bluuebunny/crossref_metadata_2025 huggingface dataset:
Adeno-Associated Virus Type 2 Capsids with Externalized VP1/VP2 Trafficking Domains Are Generated prior to Passage through the Cytoplasm and Are Maintained until Uncoating Occurs in the Nucleus
Authors: Sonntag Florian , Bleker Svenja , Leuchs Barbara , Fischer Roger , Kleinschmidt Jürgen A.
Published: October 2006

Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A2catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.
Characterization of the Functional Activities of the Bovine Papillomavirus Type 1 E2 Protein Single-Chain Heterodimers
Authors: Kurg Reet , Tekkel Helena , Abroi Aare , Ustav Mart
Published: October 2006

Papillomaviruses are small DNA viruses which establish persistent infection in the epithelial tissue of various animal species. Three papillomavirus proteins encoded by the bovine papillomavirus type 1 E2 open reading frame have a common C-terminal DNA binding and dimerization domain and function as dimeric proteins in the regulation of viral gene expression, genome replication, and maintenance. The full-length E2 protein, expressed usually at the lowest level of the three, is an activator, while shorter forms of E2, lacking the transactivation domain, serve as repressors of replication and transcription. In virally infected cells, the full-length E2 protein forms heterodimers with repressor forms of the E2 protein and the biological activities of such heterodimers are poorly known. In order to study the functionality of E2 heterodimers, we joined the full-length E2 protein and E2 repressor by a flexible polypeptide hinge so that they formed a single-chain intramolecular dimer. The single-chain E2 heterodimers folded correctly to form genuine pseudodimers capable of binding to the specific E2 protein binding site with high affinity. Characterization of the activities of this protein in transcription showed that it functions as an effective transcriptional activator, which is comparable to what was found for the full-length E2 protein. The single-chain heterodimer is dependent to some extent on Brd4 protein and is able to support papillomavirus origin replication; however, it does not support the partitioning of the multimeric E2 binding site containing plasmids in dividing cells. Our results suggest that E2 heterodimers serve as activators of transcription and replication during the viral life cycle.
Tracking of Peptide-Specific CD4+T-Cell Responses after an Acute Resolving Viral Infection: a Study of Parvovirus B19
Authors: Kasprowicz Victoria , Isa Adiba , Tolfvenstam Thomas , Jeffery Katie , Bowness Paul , Klenerman Paul
Published: October 2006

The evolution of peptide-specific CD4+T-cell responses to acute viral infections of humans is poorly understood. We analyzed the response to parvovirus B19 (B19), a ubiquitous and clinically significant pathogen with a compact and conserved genome. The magnitude and breadth of the CD4+T-cell response to the two B19 capsid proteins were investigated using a set of overlapping peptides and gamma interferon-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells (PBMCs) from a cohort of acutely infected individuals who presented with acute arthropathy. These were compared to those for a cohort of B19-specific immunoglobulin M-negative (IgM−), IgG+remotely infected individuals. Both cohorts of individuals were found to make broad CD4+responses. However, while the responses following acute infection were detectable ex vivo, responses in remotely infected individuals were only detected after culture. One epitope (LASEESAFYVLEHSSFQLLG) was consistently targeted by both acutely (10/12) and remotely (6/7) infected individuals. This epitope was DRB1*1501 restricted, and a major histocompatibility complex peptide tetramer stained PBMCs from acutely infected individuals in the range of 0.003 to 0.042% of CD4+T cells. Tetramer-positive populations were initially CD62Llo; unlike the case for B19-specific CD8+T-cell responses, however, CD62L was reexpressed at later times, as responses remained stable or declined slowly. This first identification of B19 CD4+T-cell epitopes, including a key immunodominant peptide, provides the tools to investigate the breadth, frequency, and functions of cellular responses to this virus in a range of specific clinical settings and gives an important reference point for analysis of peptide-specific CD4+T cells during acute and persistent virus infections of humans.
Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH
Authors: Schowalter Rachel M. , Smith Stacy E. , Dutch Rebecca Ellis
Published: October 2006

Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.
Persistent Hepatitis C Virus Infection In Vitro: Coevolution of Virus andHost
Authors: Zhong Jin , Gastaminza Pablo , Chung Josan , Stamataki Zania , Isogawa Masanori , Cheng Guofeng , McKeating Jane A. , Chisari Francis V.
Published: October 2006

