ECVs to this large collection of
Candida
isolates suggests the potential to develop 24-h species-specific clinical breakpoints for both posaconazole and voriconazole.
Loss of Asparagine-Linked Glycosylation Sites in Variable Region 5 of Human Immunodeficiency Virus Type 1 Envelope Is Associated with Resistance to CD4 Antibody Ibalizumab
Authors: Toma Jonathan , Weinheimer Steven P. , Stawiski Eric , Whitcomb Jeannette M. , Lewis Stanley T. , Petropoulos Christos J. , Huang Wei
Published: February 2011

Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical trials with HIV-infected patients have demonstrated the antiviral activity, safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding ibalizumab monotherapy to failing drug regimens led to transient reductions in HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility to ibalizumab. This report characterizes these variants by comparing the phenotypic susceptibilities and envelope (env) sequences of (i) paired baseline and on-treatment virus populations, (ii) individualenvclones from selected paired samples, and (iii)envclones containing site-directed mutations. Viruses with reduced susceptibility to ibalizumab were found to exhibit reduced susceptibility to the anti-CD4 antibody RPA-T4. Conversely, susceptibility to soluble CD4, which targets the HIV-1 gp120 envelope protein, was enhanced. No changes in susceptibility to the fusion inhibitor enfuvirtide or the CCR5 antagonist maraviroc were observed. Functionally, viruses with reduced ibalizumab susceptibility also displayed high levels of infectivity relative to those of paired baseline viruses. Individualenvclones exhibiting reduced ibalizumab susceptibility contained multiple amino acid changes in different regions relative to the paired baseline clones. In particular, clones with reduced susceptibility to ibalizumab contained fewer potential asparagine-linked glycosylation sites (PNGSs) in variable region 5 (V5) than did paired ibalizumab-susceptible clones. The reduction in ibalizumab susceptibility due to the loss of V5 PNGSs was confirmed by site-directed mutagenesis. Taken together, these findings provide important insights into resistance to this new class of antiretroviral drug.
Key Genomic Changes Necessary for an
In Vivo
Lethal Mouse Marburgvirus Variant Selection Process
Authors: Lofts Loreen L. , Wells Jay B. , Bavari Sina , Warfield Kelly L.
Published: February 2011

Marburgvirus (MARV) infections are generally lethal in humans and nonhuman primates but require
in vivo
lethal mouse variant selection by the serial transfer (passage) of the nonlethal virus into naïve mice to propagate a lethal infection. The passage of progenitor (wild-type) MARV or Ravn virus (RAVV) from infected
scid
BALB/c mouse liver homogenates into immunocompetent BALB/c mice results in the selection of lethal mouse viruses from within the quasispecies sufficient to establish lethality in immunocompetent mice. Genomic analysis in conjunction with the passage history of each mutation detailed the altered primary and secondary structures of the viral genomic RNA throughout the process. Key findings included the following: (i) a VP40:D184N mutation previously identified in the lethal guinea pig MARV genome was the first mutation to occur during the passage of both the MARV and RAVV variants; (ii) there was biased hypermutagenesis in the RAVV variant genome; (iii) there were two identical mutations in lethal mouse MARV and RAVV variants, VP40:Y19H in the PPPY motif and VP40:D184N in a loop structure between the two VP40 domains; (iv) the passage of wild-type MARV and RAVV in mice resulted in the selection of viral variants from among the quasispecies with different genotypes than those of the wild-type viruses; and (v) a lethal mouse RAVV variant had different tissue tropisms distinct from those of its wild-type virus. These studies provide insights into how marburgviruses manipulate the host for enzymes, metabolites, translation regulators, and effectors of the innate immune response to serve as potential viral countermeasures.
Gag Localization and Virus-Like Particle Release Mediated by the Matrix Domain of Human T-Lymphotropic Virus Type 1 Gag Are Less Dependent on Phosphatidylinositol-(4,5)-Bisphosphate than Those Mediated by the Matrix Domain of HIV-1 Gag
Authors: Inlora Jingga , Chukkapalli Vineela , Derse David , Ono Akira
Published: February 2011

