bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Real-Time PCR Assays for Detection of the
Streptococcus milleri
Group from Cystic Fibrosis Clinical Specimens by Targeting the
cpn60
and 16S rRNA Genes
Authors: Olson A. B. , Sibley C. D. , Schmidt L. , Wilcox M. A. , Surette M. G. , Corbett C. R.
Published: February 2010

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the
Streptococcus anginosus
group (
S. anginosus
,
S. constellatus
, and
S. intermedius
), herein referred to as the “
Streptococcus milleri
group” (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed “McKay agar.” McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The
cpn60
gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the
S. constellatus
(96%),
S. intermedius
(94%), and
S. anginosus
(60%) strains to be correctly identified. To increase specificity for
S. anginosus
, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for
S. anginosus
(100%), while the 16s_SCI assay is specific for
S. constellatus
and
S. intermedius
(100%). These assays can detect <10 genome equivalents in pure culture and >10
4
genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.
Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-Year Analysis of Susceptibilities of
Candida
Species to Fluconazole and Voriconazole as Determined by CLSI Standardized Disk Diffusion
Authors: Pfaller M. A. , Diekema D. J. , Gibbs D. L. , Newell V. A. , Ellis D. , Tullio V. , Rodloff A. , Fu W. , Ling T. A.
Published: February 2010

Fluconazole
in vitro
susceptibility test results for 256,882 isolates of
Candida
spp. were collected from 142 sites in 41 countries from June 1997 to December 2007. Data were collected for 197,619 isolates tested with voriconazole from 2001 to 2007. A total of 31 different species of
Candida
were isolated. Increased rates of isolation of the common non-
albicans
species
C. glabrata
(10.2% to 11.7%),
C. tropicalis
(5.4% to 8.0%), and
C. parapsilosis
(4.8% to 5.6%) were noted when the time periods 1997 to 2000 and 2005 to 2007 were compared. Investigators tested clinical isolates of
Candida
spp. by the CLSI M44-A disk diffusion method. Overall, 90.2% of
Candida
isolates tested were susceptible (S) to fluconazole; however, 13 of 31 species identified exhibited decreased susceptibility (<75% S), similar to that seen with the resistant (R) species
C. glabrata
and
C. krusei
. Among 197,619 isolates of
Candida
spp. tested against voriconazole, 95.0% were S and 3% were R. About 30% of fluconazole-R isolates of
C. albicans
,
C. glabrata
,
C. tropicalis
,
C. rugosa
,
C. lipolytica
,
C. pelliculosa
,
C. apicola
,
C. haemulonii
,
C. humicola
,
C. lambica
, and
C. ciferrii
remained S to voriconazole. An increase in fluconazole resistance over time was seen with
C. parapsilosis
,
C. guilliermondii
,
C. lusitaniae
,
C. sake
, and
C. pelliculosa
. Among the emerging fluconazole-R species were
C. guilliermondii
(11.4% R),
C. inconspicua
(53.2% R),
C. rugosa
(41.8% R), and
C. norvegensis
(40.7% R). The rates of isolation of
C. rugosa
,
C. inconspicua
, and
C. norvegensis
increased by 5- to 10-fold over the 10.5-year study period.
C. guilliermondii
and
C. rugosa
were most prominent in Latin America, whereas
C. inconspicua
and
C. norvegensis
were most common in Eastern European countries. This survey identifies several less-common species of
Candida
with decreased susceptibility to azoles. These organisms may pose a future threat to optimal antifungal therapy and underscore the importance of prompt and accurate species identification and antifungal susceptibility testing.
Recruitment of Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 to the Virus Particle
Authors: Maringer Kevin , Elliott Gillian
Published: February 2010

Although the herpes simplex virus type 1 (HSV-1) tegument is comprised of a large number of viral and cellular proteins, how and where in the cell these proteins are recruited into the virus structure is poorly understood. We have shown previously that the immediate-early gene product ICP0 is packaged by a mechanism dependent on the major tegument protein VP22, while others have shown a requirement for ICP27. We now extend our studies to show that ICP0 packaging correlates directly with the ability of ICP0 to complex with VP22 in infected cells. ICP27 is not, however, present in this VP22-ICP0 complex but is packaged into the virion in a VP22- and ICP0-independent manner. Biochemical fractionation of virions indicated that ICP0 associates tightly with the virus capsid, but intranuclear capsids contained no detectable ICP0. The RING finger domain of ICP0 and the N terminus of VP22 were both shown to be essential but not sufficient for ICP0 packaging and complex formation. Strikingly, however, the N-terminal region of VP22, while unable to form a complex with ICP0, inhibited its translocation from the nucleus to the cytoplasm. PML degradation by ICP0 was efficient in cells infected with this VP22 mutant virus, confirming that ICP0 retains activity. Hence, we would suggest that VP22 is an important molecular partner of ICP0 that controls at least one of its activities: its assembly into the virion. Moreover, we propose that the pathway by which VP22 recruits ICP0 to the virion may begin in the nucleus prior to ICP0 translocation to its final site of assembly in the cytoplasm.
Human T-Lymphotropic Virus Type 1 Transcription and Chromatin-Remodeling Complexes
Authors: Easley Rebecca , Carpio Lawrence , Guendel Irene , Klase Zachary , Choi Soyun , Kehn-Hall Kylene , Brady John N. , Kashanchi Fatah
Published: February 2010