The
virological and cellular consequences of persistent hepatitis C virus
(HCV) infection have been elusive due to the absence of the requisite
experimental systems. Here, we report the establishment and the
characteristics of persistent in vitro infection of human
hepatoma-derived cells by a recently described HCV genotype 2a
infectious molecular clone. Persistent in vitro infection was
characterized by the selection of viral variants that displayed
accelerated expansion kinetics, higher peak titers, and increased
buoyant densities. Sequencing analysis revealed the selection of a
single adaptive mutation in the HCV E2 envelope protein that was
largely responsible for the variant phenotype. In parallel, as the
virus became more aggressive, cells that were resistant to infection
emerged, displaying escape mechanisms operative at the level of viral
entry, HCV RNA replication, or both. Collectively, these results reveal
the existence of coevolutionary events during persistent HCV infection
that favor survival of both virus and
host.
Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins
Authors: Trask Shane D. , Dormitzer Philip R.
Published: October 2006

Assembly of the rotavirus outer capsid is the final step of a complex pathway. In vivo, the later steps include a maturational membrane penetration that is dependent on the scaffolding activity of a viral nonstructural protein. In vitro, simply adding the recombinant outer capsid proteins VP4 and VP7 to authentic double-layered rotavirus subviral particles (DLPs) in the presence of calcium and acidic pH increases infectivity by a factor of up to 10
7
, yielding particles as infectious as authentic purified virions. VP4 must be added before VP7 for high-level infectivity. Steep dependence of infectious recoating on VP4 concentration suggests that VP4-VP4 interactions, probably oligomerization, precede VP4 binding to particles. Trypsin sensitivity analysis identifies two populations of VP4 associated with recoated particles: properly mounted VP4 that can be specifically primed by trypsin, and nonspecifically associated VP4 that is degraded by trypsin. A full complement of properly assembled VP4 is not required for efficient infectivity. Minimal dependence of recoating on VP7 concentration suggests that VP7 binds DLPs with high affinity. The parameters for efficient recoating and the characterization of recoated particles suggest a model in which, after a relatively weak interaction between oligomeric VP4 and DLPs, VP7 binds the particles and locks VP4 in place. Recoating will allow the use of infectious modified rotavirus particles to explore rotavirus assembly and cell entry and could lead to practical applications in novel immunization strategies.
In Vitro Resistance to the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PA-457 (Bevirimat)
Authors: Adamson Catherine S. , Ablan Sherimay D. , Boeras Ioana , Goila-Gaur Ritu , Soheilian Ferri , Nagashima Kunio , Li Feng , Salzwedel Karl , Sakalian Michael , Wild Carl T. , Freed Eric O.
Published: October 2006

3-
O
-(3′,3′-dimethylsuccinyl)betulinic acid (PA-457 or bevirimat) potently inhibits human immunodeficiency virus type 1 (HIV-1) maturation by blocking a late step in the Gag processing pathway, specifically the cleavage of SP1 from the C terminus of capsid (CA). To gain insights into the mechanism(s) by which HIV-1 could evolve resistance to PA-457 and to evaluate the likelihood of such resistance arising in PA-457-treated patients, we sought to identify and characterize a broad spectrum of HIV-1 variants capable of conferring resistance to this compound. Numerous independent rounds of selection repeatedly identified six single-amino-acid substitutions that independently confer PA-457 resistance: three at or near the C terminus of CA (CA-H226Y, -L231F, and -L231M) and three at the first and third residues of SP1 (SP1-A1V, -A3T, and -A3V). We determined that mutations CA-H226Y, CA-L231F, CA-L231M, and SP1-A1V do not impose a significant replication defect on HIV-1 in culture. In contrast, mutations SP1-A3V and -A3T severely impaired virus replication and inhibited virion core condensation. The replication defect imposed by SP1-A3V was reversed by a second-site compensatory mutation in CA (CA-G225S). Intriguingly, high concentrations of PA-457 enhanced the maturation of SP1 residue 3 mutants. The different phenotypes associated with mutations that confer PA-457 resistance suggest the existence of multiple mechanisms by which HIV-1 can evolve resistance to this maturation inhibitor. These findings have implications for the ongoing development of PA-457 to treat HIV-1 infection in vivo.
hsp72, a Host Determinant of Measles Virus Neurovirulence
Authors: Carsillo Thomas , Traylor Zachary , Choi Changsun , Niewiesk Stefan , Oglesbee Michael
Published: October 2006