The human immunodeficiency virus type 1 (HIV-1) Gag matrix (MA) domain facilitates Gag targeting and binding to the plasma membrane (PM) during virus assembly. Interaction with a PM phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P
2
], plays a key role in these MA functions. Previous studies showed that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P
2
, mislocalizes HIV-1 Gag to the cytosol and greatly reduces HIV-1 release efficiency. In this study, we sought to determine the role of the MA-PI(4,5)P
2
interaction in Gag localization and membrane binding of a deltaretrovirus, human T-lymphotropic virus type 1 (HTLV-1). We compared the chimeric HIV-1 Gag (HTMA), in which MA was replaced with HTLV-1 MA, with wild-type HIV-1 and HTLV-1 Gag for PI(4,5)P
2
dependence. Our results demonstrate that, unlike HIV-1 Gag, subcellular localization of and VLP release by HTLV-1 and HTMA Gag were minimally sensitive to 5ptaseIV overexpression. These results suggest that the interaction of HTLV-1 MA with PI(4,5)P
2
is not essential for HTLV-1 particle assembly. Furthermore, liposome-binding analyses showed that both HTLV-1 and HTMA Gag can bind membrane efficiently even in the absence of PI(4,5)P
2
. Efficient HTLV-1 Gag binding to liposomes was largely driven by electrostatic interaction, unlike that of HIV-1 Gag, which required specific interaction with PI(4,5)P
2
. Furthermore, membrane binding of HTLV-1 Gag
in vitro
was not suppressed by RNA, in contrast to HIV-1 Gag. Altogether, our data suggest that Gag targeting and membrane binding mediated by HTLV-1 MA does not require PI(4,5)P
2
and that distinct mechanisms regulate HIV-1 and HTLV-1 Gag membrane binding.
Interferon Signaling Remains Functional during Henipavirus Infection of Human Cell Lines
Authors: Virtue Elena R. , Marsh Glenn A. , Wang Lin-Fa
Published: February 2011

Henipaviruses encode several proteins from the P gene, of which V and W have been demonstrated by gene-based transfection studies to antagonize the innate immune response, blocking both type I interferon production and signaling. This study examines the effects of henipavirus infection on the innate immune response in human cell lines. We report that henipavirus infection does not result in interferon production, with the virus antagonizing this response. In contrast to published transfection studies, our study found that the interferon signaling pathways are only partially blocked by henipavirus infection of human cell lines.
Vif Substitution Enables Persistent Infection of Pig-Tailed Macaques by Human Immunodeficiency Virus Type 1
Authors: Thippeshappa Rajesh , Polacino Patricia , Yu Kimata Monica T. , Siwak Edward B. , Anderson David , Wang Weiming , Sherwood Laura , Arora Reetakshi , Wen Michael , Zhou Paul , Hu Shiu-Lok , Kimata Jason T.
Published: February 2011

Among Old World monkeys, pig-tailed macaques (Pt) are uniquely susceptible to human immunodeficiency virus type 1 (HIV-1), although the infection does not persist. We demonstrate that the susceptibility of Pt T cells to HIV-1 infection is due to the absence of postentry inhibition by a TRIM5 isoform. Notably, substitution of the viral infectivity factor protein, Vif, with that from pathogenic SIVmne enabled replication of HIV-1 in Pt T cells
in vitro
. When inoculated into juvenile pig-tailed macaques, the Pt-tropic HIV-1 persistently replicated for more than 1.5 to 2 years, producing low but measurable plasma viral loads and persistent proviral DNA in peripheral blood mononuclear cells. It also elicited strong antibody responses. However, there was no decline in CD4
+
T cells or evidence of disease. Surprisingly, the Pt-tropic HIV-1 was rapidly controlled when inoculated into newborn Pt macaques, although it transiently rebounded after 6 months. We identified two notable differences between the Pt-tropic HIV-1 and SIVmne. First, SIV Vif does not associate with Pt-tropic HIV-1 viral particles. Second, while Pt-tropic HIV-1 degrades both Pt APOBEC3G and APOBEC3F, it prevents their inclusion in virions to a lesser extent than pathogenic SIVmne. Thus, while SIV Vif is necessary for persistent infection by Pt-tropic HIV-1, improved expression and inhibition of APOBEC3 proteins may be required for robust viral replication
in vivo
. Additional adaptation of the virus may also be necessary to enhance viral replication. Nevertheless, our data suggest the potential for the pig-tailed macaque to be developed as an animal model of HIV-1 infection and disease.
Influence of Gag-Protease-Mediated Replication Capacity on Disease Progression in Individuals Recently Infected with HIV-1 Subtype C
Authors: Wright Jaclyn K. , Novitsky Vladimir , Brockman Mark A. , Brumme Zabrina L. , Brumme Chanson J. , Carlson Jonathan M. , Heckerman David , Wang Bingxia , Losina Elena , Leshwedi Mopo , van der Stok Mary , Maphumulo Lungile , Mkhwanazi Nompumelelo , Chonco Fundisiwe , Goulder Philip J. R. , Essex Max , Walker Bruce D. , Ndung'u Thumbi
Published: February 2011