Human T-lymphotropic virus type 1 (HTLV-1) encodes the viral protein Tax, which is believed to act as a viral transactivator through its interactions with a variety of transcription factors, including CREB and NF-κB. As is the case for all retroviruses, the provirus is inserted into the host DNA, where nucleosomes are deposited to ensure efficient packaging. Nucleosomes act as roadblocks in transcription, making it difficult for RNA polymerase II (Pol II) to proceed toward the 3′ end of the genome. Because of this, a variety of chromatin remodelers can act to modify nucleosomes, allowing for efficient transcription. While a number of covalent modifications are known to occur on histone tails in HTLV-1 infection (i.e., histone acetyltransferases [HATs], histone deacetylases [HDACs], and histone methyltransferases [HMTs]), evidence points to the use of chromatin remodelers that use energy from ATP hydrolysis to remodel nucleosomes. Here we confirm that BRG1, which is the core subunit of eight chromatin-remodeling complexes, is essential not only for Tax transactivation but also for viral replication. This is especially evident when wild-type infectious clones of HTLV-1 are used. BRG1 associates with Tax at the HTLV-1 long terminal repeat (LTR), and coexpression of BRG1 and Tax results in increased rates of transcription. The interaction of BRG1 with Tax additionally recruits the basal transcriptional machinery and removes some of the core histones from the nucleosome at the start site (Nuc 1). When using the BRG1-deficient cell lines SW13, C33A, and TSUPR1, we observed little viral transcription and no viral replication. Importantly, while these three cell lines do not express detectable levels of BRG1, much of the SWI/SNF complex remains assembled in the cells. Knockdown of BRG1 and associated SWI/SNF subunits suggests that the BRG1-utilizing SWI/SNF complex PBAF is responsible for HTLV-1 nucleosome remodeling. Finally, HTLV-1 infection of cell lines with a knockdown in BRG1 or the PBAF complex results in a significant reduction in viral production. Overall, we concluded that BRG1 is required for Tax transactivation and HTLV-1 viral production and that the PBAF complex appears to be responsible for nucleosome remodeling.
Identification and Characterization of Clevudine-Resistant Mutants of Hepatitis B Virus Isolated from Chronic Hepatitis B Patients
Authors: Kwon So Young , Park Yong Kwang , Ahn Sung Hyun , Cho Eun Sook , Choe Won Hyeok , Lee Chang Hong , Kim Byung Kook , Ko Soon Young , Choi Hyo Sun , Park Eun-Sook , Shin Gu Choul , Kim Kyun-Hwan
Published: February 2010

Clevudine (CLV) is a nucleoside analog with potent antiviral activity against chronic hepatitis B virus (HBV) infection. Viral resistance to CLV in patients receiving CLV therapy has not been reported. The aim of this study was to characterize CLV-resistant HBV in patients with viral breakthrough (BT) during long-term CLV therapy. The gene encoding HBV reverse transcriptase (RT) was analyzed from chronic hepatitis B patients with viral BT during CLV therapy. Sera collected from the patients at baseline and at the time of viral BT were studied. To characterize the mutations of HBV isolated from the patients, we subjected the HBV mutants to
in vitro
drug susceptibility assays. Several conserved mutations were identified in the RT domain during viral BT, with M204I being the most common.
In vitro
phenotypic analysis showed that the mutation M204I was predominantly associated with CLV resistance, whereas L229V was a compensatory mutation for the impaired replication of the M204I mutant. A quadruple mutant (L129M, V173L, M204I, and H337N) was identified that conferred greater replicative ability and strong resistance to both CLV and lamivudine. All of the CLV-resistant clones were lamivudine resistant. They were susceptible to adefovir, entecavir, and tenofovir, except for one mutant clone. In conclusion, the mutation M204I in HBV RT plays a major role in CLV resistance and leads to viral BT during long-term CLV treatment. Several conserved mutations may have a compensatory role in replication. Drug susceptibility assays reveal that adefovir and tenofovir are the most effective compounds against CLV-resistant mutants. These data may provide additional therapeutic options for CLV-resistant patients.
Adenovirus Protein E4orf4 Induces Premature APC
Cdc20
Activation in
Saccharomyces cerevisiae
by a Protein Phosphatase 2A-Dependent Mechanism
Authors: Mui Melissa Z. , Roopchand Diana E. , Gentry Matthew S. , Hallberg Richard L. , Vogel Jackie , Branton Philip E.
Published: February 2010