Transient hyperthermia such as that experienced during febrile episodes increases expression of the major inducible 70-kDa heat shock protein (hsp72). Despite the relevance of febrile episodes to viral pathogenesis and the multiple in vitro roles of heat shock proteins in viral replication and gene expression, the in vivo significance of virus-heat shock protein interactions is unknown. The present work determined the in vivo relationship between hsp72 levels and neurovirulence of an hsp72-responsive virus using the mouse model of measles virus (MV) encephalitis. Transgenic C57BL/6 mice were created to constitutively overexpress hsp72 in neurons, and these mice were inoculated intracranially with Edmonston MV (Ed MV) at 42 h of age. The mean viral RNA burden in brain was approximately 2 orders of magnitude higher in transgenic animals than in nontransgenic animals 2 to 4 weeks postinfection, and this increased burden was associated with a fivefold increase in mortality. Mice were also challenged with an Ed MV variant exhibiting an attenuated in vitro response to hsp72-dependent stimulation of viral transcription (Ed N-522D). This virus exhibited an attenuated neuropathogenicity in transgenic mice, where mortality and viral RNA burdens were not significantly different from nontransgenic mice infected with either Ed N-522D or parent Ed MV. Collectively, these results indicate that hsp72 levels can serve as a host determinant of viral neurovirulence in C57BL/6 mice, reflecting the direct influence of hsp72 on viral gene expression.
Virus-Specific Cellular Immune Correlates of Survival in Vaccinated Monkeys after Simian Immunodeficiency Virus Challenge
Authors: Sun Yue , Schmitz Jörn E. , Buzby Adam P. , Barker Brianne R. , Rao Srinivas S. , Xu Ling , Yang Zhi-yong , Mascola John R. , Nabel Gary J. , Letvin Norman L.
Published: October 2006

Understanding the characteristics of the virus-specific T-lymphocyte response that will confer optimal protection against the clinical progression of AIDS will inform the development of an effective cellular immunity-based human immunodeficiency virus vaccine. We have recently shown that survival in plasmid DNA-primed/recombinant adenovirus-boosted rhesus monkeys that are challenged with the simian immunodeficiency virus SIVmac251 is associated with the preservation postchallenge of central memory CD4
+
T lymphocytes and robust gamma interferon (IFN-γ)-producing SIV-specific CD8
+
and CD4
+
T-lymphocyte responses. The present studies were initiated to extend these observations to determine which virus-specific T-lymphocyte subpopulations play a primary role in controlling disease progression and to characterize the functional repertoire of these cells. We show that the preservation of the SIV-specific central memory CD8
+
T-lymphocyte population and a linked SIV-specific CD4
+
T-lymphocyte response are associated with prolonged survival in vaccinated monkeys following challenge. Furthermore, we demonstrate that SIV-specific IFN-γ-, tumor necrosis factor alpha-, and interleukin-2-producing T lymphocytes are all comparably associated with protection against disease progression. These findings underscore the contribution of virus-specific central memory T lymphocytes to controlling clinical progression in vaccinated individuals following a primate immunodeficiency virus infection.
Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes
Authors: Kanai Ryuta , Kar Kalipada , Anthony Karen , Gould L. Hannah , Ledizet Michel , Fikrig Erol , Marasco Wayne A. , Koski Raymond A. , Modis Yorgo
Published: October 2006

West Nile virus
, a member of the
Flavivirus
genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.
A High-Throughput Method for Cloning and Sequencing Human Immunodeficiency Virus Type 1 Integration Sites
Authors: Kim Sanggu , Kim Yein , Liang Teresa , Sinsheimer Janet S. , Chow Samson A.
Published: October 2006

Integration of retroviral DNA is nonspecific and can occur at many sites throughout chromosomes. However, the process is not uniformly distributed, and both hot and cold spots for integration exist. The mechanism that determines target site specificity is not well understood. Because of the nonspecific and widespread nature of integration, studies analyzing the mechanism and factors that control target site selection require the collection and analysis of a large library of human immunodeficiency virus type 1 (HIV-1) proviral clones. Such analyses are time-consuming and labor-intensive using conventional means. We have developed an efficient and high-throughput method of sequencing and mapping a large number of independent integration sites in the absence of any selection or bias. The new assay involves the use of a modified HIV-1 (NL-Mme) containing a type IIS restriction site, MmeI, at the right end of viral DNA. Digestion of genomic DNA from NL-Mme-infected cells generated viral DNA-containing fragments of a discrete size. Subsequent ligation-mediated PCR yielded short integration site fragments termed Int-tags, which were concatemerized for determining multiple integration sites in a single sequencing reaction. Analysis of chromosomal features and sequence preference associated with integration events confirmed the validity of the new high-throughput assay. The assay will aid the effort in understanding the mechanisms of target site selection during HIV-1 DNA integration, and the described methodology can be adapted easily to integration site studies involving other retroviruses and transposons.
The C-Terminal Hydrophobic Domain of Hepatitis C Virus RNA Polymerase NS5B Can Be Replaced with a Heterologous Domain of Poliovirus Protein 3A
Authors: Lee Haekyung , Liu Ying , Mejia Edison , Paul Aniko V. , Wimmer Eckard
Published: October 2006

