bluuebunny/crossref_metadata_2025 huggingface dataset:
Internal Transcribed Spacer rRNA Gene-Based Phylogenetic Reconstruction Using Algorithms with Local and Global Sequence Alignment for Black Yeasts and Their Relatives
Authors: Caligiorne R. B. , Licinio P. , Dupont J. , de Hoog G. S.
Published: June 2005

Sequences of rRNA gene internal transcribed spacer (ITS) of a standard set of black yeast-like fungal pathogens were compared using two methods: local and global alignments. The latter is based on DNA-walk divergence analysis. This method has become recently available as an algorithm (DNAWD program) which converts sequences into three-dimensional walks. The walks are compared with, or fit to, each other generating global alignments. The DNA-walk geometry defines a proper metric used to create a distance matrix appropriated for phylogenetic reconstruction. In this work, the analyses were carried out for species currently classified in
Capronia
,
Cladophialophora
,
Exophiala
,
Fonsecaea
,
Phialophora
, and
Ramichloridium
. Main groups were verified by small-subunit rRNA gene data. DNAWD applied to ITS2 alone enabled species recognition as well as phylogenetic reconstruction reflecting clades discriminated in small-subunit rRNA gene phylogeny, which was not possible with any other algorithm using local alignment for the same data set. It is concluded that DNAWD provides rapid insight into broader relationships between groups using genes that otherwise would be hardly usable for this purpose.
Use of a BJAB-Derived Cell Line for Isolation of Human Herpesvirus 8
Authors: Gasperini Paola , Barbierato Massimo , Martinelli Canio , Rigotti Paolo , Marchini Francesco , Masserizzi Giulia , Leoncini Francesco , Chieco-Bianchi Luigi , Schulz Thomas F. , Calabrò Maria Luisa
Published: June 2005

Establishment of latently infected cell lines from primary effusion lymphomas (PEL) presently is the most efficient system for the propagation of clinical strains of human herpesvirus 8 (HHV-8) in culture. Here we describe a new approach to culture productively replicating HHV-8 from patient samples. A BJAB-derived B-cell line, BBF, was found to retain HHV-8 longer, to support the latent and lytic replication programs, and to produce transmissible virus. Supernatants from
n
-butyrate-treated peripheral blood mononuclear cells of 24 HHV-8-seropositive renal transplant recipients were used to infect BBF cells, and replicating virus was detected in cultures from 11 patients. Moreover, BBF cells infected with saliva strains showed a highly productive profile regardless of the initial viral load, which confirms that infectious HHV-8 can be present in saliva and also suggests that saliva strains may exhibit a high tropism for B lymphocytes. In conclusion, we established an in vitro system that efficiently detects HHV-8 in samples with low viral loads and that produces infectious progeny. BBF cells can be used to propagate HHV-8 from different biological samples as well as to clarify important issues related to virus-cell interactions in a context distinct from endothelial and PEL-derived cell lines.
Cryptococcus neoformans
Galactoxylomannan Contains an Epitope(s) That Is Cross-Reactive with
Aspergillus
Galactomannan
Authors: Dalle Frédéric , Charles Pierre Emmanuel , Blanc Karine , Caillot Denis , Chavanet Pascal , Dromer Françoise , Bonnin Alain
Published: June 2005

We report a case of cryptococcosis in which a serum enzyme-linked immunosorbent assay (ELISA) for
Aspergillus
galactomannan was positive, with no evidence of aspergillosis. Soluble antigens from 19
Cryptococcus neoformans
strains and purified carbohydrates of
C. neoformans
capsule were thus assayed in the
Aspergillus
galactomannan ELISA. Antigens from all
C. neoformans
strains, and purified galactoxylomannan, gave a positive reaction, suggesting that
C. neoformans
galactoxylomannan contains an epitope(s) that is cross-reactive with
Aspergillus
galactomannan.
Shortcut Method for Extraction of
Staphylococcus aureus
DNA from Blood Cultures and Conventional Cultures for Use in Real-Time PCR Assays
Authors: Adams D. Nicholas
Published: June 2005

PCR allows rapid determination of both the presence of
Staphylococcus aureus
and of methicillin resistance. I present here a simple method for direct extraction from blood cultures or colonies for use in real-time PCR, eliminating tedious, time-consuming methods of DNA extraction which may otherwise deter microbiology labs from offering PCR.
Frequency of Sample Submission for Optimal Utilization of the Cell Culture Cytotoxicity Assay for Detection of
Clostridium difficile
Toxin
Authors: Borek Anita P. , Aird Deborah Z. , Carroll Karen C.
Published: June 2005

