bluuebunny/crossref_metadata_2025 huggingface dataset:
Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication
Authors: Bubeck Anja , Wagner Markus , Ruzsics Zsolt , Lötzerich Mark , Iglesias Margot , Singh Ila R. , Koszinowski Ulrich H.
Published: July 2004

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn
7
-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.
Herpes Simplex Virus Type 1 Infection Induces the Stabilization of p53 in a USP7- and ATM-Independent Manner
Authors: Boutell Chris , Everett Roger D.
Published: July 2004

The major oncoprotein p53 regulates several cellular antiproliferation pathways that can be triggered in response to a variety of cellular stresses, including viral infection. The stabilization of p53 is a key factor in the ability of cells to initiate an efficient transcriptional response after cellular stress. Here we present data demonstrating that herpes simplex virus type 1 (HSV-1) infection of HFFF-2 cells, a low-passage-number nontransformed human primary cell line, results in the stabilization of p53. This process required viral immediate-early gene expression but occurred independently of the viral regulatory protein ICP0 and viral DNA replication. No specific viral protein could be identified as being solely responsible for the effect, which appears to be a cellular response to developing HSV-1 infections. HSV-1 infection also induced the phosphorylation of p53 at residues Ser15 and Ser20, which have previously been implicated in its stabilization in response to DNA damage. However, an HSV-1 infection of ATM
−/−
cells, which lack a kinase implicated in these phosphorylation events, did not lead to the phosphorylation of p53 at these residues, but nonetheless p53 was stabilized. We also show that the wild-type p53 expressed by osteosarcoma U2OS cells can be stabilized in response to DNA damage induced by UV irradiation, but not in response to HSV-1 infection. These data suggest that multiple cellular mechanisms are initiated to stabilize p53 during an HSV-1 infection. These mechanisms occur independently of ICP0 and its ability to sequester USP7 and may differ from those initiated in response to DNA damage.
Porcine Arterivirus Infection of Alveolar Macrophages Is Mediated by Sialic Acid on the Virus
Authors: Delputte Peter L. , Nauwynck Hans J.
Published: July 2004

Recently, we showed that porcine sialoadhesin (pSn) mediates internalization of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) in alveolar macrophages (Vanderheijden et al., J. Virol. 77:8207-8215, 2003). In rodents and humans, sialoadhesin, or Siglec-1, has been described as a macrophage-restricted molecule and to specifically bind sialic acid moieties. In the current study, we investigated whether pSn is a sialic acid binding protein and, whether so, whether this property is important for its function as a PRRSV receptor. Using untreated and neuraminidase-treated sheep erythrocytes, we showed that pSn binds sialic acid. Furthermore, pSn-specific monoclonal antibody 41D3, which blocks PRRSV infection, inhibited this interaction. PRRSV attachment to and infection of porcine alveolar macrophages (PAM) were both shown to be dependent on the presence of sialic acid on the virus: neuraminidase treatment of virus but not of PAM blocked infection and reduced attachment. Enzymatic removal of all N-linked glycans on the virus with N-glycosidase F reduced PRRSV infection, while exclusive removal of nonsialylated N-linked glycans of the high-mannose type with endoglycosidase H had no significant effect. Free sialyllactose and sialic acid containing (neo)glycoproteins reduced infection, while lactose and (neo)glycoproteins devoid of sialic acids had no significant effect. Studies with linkage-specific neuraminidases and lectins indicated that α2-3- and α2-6-linked sialic acids on the virion are important for PRRSV infection of PAM. From these results, we conclude that pSn is a sialic acid binding lectin and that interactions between sialic acid on the PRRS virion and pSn are essential for PRRSV infection of PAM.
Regulation of Relative Abundance of Arterivirus Subgenomic mRNAs
Authors: Pasternak Alexander O. , Spaan Willy J. M. , Snijder Eric J.
Published: July 2004

The subgenomic (sg) mRNAs of arteriviruses (order
Nidovirales
) form a 5′- and 3′-coterminal nested set with the viral genome. Their 5′ common leader sequence is derived from the genomic 5′-proximal region. Fusion of sg RNA leader and “body” segments involves a discontinuous transcription step. Presumably during minus-strand synthesis, the nascent RNA strand is transferred from one site in the genomic template to another, a process guided by conserved transcription-regulating sequences (TRSs) at these template sites. Subgenomic RNA species are produced in different but constant molar ratios, with the smallest RNAs usually being most abundant. Factors thought to influence sg RNA synthesis are size differences between sg RNA species, differences in sequence context between body TRSs, and the mutual influence (or competition) between strand transfer reactions occurring at different body TRSs. Using an
Equine arteritis virus
infectious cDNA clone, we investigated how body TRS activity affected sg RNA synthesis from neighboring body TRSs. Flanking sequences were standardized by head-to-tail insertion of several copies of an RNA7 body TRS cassette. A perfect gradient of sg RNA abundance, progressively favoring smaller RNA species, was observed. Disruption of body TRS function by mutagenesis did not have a significant effect on the activity of other TRSs. However, deletion of body TRS-containing regions enhanced synthesis of sg RNAs from upstream TRSs but not of those produced from downstream TRSs. The results of this study provide considerable support for the proposed discontinuous extension of minus-strand RNA synthesis as a crucial step in sg RNA synthesis.
Human Immunodeficiency Virus Type 1 Infection Induces Cyclin T1 Expression in Macrophages
Authors: Liou Li-Ying , Herrmann Christine H. , Rice Andrew P.
Published: July 2004