HLA class I-mediated selection of immune escape mutations in functionally important Gag epitopes may partly explain slower disease progression in HIV-1-infected individuals with protective HLA alleles. To investigate the impact of Gag function on disease progression, the replication capacities of viruses encoding Gag-protease from 60 individuals in early HIV-1 subtype C infection were assayed in an HIV-1-inducible green fluorescent protein reporter cell line and were correlated with subsequent disease progression. Replication capacities did not correlate with viral load set points (
P
= 0.37) but were significantly lower in individuals with below-median viral load set points (
P
= 0.03), and there was a trend of correlation between lower replication capacities and lower rates of CD4 decline (
P
= 0.09). Overall, the proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was negatively associated with replication capacities (
P
= 0.04), but host HLA-B-specific polymorphisms were associated with higher viral load set points (
P
= 0.01). Further, polymorphisms associated with host-specific protective HLA alleles were linked with higher viral load set points (
P
= 0.03). These data suggest that transmission or early HLA-driven selection of Gag polymorphisms results in reduced early cytotoxic T-lymphocyte (CTL) responses and higher viral load set points. In support of the former, 46% of individuals with nonprotective alleles harbored a Gag polymorphism exclusively associated with a protective HLA allele, indicating a high rate of their transmission in sub-Saharan Africa. Overall, HIV disease progression is likely to be affected by the ability to mount effective Gag CTL responses as well as the replication capacity of the transmitted virus.
Identification of Human Parainfluenza Virus Type 2 (HPIV-2) V Protein Amino Acid Residues That Reduce Binding of V to MDA5 and Attenuate HPIV-2 Replication in Nonhuman Primates
Authors: Schaap-Nutt Anne , Higgins Caraline , Amaro-Carambot Emerito , Nolan Sheila M. , D'Angelo Christopher , Murphy Brian R. , Collins Peter L. , Schmidt Alexander C.
Published: February 2011

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-β induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-β induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth
in vitro
. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.
Promoting calls to a quitline: quantifying the influence of message theme, strong negative emotions and graphic images in television advertisements
Authors: Farrelly Matthew C , Davis Kevin C , Nonnemaker James M , Kamyab Kian , Jackson Christine
Published: February 2011

Objective

To understand the relative effectiveness of television advertisements that differ in their thematic focus and portrayals of negative emotions and/or graphic images in promoting calls to a smokers' quitline.

Methods

Regression analysis is used to explain variation in quarterly media market-level per smoker calls to the New York State Smokers' Quitline from 2001 to 2009. The primary independent variable is quarterly market-level delivery of television advertisements measured by target audience rating points (TARPs). Advertisements were characterised by their overall objective—promoting cessation, highlighting the dangers of secondhand smoke (SHS) or other—and by their portrayals of strong negative emotions and graphic images.

Results

Per smoker call volume is positively correlated with total TARPs (p<0.001), and cessation advertisements are more effective than SHS advertisements in promoting quitline call volume. Advertisements with graphic images only or neither strong negative emotions nor graphic images are associated with higher call volume with similar effect sizes. Call volume was not significantly associated with the number of TARPs for advertisements with strong negative emotions only (p=0.71) or with both graphic images and strong emotions (p=0.09).

Conclusions

Exposure to television advertisements is strongly associated with quitline call volume, and both cessation and SHS advertisements can be effective. The use of strong negative emotions in advertisements may be effective in promoting smoking cessation in the population but does not appear to influence quitline call volume. Further research is needed to understand the role of negative emotions in promoting calls to quitlines and cessation more broadly among the majority of smokers who do not call quitlines.
Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A
Authors: Wanichawan Pimthanya , Louch William E. , Hortemo Kristin H. , Austbø Bjørg , Lunde Per Kristian , Scott John D. , Sejersted Ole M. , Carlson Cathrine R.
Published: February 2011

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.
Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway
Authors: Shatanawi Alia , Romero Maritza J. , Iddings Jennifer A. , Chandra Surabhi , Umapathy Nagavedi S. , Verin Alexander D. , Caldwell Ruth B. , Caldwell R. William
Published: February 2011

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT1 receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg−1·day−1) or arginase (ABH, 8 mg·kg−1·day−1) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT1 receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.
Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells
Authors: Gallant Cynthia , Appel Sarah , Graceffa Philip , Leavis Paul , Lin Jim Jung-Ching , Gunning Peter W. , Schevzov Galina , Chaponnier Christine , DeGnore Jon , Lehman William , Morgan Kathleen G.
Published: February 2011