We reviewed the results of repeated sample submissions within a 7-day time frame for
Clostridium difficile
toxin testing. A total of 2,940 samples were tested during a 3-month period using a cell culture cytotoxicity assay (CCCA). The results from all second samples (
n
= 1,101) were concordant with the original test result. In only two cases (0.8%;
n
= 247) was a third sample positive when the first two samples were negative. In this study, submission of multiple samples for CCCA did not increase detection of
Clostridium difficile
infection.
Shared Genotypes of
Achromobacter xylosoxidans
Strains Isolated from Patients at a Cystic Fibrosis Rehabilitation Center
Authors: Van daele Sabine , Verhelst Rita , Claeys Geert , Verschraegen Gerda , Franckx Hilde , Van Simaey Leen , de Ganck Catharine , De Baets Frans , Vaneechoutte Mario
Published: June 2005

During a study examining transmission of
Pseudomonas aeruginosa
among 76 cystic fibrosis patients in a rehabilitation center, where patients stay in close contact during prolonged periods, several clusters of patients carrying genotypically identical
P. aeruginosa
, as well as two clusters of 4 and 10 patients, respectively, colonized with genotypically identical
Achromobacter xylosoxidans
strains, were discovered.
Disseminated Coinfection with
Actinomyces graevenitzii
and
Mycobacterium tuberculosis
: Case Report and Review of the Literature
Authors: Tietz Andreas , Aldridge Kenneth E. , Figueroa Julio E.
Published: June 2005

We report the first case of disseminated infection with both
Actinomyces graevenitzii
and
Mycobacterium tuberculosis
and review the medical literature. Concomitant actinomycosis and tuberculosis is very rare. The potential of the facultatively aerobic, newly described
A. graevenitzii
for disseminated invasive infection needs to be evaluated.
Pyrrolidine Dithiocarbamate Reduces Coxsackievirus B3 Replication through Inhibition of the Ubiquitin-Proteasome Pathway
Authors: Si Xiaoning , McManus Bruce M. , Zhang Jingchun , Yuan Ji , Cheung Caroline , Esfandiarei Mitra , Suarez Agripina , Morgan Andrew , Luo Honglin
Published: June 2005

Coxsackievirus B3 (CVB3) is one of the most common pathogens for viral myocarditis. The lack of effective therapeutics for CVB3-caused viral diseases underscores the importance of searching for antiviral compounds. Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and is recently reported to inhibit ubiquitin-proteasome-mediated proteolysis. Previous studies have shown that PDTC inhibits replication of rhinovirus, influenza virus, and poliovirus. In the present study, we report that PDTC is a potent inhibitor of CVB3. Coxsackievirus-infected HeLa cells treated with PDTC showed a significant reduction of CVB3 viral RNA synthesis, viral protein VP1 expression, and viral progeny release. Similar to previous observation that divalent ions mediate the function of PDTC, we further report that serum-containing copper and zinc are required for its antiviral activity. CVB3 infection resulted in massive generation of reactive oxygen species (ROS). Although PDTC alleviated ROS generation, the antiviral activity was unlikely dependent on its antioxidant effect because the potent antioxidant,N-acetyl-l-cysteine, failed to inhibit CVB3 replication. Consistent with previous reports that PDTC inhibits ubiquitin-proteasome-mediated protein degradation, we found that PDTC treatment led to the accumulation of several short-lived proteins in infected cells. We further provide evidence that the inhibitory effect of PDTC on protein degradation was not due to inhibition of proteasome activity but likely modulation of ubiquitination. Together with our previous findings that proteasome inhibition reduces CVB3 replication (H. Luo, J. Zhang, C. Cheung, A. Suarez, B. M. McManus, and D. Yang, Am. J. Pathol.163:381-385, 2003), results in this study suggest a strong antiviral effect of PDTC on coxsackievirus, likely through inhibition of the ubiquitin-proteasome pathway.
Hepatitis C Virus E2-CD81 Interaction Induces Hypermutation of the Immunoglobulin Gene in B Cells
Authors: Machida Keigo , Cheng Kevin T.-H. , Pavio Nicole , Sung Vicky M.-H. , Lai Michael M. C.
Published: June 2005

Hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases and B-lymphocyte proliferative disorders, including mixed cryoglobulinemia and B-cell lymphoma. It has been suggested that HCV infects human cells through the interaction of its envelope glycoprotein E2 with a tetraspanin molecule CD81, the putative viral receptor. Here, we show that the engagement of B cells by purified E2 induced double-strand DNA breaks specifically in the variable region of immunoglobulin (VH) gene locus, leading to hypermutation in theVHgenes of B cells. Other gene loci were not affected. Preincubation with the anti-CD81 monoclonal antibody blocked this effect. E2-CD81 interaction on B cells triggered the enhanced expression of activation-induced cytidine deaminase (AID) and also stimulated the production of tumor necrosis factor alpha. Knockdown of AID by the specific small interfering RNA blocked the E2-induced double-strand DNA breaks and hypermutation of theVHgene. These findings suggest that HCV infection, through E2-CD81 interaction, may modulate host's innate or adaptive immune response by activation of AID and hypermutation of immunoglobulin gene in B cells, leading to HCV-associated B-cell lymphoproliferative diseases.
Novel Nuclear Export Signal-Interacting Protein, NESI, Critical for the Assembly of Hepatitis Delta Virus
Authors: Wang Yun-Hsin , Chang Shin C. , Huang Cheng , Li Ya-Ping , Lee Chia-Huei , Chang Ming-Fu
Published: June 2005