Protein phosphatase 2A (PP2A) has been implicated in cell cycle progression and mitosis; however, the complexity of PP2A regulation via multiple B subunits makes its functional characterization a significant challenge. The human adenovirus protein E4orf4 has been found to induce both high Cdk1 activity and the accumulation of cells in G
2
/M in both mammalian and yeast cells, effects which are largely dependent on the B55/Cdc55 regulatory subunit of PP2A. Thus, E4orf4 represents a unique means by which the function of a specific form of PP2A can be delineated
in vivo
. In
Saccharomyces cerevisiae
, only two PP2A regulatory subunits exist, Cdc55 and Rts1. Here, we show that E4orf4-induced toxicity depends on a functional interaction with Cdc55. E4orf4 expression correlates with the inappropriate reduction of Pds1 and Scc1 in S-phase-arrested cells. The unscheduled loss of these proteins suggests the involvement of PP2A
Cdc55
in the regulation of the Cdc20 form of the anaphase-promoting complex (APC). Contrastingly, activity of the Hct1 form of the APC is not induced by E4orf4, as demonstrated by the observed stability of its substrates. We propose that E4orf4, being a Cdc55-specific inhibitor of PP2A, demonstrates the role of PP2A
Cdc55
in regulating APC
Cdc20
activity.
The Interferon-Induced Gene ISG15 Blocks Retrovirus Release from Cells Late in the Budding Process
Authors: Pincetic Andrew , Kuang Zhizhou , Seo Eun Joo , Leis Jonathan
Published: February 2010

The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process.
Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins
Authors: Plant Ewan P. , Rakauskaitė Rasa , Taylor Deborah R. , Dinman Jonathan D.
Published: February 2010

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed −1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the −1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a “golden mean” model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.
Two N-Linked Glycosylation Sites in the V2 and C2 Regions of Human Immunodeficiency Virus Type 1 CRF01_AE Envelope Glycoprotein gp120 Regulate Viral Neutralization Susceptibility to the Human Monoclonal Antibody Specific for the CD4 Binding Domain
Authors: Utachee Piraporn , Nakamura Shota , Isarangkura-na-ayuthaya Panasda , Tokunaga Kenzo , Sawanpanyalert Pathom , Ikuta Kazuyoshi , Auwanit Wattana , Kameoka Masanori
Published: February 2010

A recombinant human monoclonal antibody, IgG1 b12 (b12), recognizes a conformational epitope on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) gp120 that overlaps the CD4 binding domain. Although b12 is able to broadly neutralize HIV-1 subtype B, C, and D viruses, many HIV-1 CRF01_AE viruses are resistant to b12-mediated neutralization. In this report, we examined the molecular mechanisms underlying the low neutralization susceptibility of CRF01_AE viruses to b12, using recently established CRF01_AE Env recombinant viruses. Our results showed that two potential N-linked glycosylation (PNLG) sites in the V2 and C2 regions of Env gp120 played an important role in regulating the susceptibility of CRF01_AE Env to b12. The locations of these PNLG sites correspond to amino acid positions 186 and 197 in HXB2 Env gp120; thus, they are designated N186 and N197 in this study. Removal of N186 significantly conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones, 65CC4 and 107CC2, while the introduction of N186 reduced the b12 susceptibility of a susceptible CRF01_AE Env clone, 65CC1. In addition, removal of both N186 and N197 conferred the b12 susceptibility of 3 resistant CRF01_AE Env clones, 45PB1, 62PL1, and 101PL1, whereas the removal of either N186 or N197 was not sufficient to confer the b12 susceptibility of these CRF01_AE Env clones. Finally, removal of N197 conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones lacking N186, 55PL1 and 102CC2. Taken together, we propose that two PNLG sites, N186 and N197, in Env gp120 are important determinants of the b12 resistance of CRF01_AE viruses.
Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate
Authors: Banerjee Manidipa , Speir Jeffrey A. , Kwan Maggie H. , Huang Rick , Aryanpur Peyman P. , Bothner Brian , Johnson John E.
Published: February 2010

Divalent metal ions are components of numerous icosahedral virus capsids. Flock House virus (FHV), a small RNA virus of the family
Nodaviridae
, was utilized as an accessible model system with which to address the effects of metal ions on capsid structure and on the biology of virus-host interactions. Mutations at the calcium-binding sites affected FHV capsid stability and drastically reduced virus infectivity, without altering the overall architecture of the capsid. The mutations also altered the conformation of gamma, a membrane-disrupting, virus-encoded peptide usually sequestered inside the capsid, by increasing its exposure under neutral pH conditions. Our data demonstrate that calcium binding is essential for maintaining a pH-based control on gamma exposure and host membrane disruption, and they reveal a novel rationale for the metal ion requirement during virus entry and infectivity. In the light of the phenotypes displayed by a calcium site mutant of FHV, we suggest that this mutant corresponds to an early entry intermediate formed in the endosomal pathway.
Development of an Intergenotypic Hepatitis C Virus (HCV) Cell Culture Method To Assess Antiviral Susceptibilities and Resistance Development of HCV NS3 Protease Genes from HCV Genotypes 1 to 6
Authors: Imhof Ingrid , Simmonds Peter
Published: February 2010

