bluuebunny/crossref_metadata_2025 huggingface dataset:
A Cellular
Drosophila melanogaster
Protein with Similarity to Baculovirus F Envelope Fusion Proteins
Authors: Lung Oliver , Blissard Gary W.
Published: June 2005

Baculovirus F (fusion) proteins are found in the envelopes of budded virions. Recently a
Drosophila melanogaster
gene (CG4715) that encodes a protein with sequence similarity to baculovirus F proteins was discovered. To examine similarities and differences with baculovirus F proteins, we cloned the
D. melanogaster
cellular F (
Dm-cF
) protein gene and analyzed
Dm-cF
expression and localization. The predicted Dm-cF protein sequence lacks a furin cleavage site, and transiently expressed Dm-cF showed no protein cleavage and no detectable membrane fusion activity. In cell localization studies, transiently expressed Dm-cF was localized to intracellular organelles in
D. melanogaster
S2 cells, unlike baculovirus F proteins, which localize to cellular plasma membranes. Using reverse transcriptase PCR and Western blot analysis to examine
Dm-cF
expression in animals, we detected
Dm-cF
expression in both larval and adult
D. melanogaster
cells. However,
Dm-cF
expression was detected only in third instar larvae and adults, suggesting that
Dm-cF
expression may be developmentally regulated. We also identified genes related to
Dm-cF
in the genomes of two other
Drosophila
species,
Drosophila yakuba
and
Drosophila pseudoobscura
, and the mosquito
Anopheles gambiae
. These observations suggest that
f
genes may be present in the genomes of many insects. Conservation within and between 22 baculovirus and 4 insect F proteins was examined in detail. These studies demonstrate that
Dm-cF
is expressed in
D. melanogaster
and suggest that if baculovirus
f
genes are derived from a host cellular
f
gene, the function appears to have changed substantially upon adaptation to the baculovirus infection cycle.
Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses
Authors: Muñoz-Jordán Jorge L. , Laurent-Rolle Maudry , Ashour Joseph , Martínez-Sobrido Luis , Ashok Mundrigi , Lipkin W. Ian , García-Sastre Adolfo
Published: June 2005

Flaviviruses are insect-borne, positive-strand RNA viruses that have been disseminated worldwide. Their genome is translated into a polyprotein, which is subsequently cleaved by a combination of viral and host proteases to produce three structural proteins and seven nonstructural proteins. The nonstructural protein NS4B of dengue 2 virus partially blocks activation of STAT1 and interferon-stimulated response element (ISRE) promoters in cells stimulated with interferon (IFN). We have found that this function of NS4B is conserved in West Nile and yellow fever viruses. Deletion analysis shows that that the first 125 amino acids of dengue virus NS4B are sufficient for inhibition of alpha/beta IFN (IFN-α/β) signaling. The cleavable signal peptide at the N terminus of NS4B, a peptide with a molecular weight of 2,000, is required for IFN antagonism but can be replaced by an unrelated signal peptide. Coexpression of dengue virus NS4A and NS4B together results in enhanced inhibition of ISRE promoter activation in response to IFN-α/β. In contrast, expression of the precursor NS4A/B fusion protein does not cause an inhibition of IFN signaling unless this product is cleaved by the viral peptidase NS2B/NS3, indicating that proper viral polyprotein processing is required for anti-interferon function.
Extensive Syncytium Formation Mediated by the Reovirus FAST Proteins Triggers Apoptosis-Induced Membrane Instability
Authors: Salsman Jayme , Top Deniz , Boutilier Julie , Duncan Roy
Published: June 2005

The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by nonenveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively nonleaky process, as demonstrated by the absence of significant [
3
H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ectodomain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic nonenveloped viruses, with nonleaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.
Preliminary Physical Mapping of RNA-RNA Linkages in the Genomic RNA of Moloney Murine Leukemia Virus
Authors: Hibbert Catherine S. , Rein Alan
Published: June 2005

Retrovirus particles contain two copies of their genomic RNA, held together in a dimer by linkages which presumably consist of a limited number of base pairs. In an effort to localize these linkages, we digested deproteinized RNA from Moloney murine leukemia virus (MLV) particles with RNase H in the presence of oligodeoxynucleotides complementary to specific sites in viral RNA. The cleaved RNAs were then characterized by nondenaturing gel electrophoresis. We found that fragments composed of nucleotides 1 to 754 were dimeric, with a linkage as thermostable as that between dimers of intact genomic RNA. In contrast, there was no stable linkage between fragments consisting of nucleotides 755 to 8332. Thus, the most stable linkage between monomers is on the 5′ side of nucleotide 754. This conclusion is in agreement with earlier electron microscopic analyses of partially denatured viral RNAs and with our study (C. S. Hibbert, J. Mirro, and A. Rein, J. Virol. 78:10927-10938, 2004) of encapsidated nonviral mRNAs containing inserts of viral sequence. We obtained similar results with RNAs from immature MLV particles, in which the dimeric linkage is different from that in mature particles and has not previously been localized. The 5′ and 3′ fragments of cleaved RNA are all held together by thermolabile linkages, indicating the presence of tethering interactions between bases 5′ and bases 3′ of the cleavage site. When RNAs from mature particles were cleaved at nucleotide 1201, we detected tethering interactions spanning the cleavage site which are intramonomeric and are as strong as the most stable linkage between the monomers.
Enhanced Potency of Plasmid DNA Microparticle Human Immunodeficiency Virus Vaccines in Rhesus Macaques by Using a Priming-Boosting Regimen with Recombinant Proteins
Authors: Otten Gillis R. , Schaefer Mary , Doe Barbara , Liu Hong , Srivastava Indresh , zur Megede Jan , Kazzaz Jina , Lian Ying , Singh Manmohan , Ugozzoli Mildred , Montefiori David , Lewis Mark , Driver David A. , Dubensky Thomas , Polo John M. , Donnelly John , O'Hagan Derek T. , Barnett Susan , Ulmer Jeffrey B.
Published: June 2005