The process of host factor-mediated nucleocytoplasmic transport is critical for diverse cellular events in eukaryotes and the life cycle of viruses. We have previously identified a chromosome region maintenance 1-independent nuclear export signal (NES) at the C terminus of the large form of hepatitis delta antigen (HDAg), designated NES(HDAg-L) that is required for the assembly of hepatitis delta virus (HDV) (C.-H. Lee et al., J. Biol. Chem.
276:
8142-8148, 2001). To look for interacting proteins of the NES(HDAg-L), yeast two-hybrid screening was applied using the GAL4-binding domain fused to the NES(HDAg-L) as bait. Among the positive clones, one encodes a protein, designated NESI [NES(HDAg-L) interacting protein] that specifically interacted with the wild-type NES(HDAg-L) but not with the export/package-defective HDAg-L mutant, NES*(HDAg-L), in which Pro-205 has been replaced by Ala. Northern blot analysis revealed NESI as the gene product of a 1.9-kb endogenous mRNA transcript that is present predominantly in human liver tissue. NESI consists of 467 amino acid residues and bears a putative actin-binding site and a bipartite nuclear localization signal. Specific interaction between HDAg-L and NESI was further confirmed by coimmunoprecipitation and immunofluorescence staining. Overexpression of antisense NESI RNAs inhibited the expression of NESI and abolished HDAg-L-mediated nuclear export and assembly of HDV genomic RNA. These data indicate a critical role of NESI in the assembly of HDV through interaction with HDAg-L.
Characterization of a Previously Unidentified Viral Protein in Porcine Circovirus Type 2-Infected Cells and Its Role in Virus-Induced Apoptosis
Authors: Liu Jue , Chen Isabelle , Kwang Jimmy
Published: June 2005

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs. In this study, transcription and translation of a novel viral gene (termed ORF3 here) was detected during productive infection of PCV2 in PK15 cells. The results of infection with ORF3-deficient PCV2 by site-directed mutagenesis indicated that the protein is not essential for viral replication. To investigate the underlying mechanism of cell death caused by replication of PCV2, apoptosis characterized by chromosomal condensation and fragmentation, formation of apoptotic bodies, and significant increase in hypodiploids were detected in infected cells. We further demonstrated that PCV2-induced apoptosis required the activation of caspase-8 but not caspase-9. The activation of caspase-8 results in the activation of caspase-3 as shown by an increase in the cleavage of the caspase substrate in the infected cells. To determine whether ORF3 protein could trigger apoptosis, ORF3 as well as ORF1 and ORF2 genes were transiently expressed in PK15 and Cos-7 cells for apoptotic activity assay. Transfection of cells with the ORF3 alone induced apoptosis using a pathway similar to that described in the context of viral infection. This is further confirmed by a significant decrease in apoptotic activity of infected cells in the absence of the ORF3 expression, suggesting that the protein plays a major role in the induction of virus-induced apoptosis. Altogether, these results indicate that ORF3 is a novel PCV2 protein that is not essential for viral replication in cultured cells but is involved in PCV2-induced apoptosis by activating caspase-8 and caspase-3 pathways.
Seroprevalence and Genomic Divergence of Circulating Strains of Feline Immunodeficiency Virus among
Felidae
and
Hyaenidae
Species
Authors: Troyer Jennifer L. , Pecon-Slattery Jill , Roelke Melody E. , Johnson Warren , VandeWoude Sue , Vazquez-Salat Nuria , Brown Meredith , Frank Laurence , Woodroffe Rosie , Winterbach Christiaan , Winterbach Hanlie , Hemson Graham , Bush Mitch , Alexander Kathleen A. , Revilla Eloy , O'Brien Stephen J.
Published: June 2005