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication and activates RNA polymerase II transcriptional elongation through the association with a cellular protein kinase composed of Cdk9 and cyclin T1. Tat binds to this kinase complex through a direct protein-protein interaction with cyclin T1. Monocytes/macrophages are important targets of HIV-1 infection, and previous work has shown that cyclin T1 but not Cdk9 protein expression is low in monocytes isolated from blood. While Cdk9 expression is expressed at a high level during monocyte differentiation to macrophages in vitro, cyclin T1 expression is induced during the first few days of differentiation and is shut off after 1 to 2 weeks. We show here that the shutoff of cyclin T1 expression in late-differentiated macrophages involves proteasome-mediated proteolysis. We also show that cyclin T1 can be reinduced by a number of pathogen-associated molecular patterns that activate macrophages, indicating that up-regulation of cyclin T1 is part of an innate immune response. Furthermore, we found that HIV-1 infection early in macrophage differentiation results in sustained cyclin T1 expression, while infection at late times in differentiation results in the reinduction of cyclin T1. Expression of the viral Nef protein from an adenovirus vector suggests that Nef contributes to the HIV-1 induction of cyclin T1. These findings suggest that HIV-1 infection hijacks a component of the innate immune response in macrophages that results in enhancement rather than inhibition of viral replication.
By Inhibiting Replication, the Large Hepatitis Delta Antigen Can Indirectly Regulate Amber/W Editing and Its Own Expression
Authors: Sato Shuji , Cornillez-Ty Cromwell , Lazinski David W.
Published: July 2004

Hepatitis delta virus (HDV) expresses two essential proteins with distinct functions. The small hepatitis delta antigen (HDAg-S) is expressed throughout replication and is needed to promote that process. The large form (HDAg-L) is farnesylated, is expressed only at later times via RNA editing of the amber/W site, and is required for virion assembly. When HDAg-L is artificially expressed at the onset of replication, it strongly inhibits replication. However, there is controversy concerning whether HDAg-L expressed naturally at later times as a consequence of editing and replication can similarly inhibit replication. Here, by stabilizing the predicted secondary structure downstream from the amber/W site, a replication-competent HDV mutant that exhibited levels of editing higher than those of the wild type was created. This mutant expressed elevated levels of HDAg-L early during replication, and at later times, its replication aborted prematurely. No further increase in amber/W editing was observed following the cessation of replication, indicating that editing was coupled to replication. A mutation in HDAg-L and a farnesyl transferase inhibitor were both used to abolish the ability of HDAg-L to inhibit replication. Such treatments rescued the replication defect of the overediting mutant, and even higher levels of amber/W editing resulted. It was concluded that when expressed naturally during replication, HDAg-L is able to inhibit replication and thereby inhibit amber/W editing and its own synthesis. In addition, the structure adjacent to the amber/W site is suboptimal for editing, and this creates a window of time in which replication can occur in the absence of HDAg-L.
Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells
Authors: Bose Santanu , Basu Mausumi , Banerjee Amiya K.
Published: July 2004

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i) pretreatment of cells with antinucleolin antibodies and (ii) preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.
Increased Levels of Wee-1 Kinase in G
2
Are Necessary for Vpr- and Gamma Irradiation-Induced G
2
Arrest
Authors: Yuan Huidong , Kamata Masakazu , Xie Yi-Ming , Chen Irvin S. Y.
Published: July 2004

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle arrest at the G
2
/M transition and subsequently apoptosis. Here we examined the potential involvement of Wee-1 in Vpr-induced G
2
arrest. Wee-1 is a cellular protein kinase that inhibits Cdc2 activity, thereby preventing cells from proceeding through mitosis. We previously showed that the levels of Wee-1 correlate with Vpr-mediated apoptosis. Here, we demonstrate that Vpr-induced G
2
arrest correlated with delayed degradation of Wee-1 at G
2
/M. Experimental depletion of Wee-1 by a small interfering RNA directed to
wee-1
mRNA alleviated Vpr-induced G
2
arrest and allowed apparently normal progression through M into G
1
. Similar results were observed when cells were arrested at G
2
following gamma irradiation. Thus, Wee-1 is integrally involved as a key cellular regulatory protein in the signal transduction pathway for HIV-1 Vpr-induced cell cycle arrest.
Analysis of Splice Variants of the Immediate-Early 1 Region of Human Cytomegalovirus
Authors: Awasthi Sita , Isler Jennifer A. , Alwine James C.
Published: July 2004