Tropomyosin (Tm) is known to be an important gatekeeper of actin function. Tm isoforms are encoded by four genes, and each gene produces several variants by alternative splicing, which have been proposed to play roles in motility, proliferation, and apoptosis. Smooth muscle studies have focused on gizzard smooth muscle, where a heterodimer of Tm from the α-gene (Tmsm-α) and from the β-gene (Tmsm-β) is associated with contractile filaments. In this study we examined Tm in differentiated mammalian vascular smooth muscle (dVSM). Liquid chromatography-tandem mass spectrometry (LC MS/MS) analysis and Western blot screening with variant-specific antibodies revealed that at least five different Tm proteins are expressed in this tissue: Tm6 (Tmsm-α) and Tm2 from the α-gene, Tm1 (Tmsm-β) from the β-gene, Tm5NM1 from the γ-gene, and Tm4 from the δ-gene. Tm6 is by far most abundant in dVSM followed by Tm1, Tm2, Tm5NM1, and Tm4. Coimmunoprecipitation and coimmunofluorescence studies demonstrate that Tm1 and Tm6 coassociate with different actin isoforms and display different intracellular localizations. Using an antibody specific for cytoplasmic γ-actin, we report here the presence of a γ-actin cortical cytoskeleton in dVSM cells. Tm1 colocalizes with cortical cytoplasmic γ-actin and coprecipitates with γ-actin. Tm6, on the other hand, is located on contractile bundles. These data indicate that Tm1 and Tm6 do not form a classical heterodimer in dVSM but rather describe different functional cellular compartments.
Protein composition and function of red and white skeletal muscle mitochondria
Authors: Glancy Brian , Balaban Robert S.
Published: February 2011

Red and white muscles are faced with very different energetic demands. However, it is unclear whether relative mitochondrial protein expression is different between muscle types. Mitochondria from red and white porcine skeletal muscle were isolated with a Percoll gradient. Differences in protein composition were determined using blue native (BN)-PAGE, two-dimensional differential in gel electrophoresis (2D DIGE), optical spectroscopy, and isobaric tag for relative and absolute quantitation (iTRAQ). Complex IV and V activities were compared using BN-PAGE in-gel activity assays, and maximal mitochondrial respiration rates were assessed using pyruvate (P) + malate (M), glutamate (G) + M, and palmitoyl-carnitine (PC) + M. Without the Percoll step, major cytosolic protein contamination was noted for white mitochondria. Upon removal of contamination, very few protein differences were observed between red and white mitochondria. BN-PAGE showed no differences in the subunit composition of Complexes I–V or the activities of Complexes IV and V. iTRAQ analysis detected 358 mitochondrial proteins, 69 statistically different. Physiological significance may be lower: at a 25% difference, 48 proteins were detected; at 50%, 14 proteins were detected; and 3 proteins were detected at a 100%. Thus any changes could be argued to be physiologically modest. One area of difference was fat metabolism where four β-oxidation enzymes were ∼25% higher in red mitochondria. This was correlated with a 40% higher rate of PC+M oxidation in red mitochondria compared with white mitochondria with no differences in P+M and G+M oxidation. These data suggest that metabolic demand differences between red and white muscle fibers are primarily matched by the number of mitochondria and not by significant alterations in the mitochondria themselves.
Fibrosis is regulated by Th2 and Th17 responses and by dynamic interactions between fibroblasts and macrophages
Authors: Barron Luke , Wynn Thomas A.
Published: February 2011

Dysregulated wound healing leads to fibrosis, whereby fibroblasts synthesize excess extracellular matrix and scarring impairs proper organ function. Although fibrotic diseases arise from diverse causes and display heterogeneous features, fibrosis commonly associates with chronic inflammation. Recent discoveries reinforce the idea that communication between fibroblasts, macrophages, and CD4 T cells integrates the processes of wound healing and host defense. Signals between macrophages and fibroblasts can exacerbate, suppress, or reverse fibrosis. Fibroblasts and macrophages are activated by T cells, but their activation also engages negative feedback loops that reduce fibrosis by restraining the immune response, particularly when the Th2 cytokine IL-13 contributes to pathology. Thus the interactions among fibroblasts, macrophages, and CD4 T cells likely play general and critical roles in initiating, perpetuating, and resolving fibrosis in both experimental and clinical conditions.
Inhibition of heme oxygenase augments tubular sodium reabsorption
Authors: Jackson Keith E. , Jackson Debra W. , Quadri Syed , Reitzell Marshall J. , Navar L. Gabriel
Published: February 2011