Feline immunodeficiency virus (FIV) infects numerous wild and domestic feline species and is closely related to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Species-specific strains of FIV have been described for domestic cat (
Felis catus
), puma (
Puma concolor
), lion (
Panthera leo
), leopard (
Panthera pardus
), and Pallas' cat (
Otocolobus manul
). Here, we employ a three-antigen Western blot screening (domestic cat, puma, and lion FIV antigens) and PCR analysis to survey worldwide prevalence, distribution, and genomic differentiation of FIV based on 3,055 specimens from 35
Felidae
and 3
Hyaenidae
species. Although FIV infects a wide variety of host species, it is confirmed to be endemic in free-ranging populations of nine
Felidae
and one
Hyaenidae
species. These include the large African carnivores (lion, leopard, cheetah, and spotted hyena), where FIV is widely distributed in multiple populations; most of the South American felids (puma, jaguar, ocelot, margay, Geoffroy's cat, and tigrina), which maintain a lower FIV-positive level throughout their range; and two Asian species, the Pallas' cat, which has a species-specific strain of FIV, and the leopard cat, which has a domestic cat FIV strain in one population. Phylogenetic analysis of FIV proviral sequence demonstrates that most species for which FIV is endemic harbor monophyletic, genetically distinct species-specific FIV strains, suggesting that FIV transfer between cat species has occurred in the past but is quite infrequent today.
Characterization of
Hortulanus
Endogenous Murine Leukemia Virus, an Endogenous Provirus That Encodes an Infectious Murine Leukemia Virus of a Novel Subgroup
Authors: Tipper Christopher H. , Bencsics Craig E. , Coffin John M.
Published: June 2005

Simple retroviruses present a unique opportunity for examining the host-virus relationship. Following exogenous infection and integration into the germ line, copies of these viruses can become fixed within the genome. The resulting endogenous proviral “fossils” represent a record of past retroviral infections and forms. Previous work in our laboratory has been directed at dissecting the extensive nonecotropic murine leukemia virus content of the mouse genome. One such provirus,
hortulanus
endogenous murine leukemia virus (HEMV), found in a single copy in the genome of
Mus spicilegus
, was remarkable for characteristics that suggested that it was ancient and related to the hypothetical common ancestor of murine leukemia viruses (MLVs) and other gammaretroviral species. In the present study, we have analyzed its functional properties. Transfection of a molecular clone of the HEMV provirus into mouse-derived cell lines revealed that it is replication competent. Furthermore, host range and interference studies revealed a strictly ecotropic host range and the use of a receptor distinct from those used by other classical MLVs. The identity of nucleotide sequence of the long terminal repeats (LTRs) further suggested that HEMV is a relatively recent insertion into the
M. spicilegus
genome at the distal end of chromosome 7. Although unique to
M. spicilegus
, its presence in a homozygous state in three individuals obtained from different regions implies that it has been present long enough to become fixed in this species. Exhaustive phylogenetic analysis of all regions of the HEMV genome supported the previously assigned ancestral position of HEMV relative to other MLV-related viruses. Thus, HEMV is a relatively recent introduction into the
Mus
germ line but is representative of a relatively ancestral MLV group.
Wild-Type-Like Viral Replication Potential of Human Immunodeficiency Virus Type 1 Envelope Mutants Lacking Palmitoylation Signals
Authors: Chan Woan-Eng , Lin Hui-Hua , Chen Steve S.-L.
Published: June 2005

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4
+
T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4
+
T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.
Attenuation of Recombinant Yellow Fever 17D Viruses Expressing Foreign Protein Epitopes at the Surface
Authors: Bonaldo Myrna C. , Garratt Richard C. , Marchevsky Renato S. , Coutinho Evandro S. F. , Jabor Alfredo V. , Almeida Luís F. C. , Yamamura Anna M. Y. , Duarte Adriana S. , Oliveira Prisciliana J. , Lizeu Jackeline O. P. , Camacho Luiz A. B. , Freire Marcos S. , Galler Ricardo
Published: June 2005

The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein
fg
loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the
fg
loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein
fg
loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines.
Role of Papillomavirus E1 Initiator Dimerization in DNA Replication
Authors: Schuck Stephen , Stenlund Arne
Published: June 2005

Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.
In Vitro Assembly of PB2 with a PB1-PA Dimer Supports a New Model of Assembly of Influenza A Virus Polymerase Subunits into a Functional Trimeric Complex
Authors: Deng Tao , Sharps Jane , Fodor Ervin , Brownlee George G.
Published: June 2005

Influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex of PB1, PB2, and PA. We show that the individually expressed PB2 subunit can be assembled with the coexpressed PB1-PA dimer in vitro into a transcriptionally active complex. Furthermore, we demonstrate that a model viral RNA promoter can bind to the PB1-PA dimer prior to assembly with PB2. Our results are consistent with a recently proposed model for the sequential assembly of viral RNA polymerase complex in which the PB1-PA dimeric complex and the PB2 monomer are transported into the nucleus separately and then assembled in the nucleus.
False dichotomies: EBM, clinical freedom, and the art of medicine
Authors: Parker M
Published: June 2005