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.
Adrenomedullin inhibits angiotensin II-induced oxidative stress via Csk-mediated inhibition of Src activity
Authors: Liu Jing , Shimosawa Tatsuo , Matsui Hiromitsu , Meng Fanyin , Supowit Scott C. , DiPette Donald J. , Ando Katsuyuki , Fujita Toshiro
Published: October 2006

We have demonstrated that adrenomedullin (AM) protects against angiotensin II (ANG II)-induced cardiovascular damage through the attenuation of increased oxidative stress observed in AM-deficient mice. However, the mechanism(s) that underlie this activity remain unclear. To address this question, we investigated the effect of AM on ANG II-stimulated reactive oxygen species (ROS) production in cultured rat aortic vascular smooth muscle cells (VSMCs). ANG II markedly increased ROS production through activation of NADPH oxidase. This effect was significantly attenuated by AM in a concentration-dependent manner. This effect was mimicked by dibutyl-cAMP and blocked by pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89), a protein kinase A inhibitor, and CGRP8–37, an AM/CGRP receptor antagonist. This inhibitory effect of AM was also lost following the expression of a constitutively active Src. Moreover, AM intersected ANG II signaling by inducing COOH-terminal Src kinase (Csk) activation that, in turn, inhibits Src activation. These data, for the first time, demonstrate that AM attenuates the ANG II-induced increase in ROS in VSMCs via activation of Csk, thereby inhibiting Src activity.
FGF-10 is decreased in bronchopulmonary dysplasia and suppressed by Toll-like receptor activation
Authors: Benjamin John T. , Smith Rebekah J. , Halloran Brian A. , Day Timothy J. , Kelly David R. , Prince Lawrence S.
Published: October 2006

Many extremely preterm infants continue to suffer from bronchopulmonary dysplasia, which results from abnormal saccular-stage lung development. Here, we show that fibroblast growth factor-10 (FGF-10) is required for saccular lung development and reduced in the lung tissue of infants with bronchopulmonary dysplasia. Although exposure to bacteria increases the risk of bronchopulmonary dysplasia, no molecular target has been identified connecting inflammatory stimuli and abnormal lung development. In an experimental mouse model of saccular lung development, activation of Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) inhibited FGF-10 expression, leading to abnormal saccular airway morphogenesis. In addition, Toll-mediated FGF-10 inhibition disrupted the normal positioning of myofibroblasts around saccular airways, similar to the mislocalization of myofibroblasts seen in patients with bronchopulmonary dysplasia. Reduced FGF-10 expression may therefore link the innate immune system and impaired lung development in bronchopulmonary dysplasia.
Truncating Variants in p53AIP1 Disrupting DNA Damage–Induced Apoptosis Are Associated with Prostate Cancer Risk
Authors: Wang Xianshu , Wang Fengwei , Taniguchi Ken , Seelan Ratnam S. , Wang Liang , Zarfas Katherine E. , McDonnell Shannon K. , Qian Chiping , Pan Kaifeng , Lu Youyong , Shridhar Viji , Couch Fergus J. , Tindall Donald J. , Beebe-Dimmer Jennifer L. , Cooney Kathleen A. , Isaacs William B. , Jacobsen Steven J. , Schaid Daniel J. , Thibodeau Stephen N. , Liu Wanguo
Published: November 2006