Heme oxygenase (HO) catalyzes the degradation of heme to form iron, biliverdin, and carbon monoxide (CO). The vascular actions of CO include direct vasodilation of vascular smooth muscle and indirect vasoconstriction through inhibition of nitric oxide synthase (NOS). This study was performed to examine the effects in the kidney of inhibition of heme oxygenase alone or combined with NOS inhibition. Chromium mesoporphyrin (CrMP; 45 μmol/kg ip), a photostable HO inhibitor, was given to control rats and NG-nitro-l-arginine methyl ester (l-NAME)-treated hypertensive rats (50 mg·kg−1·day−1, 12 h, 4 days). In control animals, CrMP decreased CO levels, renal HO-1 levels, urine volume, and sodium excretion, but had no effect on arterial pressure, renal blood flow (RBF), plasma renin activity (PRA), or glomerular filtration rate (GFR). In l-NAME-treated hypertensive rats, CrMP decreased endogenous CO and renal HO-1 levels and had no effect on arterial pressure, RBF, or GFR but decreased sodium and water excretion in a similar manner to control animals. An increase in PRA was observed in untreated rats but not in l-NAME-infused rats, indicating that this effect is associated with an absent NO system. The results suggest that inhibition of HO promotes water and sodium excretion by a direct tubular action that is independent of renal hemodynamics or the NO system.
Role of smooth muscle protein SM22α in glomerular epithelial cell injury
Authors: Marshall Caroline B. , Krofft Ron D. , Blonski Mary J. , Kowalewska Jolanta , Logar Christine M. , Pippin Jeffrey W. , Kim Francis , Feil Robert , Alpers Charles E. , Shankland Stuart J.
Published: February 2011

Podocytes are considered terminally differentiated cells in the mature kidney under normal conditions. In the face of injury, podocytes may proceed along several possible pathways, including dedifferentiation and proliferation, persistent cell cycle arrest, hypertrophy, apoptosis, or necrosis. There is mounting evidence that transdifferentiation into a dysregulated phenotype may also be a potential cell fate. We have previously reported that the transcript of SM22α, an actin-binding protein considered one of the earliest markers of smooth muscle differentiation, is upregulated nearly 70-fold in glomeruli of rats with passive Heymann nephritis (PHN). In contrast, the SM22α transcript is absent in normal adult rat glomeruli. The purpose of this study was to define SM22α's expression during kidney development and its role in glomerular diseases characterized by podocyte injury and proteinuria. During glomerulogenesis and podocyte differentiation, SM22α was expressed in glomeruli. This expression disappeared with glomerular maturation. Along with SM22α induction in PHN, confirmed at both mRNA and protein levels, SM22α was also induced across a broad range of proteinuric diseases, including experimental animal models (puromycin aminonucleoside nephropathy, adriamycin nephropathy, passive nephrotoxic nephritis, and diet-induced obesity) and human diseases (collapsing glomerulopathy, diabetic nephropathy, classic focal segmental glomerulosclerosis, IgA nephropathy, minimal-change disease, membranous nephropathy, and membranoproliferative glomerulonephritis). Crescentic glomerulonephritis was induced in SM22α +/+ and SM22α −/− mice by intraperitoneal injection of sheep anti-rabbit glomeruli antibody 12.5 mg/20 g body wt × 2 doses ( n = 12–15/group), with mice euthanized at 7 and 14 days. Compared with SM22α −/− mice, SM22α +/+ mice demonstrated worse disease by histopathological parameters. In addition, there was greater apoptosis (cleaved caspase-3 immunostaining), fewer podocytes (Wilms' tumor-1 immunostaining), and less proliferation (Ki-67 immunostaining) in diseased SM22α +/+ mice. Furthermore, there was decreased activation of Erk1/2 in diseased SM22α +/+ mice. We conclude that the de novo expression of SM22α in glomerular epithelial cells affects the course of crescentic glomerulonephritis.
Alterations in chromatin are associated with increases in collagen III expression in aging nephropathy
Authors: Abrass Christine K. , Hansen Kim , Popov Viorica , Denisenko Oleg
Published: February 2011