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.
Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells
Authors: Sinha Sapna , Grandgenett Duane P.
Published: June 2005

Retrovirus preintegration complexes (PIC) in virus-infected cells contain the linear viral DNA genome (∼10 kbp), viral proteins including integrase (IN), and cellular proteins. After transport of the PIC into the nucleus, IN catalyzes the concerted insertion of the two viral DNA ends into the host chromosome. This successful insertion process is termed “full-site integration.” Reconstitution of nucleoprotein complexes using recombinant human immunodeficiency virus type 1 (HIV-1) IN and model viral DNA donor substrates (∼0.30 to 0.48 kbp in length) that are capable of catalyzing efficient full-site integration has proven difficult. Many of the products are half-site integration reactions where either IN inserts only one end of the viral donor substrate into a circular DNA target or into other donors. In this report, we have purified recombinant HIV-1 IN at pH 6.8 in the presence of MgSO
4
that performed full-site integration nearly as efficiently as HIV-1 PIC. The size of the viral DNA substrate was significantly increased to 4.1 kbp, thus allowing for the number of viral DNA ends and the concentrations of IN in the reaction mixtures to be decreased by a factor of ∼10. In a typical reaction at 37°C, recombinant HIV-1 IN at 5 to 10 nM incorporated 30 to 40% of the input DNA donor into full-site integration products. The synthesis of full-site products continued up to ∼2 h, comparable to incubation times used with HIV-1 PIC. Approximately 5% of the input donor was incorporated into the circular target producing half-site products with no significant quantities of other integration products produced. DNA sequence analysis of the viral DNA-target junctions derived from wild-type U3 and U5 coupled reactions showed an ∼70% fidelity for the HIV-1 5-bp host site duplications. Recombinant HIV-1 IN successfully utilized a mutant U5 end containing additional nucleotide extensions for full-site integration demonstrating that IN worked properly under nonideal active substrate conditions. The fidelity of the 5-bp host site duplications was also high with these coupled mutant U5 and wild-type U3 donor ends. These studies suggest that recombinant HIV-1 IN is at least as capable as native IN in virus particles and approaching that observed with HIV-1 PIC for catalyzing full-site integration.
Lentivirus-Specific Cytotoxic T-Lymphocyte Responses Are Rapidly Lost in Thymectomized Cats Infected with Feline Immunodeficiency Virus
Authors: Hayes Kathleen A. , Köksoy Sadi , Phipps Andrew J. , Buck Wayne R. , Kociba Gary J. , Mathes Lawrence E.
Published: June 2005

To what extent the thymus is needed to preserve the virus-specific cytotoxic T-lymphocyte (CTL) response of lentivirus-infected adults is unclear. Presented here is the first definitive study using thymectomized (ThX) animals to directly evaluate the contribution of thymic function to lentivirus-specific CTL response and the control of lentivirus infections. ThX and mock-ThX cats were inoculated with feline immunodeficiency virus (FIV) and monitored for their FIV-specific CTL responses. Early in infection, both FIV-ThX and FIV-mock-ThX cats produced similar CTL responses, but surprisingly, after 20 weeks, the FIV-ThX cats showed a statistically significant loss of FIV-specific CTL activity, while FIV-infected cats with intact thymuses continued to maintain FIV-specific CTL. The loss of CTL did not affect plasma virus load, which remained elevated for both groups. These results emphasize the importance of thymic integrity in maintaining immunity to lentiviruses, but also bring into question the notion that virus load is regulated predominantly by the virus-specific CTL response.
In-Depth, Longitudinal Analysis of Viral Quasispecies from an Individual Triply Infected with Late-Stage Human Immunodeficiency Virus Type 1, Using a Multiple PCR Primer Approach
Authors: Gerhardt M. , Mloka D. , Tovanabutra S. , Sanders-Buell E. , Hoffmann O. , Maboko L. , Mmbando D. , Birx D. L. , McCutchan F. E. , Hoelscher M.
Published: June 2005