Germ line mutations in several genes (BRCA1, BRCA2, and CHEK2) whose products are involved in the DNA damage–signaling pathway have been implicated in prostate cancer risk. To identify additional genes in this pathway that might confer susceptibility to this cancer, we analyzed a recently identified DNA damage–response gene, p53AIP1 (a gene encoding for p53-regulated apoptosis-inducing protein 1), for genetic variants in prostate cancer. Five novel germ line variants were identified. The two truncating variants (Ser32Stop and Arg21insG) were found in 3% (4 of 132) of unselected prostate tumor samples. Genotyping of the two variants in an additional 393 men with sporadic prostate cancer showed a frequency of 3.1% (12 of 393) in contrast to 0.6% (2 of 327) in 327 unaffected men (Fisher's exact test, P = 0.018), with an odds ratio (OR) of 5.1 [95% confidence interval (95% CI), 1.1-23.0]. In addition, two of six tumors carrying the truncating variants were associated with loss of heterozygosity of the wild-type alleles, suggesting that p53AIP1 may act as a tumor suppressor. We also showed that the truncated p53AIP1 was unable to induce apoptosis and suppress cell growth in HeLa and COS-7 cells. These results suggest that loss-of-function variants in p53AIP1 associated with the risk of sporadic prostate cancer and further support the concept that the genetic defects in the DNA damage–response genes play an important role in the development of prostate cancer. (Cancer Res 2006; 66(21): 10302-7)
Examination of a CpG Island Methylator Phenotype and Implications of Methylation Profiles in Solid Tumors
Authors: Marsit Carmen J. , Houseman E. Andres , Christensen Brock C. , Eddy Karen , Bueno Raphael , Sugarbaker David J. , Nelson Heather H. , Karagas Margaret R. , Kelsey Karl T.
Published: November 2006

The CpG island methylator phenotype (CIMP), thoroughly described in colorectal cancer and to a lesser extent in other solid tumors, is important in understanding epigenetics in carcinogenesis and may be clinically useful for classification of neoplastic disease. Therefore, we investigated whether this putative phenotype exists in exposure-related solid tumors, where somatic gene alterations and enhanced clonal growth are selected for by carcinogens, and examined the ability of methylation profiles to classify malignant disease. We studied promoter hypermethylation of 16 tumor suppressor genes and 3 MINT loci (acknowledged classifiers of CIMP) in 344 bladder cancers, 346 head and neck squamous cell carcinomas (HNSCC), 146 non–small-cell lung cancer (NSCLC), and 71 malignant pleural mesotheliomas (MPM). We employed rigorous statistical methods to examine the distribution of promoter methylation and the usefulness of these profiles for disease classification. In bladder cancer, HNSCC, and NSCLC, there was a significant correlation (P < 0.0001) between methylation of the three MINT loci and methylation index, although the distribution of methylated loci varied significantly across these disease. Although there was a significant (P < 0.001) association between gene methylation profile and disease, rates of misclassification of each disease by their methylation profile ranged from 28% to 32%, depending on the classification scheme used. These data suggest that a form of CIMP exists in these solid tumors, although its etiology remains elusive. Whereas the gene profiles of hypermethylation among examined loci could not unequivocally distinguish disease type, the existence of CIMP and the relative preponderance of hypermethylation in these cancers suggest that methylation analysis may be clinically useful as a targeted screening tool. (Cancer Res 2006; 66(21): 10621-9)
Cells of Proopiomelanocortin Lineage from the Rodent Anterior Pituitary Lack Sexually Dimorphic Expression of Neurofilaments
Authors: Fiordelisio Tatiana , Millán-Aldaco Diana , Hernández-Cruz Arturo
Published: November 2006

Lactotrophs, gonadotrophs, thyrotrophs and somatotrophs of the rat anterior pituitary (AP) express 68-kDa neurofilaments (NF68) and other neuronal markers. NF68 expression in the AP appears to be estrogen-dependent, but its significance is unknown. The aims of this work were: (1) to establish the expression pattern of NF68 immunoreactivity in the mouse AP, and (2) discover if corticotrophs and melanotrophs from both rodent species also express NF68. Primary cultures and frozen sections of AP from sexually mature mice were immunolabeled with anti-NF68 antibodies. In separate experiments, samples were immunostained for NF68 and AP hormones. Here we report that mouse lactotrophs, gonadotrophs, thyrotrophs and somatotrophs also express NF68 in a sexually dimorphic manner. The percentages of non-expressing, weakly expressing and strongly expressing cells were similar between both rodent species, although NF68+ cells were about 50% less abundant in the mouse compared to the rat pituitary. Remarkably, our study shows for the first time that rodent pituitary cells from the proopiomelanocortin lineage nearly completely lack NF68 immunoreactivity. In this regard, they differ from the rest of the AP population. Our findings establish a foundation for experiments aimed at investigating the functional significance of estrogen-dependent regulation of NF68 expression in rodent AP cells.
Friedrich Feyrter: A Precise Intellect in a Diffuse System
Authors: Champaneria Manish C. , Modlin Irvin M. , Kidd Mark , Eick Geeta N.
Published: November 2006