According to numerous commentators, clinical freedom, the art of medicine, and, by implication, a degree of patient welfare, are threatened by evidence based medicine (EBM). As EBM has developed over the last fifteen years, claims about better evidence for medical treatments, and improvements in healthcare delivery, have been matched by critiques of EBM’s reductionism and uniformity, its problematic application to individual patients, and its alleged denial of the continuing need for clinical interpretation, insight, and judgment. Most of these attacks on EBM and defences of clinical freedom fail. They are based on erroneous understandings of the relationships between inductive knowledge, clinical uncertainty, and action. Evidence based medicine is anecessary conditionfor clinical freedom, not a threat to it, and EBM is not something to bebalancedwith either clinical experience or patient preferences. The art and science of medicine are more conceptually and practically connected than the defenders of clinical freedom, whatever they conceive that to be, are willing to admit.
RELATIVISTIC QUANTUM FIELD THEORY FOR CONDENSED SYSTEMS-(III): AN EXPLICIT EXPRESSION OF ATOMIC SCHWINGER–DYSON METHOD
Authors: MATSUURA HIROYUKI , NAKANO MASAHIRO
Published: June 2005

A new explicit expressions of self-energies Πμν and Σ are introduced for photons and electrons based on the particle-hole-antiparticle representation (PHA) of Atomic Schwinger–Dyson formalism (ASD). The PHA representation describes exactly the physical processes such as particle-hole excitations (electron-hole) and particle-antiparticle excitations (electron-positron). The self-energy Σ includes both the quantum component and the classical component (classical external field and Coulomb's field), which are divided into the scalar part Σs and 4-dimensional vector parts Σ0, Σj. The electron propagators are composed of the particle part, hole part and antiparticle part in PHA representation. The general representation of photon self-energy Πμν with 16 elements is expressed in terms of only two components (transverse and longitudinal) Πt and Πl. The general form of the photon propagators are written in terms of free propagator D0 and two independent propagators Dl and Dt, which include two independent photon self-energies. The tensor part of the electron self-energy does not appear in ASD formalism which makes perfectly the closed self-consistent system, when we take the bare vertex approximation, Γμ→γμ.
Role of high-voltage-activated calcium channels in glucose-regulated β-cell calcium homeostasis and insulin release
Authors: Taylor James T. , Huang Luping , Keyser Brian M. , Zhuang Hean , Clarkson Craig W. , Li Ming
Published: June 2005

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, LC-type, and LD-type) in rat pancreatic β-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.
Dynamic myocardial contractile parameters from left ventricular pressure-volume measurements
Authors: Campbell K. B. , Wu Y. , Simpson A. M. , Kirkpatrick R. D. , Shroff S. G. , Granzier H. L. , Slinker B. K.
Published: June 2005

A new dynamic model of left ventricular (LV) pressure-volume relationships in beating heart was developed by mathematically linking chamber pressure-volume dynamics with cardiac muscle force-length dynamics. The dynamic LV model accounted for >80% of the measured variation in pressure caused by small-amplitude volume perturbation in an otherwise isovolumically beating, isolated rat heart. The dynamic LV model produced good fits to pressure responses to volume perturbations, but there existed some systematic features in the residual errors of the fits. The issue was whether these residual errors would be damaging to an application where the dynamic LV model was used with LV pressure and volume measurements to estimate myocardial contractile parameters. Good agreement among myocardial parameters responsible for response magnitude was found between those derived by geometric transformations of parameters of the dynamic LV model estimated in beating heart and those found by direct measurement in constantly activated, isolated muscle fibers. Good agreement was also found among myocardial kinetic parameters estimated in each of the two preparations. Thus the small systematic residual errors from fitting the LV model to the dynamic pressure-volume measurements do not interfere with use of the dynamic LV model to estimate contractile parameters of myocardium. Dynamic contractile behavior of cardiac muscle can now be obtained from a beating heart by judicious application of the dynamic LV model to information-rich pressure and volume signals. This provides for the first time a bridge between the dynamics of cardiac muscle function and the dynamics of heart function and allows a beating heart to be used in studies where the relevance of myofilament contractile behavior to cardiovascular system function may be investigated.
Increased expression of ENaC subunits and increased apical targeting of AQP2 in the kidneys of spontaneously hypertensive rats
Authors: Kim Soo Wan , Wang Weidong , Kwon Tae-Hwan , Knepper Mark A. , Frøkiær Jørgen , Nielsen Søren
Published: June 2005