Aging nephropathy is a slowly progressive fibrotic process that affects all compartments of the kidney and eventually impairs kidney function; however, little is known about the mechanisms that contribute to this process. These studies examined the epigenetic control of expression of collagen III (Col3a1), a matrix protein that contributes to kidney fibrosis. Using real-time PCR, Western blotting, and chromatin immunoprecipitation assay of kidneys harvested from 4- and 24-mo-old ad libitum-fed F344 rats, we found increased transcription of Col3a1 that was associated with increased RNA polymerase II recruitment despite elevated posttranslational histone modification (H3K27me3) normally associated with gene silencing. A reduction in the density of another repressive modification (H3K9me3) at the Col3a1 locus in aged rats suggests that cooperation between Polycomb- and heterochromatin-mediated systems are required to maintain repression of the Col3a1 gene. These findings demonstrate alterations in epigenetic control of gene expression in association with the fibrosis of aging nephropathy.
Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPARγ, and HO-1
Authors: Wu Qing Qing , Wang Yanxia , Senitko Martin , Meyer Colin , Wigley W. Christian , Ferguson Deborah A. , Grossman Eric , Chen Jianlin , Zhou Xin J. , Hartono John , Winterberg Pamela , Chen Bo , Agarwal Anapam , Lu Christopher Y.
Published: February 2011

Ischemic acute kidney injury (AKI) triggers expression of adaptive (protective) and maladaptive genes. Agents that increase expression of protective genes should provide a therapeutic benefit. We now report that bardoxolone methyl (BARD) ameliorates ischemic murine AKI as assessed by both renal function and pathology. BARD may exert its beneficial effect by increasing expression of genes previously shown to protect against ischemic AKI, NF-E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor-γ (PPARγ), and heme oxygenase 1 (HO-1). Although we found that BARD alone or ischemia-reperfusion alone increased expression of these genes, the greatest increase occurred after the combination of both ischemia-reperfusion and BARD. BARD had a different mode of action than other agents that regulate PPARγ and Nrf2. Thus we report that BARD regulates PPARγ, not by acting as a ligand but by increasing the amount of PPARγ mRNA and protein. This should increase ligand-independent effects of PPARγ. Similarly, BARD increased Nrf2 mRNA; this increased Nrf2 protein by mechanisms in addition to the prolongation of Nrf2 protein half-life previously reported. Finally, we localized expression of these protective genes after ischemia and BARD treatment. Using double-immunofluorescence staining for CD31 and Nrf2 or PPARγ, we found increased Nrf2 and PPARγ on glomerular endothelia in the cortex; Nrf2 was also present on cortical peritubular capillaries. In contrast, HO-1 was localized to different cells, i.e., tubules and interstitial leukocytes. Although Nrf2-dependent increases in HO-1 have been described, our data suggest that BARD's effects on tubular and leukocyte HO-1 during ischemic AKI may be Nrf2 independent. We also found that BARD ameliorated cisplatin nephrotoxicity.
Protein kinase C-ε activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules
Authors: Nowak Grazyna , Bakajsova Diana , Samarel Allen M.
Published: February 2011

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307–F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F0F1-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F0F1-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC.
HB-EGF release mediates glucose-induced activation of the epidermal growth factor receptor in mesangial cells
Authors: Uttarwar L. , Peng F. , Wu D. , Kumar S. , Gao B. , Ingram A. J. , Krepinsky J. C.
Published: February 2011

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We showed that transactivation of the epidermal growth factor receptor (EGFR) is an important mediator of matrix upregulation in mesangial cells (MC) in response to high glucose (HG). Here, we study the mechanism of EGFR transactivation. In primary MC, EGFR transactivation by 1 h of HG (30 mM) was unaffected by inhibitors of protein kinase C, reactive oxygen species, or the angiotensin II AT1 receptor. However, general metalloprotease inhibition, as well as specific inhibitors of heparin-binding EGF-like growth factor (HB-EGF), prevented both EGFR and downstream Akt activation. HB-EGF was released into the medium by 30 min of HG, and this depended on metalloprotease activity. One of the metalloproteases shown to cleave proHB-EGF is ADAM17 (TACE). HG, but not an osmotic control, activated ADAM17, and its inhibition prevented EGFR and Akt activation and HB-EGF release into the medium. siRNA to either ADAM17 or HB-EGF prevented HG-induced EGFR transactivation. We previously showed that EGFR/Akt signaling increases transforming growth factor (TGF)-β1 transcription through the transcription factor activator protein (AP)-1. HG-induced AP-1 activation, as assessed by EMSA, was abrogated by inhibitors of metalloproteases, HB-EGF and ADAM17. HB-EGF and ADAM17 siRNA also prevented AP-1 activation. Finally, these inhibitors and siRNA prevented TGF-β1 upregulation by HG. Thus, HG-induced EGFR transactivation in MC is mediated by the release of HB-EGF, which requires activity of the metalloprotease ADAM17. The mechanism of ADAM17 activation awaits identification. Targeting upstream mediators of EGFR transactivation including HB-EGF or ADAM17 provides novel therapeutic targets for the treatment of diabetic nephropathy.
Small interfering RNA targeting IKKβ prevents renal ischemia-reperfusion injury in rats
Authors: Wan Xin , Fan Li , Hu Bo , Yang Jing , Li Xing , Chen Xin , Cao Changchun
Published: February 2011