Prior to the contributions of Friedrich Feyrter (1895–1973), the regulation of gastrointestinal function was an ill-understood field that was polarized by a combination of the inability of clinical scientists to perceive the relationship between the cellular elements of ‘nervism’ and the newly recognized chemical messenger system. Feyrter, an Austrian pathologist of luminescent intellect and possessed of rigorous analytic capacity, recognized the interface of the divergent elements (neural and endocrine) and established the concept of the diffuse neuroendocrine system. His pathological descriptions of the specialized neuroendocrine cells producing biologically active substances and regulating homeostasis by a network functioning via endocrine, paracrine, and neuracrine mechanisms laid the basis for contemporary understanding of gut function. In 1938, Feyrter identified Helle Zellen (clear cells) of the pancreas and gastrointestinal tract, which was later incorporated into the amine precursor uptake decarboxylation concept of endocrine cells by A.G.E. Pearse (1916–2003). Feyrter proposed a diffuse network as a functional regulatory system as opposed to the then current doctrine of ‘organ’ regulation in his 1938 manuscript Über diffuse endokrine epitheliale Organe. In addition to this seminal contribution, the prodigious intellect of Feyrter produced an array of novel observations including benign and malignant tumors of the skin, gastrointestinal tract, and eyes, carcinoid tumors and the carcinoid syndrome, the genesis of the nevus, the transformation of lipids and disorders of cellular metabolism. Sadly, the contributions of Feyrter were obscured in the catastrophe of wartime Germany and his accomplishments little recognized. We describe the life and times of this gifted scientist, teacher, and pathologist, often referred to as the ‘Father of Neuroendocrinology’.
Apolipoprotein E Genotype Influences Presence and Severity of Delusions and Aggressive Behavior in Alzheimer Disease
Authors: van der Flier Wiesje M. , Staekenborg Salka , Pijnenburg Yolande A.L. , Gillissen Freek , Romkes Rolinka , Kok Astrid , Bouwman Femke H. , Scheltens Philip
Published: November 2006

Aim: We investigated differences in the prevalence and severity of 10 neuropsychiatric and behavioral symptoms according to apolipoprotein E (APOE) genotype and dementia severity in Alzheimer disease (AD). Methods: Neuropsychiatric and behavioral symptoms of 110 AD patients were assessed using the Neuropsychatric Inventory. Dementia severity was assessed using the Mini Mental State Examination (MMSE). Results: There were 27 APOE-Ε4-negative patients, 65 heterozygous patients and 18 homozygous patients. There was a significant association between the number of APOE Ε4 alleles and prevalence and severity of neuropsychiatric and behavioral symptoms that was mainly attributable to delusions and agitation/aggression, which were more common and severer among homozygous APOE Ε4 carriers. In addition, the presence of hallucinations, anxiety, apathy and aberrant motor behavior increased with deteriorating MMSE score, independently of APOE Ε4 status. Conclusions: The present study showed that the APOE Ε4 genotype modifies neuropsychiatric and behavioral phenotype in AD. In particular, it was shown that delusions and agitation/aggression were more common and severer among homozygous APOE Ε4 carriers than among heterozygous or APOE-Ε4-negative patients.
Topological basis of signal integration in the transcriptional-regulatory network of the yeast, Saccharomyces cerevisiae
Authors: Farkas Illés J , Wu Chuang , Chennubhotla Chakra , Bahar Ivet , Oltvai Zoltán N
Published: October 2006

Background

Signal recognition and information processing is a fundamental cellular function, which in part involves comprehensive transcriptional regulatory (TR) mechanisms carried out in response to complex environmental signals in the context of the cell's own internal state. However, the network topological basis of developing such integrated responses remains poorly understood.

Results

By studying the TR network of the yeast Saccharomyces cerevisiae we show that an intermediate layer of transcription factors naturally segregates into distinct subnetworks. In these topological units transcription factors are densely interlinked in a largely hierarchical manner and respond to external signals by utilizing a fraction of these subnets.

Conclusion

As transcriptional regulation represents the 'slow' component of overall information processing, the identified topology suggests a model in which successive waves of transcriptional regulation originating from distinct fractions of the TR network control robust integrated responses to complex stimuli.
Bifidobacterium infantisstrains with and without a combination of Oligofructose and Inulin (OFI) attenuate inflammation in DSS-induced colitis in rats
Authors: Osman Nadia , Adawi Diya , Molin Göran , Ahrne Siv , Berggren Anna , Jeppsson Bengt
Published: October 2006

Background

Pathogenesis of inflammatory bowel disease is thought to be through different factors and there is a relationship between the gut flora and the risk of its development. Probiotics can manipulate the microflora in chronic inflammation and may be effective in treating inflammation. Bifidobacterium are saccharolytic and their growth in the gut can be promoted by non-absorbable carbohydrates and its increase in the colon appears to be of benefit.