Coinfections with more than one human immunodeficiency virus type 1 (HIV-1) subtype appear to be the source of new recombinant strains and may be commonplace in high-risk cohorts exposed to multiple subtypes. Many potential dual infections have been identified during the HIV Superinfection Study in Mbeya, Tanzania, where 600 female bar workers who are highly exposed to subtypes A, C, and D have been evaluated every 3 months for over 3 years by use of the MHAacd HIV-1 genotyping assay. Here we describe an in-depth, longitudinal analysis of the viral quasispecies in a woman who was triply infected with HIV-1 and who developed AIDS and passed away 15 months after enrollment. The MHA results obtained at 0, 3, 6, 9, and 12 months revealed dual-probe reactivities and shifts in subtype over time, indicating a potential dual infection and prompting further investigation. The multiple infection was confirmed by PCR amplification of three genome regions by a multiple primer approach, followed by molecular cloning and sequencing. A highly complex viral quasispecies was found, including several recombinant forms, with
vpu
/gp120 being the most diverse region. A significant fluctuation in molecular forms over time was observed, showing that the serial sample format is highly desirable, if not essential, for the identification of multiple infections. In a separate experiment, we confirmed that the detection of coinfections is more efficient with the use of multiple amplification primers to overcome the primer bias that results from the enormous diversity in the HIV-1 genome.
Torovirus Non-Discontinuous Transcription: Mutational Analysis of a Subgenomic mRNA Promoter
Authors: Smits Saskia L. , van Vliet Arno L. W. , Segeren Katja , el Azzouzi Hamid , van Essen Maarten , de Groot Raoul J.
Published: June 2005

Toroviruses (order
Nidovirales
) are enveloped positive-strand RNA viruses of mammals. The prototype torovirus, equine torovirus strain Berne (Berne virus [BEV]), uses two different transcription strategies to produce a 3′-coterminal nested set of subgenomic (sg) mRNAs. Its mRNA 2 carries a leader sequence derived from the 5′ end of the genome and is produced via discontinuous transcription. The remaining three sg mRNAs, 3 to 5, are colinear with the 3′ end of the genome and are made via non-discontinuous RNA synthesis. Their synthesis is supposedly regulated by short conserved sequence motifs, 5′-ACN
3-4
CUUUAGA-3′, within the noncoding intergenic regions that precede the M, HE, and N genes (A. L. van Vliet, S. L. Smits, P. J. Rottier, and R. J. de Groot, EMBO J.
21:
6571-6580, 2002). We have now studied the—for nidoviruses unusual—non-discontinuous transcription mechanism in further detail by probing the role of the postulated transcription-regulating sequences (TRSs). To this end, we constructed a synthetic defective interfering (DI) RNA, carrying a 24-nucleotide segment of the intergenic region between the HE and N genes. We demonstrate that this DI RNA, when introduced into BEV-infected cells, directs the synthesis of a sg DI RNA species; in fact, a 16-nucleotide cassette containing the TRS already proved sufficient. Synthesis of this sg DI RNA, like that of mRNAs 3 to 5 of the standard virus, initiated at the 5′-most adenylate of the TRS. An extensive mutational analysis of the TRS is presented. Our results provide first and formal experimental evidence that the conserved motifs within the BEV intergenic sequences indeed drive sg RNA synthesis.
Control of Translation by the 5′- and 3′-Terminal Regions of the Dengue Virus Genome
Authors: Chiu Wei-Wei , Kinney Richard M. , Dreher Theo W.
Published: June 2005

The genomic RNAs of flaviviruses such as dengue virus (DEN) have a 5′ m
7
GpppN cap like those of cellular mRNAs but lack a 3′ poly(A) tail. We have studied the contributions to translational expression of 5′- and 3′-terminal regions of the DEN serotype 2 genome by using luciferase reporter mRNAs transfected into Vero cells. DCLD RNA contained the entire DEN 5′ and 3′ untranslated regions (UTRs), as well as the first 36 codons of the capsid coding region fused to the luciferase reporter gene. Capped DCLD RNA was as efficiently translated in Vero cells as capped GLGpA RNA, a reporter with UTRs from the highly expressed α-globin mRNA and a 72-residue poly(A) tail. Analogous reporter RNAs with regulatory sequences from West Nile and Sindbis viruses were also strongly expressed. Although capped DCLD RNA was expressed much more efficiently than its uncapped form, uncapped DCLD RNA was translated 6 to 12 times more efficiently than uncapped RNAs with UTRs from globin mRNA. The 5′ cap and DEN 3′ UTR were the main sources of the translational efficiency of DCLD RNA, and they acted synergistically in enhancing translation. The DEN 3′ UTR increased mRNA stability, although this effect was considerably weaker than the enhancement of translational efficiency. The DEN 3′ UTR thus has translational regulatory properties similar to those of a poly(A) tail. Its translation-enhancing effect was observed for RNAs with globin or DEN 5′ sequences, indicating no codependency between viral 5′ and 3′ sequences. Deletion studies showed that translational enhancement provided by the DEN 3′ UTR is attributable to the cumulative contributions of several conserved elements, as well as a nonconserved domain adjacent to the stop codon. One of the conserved elements was the conserved sequence (CS) CS1 that is complementary to cCS1 present in the 5′ end of the DEN polyprotein open reading frame. Complementarity between CS1 and cCS1 was not required for efficient translation.
Human Cytomegalovirus UL130 Protein Promotes Endothelial Cell Infection through a Producer Cell Modification of the Virion
Authors: Patrone Marco , Secchi Massimiliano , Fiorina Loretta , Ierardi Mariagrazia , Milanesi Gabriele , Gallina Andrea
Published: June 2005