Protease inhibitors (PIs) of hepatitis C virus (HCV) provide an additional or alternative therapy for chronic infection. However, assessment of their efficacy and ability to inhibit replication of different genotypes is hampered by the lack of a convenient animal model or a method for
in vitro
culture of HCV other than the type 1/2-based replicons and the infectious genotype 2a clone JFH1. To address this problem, we constructed a panel of replication-competent chimeric Jc1 (pFK JFH1/J6/C-846) clones containing protease and NS4A coding sequences from all six major genotypes, enabling the determination of replication and the susceptibility to PIs. Chimeras showed substantial variability in replication kinetics, attributable in part to naturally occurring polymorphisms and differing requirements for adaptive mutations in NS3 and NS4A. Through calculation of 50% inhibitory concentrations (IC
50
s) of BILN 2061, measuring reduction in the number of focus-forming units/ml (FFU/ml) and replication inhibition, consistent genotype-associated differences in antiviral susceptibilities were observed. IC
50
s for genotype 1b, 4a, and 6a-derived chimeras (1 to 3 nM) were approximately 100-fold lower than those for genotypes 2a, 3a, and 5a (range, 80 to 720 nM), implying major differences in response to therapy.
In vitro
passage in increasing concentrations of BILN 2061 rapidly induced resistance-associated mutations at position 168 in chimeras of all 6 genotypes and at position 156 in genotypes 1b and 4a, each with substantial variability in the identity of substituted amino acids. The system will allow future comprehensive phenotypic characterization of naturally occurring and treatment-induced mutations for PIs in trial or entering clinical use.
ON LIU'S INFERENCE RULE FOR UNCERTAIN SYSTEMS
Authors: GAO XIN , GAO YUAN , RALESCU DAN A.
Published: February 2010

Liu's inference is a process of deriving consequences from uncertain knowledge or evidence via the tool of conditional uncertainty. Using membership functions, this paper derives some expressions of Liu's inference rule for uncertain systems. This paper also discusses Liu's inference rule with multiple antecedents and with multiple if-then rules.
HIERARCHICAL BAYESIAN FUZZY INFERENCE NETS FOR INTERNAL FAULT DIAGNOSIS OF THREE-PHASE SQUIRREL CAGE INDUCTION MOTOR
Authors: SEKAR BOOMA DEVI , CHEANG TAK SON , DONG MING CHUI , CHAN SI LEONG
Published: February 2010

A generalized Bayesian inference nets model (GBINM) and a new approach for internal fault detection and diagnosis of three-phase induction motor is proposed. The input data set for designing and testing the fault diagnostic system are acquired through on-site experiment. The preprocessed input data and experts' rich diagnostic experience/knowledge are used to define the membership functions and fault fuzzy sets. With GBINM and the defined fault fuzzy sets, the hierarchical Bayesian fuzzy inference nets are constructed to carry out the complex motor fault diagnostic procedure. The propagation of probability is used to address the uncertainties involved in detecting and diagnosing the motor incipient faults. The immense difficulties of defining and assigning statistical parameters, required for calculating the propagation of probability are effectively solved. The validity and effectiveness of the proposed approach is witnessed clearly from the testing results obtained.
LEARNING BAYESIAN NETWORKS FOR REGRESSION FROM INCOMPLETE DATABASES
Authors: FERNÁNDEZ ANTONIO , NIELSEN JENS D. , SALMERÓN ANTONIO
Published: February 2010

In this paper we address the problem of inducing Bayesian network models for regression from incomplete databases. We use mixtures of truncated exponentials (MTEs) to represent the joint distribution in the induced networks. We consider two particular Bayesian network structures, the so-called naïve Bayes and TAN, which have been successfully used as regression models when learning from complete data. We propose an iterative procedure for inducing the models, based on a variation of the data augmentation method in which the missing values of the explanatory variables are filled by simulating from their posterior distributions, while the missing values of the response variable are generated using the conditional expectation of the response given the explanatory variables. We also consider the refinement of the regression models by using variable selection and bias reduction. We illustrate through a set of experiments with various databases the performance of the proposed algorithms.
STUDYING INTEREST MEASURES FOR ASSOCIATION RULES THROUGH A LOGICAL MODEL
Authors: DELGADO MIGUEL , RUIZ M. DOLORES , SÁNCHEZ DANIEL
Published: February 2010

Many papers have addressed the task of proposing a set of convenient axioms that a good rule interestingness measure should fulfil. We provide a new study of the principles proposed until now by means of the logic model proposed by Hájek et al.14 In this model association rules can be viewed as general relations of two itemsets quantified by means of a convenient quantifier.28 Moreover, we propose and justify the addition of two new principles to the three proposed by Piatetsky-Shapiro.27 We also use the logic approach for studying the relation between the different classes of quantifiers and these axioms. We define new classes of quantifiers according to the notions of strong and very strong rules, and we present a quantifier based on the certainty factor measure,317 studying its most salient features.
Calcineurin activity augments cAMP/PKA-dependent activation of V-ATPase in blowfly salivary glands
Authors: Voss Martin , Fechner Lennart , Walz Bernd , Baumann Otto
Published: February 2010