The transcription factor NF-κB has been found critical to the pathogenesis of renal ischemia-reperfusion injury, which is a major cause of acute kidney injury (AKI). Activation of NF-κB is dependent upon the activation of the specific inhibitory κB kinase (IKK) subunit IKKβ. Here, we investigate whether small interfering RNA (siRNA) targeting IKKβ protects rats from renal ischemia- reperfusion injury in vivo. Renal ischemia-reperfusion injury was induced by clamping the renal artery for 45 min. Rats were treated before ischemia with IKKβ siRNA or scrambled siRNA, administered by renal artery injection. Treated animals were evaluated for renal IKKβ protein and mRNA expression, blood biochemistry, tissue histopathology, NF-κB/DNA binding activity, and expression of two downstream inflammatory cytokines, neutrophil gelatinase-associated lipocalin (NGAL) and IL-18. A local injection of IKKβ siRNA resulted in inhibition of renal IKKβ gene expression, NF-κB/DNA binding activity, and expression of NGAL and IL-18. Rats pretreated with IKKβ siRNA had significantly less blood urea nitrogen and serum creatinine levels and less renal tubular damage scores. Consequently, our data confirm that targeted silencing of IKKβ using siRNA substantially diminishes kidney injury and inflammation following ischemia-reperfusion.
Temporal complexity in clinical manifestations of lung disease
Authors: Frey Urs , Maksym Geoffrey , Suki Béla
Published: February 2011

In this review, we summarize results of recent research on the temporal variability of lung function, symptoms, and inflammatory biomarkers. Specifically, we demonstrate how fluctuation analysis borrowed from statistical physics can be used to gain insight into neurorespiratory control and complex chronic dynamic diseases such as asthma viewed as a system of interacting components (e.g., inflammatory, immunological, and mechanical). Fluctuation analysis tools are based on quantifying the distribution and the short- and long-term temporal history of tidal breathing and lung function parameters to assess neurorespiratory control and monitor chronic disease. The latter includes the assessment of severity and disease control, the impact of treatment and environmental triggers, the temporal characterization of disease phenotypes, and the individual risk of exacerbation. While in many cases specific mechanistic insight into the fluctuations still awaits further research, appropriate analyses of the fluctuations already impact on clinical science and practice.
Temporally selective processing of communication signals by auditory midbrain neurons
Authors: Elliott Taffeta M. , Christensen-Dalsgaard Jakob , Kelley Darcy B.
Published: February 2011

Perception of the temporal structure of acoustic signals contributes critically to vocal signaling. In the aquatic clawed frog Xenopus laevis, calls differ primarily in the temporal parameter of click rate, which conveys sexual identity and reproductive state. We show here that an ensemble of auditory neurons in the laminar nucleus of the torus semicircularis (TS) of X. laevis specializes in encoding vocalization click rates. We recorded single TS units while pure tones, natural calls, and synthetic clicks were presented directly to the tympanum via a vibration-stimulation probe. Synthesized click rates ranged from 4 to 50 Hz, the rate at which the clicks begin to overlap. Frequency selectivity and temporal processing were characterized using response-intensity curves, temporal-discharge patterns, and autocorrelations of reduplicated responses to click trains. Characteristic frequencies ranged from 140 to 3,250 Hz, with minimum thresholds of −90 dB re 1 mm/s at 500 Hz and −76 dB at 1,100 Hz near the dominant frequency of female clicks. Unlike units in the auditory nerve and dorsal medullary nucleus, most toral units respond selectively to the behaviorally relevant temporal feature of the rate of clicks in calls. The majority of neurons (85%) were selective for click rates, and this selectivity remained unchanged over sound levels 10 to 20 dB above threshold. Selective neurons give phasic, tonic, or adapting responses to tone bursts and click trains. Some algorithms that could compute temporally selective receptive fields are described.
Specializations of a pheromonal glomerulus in the Drosophila olfactory system
Authors: Agarwal Gautam , Isacoff Ehud
Published: February 2011