In models of genetic hypertension, renal tubular dysfunction could be involved in the increased sodium and water reabsorption. However, the molecular basis for the increased renal sodium and water retention remains largely undefined in spontaneously hypertensive rats (SHR). We hypothesized that dysregulation of renal epithelial sodium channels (ENaC), sodium (co)transporters, or aquaporin-2 (AQP2) could be involved in the pathogenesis of hypertension in SHR. Six-week-old or twelve-week-old SHR and corresponding age-matched Wistar-Kyoto control rats (WKY) were studied. In both SHR groups, systolic blood pressure was markedly increased, whereas urine output, creatinine clearance, and urinary sodium excretion were decreased compared with corresponding WKY. Moreover, urine osmolality and urine-to-plasma osmolality ratio were increased compared with WKY. Semiquantitative immunoblotting demonstrated that the protein abundance of β- and γ-subunits of ENaC was increased in the cortex and outer stripe of the outer medulla and inner stripe of the outer medulla (ISOM) in SHR, whereas α-ENaC abundance was increased in ISOM. Immunoperoxidase microscopy confirmed the increased labeling of β-ENaC and γ-ENaC subunits in the late distal convoluted tubule, connecting tubule, and cortical and outer medullary collecting duct segments. In contrast, subcellular localization of α-ENaC, β-ENaC, and γ-ENaC was not changed. Expression of sodium/hydrogen exchanger type 3, bumetanide-sensitive Na-K-2Cl cotransporter, and thiazide-sensitive Na-Cl cotransporter was not altered in SHR. AQP2 levels were increased in the ISOM in SHR, and immunoperoxidase microscopy demonstrated an increased apical labeling of AQP2 in the inner medullary collecting duct in SHR. These results suggest that the increased protein abundance of ENaC subunits as well as the increased apical targeting of AQP2 may contribute to renal sodium and water retention observed during the development of hypertension in SHR.
Effect of COX inhibitors and NO on renal hemodynamics following ischemia-reperfusion injury in normotensive and hypertensive rats
Authors: Knight Sarah , Johns Edward J.
Published: June 2005

The processes involved in the renal damage resulting from ischemia-reperfusion injury are poorly understood. This study examined the contribution of prostaglandins and nitric oxide (NO) in the vascular responses to ischemia-reperfusion injury in the kidneys of normotensive and hypertensive rats. Groups of Wistar and stroke-prone spontaneously hypertensive rats (SHRSP) were dosed with polyethylene glycol vehicle, aspirin (53.5 mg·kg−1·day−1), NO-aspirin (100 mg·kg−1·day−1), or celecoxib (10 mg·kg−1·day−1) for 7 days. On day 7, rats were anesthetized with chloralose/urethane and the left kidney was exposed to a 30-min period of ischemia followed by 90-min reperfusion. Renal cortical and medullary perfusions were monitored throughout using laser-Doppler flowmetry. In the vehicle- and celecoxib-treated Wistar rats, cortical and medullary postischemic perfusion was reduced to 66 and 62% and 53 and 62%, respectively (all P < 0.05), of baseline. The ischemia-induced reductions in cortical and medullary flux were ameliorated in the aspirin and NO-aspirin groups where flux fell to 96 and 78% and 105 and 83%, respectively ( P < 0.05). There was a fall in cortical and medullary flux in the postischemic period in the vehicle-treated SHRSP to 82 and 77% ( P < 0.05). These findings show that nonselective cyclooxygenase (COX) inhibition, and to an even greater extent NO donation, provided protection to the renal vasculature from ischemic injury in the Wistar rat but not in the SHRSP. This would suggest that prostaglandins are less important in the development of renal ischemia-reperfusion injury during hypertension and both COX isoforms must be inhibited to offset the decrease in renal hemodynamics.
Effects of Salicylate on Plasma Membrane Mechanics
Authors: Ermilov Sergey A. , Murdock David R. , El-Daye Dania , Brownell William E. , Anvari Bahman
Published: June 2005

High concentrations of the amphipathic drug salicylate (Sal) block outer hair cell (OHC) electromotility resulting in reversible hearing loss. We used optical tweezers to study the effects of Sal on the mechanics of the cell plasma membrane. Membrane tethers were formed from guinea pig OHCs and cultured human embryonic kidney (HEK) cells as controls. HEK cells are commonly used in functional expression studies of electromotility. Effective tether viscosity (ηeff), steady-state tethering force extrapolated to zero pulling rate Fss(0), and time constant for tether growth (τtg) were estimated from the measurements of the instantaneous tethering force at different tether pulling rates. Average values of ηeff, Fss(0), and τtg for the OHC lateral wall plasma membrane and control cell plasma membrane remained the same after Sal perfusion, which is consistent with the hypothesis that Sal-induced reversible hearing loss appears to be more the result of its competition with essential anions and less the result of a change in plasma membrane mechanics.
WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum
Authors: Wouters Mira , Smans Karine , Vanderwinden Jean-Marie
Published: June 2005