We have examined the role of the Ca2+-dependent protein phosphatase 2B (calcineurin) in the regulation of the vacuolar H+-ATPase (V-ATPase) in blowfly salivary glands. In response to the neurohormone serotonin [5-hydroxytryptamine (5-HT)] and under the mediation of the cAMP/PKA signaling pathway, the secretory cells assemble and activate V-ATPase molecules at the apical membrane. We demonstrate that the inhibition of calcineurin activity by cyclosporin A, by FK-506, or by prevention of the elevation of Ca2+ diminishes the 5-HT-induced assembly and activation of V-ATPase. The effect of calcineurin on V-ATPase is mediated by the cAMP/PKA signaling pathway, with calcineurin acting upstream of PKA, because 1) cyclosporin A does not influence the 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (8-CPT-cAMP)-induced activation of V-ATPase, and 2) the 5-HT-induced rise in cAMP is highly reduced in the presence of cyclosporin A. Moreover, a Ca2+ rise evoked by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid leads to an increase in intracellular cAMP concentration and a calcineurin-mediated PKA-dependent activation of V-ATPase. We propose that calcineurin activity mediates cross talk between the inositol 1,4,5-trisphosphate/Ca2+ and the cAMP/PKA signaling pathways, thereby augmenting the 5-HT-induced rise in cAMP and thus the cAMP/PKA-mediated activation of V-ATPase.
Glucose downregulation of PKG-I protein mediates increased thrombospondin1-dependent TGF-β activity in vascular smooth muscle cells
Authors: Wang Shuxia , Lincoln Thomas M. , Murphy-Ullrich Joanne E.
Published: February 2010

Diabetes is a major predictor of in-stent restenosis, which is associated with fibroproliferative remodeling of the vascular wall due to increased transforming growth factor-β (TGF-β) action. It is well established that thrombospondin1 (TSP1) is a major regulator of TGF-β activation in renal and cardiac complications of diabetes. However, the role of the TSP1-TGF-β pathway in macrovascular diabetic complications, including restenosis, has not been addressed. In mesangial cells, high glucose concentrations depress protein kinase G (PKG) activity, but not PKG-I protein, thereby downregulating transcriptional repression of TSP1. Previously, we showed that high glucose downregulates PKG-I protein expression by vascular smooth muscle cells (VSMCs) through altered NADPH oxidase signaling. In the present study, we investigated whether high glucose regulation of PKG protein and activity in VSMCs similarly regulates TSP1 expression and downstream TGF-β activity. These studies showed that high glucose stimulates both TSP1 expression and TGF-β bioactivity in primary murine aortic smooth muscle cells (VSMCs). TSP1 is responsible for the increased TGF-β bioactivity under high glucose conditions, because treatment with anti-TSP1 antibody, small interfering RNA-TSP1, or an inhibitory peptide blocked glucose-mediated increases in TGF-β activity and extracellular matrix protein (fibronectin) expression. Overexpression of constitutively active PKG, but not the PKG-I protein, inhibited glucose-induced TSP1 expression and TGF-β bioactivity, suggesting that PKG protein expression is insufficient to regulate TSP1 expression. Together, these data establish that glucose-mediated downregulation of PKG levels stimulates TSP1 expression and enhances TGF-β activity and matrix protein expression, which can contribute to vascular remodeling in diabetes.
Inactivation of L-type calcium channel modulated by HCN2 channel
Authors: Lin Yen-Chang , Huang Jianying , Zhang Qi , Hollander John M. , Frisbee Jefferson C. , Martin Karen H. , Nestor Casey , Goodman Robert , Yu Han-Gang
Published: February 2010

Ca2+ entry is delicately controlled by inactivation of L-type calcium channel (LTCC) composed of the pore-forming subunit α1C and the auxiliary subunits β1 and α2δ. Calmodulin is the key protein that interacts with the COOH-terminal motifs of α1C, leading to the fine control of LTCC inactivation. In this study we show evidence that a hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, can act as a nonchannel regulatory protein to narrow the L-type Ca2+ channel current-voltage curve. In the absence of LTCC auxiliary subunits, HCN2 can induce α1C inactivation. Without α2δ, HCN2-induced fast inactivation of α1C requires calmodulin. With α2δ, the α1C/HCN2/α2δ channel inactivation does not require calmodulin. In contrast, β1-subunit plays a relatively minor role in the interaction of α1C with HCN2. The NH2 terminus of HCN2 and the IQ motif of α1C subunit are required for α1C/HCN2 channel interaction. Ca2+ channel inactivation is significantly slowed in hippocampus neurons (HNs) overexpressing HCN2 mutant lacking NH2 terminus and accelerated in HNs overexpressing the wild-type HCN2 compared with HN controls. Collectively, these results revealed a potentially novel protection mechanism for achieving the LTCC inactivation via interaction with HCN2.
Influence of the extracellular matrix and integrins on volume-sensitive osmolyte anion channels in C2C12 myoblasts
Authors: Neveux Iva , Doe Jinger , Leblanc Normand , Valencik Maria L.
Published: February 2010