Insect pheromonal glomeruli are thought to track the fine spatiotemporal features of one or a few odorants to aid conspecific localization. However, it is not clear whether they function differently from generalist glomeruli, which respond to many odorants. In this study, we test how DA1, a model pheromonal glomerulus in the fruit fly, represents the spatial and temporal properties of its input, compared with other glomeruli. We combine calcium imaging and electrical stimulation in an isolated brain preparation for a simultaneous, unbiased comparison of the functional organization of many glomeruli. In contrast to what is found in other glomeruli, we find that ipsilateral and contralateral stimuli elicit distinct spatial patterns of activity within DA1. DA1's output shows a greater preference for ipsilateral stimuli in males than in females. DA1 experiences greater and more rapid inhibition than other glomeruli, allowing it to report slight interantennal delays in stimulus onset in a “winner-take-all” manner. DA1's ability to encode spatiotemporal input features distinguishes it from other glomeruli in the fruit fly antennal lobe but relates it to pheromonal glomeruli in other insect species. We propose that DA1 is specialized to help the fly localize and orient with respect to pheromone sources.
Kinetic modeling of Nav1.7 provides insight into erythromelalgia-associated F1449V mutation
Authors: Gurkiewicz Meron , Korngreen Alon , Waxman Stephen G. , Lampert Angelika
Published: February 2011

Gain-of-function mutations of the voltage-gated sodium channel (VGSC) Nav1.7 have been linked to human pain disorders. The mutation F1449V, located at the intracellular end of transmembrane helix S6 of domain III, induces the inherited pain syndrome erythromelalgia. A kinetic model of wild-type (WT) and F1449V Nav1.7 may provide a basis for predicting putative intraprotein interactions. We semiautomatically constrained a Markov model using stochastic search algorithms and whole cell patch-clamp recordings from human embryonic kidney cells transfected with Nav1.7 and its F1449V mutation. The best models obtained simulated known differences in action potential thresholds and firing patterns in spinal sensory neurons expressing WT and F1449V. The most suitable Markov model consisted of three closed, one open, and two inactivated states. The model predicted that the F1449V mutation shifts occupancy of the closed states closer to the open state, making it easier for the channel pore to open. It also predicted that F1449V's second inactivated state is more than four times more likely to be occupied than the equivalent state in WT at hyperpolarized potentials, although the mutation still lowered the firing threshold of action potentials. The differences between WT and F1449V were not limited to a single transition. Thus a point mutation in position F1449, while phenotypically most probably affecting the activation gate, may also modify channel functions mediated by structures in more distant areas of the channel protein.
Parasagittally aligned, mGluR1-dependent patches are evoked at long latencies by parallel fiber stimulation in the mouse cerebellar cortex in vivo
Authors: Wang Xinming , Chen Gang , Gao Wangcai , Ebner Timothy J.
Published: February 2011

The parallel fibers (PFs) in the cerebellar cortex extend several millimeters along a folium in the mediolateral direction. The PFs are orthogonal to and cross several parasagittal zones defined by the olivocerebellar and corticonuclear pathways and the expression of molecular markers on Purkinje cells (PCs). The functions of these two organizations remain unclear, including whether the bands respond similarly or differentially to PF input. By using flavoprotein imaging in the anesthetized mouse in vivo, this study demonstrates that high-frequency PF stimulation, which activates a beamlike response at short latency, also evokes patches of activation at long latencies. These patches consist of increased fluorescence along the beam at latencies of 20–25 s with peak activation at 35 s. The long-latency patches are completely blocked by the type 1 metabotropic glutamate receptor (mGluR1) antagonist LY367385. Conversely, the AMPA and NMDA glutamate receptor antagonists DNQX and APV have little effect. Organized in parasagittal bands, the long-latency patches align with zebrin II-positive PC stripes. Additional Ca2+imaging demonstrates that the patches reflect increases in intracellular Ca2+. Both the PLCβ inhibitor U73122 and the ryanodine receptor inhibitor ryanodine completely block the long-latency patches, indicating that the patches are due to Ca2+release from intracellular stores. Robust, mGluR1-dependent long-term potentiation (LTP) of the patches is induced using a high-frequency PF stimulation conditioning paradigm that generates LTP of PF-PC synapses. Therefore, the parasagittal bands, as defined by the molecular compartmentalization of PCs, respond differentially to PF inputs via mGluR1-mediated release of internal Ca2+.