Methods

Oligofructose and inulin (OFI) alone and the two B. infantis DSM 15158 and DSM 15159 with and without OFI, were fed to Sprague-Dawley rats for 7 days prior to colitis induction and administrations continued for another 7 days with the DSS. Colitis severity assessed using a Disease Activity Index. Samples were collected 7 days after colitis induction, for intestinal bacterial flora, bacterial translocation, short chain fatty acids (SCFAs), myeloperoxidase (MPO), cytokines (IL-1β, TNF-α, IL-10 and TGF-β) and malondialdehyde (MDA).

Results

OFI alone or the B. infantis strains with and without OFI improved significantly the DAI and decreased colonic MPO activity. Colonic tissue IL-1β decreased significantly in all treated groups except B. infantis DSM 15158. MDA decreased significantly in B. infantis DSM 15159 with and without OFI compared to colitis control. Succinic acid increased significantly in OFI group with and without DSM 15159 compared to all groups. Sum values of propionic, succinic acid and butyric acid increased significantly in all groups compare to the colitis control. Bacterial translocation to mesenteric lymph nodes decreased significantly in all groups compared to colitis control. Translocation to the liver decreased significantly in all groups compare to the colitis control and OFI + B. infantis DSM 15158 groups.

Conclusion

Administrations of OFI and Bifidobacterium improve DSS-induced acute colitis and have an anti-inflammatory effect. Major differences in effect were observed between the two B. infantis strains as indicated in MDA and succinic acid concentration as well as bacterial translocation rate in synbiotic combinations.
Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes
Authors: Goodwin Timothy JD , Butler Margaret I , Poulter Russell TM
Published: October 2006

Background

Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi.

Results

We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2) from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2) of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2), one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi.

These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins.

Conclusion

The identification of these new inteins increases the known host range of intein sequences in eukaryotes, and provides fresh insights into their origins and evolution. We conclude that inteins are ancient eukaryote elements once found widely among microbial eukaryotes. They persist as rarities in the genomes of a sporadic array of microorganisms, occupying highly conserved sites in diverse proteins.
Efficacy and safety of a low-flow veno-venous carbon dioxide removal device: results of an experimental study in adult sheep
Authors: Livigni Sergio , Maio Mariella , Ferretti Enrica , Longobardo Annalisa , Potenza Raffaele , Rivalta Luca , Selvaggi Paola , Vergano Marco , Bertolini Guido
Published: October 2006

Introduction

Extracorporeal lung assist, an extreme resource in patients with acute respiratory failure (ARF), is expanding its indications since knowledge about ventilator-induced lung injury has increased and protective ventilation has become the standard in ARF.

Methods

A prospective study on seven adult sheep was conducted to quantify carbon dioxide (CO2) removal and evaluate the safety of an extracorporeal membrane gas exchanger placed in a veno-venous pump-driven bypass. Animals were anaesthetised, intubated, ventilated in order to reach hypercapnia, and then connected to the CO2 removal device. Five animals were treated for three hours, one for nine hours, and one for 12 hours. At the end of the experiment, general anaesthesia was discontinued and animals were extubated. All of them survived.

Results

No significant haemodynamic variations occurred during the experiment. Maintaining an extracorporeal blood flow of 300 ml/minute (4.5% to 5.3% of the mean cardiac output), a constant removal of arterial CO2, with an average reduction of 17% to 22%, was observed. Arterial partial pressure of carbon dioxide (PaCO2) returned to baseline after treatment discontinuation. No adverse events were observed.

Conclusion

We obtained a significant reduction of PaCO2 using low blood flow rates, if compared with other techniques. Percutaneous venous access, simplicity of circuit, minimal anticoagulation requirements, blood flow rate, and haemodynamic impact of this device are more similar to renal replacement therapy than to common extracorporeal respiratory assistance, making it feasible not only in just a few dedicated centres but in a large number of intensive care units as well.
Out on a limb: the differential effect of substrate diameter on acceleration capacity inAnolislizards
Authors: Vanhooydonck Bieke , Herrel Anthony , Irschick Duncan J.
Published: November 2006