The purpose of this study was to determine whether extracellular matrix (ECM) composition through integrin receptors modulated the volume-sensitive osmolyte anion channels (VSOACs) in skeletal muscle-derived C2C12 cells. Cl− currents were recorded in whole cell voltage-clamped cells grown on laminin (LM), fibronectin (FN), or in the absence of a defined ECM (NM). Basal membrane currents recorded in isotonic media (300 mosmol/kg) were larger in cells grown on FN (3.8-fold at +100 mV) or LM (8.8-fold at +100 mV) when compared with NM. VSOAC currents activated by cell exposure to hypotonic solution were larger in cells grown on LM (1.72-fold at +100 mV) or FN (1.75-fold at +100 mV) compared with NM. Additionally, the kinetics of VSOAC activation was ≈27% quicker on FN and LM. These currents were tamoxifen sensitive, displayed outward rectification, reversed at the equilibrium potential of Cl− and inactivated at potentials >+60 mV. Specific knockdown of β1-integrin by short hairpin RNA interference strongly inhibited the VSOAC Cl− currents in cells plated on FN. In conclusion, ECM composition and integrins profoundly influence the biophysical properties and mechanisms of onset of VSOACs.
The contribution of AKAP5 in amylase secretion from mouse parotid acini
Authors: Wu Ching-Yi , DiJulio Dennis H. , Jacobson Kerry L. , McKnight G. Stanley , Watson Eileen L.
Published: February 2010

A-kinase (PKA) anchoring proteins (AKAPs) are essential for targeting type II PKA to specific locales in the cell to control function. In the present study, AKAP5 (formerly AKAP150) and AKAP6 were identified in mouse parotid acini by type II PKA regulatory subunit (RII) overlay assay and Western blot analysis of mouse parotid cellular fractions, and the role of AKAP5 in mouse parotid acinar cell secretion was determined. Mice were euthanized with CO2. Immunofluorescence staining of acinar cells localized AKAP5 to the basolateral membrane, whereas AKAP6 was associated with the perinuclear region. In functional studies, amylase secretion from acinar cells of AKAP5 mutant [knockout (KO)] mice treated with the β-adrenergic agonist, isoproterenol, was reduced overall by 30–40% compared with wild-type (WT) mice. In contrast, amylase secretion in response to the adenylyl cyclase (AC) activator, forskolin, and the cAMP-dependent protein kinase (PKA) activator, N6-phenyl-cAMP, was not statistically different in acini from WT and AKAP5 KO mice. Treatment of acini with isoproterenol mimicked the effect of the Epac activator, 8-(4-methoxyphenylthio)-2′- O-methyladenosine-3′,5′-cyclic monophosphate (8-pMeOPT-2′- O-Me-cAMP), in stimulating Rap1. However, in contrast to isoproterenol, treatment of acini with 8-pMeOPT-2′- O-Me-cAMP resulted in stimulation of amylase secretion from both AKAP5 KO and WT acinar cells. As a scaffolding protein, AKAP5 was found to coimmunoprecipitate with AC6, but not AC8. Data suggest that isoproterenol-stimulated amylase secretion occurs via both an AKAP5/AC6/PKA complex and a PKA-independent, Epac pathway in mouse parotid acini.
l-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture
Authors: Washington Jennifer M. , Rathjen Joy , Felquer Fernando , Lonic Ana , Bettess Michael D. , Hamra Nancy , Semendric Ljiljana , Tan Boon Siang Nicholas , Lake Julie-Anne , Keough Rebecca A. , Morris Michael B. , Rathjen Peter D.
Published: February 2010

The development of cell therapeutics from embryonic stem (ES) cells will require technologies that direct cell differentiation to specific somatic cell lineages in response to defined factors. The initial step in formation of the somatic lineages from ES cells, differentiation to an intermediate, pluripotent primitive ectoderm-like cell, can be achieved in vitro by formation of early primitive ectoderm-like (EPL) cells in response to a biological activity contained within the conditioned medium MEDII. Fractionation of MEDII has identified two activities required for EPL cell formation, an activity with a molecular mass of <3 kDa and a second, much larger species. Here, we have identified the low-molecular-weight activity as l-proline. An inhibitor of l-proline uptake, glycine, prevented the differentiation of ES cells in response to MEDII. Supplementation of the culture medium of ES cells with >100 M l-proline and some l-proline-containing peptides resulted in changes in colony morphology, cell proliferation, gene expression, and differentiation kinetics consistent with differentiation toward a primitive ectoderm-like cell. This activity appeared to be associated with l-proline since other amino acids and analogs of proline did not exhibit an equivalent activity. Activation of the mammalian target of rapamycin (mTOR) signaling pathway was found to be necessary but not sufficient for l-proline activity; addition of other activators of the mTOR signaling pathway failed to alter the ES cell phenotype. This is the first report describing a role for amino acids in the regulation of pluripotency and cell differentiation and identifies a novel role for the imino acid l-proline.
Sex-related resistance to myocardial ischemia-reperfusion injury is associated with high constitutive ARC expression
Authors: Bouma Wobbe , Noma Mio , Kanemoto Shinya , Matsubara Muneaki , Leshnower Bradley G. , Hinmon Robin , Gorman Joseph H. , Gorman Robert C.
Published: February 2010