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in
trans
to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in
trans
the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.
Herpes Simplex Virus 1 ICP22 Regulates the Accumulation of a Shorter mRNA and of a Truncated U
S
3 Protein Kinase That Exhibits Altered Functions
Authors: Poon Alice P. W. , Roizman Bernard
Published: June 2005

The U
S
3 open reading frame of herpes simplex virus 1 (HSV-1) was reported to encode two mRNAs each directing the synthesis of the same protein. We report that the U
S
3 gene encodes two proteins. The predominant U
S
3 protein is made in wild-type HSV-1-infected cells. The truncated mRNA and a truncated protein designated U
S
3.5 and initiating from methionine 77 were preeminent in cells infected with a mutant lacking the gene encoding ICP22. Both the wild-type and truncated proteins also accumulated in cells transduced with a baculovirus carrying the entire U
S
3 open reading frame. The U
S
3.5 protein accumulating in cells infected with the mutant lacking the gene encoding ICP22 mediated the phosphorylation of histone deacetylase 1, a function of U
S
3 protein, but failed to block apoptosis of the infected cells. The U
S
3.5 and U
S
3 proteins differ with respect to the range of functions they exhibit.
Actin Is a Component of the Compensation Mechanism in Pseudorabies Virus Virions Lacking the Major Tegument Protein VP22
Authors: del Rio T. , DeCoste C. J. , Enquist L.W.
Published: June 2005

Despite being a major component of the pseudorabies virus tegument, VP22 is not required for PRV replication, virulence, or neuroinvasion (T. del Rio, H. C. Werner, and L. W. Enquist, J. Virol.
76:
774-782, 2002). In the absence of VP22, tegument assembly compensates in a limited fashion with increased incorporation of cellular actin. Infection of epithelial cell lines expressing fluorescent actin fusion proteins resulted in the incorporation of filamentous and nonfilamentous actin into individual virions that were predominately light, noninfectious particles. We conclude that cellular actin is incorporated in the tegument of wild-type virions and is part of a compensation mechanism for VP22-null virions.
nTH-ORDER SQUEEZING OF THE FIELD AMPLITUDE IN RAMAN PROCESS AS A GENERALIZATION OF THE HIGHER-ORDER SQUEEZING
Authors: GIRI DILIP KUMAR , GUPTA P. S.
Published: June 2005

The concept of nth-order amplitude squeezing is introduced in the fundamental mode in Raman process as a generalization of the higher-order squeezing under short-time approximation based on a fully quantum mechanical approach. The condition for occurrence of nth-order squeezing is obtained from which higher-order squeezing up to n = 5 are studied. The coupled Heisenberg equation of motion involving real and imaginary parts of the quadrature operators is established. Dependence of squeezing on photon number is also established.
VORTICES IN MULTICOMPONENT BOSE–EINSTEIN CONDENSATES
Authors: KASAMATSU KENICHI , TSUBOTA MAKOTO , UEDA MASAHITO
Published: June 2005

We review the topic of quantized vortices in multicomponent Bose–Einstein condensates of dilute atomic gases, with an emphasis on the two-component condensates. First, we review the fundamental structure, stability and dynamics of a single vortex state in a slowly rotating two-component condensates. To understand recent experimental results, we use the coupled Gross–Pitaevskii equations and the generalized nonlinear sigma model. An axisymmetric vortex state, which was observed by the JILA group, can be regarded as a topologically trivial skyrmion in the pseudospin representation. The internal, coherent coupling between the two components breaks the axisymmetry of the vortex state, resulting in a stable vortex molecule (a meron pair). We also mention unconventional vortex states and monopole excitations in a spin-1 Bose–Einstein condensate. Next, we discuss a rich variety of vortex states realized in rapidly rotating two-component Bose–Einstein condensates. We introduce a phase diagram with axes of rotation frequency and the intercomponent coupling strength. This phase diagram reveals unconventional vortex states such as a square lattice, a double-core lattice, vortex stripes and vortex sheets, all of which are in an experimentally accessible parameter regime. The coherent coupling leads to an effective attractive interaction between two components, providing not only a promising candidate to tune the intercomponent interaction to study the rich vortex phases but also a new regime to explore vortex states consisting of vortex molecules characterized by anisotropic vorticity. A recent experiment by the JILA group vindicated the formation of a square vortex lattice in this system.
CORRELATION OF SUPERCURRENTS IN TWO DISTANT MESOSCOPIC SQUID RINGS IN THE PRESENCE OF TWO-MODE NONCLASSICAL FIELD
Authors: PENG Z. H. , ZOU J. , SHAO B. , CAI J. F.
Published: June 2005