We investigated how substrate diameter affects acceleration performance in three Anolis lizard species (A. sagrei, A. carolinensis and A. valencienni), representing three different ecomorphs (trunk-ground, trunk-crown, and twig, respectively). We did so by measuring maximal acceleration capacity of the three species on a broad and narrow dowel. In addition to acceleration capacity, we quantified maximal sprint speed on both dowels. Both acceleration capacity and sprint speed are affected by substrate diameter, but the way in which they are, differs among species. Acceleration capacity in the trunk-ground anole, A. sagrei,was least affected by dowel diameter, whereas it was greatly reduced on the narrow dowel in the twig anole, A. valencienni. Sprint speed on the narrow dowel, however, was reduced to the greatest extent in the fastest running species, A. sagrei, whereas sprint speed was hardly affected by dowel diameter in the slow A. valencienni. The differential effect of dowel diameter on maximal acceleration capacity cannot be explained by differences in the timing of reaching maximal acceleration, but may be due to interspecific differences in the relative positioning of the limbs on the different dowels. The differential effect of dowel diameter on sprint speed,on the other hand, may be based on interspecific differences in the relative contribution of subsequent acceleratory bouts to maximal sprint speed on the broad and narrow dowel.
Electrical and Mechanical Properties of Conducive Carbon Black/Polyolefin Composites Mixed With Carbon Fiber
Authors: Farshidfar A , Haddadi-Asl V , Nazokdast H
Published: November 2006

The electrical conductivity of polymers can be increased by the addition of conductive fillers, including forms of carbon fibers and carbon black. The resulting composites can be used in applications where metals have typically been the materials of choice. The advantages of using these materials include lighter weight, resistance to corrosion, and the ability to be readily adapted to the needs of a specific application. One of the most significant applications for conductive polymer composites are conductive carbon-polymer composite electrodes. As many properties such as conductivity, mechanical integrity, low permeability, electrochemical activity, and stability in the electrolytes are required of materials to be used as electrodes, so “material selection” plays a crucial role in fabricating these materials. In this work it was found that high density polyethylene (HDPE)/ethylene-propylene-diene monomer(EPDM) blend ratio (70/30) has lower percolation threshold and volume resistivity than individually carbon black filled HDPE and EPDM due to “double percolation” effect. Carbon fibers were also added to the polymer-carbon black mixtures to enhance the conductivity and mechanical properties. The electrical conductivity of composites with different ratios of carbon black (CB) content to carbon fiber (CF) content was studied. The CB content is the main factor to determine the resistivity of the composites filled with CB and CF. Mechanical properties, including tensile strength, elongation at break, and impact strength of the conductive composites were evaluated. The results showed that incorporation of CB and CF in the composites will enhance tensile strength, but decrease elongation at break and impact strength of the composites. In mechanical properties, CF content has a greater effect than CB content. From the comparison of the resistivity and mechanical properties of the composites filled with CB and CF with that of the composites filled with CB only, it is concluded that using CF as a substitute for part of the CB in CB-filled composites can enhance electrical and mechanical properties.
Rescuing qkvDysmyelination by a Single Isoform of the Selective RNA-Binding Protein QKI
Authors: Zhao Lixia , Tian Donghua , Xia Mingjing , Macklin Wendy B. , Feng Yue
Published: November 2006

Alternative splicing of theqkItranscript generates multiple isoforms of the selective RNA-binding protein QKI, which play key roles in controlling the homeostasis of their mRNA targets. QKI deficiency in oligodendrocytes of homozygous quakingviable (qkv/qkv) mutant mice results in severe hypomyelination, indicating the essential function of QKI in myelinogenesis. However, the molecular mechanisms by which QKI controls myelination remain elusive. We report here that QKI-6 is the most abundant isoform in brain and is preferentially reduced in the qkv/qkvmutant during normal myelinogenesis. To test whether QKI-6 is the predominant isoform responsible for advancing CNS myelination, we developed transgenic mice that express Flag-QKI-6 specifically in the oligodendroglia lineage, driven by the proteolipid protein (PLP) promoter. When introduced into the qkv/qkvmutant, the QKI-6 transgene rescues the severe tremor and hypomyelination phenotype. Electron microscopic studies further revealed that the Flag-QKI-6 transgene is sufficient for restoring compact myelin formation with normal lamellar periodicity and thickness. Interestingly, Flag-QKI-6 preferentially associates with the mRNA encoding the myelin basic protein (MBP) and rescues MBP expression from the beginning of myelinogenesis. In contrast, Flag-QKI-6 binds the PLP mRNA with lower efficiency and has a minimal impact on PLP expression until much later, when the expression level of QKI-6 in the transgenic animal significantly exceeds what is needed for normal myelination. Together, our results demonstrate that QKI-6 is the major isoform responsible for CNS myelination, which preferentially promotes MBP expression in oligodendrocytes.