The female sex has been associated with improved myocardial salvage after ischemia and reperfusion (I/R). Estrogen, specifically 17β-estradiol, has been demonstrated to mediate this phenomenon by limiting cardiomyocyte apoptosis. We sought to quantitatively assess the effect of sex, ovarian hormone loss, and I/R on myocardial Bax, Bcl-2, and apoptosis repressor with caspase recruitment domain (ARC) expression. Male ( n = 48), female ( n = 26), and oophorectomized female ( n = 20) rabbits underwent 30 min of regional ischemia and 3 h of reperfusion. The myocardial area at risk and infarct size were determined using a double-staining technique and planimetry. In situ oligo ligation was used to assess apoptotic cell death. Western blot analysis was used to determine proapoptotic (Bax) and antiapoptotic (Bcl-2 and ARC) protein levels in all three ischemic groups and, additionally, in three nonischemic groups. Infarct size (43.7 ± 3.2%) and apoptotic cell death (0.51 ± 0.10%) were significantly attenuated in females compared with males (56.4 ± 1.6%, P < 0.01, and 4.29 ± 0.95%, P < 0.01) and oophorectomized females (55.7 ± 3.4%, P < 0.05, and 4.36 ± 0.51%, P < 0.01). Females expressed significantly higher baseline ARC levels (3.62 ± 0.29) compared with males (1.78 ± 0.18, P < 0.01) and oophorectomized females (1.08 ± 0.26, P < 0.01). Males expressed a significantly higher baseline Bax-to-Bcl-2 ratio (4.32 ± 0.99) compared with females (0.65 ± 0.13, P < 0.01) and oophorectomized females (0.42 ± 0.10, P < 0.01). I/R significantly reduced Bax-to-Bcl-2 ratios in males. In all other groups, ARC levels and Bax-to-Bcl-2 ratios did not significantly change. These results support the conclusion that in females, endogenous estrogen limits I/R-induced cardiomyocyte apoptosis by producing a baseline antiapoptotic profile, which is associated with estrogen-dependent high constitutive myocardial ARC expression.
Chronic administration of brain-derived neurotrophic factor in the hypothalamic paraventricular nucleus reverses obesity induced by high-fat diet
Authors: Wang ChuanFeng , Godar Rebecca J. , Billington Charles J. , Kotz Catherine M.
Published: February 2010

An acute injection of brain-derived neurotrophic factor (BDNF) in the hypothalamic paraventricular nucleus (PVN) reduces body weight by decreasing feeding and increasing energy expenditure (EE), in animals on standard laboratory chow. Animals have divergent responses to a high-fat diet (HFD) exposure, with some developing obesity and others remaining lean. In the current study, we tested two hypotheses: 1) BDNF in the PVN reverses HFD-induced obesity, and 2) animals with higher body fat have a greater physiological response to BDNF than those with less body fat. Eighty-four 10-wk old rats were allowed HFD ad libitum for 9 wk and then prepared with bilateral PVN cannulas. Animals were then divided into tertiles based on their body fat rank: high, intermediate, and low (H, I, and L). Each group was further divided into 2 subgroups and then PVN injected with BDNF or control (artificial cerebrospinal fluid, aCSF) every other day for 3 wk. Energy intake (EI), body weight, and body composition were measured. At study's end, rats were killed to allow measurement of other metabolic indices. In parallel, another 12 rats were fed control diet (CD), PVN-cannulated and injected with aCSF. HFD exposure induced obesity, particularly in the H body fat group, with a significant increase in EI, body weight, fat mass, liver size, and serum glucose, triglycerides, insulin, and leptin. BDNF significantly reduced EI, body weight, body fat, lean mass, and serum metabolic indices. These BDNF effects were greatest in the H body fat group. These data indicate that BDNF reduced HFD-induced obesity and metabolic syndrome-like measures, and the animals with the most body fat had the most significant response to BDNF.
The pontine respiratory group, particularly the Kölliker-Fuse nucleus, mediates phases of the hypoxic ventilatory response in unanesthetized goats
Authors: Bonis J. M. , Neumueller S. E. , Krause K. L. , Kiner T. , Smith A. , Marshall B. D. , Qian B. , Pan L. G. , Forster H. V.
Published: February 2010

The objective of the present study was to test the hypothesis that, in the in vivo awake goat model, perturbation/lesion in the pontine respiratory group (PRG) would decrease the sensitivity to hypercapnia and hypoxia. The study reported herein was part of two larger studies in which cholinergic modulation in the PRG was attenuated by microdialysis of atropine and subsequently ibotenic acid injections neurotoxically lesioned the PRG. In 14 goats, cannula were bilaterally implanted into either the lateral ( n = 4) or medial ( n = 4) parabrachial nuclei or the Kölliker-Fuse nucleus (KFN, n = 6). Before and after cannula implantation, microdialysis of atropine, and injection of ibotenic acid, hypercapnic and hypoxic ventilatory sensitivities were assessed. Hypercapnic sensitivity was assessed by three 5-min periods at 3, 5, and 7% inspired CO2. In all groups of goats, CO2 sensitivity was unaffected ( P > 0.05) by any PRG perturbations/lesions. Hypoxic sensitivity was assessed with a 30-min period at 10.8% inspired O2. The response to hypoxia was typically triphasic, with a phase 1 increase in pulmonary ventilation, a phase 2 roll-off, and a phase 3 prolonged increase associated with shivering and increased metabolic rate and body temperature. In all groups of goats, the phase 1 of the hypoxic ventilatory responses was unaffected by any PRG perturbations/lesions, and there were no consistent effects on the phase 2 responses. However, in the KFN group of goats, the phase 3 ventilatory, shivering, metabolic rate, and temperature responses were markedly attenuated after the atropine dialysis studies, and the attenuation persisted after the ibotenic acid studies. These findings support an integrative or modulatory role for the KFN in the phase 3 responses to hypoxia.
Acinar determinants of the apparent diffusion coefficient for helium-3
Authors: Verbanck Sylvia , Paiva Manuel
Published: February 2010