In this paper we consider two distant mesoscopic superconducting quantum interference device (SQUID) rings A and B in the presence of two-mode squeezed vacuum state field, and aim to see the effect of the field on the correlation of supercurrents in the two rings. We know the degree of entanglement of the two-mode squeezed vacuum state increases with the squeezing parameter r. In this paper we find that the correlation of the supercurrents increases with the parameter r too, meaning that the correlation of the supercurrents increases with the degree of entanglement of the field.
Increased cathepsin D release byHypmouse osteoblast cells
Authors: Matsumoto Naoko , Jo Oak D. , Shih Remi N. J. , Brochmann Elsa J. , Murray Samuel S. , Hong Victor , Yanagawa Jane , Yanagawa Norimoto
Published: June 2005

The X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism but also by factor(s) locally released by osteoblast cells (ObCs). The identity of these ObC-derived pathogenic factors remains unclear. In our present study, we report our finding of a prominent protein in the culture media derived from ObC of the hypophosphatemic ( Hyp) mice, a murine homolog of human XLH, which was identified as the murine procathepsin D (Cat D). By metabolic labeling studies, we further confirmed that Hyp mouse ObCs released greater amount of Cat D into culture media. This increased Cat D release by Hyp mouse ObCs was unlikely to be due to nonspecific cell damage or heterogeneous cell population and was found to be associated with an increased Cat D expression at the protein level, possibly due to a reduced Cat D degradation. However, we were not able to detect a direct effect of PHEX protein on Cat D cleavage. In support of the involvement of Cat D in mediating the inhibitory effect of Hyp mouse ObC-conditioned media on ObC calcification, we found that exposure to Cat D inhibited ObC45Ca incorporation and that inhibition of Cat D abolished the inhibitory effect of Hyp mouse-conditioned media on ObC calcification. In conclusion, results from our present study showed that Hyp mouse ObCs release a greater amount of Cat D, which may contribute to the inhibitory effect of Hyp mouse ObC-conditioned media on ObC mineralization.
Does load-induced ventricular hypertrophy progress to systolic heart failure?
Authors: Berenji Kambeez , Drazner Mark H. , Rothermel Beverly A. , Hill Joseph A.
Published: June 2005

Ventricular hypertrophy develops in response to numerous forms of cardiac stress, including pressure or volume overload, loss of contractile mass from prior infarction, neuroendocrine activation, and mutations in genes encoding sarcomeric proteins. Hypertrophic growth is believed to have a compensatory role that diminishes wall stress and oxygen consumption, but Framingham and other studies established ventricular hypertrophy as a marker for increased risk of developing chronic heart failure, suggesting that hypertrophy may have maladaptive features. However, the relative contribution of comorbid disease to hypertrophy-associated systolic failure is unknown. For instance, coronary artery disease is induced by many of the same risk factors that cause hypertrophy and can itself lead to systolic dysfunction. It is uncertain, therefore, whether ventricular hypertrophy commonly progresses to systolic dysfunction without the contribution of intervening ischemia or infarction. In this review, we summarize findings from epidemiologic studies, preclinical experiments in animals, and clinical trials to lay out what is known—and not known—about this important question.
Comments on “Do fever and anapyrexia exist? Analysis of set point-based definitions”
Authors: Caputa Michal
Published: June 2005

The following is the abstract of the article discussed in the subsequent letter:

Fever and anapyrexia are the most studied thermoregulatory responses. They are defined as a body temperature (Tb) increase and decrease, respectively, occurring because of a shift in the set point (SP) and characterized by active defense of the new Tb. Although models of Tb control with a single SP (whether obvious or hidden) have been criticized, the SP-based definitions have remained unchallenged. In this article, the SP-based definitions of fever and anapyrexia were subjected to two tests. In test 1, they were compared with experimental data on changes in thresholds for activation of different thermoeffectors. Changes in thresholds were found compatible with an SP increase in some (but not all) cases of fever. In all cases of what is called anapyrexia, its mechanism (dissociation of thresholds of different effectors) was found incompatible with a decrease in a single SP. In test 2, experimental data on the dependence of Tb on ambient temperature (Ta) were analyzed. It was found that the febrile level of Tb is defended in some (but not all) cases. However, strong dependence on Ta was found in all cases of anapyrexia, which agrees with threshold dissociation but not with a decrease of the SP. It is concluded that fever (as defined) has only limited experimental support, whereas anapyrexia (as defined) does not exist. Two solutions are offered. A palliative is to accept that SP-based terms (anapyrexia, cryexia, regulated hypothermia, and such) are inadequate and should be abandoned. A radical solution is to transform all definitions based on comparing Tb with the SP into definitions based on balancing active and passive processes of Tb control.
High-soy diet decreases infarct size after permanent middle cerebral artery occlusion in female rats
Authors: Schreihofer Derek A. , Do Khoi D. , Schreihofer Ann M.
Published: June 2005