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called WZsGreen. In the gastrointestinal tract of heterozygous WZsGreen/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel WZsGreen/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.
Clinical Research in Primary Stroke Prevention: Needs, Opportunities, and Challenges
Authors: Radziszewska Barbara , Hart Robert G. , Wolf Philip A. , D’Agostino, Sr. Ralph B. , Cutler Jeffrey A.
Published: June 2005

Most (∼70%) of strokes are first-ever strokes, and hence to substantially reduce the neurological burden, primary prevention is crucial. Here, highlights of the National Institute of Neurological Disorders and Stroke workshop ‘Stroke Risk Assessment and Future Stroke Primary Prevention Trials’ held January 12–13, 2004 are summarized. The Workshop discussions focused on stroke risk assessment; the high-risk vs. population-based approaches to primary prevention; desirable characteristics of candidate treatments and potential novel treatments, such as the ‘polypill’; subclinical disease as risk assessment tool and as surrogate outcome, and methodological issues in stroke primary prevention trials. The importance of assessing cognitive decline as an important consequence of covert and overt vascular injury of the brain was emphasized. The scientific or logistic barriers to stroke primary prevention trials are challenging, but are not insurmountable.
Survival of Patients with Amyotrophic Lateral Sclerosis in a Population-Based Registry
Authors: Millul A. , Beghi E. , Logroscino G. , Micheli A. , Vitelli E. , Zardi A.
Published: June 2005

Objective: To evaluate the survival of patients with amyotrophic lateral sclerosis (ALS) in an Italian population and to assess the effects of selected prognostic indicators on survival. Background: Median survival of ALS patients has been reported to range between 12 and 23 months from diagnosis and between 23 and 36 months from onset of symptoms. Although several negative prognostic factors have been identified, the overall picture still needs clarification. Methods: We included patients enrolled in an Italian ALS Regional Register (population 4,529,003) during the calendar year 1998. The diagnosis was confirmed by an ad hoc committee using the original El Escorial criteria. Each case was regularly followed up until death or December 31, 2002, whichever came first. Survival was assessed with the Kaplan-Meier method in the whole sample, by level of diagnostic certainty, and by selected prognostic indicators (age, sex, bulbar or spinal onset, and disease duration). Multivariate analysis was done with the Cox proportional hazard function. Results: The sample comprised 79 patients (33 female; 46 male) aged 28–85 years (mean age 64.4 years). Onset of symptoms was bulbar in 30% of cases. Mean symptom duration at diagnosis was 13.3 months. ALS was definite in 43%, probable in 29%, possible in 6%, and suspected in 22%. By December 31, 2002, 56 cases (71%) had died. The cumulative probability of surviving after diagnosis was 78% at 12 months, 56% at 24 months, and 32% at 48 months. Median survival from onset was 39.2 months and from diagnosis 30.6 months. Multivariate analysis confirmed definite ALS at diagnosis and older age as adverse prognostic factors. Conclusions: Survival of ALS patients in the present sample was slightly longer than previously reported. Better palliative care and supportive treatment may explain the difference. Older age and the presence of definite ALS at diagnosis are poor prognostic predictors.
Assessment of Relations between Clinical Outcome of Ischemic Stroke and Activity of Inflammatory Processes in the Acute Phase Based on Examination of Selected Parameters
Authors: Palasik W. , Fiszer U. , Lechowicz W. , Czartoryska B. , Krzesiewicz M. , Lugowska A.
Published: June 2005

Background and Purposes: Inflammatory factors play an important role in the pathogenesis of ischemic stroke. They may influence circulation during the acute phase of stroke and enhance the ischemic region. Materials and Methods: We examined 51 patients – 36 patients in the early stage of stroke, i.e. the first 24 h after onset. Of these, 15 patients had infection and 21 had no infection during the week preceding stroke. There were 15 patients with noninflammatory diseases in the control group. We analyzed parameters of inflammation such as: activity of serum chitotriosidase by fluorimetric assay, C-reactive proteins (CRP), number of white body cells (WBCs), IgG and fibrinogen. We also assessed the neurological stage according to the Scandinavian Stroke Scale (SSS). Results: In our study, we observed a statistically significant difference (p < 0.05) in the activity of most parameters of inflammation. This difference could be seen in the levels of CRP, number of WBCs and the activity of chitotriosidase, apart from IgG and fibrinogen, between the control group and groups with versus without infection. A significantly increased level of CRP (p < 0.0005) and fibrinogen (p > 0.01) was found on the first day in the stroke group as compared to the control group. The neurological stage on day 4 after stroke, assessed according to the SSS, was significantly worse in the group of patients with infection before stroke than in stroke patients without infection (p < 0.008). Conclusion: These results suggest the importance of active inflammatory processes in the pathogenesis of stroke. We observed increased activity of chitotioridase, a parameter of the inflammatory process, in stroke. This study is one more proof that inflammatory processes caused by infection may influence the occurrence of stroke and worsen its outcome. It could be another step towards understanding immunological processes during the acute phase of stroke. The study may also help establish new diagnostic and therapeutic strategies and could be a useful tool for prophylaxis.
Local Expression of Platelet-Activating Factor-Acetylhydrolase Reduces Accumulation of Oxidized Lipoproteins and Inhibits Inflammation, Shear Stress-Induced Thrombosis, and Neointima Formation in Balloon-Injured Carotid Arteries in Nonhyperlipidemic Rabbits
Authors: Arakawa Hideki , Qian Jian-Yong , Baatar Dolgor , Karasawa Ken , Asada Yujiro , Sasaguri Yasuyuki , Miller Elizabeth R. , Witztum Joseph L. , Ueno Hikaru
Published: June 2005