The apparent diffusion coefficient (ADC) obtained by helium-3 magnetic resonance imaging over several seconds is thought to reflect diffusion impairment due to both intra- and interacinar structure. In this study, numerical simulations of intra-acinar gas mixing and effective diffusion were performed in a multiple-branch-point model of the human acinus. Using a previously described method, we computed the instantaneous effective diffusion resulting from the diffusive impairment imposed by intra-acinar branching for varying times up to 5 s. We also tested the influence on effective diffusion of intra-acinar collateral channels in the fully alveolated intra-acinar airways to mimic the effect of emphysema. Randomly connecting two or four pairs of airways per generation (in generations 19–25) led to a 40 and 142% increase, respectively, in effective diffusion coefficient cumulated over the time interval of 0.2–5 s. Finally, we also used a system of two coupled multiple branch-point models to simulate diffusive attenuation over a 50-s interval in cases of purely acinar tagging (i.e., the initial gas concentration = 1 in one acinus and 0 in the other) and of partial tagging astride on two acini. It is shown that, in the latter case, the decay rate cannot be approximated by a mono-exponential with a several-fold faster decay for times below 10 s due to intra-acinar diffusion. We conclude that both the characteristic biphasic time dependence of simulated effective diffusion and its sensitivity to intra-acinar structural change mimic experimental ADC behavior. Additional simulations of combined inter- and intra-acinar diffusion strongly suggest that neglecting intra-acinar branching would in fact lead to considerable error of simulated ADC.
Activity in the Barrel Cortex During Active Behavior and Sleep
Authors: Vijayan Sujith , Hale Greg J. , Moore Christopher I. , Brown Emery N. , Wilson Matthew
Published: February 2010

The rate at which neurons fire has wide-reaching implications for the coding schemes used by neural systems. Despite the extensive use of the barrel cortex as a model system, relatively few studies have examined the rate of sensory activity in single neurons in freely moving animals. We examined the activity of barrel cortex neurons in behaving animals during sensory cue interaction, during non–stimulus-related activity, during various states of sleep, and during the administration of isoflurane. The activity of regular-spiking units (RSUs: predominantly excitatory neurons) and fast spiking units (FSUs: a subtype of inhibitory interneurons) was examined separately. We characterized activity by calculating neural firing rates, because several reports have emphasized the low firing rates in this system, reporting that both baseline activity and stimulus evoked activity is <1 Hz. We report that, during sensory cue interaction or non–stimulus-related activity, the majority of RSUs in rat barrel cortex fired at rates significantly >1 Hz, with 27.4% showing rates above 10 Hz during cue interaction. Even during slow wave sleep, which had the lowest mean and median firing rates of any nonanesthetized state observed, 80.0% of RSUs fired above 1 Hz. During all of the nonanesthetized states observed 100% of the FSUs fired well above 1 Hz. When rats were administered isoflurane and at a depth of anesthesia used in standard in vivo electrophysiological preparations, all of the RSUs fired below 1 Hz. We also found that >80% of RSUs either upmodulated or downmodulated their firing during cue interaction. These data suggest that low firing rates do not typify the output of the barrel cortex during awake activity and during sleep and indicate that sensory coding at both the individual and population levels may be nonsparse.
Intrinsic Properties of Mouse Lumbar Motoneurons Revealed by Intracellular Recording In Vivo
Authors: Meehan C. F. , Sukiasyan N. , Zhang M. , Nielsen J. B. , Hultborn H.
Published: February 2010

We have developed an in vivo model for intracellular recording in the adult anesthetized mouse using sharp microelectrode electrodes as a basis for investigations of motoneuron properties in transgenic mouse strains. We demonstrate that it is possible to record postsynaptic potentials underlying identified circuits in the spinal cord. Forty-one motoneurons with antidromic spike potentials (>50 mV) from the sciatic nerve were investigated. We recorded the intrinsic properties of the neurons, including input resistance (mean: 2.4 ± 1.2 MΩ), rheobase (mean: 7.1 ± 5.9 nA), and the duration of the afterhyperpolarization (AHP; mean: 55.3 ± 14 ms). We also measured the minimum firing frequencies (Fmin, mean 23.5 ± 5.7 SD Hz), the maximum firing frequencies (Fmax; >300 Hz) and the slope of the current–frequency relationship ( f–I slope) with increasing amounts of current injected (mean: 13 ± 5.7 Hz/nA). Signs of activation of persistent inward currents (PICs) were seen, such as accelerations of firing frequency or jumps in the membrane potential with increasing amounts of injected current. It is likely that the particular anesthetic regime with a mixture of Hypnorm and midazolam is essential for the possibility to evoke PICs. The data demonstrate that mouse spinal motoneurons share many of the same properties that have been demonstrated previously for cat, rat, and human motoneurons. The shorter AHP duration, steeper f–I slopes, and higher Fmin and Fmax than those in rats, cats, and humans are likely to be tailored to the characteristics of the mouse muscle contraction properties.