Estrogen is a powerful neuroprotective agent in rodent models of ischemic stroke. However, in humans, estrogen treatment can increase risk of stroke. Health risks associated with hormone replacement have led many women to consider alternative therapies including high-soy diets or supplements containing soy isoflavones, which act as estrogen receptor ligands to selectively mimic some of estrogen's actions. We hypothesized that a high-soy diet would share the neuroprotective actions of estrogen in focal cerebral ischemia. Female Sprague-Dawley rats were ovariectomized and divided into three groups: isoflavone-free diet + placebo (IF-P), isoflavone-free diet + estradiol (IF-E), or high-soy diet + placebo (S-P). Two weeks after being placed on diets, rats underwent left permanent middle cerebral artery occlusion (MCAO). Reductions in ipsilateral cerebral blood flow were equivalent across groups (∼50%). Twenty-four hours later neurological deficit was determined, and brains were collected for assay of cerebral infarct by TTC staining. In the IF-P rats MCAO produced a 50 ± 4% cerebral infarct. Estrogen and high-soy diet both significantly reduced the size of the infarcts to 26 ± 5% in IF-E rats and to 37 ± 5% in S-P rats. Analysis at five rostro-caudal levels revealed that estrogen treatment was slightly more effective at reducing infarct size than high soy diet. Overall neurological deficit scores at 24 h correlated with infarct size; however, there were no statistically significant differences among the treatment groups. These data show that 2 wk of a high-soy diet is an effective prophylactic strategy for reducing stroke size in a rat model of focal cerebral ischemia.
Diabetes causes inhibition of glucose-6-phosphate dehydrogenase via activation of PKA, which contributes to oxidative stress in rat kidney cortex
Authors: Xu Yizhen , Osborne Brent W. , Stanton Robert C.
Published: June 2005

The incidence of diabetic nephropathy has been increasing. Studies have shown that oxidative stress (due to increased oxidant production and/or decreased antioxidant activity) is a critical underlying mechanism. The principal intracellular reductant is NADPH whose production is mainly dependent on glucose-6-phosphate dehydrogenase (G6PD) activity. Our work in cultured cells previously showed that high glucose caused activation of protein kinase A (PKA) and subsequent phosphorylation and inhibition of G6PD activity and hence decreased NADPH (Zhang Z, Apse K, Pang J, and Stanton RC. J Biol Chem 275:40042-40047, 2000). The purpose of this study was to determine whether these findings occur in diabetic rats (induced by streptozotocin) compared with control. G6PD activity and accordingly NADPH levels and glutathione levels were significantly decreased in diabetic kidneys compared with control kidneys. Lipid peroxidation was significantly increased, which correlated with decreased G6PD activity ( r = 0.48). G6PD expression was significantly reduced, which correlated with decreased G6PD activity ( r = 0.72). PKA activity and serine phosphorylation of G6PD were significantly increased and were closely correlated with decreased G6PD activity ( r = 0.51 for PKA activity; r = 0.93 for serine phosphorylation of G6PD). Insulin treatment and/or correction of hyperglycemia ameliorated the changes caused by diabetes. In conclusion, chronic hyperglycemia caused inhibition of G6PD activity via decreased expression and increased phosphorylation of G6PD, which therefore led to increased oxidative stress.
Hypoxia-inducible factor modulates tubular cell survival in cisplatin nephrotoxicity
Authors: Tanaka Tetsuhiro , Kojima Ichiro , Ohse Takamoto , Inagi Reiko , Miyata Toshio , Ingelfinger Julie R. , Fujita Toshiro , Nangaku Masaomi
Published: June 2005

Hypoxia-inducible factor (HIF)-1 is a transcription factor mediating cellular response to hypoxia. Although it is expressed in tubular cells of the ischemic kidney, its functional role is not fully clarified in the pathological context. In this study, we investigated a role of HIF in tubular cell apoptosis induced by cisplatin. HIF-1α was expressed in tubular cells in the outer medulla 3 days after cisplatin (6 mg/kg) administration. With the in vivo administration of cobalt to activate HIF, the number of apoptotic renal tubular cells became much smaller in the outer medulla, compared with the vehicle group. We also examined the functional role of HIF-1 in vitro using immortalized rat proximal tubular cells (IRPTC). In hypoxia, IRPTC that express dominant-negative (dn) HIF-1α showed impaired survival in cisplatin injury at variable doses (25–100 μM, 24 h), which was not obvious in normoxia. The observed difference in cell viability in hypoxia was associated with the increased number of apoptotic cells in dnHIF-1α clones (Hoechst 33258 staining). Studies on intracellular signaling revealed that the degree of cytochrome c release, dissipation of mitochondrial membrane potentials, and caspase-9 activity were all more prominent in dnHIF-1α clones than in control IRPTC, pointing to the accelerated signaling of mitochondrial pathways. We propose that HIF-1 mediates cytoprotection against cisplatin injury in hypoxic renal tubular cells, by reducing the number of apoptotic cells through stabilization of mitochondrial membrane integrity and suppression of apoptosis signaling. A possibility was suggested that activation of HIF-1 could be a new promising therapeutic target for hypoxic renal diseases.
Association of CD2AP with dynamic actin on vesicles in podocytes
Authors: Welsch Thilo , Endlich Nicole , Gökce Gökmen , Doroshenko Elena , Simpson Jeremy C. , Kriz Wilhelm , Shaw Andrey S. , Endlich Karlhans
Published: June 2005