Background—

Platelet-activating factor (PAF) and PAF-like phospholipids are inactivated by PAF-acetylhydrolase (PAF-AH). Using nonhyperlipidemic animals, we tested whether local expression of PAF-AH into injured arteries might induce antithrombotic and antiinflammatory effects.

Method and Results—

Balloon-injured rabbit carotid arteries were infected at the time of injury with an adenovirus expressing either human plasma PAF-AH (AdPAF-AH) or bacterial β-galactosidase (AdLacZ) or infused with saline. Seven days later, shear stress-induced thrombosis was observed in all AdLacZ-infected and saline-infused arteries (controls) but eliminated in AdPAF-AH-treated contralateral arteries, even in the presence of epinephrine or an inhibitor of NO production. Injury-induced expression of tissue factor was also significantly suppressed. In AdPAF-AH-treated arteries compared with controls, the expressions of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and macrophage infiltration were decreased by 66%, 66%, and 71%, respectively (
P
<0.01), and intimal area and intima/media ratio were decreased on day 21 by 43% and 52%, respectively (
P
<0.05). Within 1 week after injury, oxidized lipoproteins (OxLDL) had readily accumulated in the arterial wall. However, this was markedly reduced in the AdPAF-AH-treated arteries. No differences in the titers of autoantibodies to OxLDL or total cholesterol in blood were found between controls and AdPAF-AH-treated rabbits.

Conclusions—

Our results show for the first time that OxLDL accumulates in arteries in nonhyperlipidemic animals within 1 week after injury and that local expression of PAF-AH reduces this accumulation and exerts antiinflammatory, antithrombotic, and antiproliferative effects without changing the plasma levels of PAF-AH activity or titers of autoantibodies to OxLDL.
Hypothermia Improves Defibrillation Success and Resuscitation Outcomes From Ventricular Fibrillation
Authors: Boddicker Kimberly A. , Zhang Yi , Zimmerman M. Bridget , Davies Loyd R. , Kerber Richard E.
Published: June 2005

Background—

Induced hypothermia is recommended to improve neurological outcomes in unconscious survivors of out-of-hospital ventricular fibrillation (VF) cardiac arrest. Patients resuscitated from a VF arrest are at risk of refibrillation, but there are few data on the effects of already existing hypothermia on defibrillation and resuscitation.

Methods and Results—

Thirty-two swine (mean±SE weight, 23.0±0.6 kg) were divided into 4 groups: normothermia (n=8), mild hypothermia (35°C) (n=8), moderate hypothermia (33°C) (n=8), and severe hypothermia (30°C) (n=8). Hypothermia was induced by surrounding the animal with ice, and VF was electrically induced. After 8 minutes of unsupported VF (no CPR), the swine were defibrillated (biphasic waveform) with successive shocks as needed and underwent CPR until resumption of spontaneous circulation or no response (≥10 minutes). First-shock defibrillation success was higher in the moderate hypothermia group (6 of 8 hypothermia versus 1 of 8 normothermia;
P
=0.04). The number of shocks needed for late defibrillation (≥1 minute after initial shock) was less in all 3 hypothermia groups compared with normothermia (all
P
<0.05). None of the 8 animals in the normothermia group achieved resumption of spontaneous circulation compared with 3 of 8 mild hypothermia (
P
=NS), 7 of 8 moderate hypothermia (
P
=0.001), and 5 of 8 severe hypothermia (
P
=0.03) animals. Coronary perfusion pressure during CPR was not different between the groups.

Conclusions—

When VF was induced in the setting of moderate or severe hypothermia, resuscitative measures were facilitated with significantly improved defibrillation success and resuscitation outcome. The beneficial effect of hypothermia was not due to alteration of coronary perfusion pressure, which suggests that changes in the mechanical, metabolic, or electrophysiological properties of the myocardium may be responsible.