The docking protein CD2AP (CD2-associated protein) serves a nonredundant function in podocytes as CD2AP knockout mice die of renal failure at the age of 6–7 wk. Furthermore, haploinsufficiency due to mutation of the CD2AP gene is associated with focal segmental glomerulosclerosis in humans. Although CD2AP has been shown to interact with proteins regulating actin polymerization, with proteins of the slit diaphragm, and with the endocytic machinery, its critical function in podocytes remains unclear. In conditionally immortalized mouse podocytes, we demonstrate that CD2AP colocalizes with cortactin and F-actin in spots of ≤0.5-μm diameter. Confocal time-lapse microscopy in living podocytes expressing GFP-CD2AP or GFP-actin revealed that spots are motile, possess a limited lifetime, and are frequently associated with vesicles. A significant portion of spot-associated vesicles belongs to a later endosomal-sorting compartment, characterized by delayed uptake of fluorescent dextran (10 kDa) and by colocalization with Rab4, but not Rab5 and AP-2. Rapid accumulation of microinjected G-actin in spots and abrogation of spot motility by jasplakinolide demonstrate that spot movements depend on actin polymerization. Furthermore, a high turnover (half-time < 10 s) of CD2AP in spots was demonstrated by FRAP (fluorescence recovery after photobleaching). Our results demonstrate that CD2AP is associated with dynamic actin in a specific late endosomal compartment in podocytes, suggesting that CD2AP might be crucially involved in endosomal sorting and/or trafficking via regulation of actin assembly on vesicles.
Interlimb Coordination During Locomotion: What Can be Adapted and Stored?
Authors: Reisman Darcy S. , Block Hannah J. , Bastian Amy J.
Published: June 2005

Interlimb coordination is critically important during bipedal locomotion and often must be adapted to account for varying environmental circumstances. Here we studied adaptation of human interlimb coordination using a split-belt treadmill, where the legs can be made to move at different speeds. Human adults, infants, and spinal cats can alter walking patterns on a split-belt treadmill by prolonging stance and shortening swing on the slower limb and vice versa on the faster limb. It is not known whether other locomotor parameters change or if there is a capacity for storage of a new motor pattern after training. We asked whether adults adapt both intra- and interlimb gait parameters during split-belt walking and show aftereffects from training. Healthy subjects were tested walking with belts tied (baseline), then belts split (adaptation), and again tied (postadaptation). Walking parameters that directly relate to the interlimb relationship changed slowly during adaptation and showed robust aftereffects during postadaptation. These changes paralleled subjective impressions of limping versus no limping. In contrast, parameters calculated from an individual leg changed rapidly to accommodate split-belts and showed no aftereffects. These results suggest some independence of neural control of intra- versus interlimb parameters during walking. They also show that the adult nervous system can adapt and store new interlimb patterns after short bouts of training. The differences in intra- versus interlimb control may be related to the varying complexity of the parameters, task demands, and/or the level of neural control necessary for their adaptation.
Mice Vaccinated with Allergen-Pulsed Myeloid Dendritic Cells Are Not Protected from Developing Allergen-Induced Th2 Responses
Authors: Trujillo-Vargas Claudia Milena , Ramirez-Pineda José Robinson , Palmetshofer Alois , Grunewald Susanne , Moll Heidrun , Berberich Christoph , Erb Klaus Joseph
Published: June 2005

Background: Dendritic cells (DC) play a decisive role in the induction of allergen-induced Th1 and Th2 responses. Since the induction of allergen-specific Th1 responses has shown to inhibit allergen-induced Th2-type inflammation, in this study we investigated whether manipulated myeloid-derived DC pulsed with the specific allergen would predominantly induce allergen-specific Th1 responses thereby reducing the development of Th2 responses. Methods: Murine bone marrow (BM)-DC were generated and pulsed with ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG-ODN). Langerhans cells (LC) were also isolated and pulsed in vitro with OVA. Subsequently, mice were vaccinated intravenously with either CpG/OVA-pulsed BM-DC or OVA-pulsed LC, and the protocol to induce OVA-specific Th2 responses using OVA/alum sensitization was initiated. Airway inflammation and OVA-specific serum antibody levels were evaluated 6 days after the intranasal challenge with OVA. Results: The application ofCpG/OVA-pulsed BM-DC was unable to reduce airway eosinophilia and inflammation in OVA/alum-immunized mice. OVA-specific IgG1 or IgE serum levels were also not reduced. The experiments using LC pulsed with OVA yielded similar results. However, mice vaccinated with CpG/OVA-pulsed BM-DC had greatly enhanced levels of serum OVA-specific IgG2a, suggesting the induction of allergen-specific Th1 responses in vivo. Moreover, allergen-induced mast cell degranulation was decreased using this approach. Conclusions: Taken together, our results demonstrated that the vaccination with OVA-pulsed BM-DC matured with CpG-ODN or OVA-pulsed LC did not result in a reduction in allergen-specific Th2 responses in a murine model of severe atopic asthma. Other DC-based vaccination strategies should be evaluated in order to prevent the development of allergic disorders.
Intravenous Calcitriol Therapy Increases Serum Concentrations of Fibroblast Growth Factor-23 in Dialysis Patients with Secondary Hyperparathyroidism
Authors: Nishi Hiroshi , Nii-Kono Tomoko , Nakanishi Shohei , Yamazaki Yuji , Yamashita Takeyoshi , Fukumoto Seiji , Ikeda Kazuto , Fujimori Akira , Fukagawa Masafumi
Published: June 2005

Background/Aims: Fibroblast growth factor-23 (FGF-23) is a recently discovered phosphaturic factor. Although increased levels of serum FGF-23 have been reported in dialysis patients, the role of high FGF-23 levels remains unclear. Since FGF-23 is associated also with vitamin D metabolism, we examined the changes of serum FGF-23 levels in chronic dialysis patients treated with intravenous calcitriol therapy. Methods: Thirty patients with severe secondary hyperparathyroidism were treated with intravenous calcitriol (0.5–1.0 µg) two or three times per week for 6 months. The changes of serum levels of calcium, phosphate, intact PTH, and FGF-23 were evaluated. Results: Baseline serum FGF-23 levels were markedly high. By intravenous calcitriol therapy, intact PTH levels decreased effectively in the first month (p < 0.001). In contrast, FGF-23 levels increased gradually during the study period (p = 0.027). The Δ serum FGF-23 level was significantly correlated with the total doses of calcitriol injected intravenously in 6 months in patients with refractory secondary hyperparathyroidism (R2 = 0.147; p = 0.036). Conclusions: Intravenous calcitriol decreased serum intact PTH level and increased serum FGF-23 levels significantly. Extremely high levels of serum FGF-23 in these patients may be attributed, at least in part, to the cumulative dose of vitamin D.
Endothelin-1 (1-31) Is an Intermediate in the Production of Endothelin-1 After Big Endothelin-1 Administration In Vivo
Authors: Fecteau Marie-Hélène , Honoré Jean-Claude , Plante Mirco , Labonté Julie , Rae Giles A. , D’Orléans-Juste Pedro
Published: June 2005

The precursor of endothelin-1, big endothelin-1, can be hydrolyzed by chymase to generate endothelin-1 (1-31) in vitro. In the present study, we explored the processes involved in the production of endothelin-1 (1-31) as well as its pharmacodynamic characteristics in the rabbit in vivo. Endothelin-1 (1-31) (1 nmol/kg, injected into the left cardiac ventricle) induced a monophasic increase of mean arterial blood pressure similarly to big endothelin-1 (1-38), whereas endothelin-1 induces a biphasic response. Phosphoramidon, a dual neutral endopeptidase and endothelin-converting enzyme inhibitor, blocked both pressor responses to endothelin-1 (1-31) and big endothelin-1 but not those afforded by endothelin-1. Thiorphan, a neutral endopeptidase inhibitor, markedly inhibited the response to endothelin-1 (1-31) but only weakly reduced that of big endothelin-1. In contrast, CGS 35066, an endothelin-converting enzyme inhibitor, was significantly more efficient against the pressor response to big endothelin-1 than to endothelin-1 (1-31). Furthermore, injection of big endothelin-1 concomitantly with phosphoramidon induced an increase in endothelin-1 (1-31) plasma levels. Finally, intracardiac-administered endothelin-1 (1-31) induced an increase of endothelin-1 plasma levels, which are markedly reduced by phosphoramidon and thiorphan but not by CGS 35066. Our results thus demonstrate that endothelin-1 (1-31) is an alternate intermediate in the production of endothelin-1 after big endothelin-1 administration in the rabbit in vivo.
Modest Salt Reduction Lowers Blood Pressure in Isolated Systolic Hypertension and Combined Hypertension
Authors: He Feng J. , Markandu Nirmala D. , MacGregor Graham A.
Published: June 2005

Many randomized trials have shown that a reduction in salt intake lowers blood pressure in hypertensive individuals. However, few have looked at the effects according to hypertension category. A recent analysis of the third and fourth National Health and Nutrition Examination Survey suggests that salt intake may not be related to blood pressure in isolated systolic or combined hypertension. To look at this further, we reanalyzed the data of our previous salt reduction trials. Hypertensive individuals were studied in randomized double-blind crossover studies: 1 month of usual salt intake compared with 1 month of reduced salt intake. In isolated systolic hypertension (n=24), blood pressure was reduced from 166±19/86±7 to 156±20/85±7 mm Hg (systolic
P
<0.001; diastolic
P
=0.459) with a reduction in urinary sodium from 175±51 to 87±38 mmol per 24-hour period (10.3 to 5.1 g per day of salt). In combined hypertension (n=88), blood pressure was reduced from 161±16/100±9 to 154±17/96±9 mm Hg (
P
<0.001) with a reduction urinary sodium from 176±65 to 98±51 mmol per 24-hour period (10.4 to 5.8 g per day of salt). These results demonstrate that salt reduction has a significant effect on blood pressure in isolated systolic and combined hypertension. The fall in systolic observed in isolated systolic hypertension would be predicted to reduce stroke by approximately one third, ischemic heart disease by one quarter, and heart failure by one quarter in the population between 60 and 80 years of age, in whom isolated systolic hypertension is the predominate form of hypertension and carries the highest risk. These results provide strong support for universal salt reduction in all hypertensives.