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) produces multiple mRNAs through differential splicing and polyadenylation. Reverse transcriptase PCR was used to characterize transcripts from exons 1, 2, 3, and 4 (immediate-early 1 [IE1]). The expected IE72 and IE19 mRNAs were detected, as well as two heretofore-uncharacterized transcripts designated IE17.5 and IE9. The IE72, IE19, and IE17.5 transcripts utilized the same 5′-splice site in exon 3. IE9 utilized a cryptic 5′-splice site within exon 3. The IE19, IE17.5, and IE9 transcripts all used different 3′-splice sites within exon 4. These spliced species occur in infected human foreskin fibroblast (HFF) cells, with accumulation kinetics similar to those of IE72 mRNA. IE19 and IE9 RNAs were much more abundant than IE17.5 RNA. Transfection of CV-1 cells with cDNAs resulted in IE19 and IE17.5 proteins detectable by antibodies to either N-terminal or C-terminal epitopes. No IE9 protein product has been detected. We have not been able to detect IE19, IE17.5, or IE9 proteins during infection of HFF, HEL, or U373MG cells. Failure to detect IE19 protein contrasts with a previous report (M. Shirakata, M. Terauchi, M. Ablikin, K. Imadome, K. Hirai, T. Aso, and Y. Yamanashi, J. Virol. 76:3158-3167, 2002) of IE19 protein expression in HCMV-infected HEL cells. Our analysis suggests that an N-terminal breakdown product of IE72 may be mistaken for IE19. Expression of IE19 or IE17.5 from its respective cDNA results in repression of viral gene expression in infected cells. We speculate that expression of these proteins during infection may be restricted to specific conditions or cell types.
Oral Immunization with RecombinantListeria monocytogenesControls Virus Load after Vaginal Challenge with Feline Immunodeficiency Virus
Authors: Stevens Rosemary , Howard Kristina E. , Nordone Sushila , Burkhard MaryJo , Dean Gregg A.
Published: July 2004

RecombinantListeria monocytogeneshas many attractive characteristics as a vaccine vector against human immunodeficiency virus (HIV). Wild-type and attenuatedListeriastrains expressing HIV Gag have been shown to induce long-lived mucosal and systemic T-cell responses in mice. Using the feline immunodeficiency virus (FIV) model of HIV we evaluated recombinantL. monocytogenesin a challenge system. Five cats were immunized with recombinantL. monocytogenesthat expresses the FIV Gag and delivers an FIV Env-expressing DNA vaccine (LMgag/pND14-Lc-env). Control cats were either sham immunized or immunized with wild-typeL. monocytogenes(LM-wt). At 1 year after vaginal challenge, provirus could not be detected in any of the nine tissues evaluated from cats immunized with the recombinant bacteria but was detected in at least one tissue in 8 of 10 control animals. Virus was isolated from bone marrow of four of five LMgag/pND14-Lc-env-immunized cats by use of a stringent coculture system but required CD8+T-cell depletion, indicating CD8+T-cell suppression of virus replication. Control animals had an inverted CD4:CD8 ratio in mesenteric lymph node and were depleted of both CD4+and CD8+intestinal epithelial T cells, while LMgag/pND14-Lc-env-immunized animals showed no such abnormalities. Vaginal FIV-specific immunoglobulin A was present at high titer in three LMgag/pND14-Lc-env-immunized cats before challenge and in all five at 1 year postchallenge. This study demonstrates that recombinantL. monocytogenesconferred some control of viral load after vaginal challenge with FIV.
p53 Targets Simian Virus 40 Large T Antigen for Acetylation by CBP
Authors: Poulin Danielle L. , Kung Andrew L. , DeCaprio James A.
Published: July 2004

Simian virus 40 (SV40) large T antigen (T Ag) interacts with the tumor suppressor p53 and the transcriptional coactivators CBP and p300. Binding of these cellular proteins in a ternary complex has been implicated in T Ag-mediated transformation. It has been suggested that the ability of CBP/p300 to modulate p53 function underlies p53's regulation of cell proliferation and tumorigenesis. In this study, we provide further evidence that CBP activity may be mediated through its synergistic action with p53. We demonstrate that SV40 T Ag is acetylated in vivo in a p53-dependent manner and T Ag acetylation is largely mediated by CBP. The acetylation of T Ag is dependent on its interaction with p53 and on p53's interaction with CBP. We have mapped the site of acetylation on T Ag to the C-terminal lysine residue 697. This acetylation site is conserved between the T antigens of the human polyomaviruses JC and BK, which are also known to interact with p53. We show that both JC and BK T antigens are also acetylated at corresponding sites in vivo. While other proteins are known to be acetylated by CBP/p300, none are known to depend on p53 for acetylation. T Ag acetylation may provide a regulatory mechanism for T Ag binding to a cellular factor or play a role in another aspect of T Ag function.
Common E Protein Determinants for Attenuation of Glycosaminoglycan-Binding Variants of Japanese Encephalitis and West Nile Viruses
Authors: Lee Eva , Hall Roy A. , Lobigs Mario
Published: July 2004

Natural isolates and laboratory strains of West Nile virus (WNV) and Japanese encephalitis virus (JEV) were attenuated for neuroinvasiveness in mouse models for flavivirus encephalitis by serial passage in human adenocarcinoma (SW13) cells. The passage variants displayed a small-plaque phenotype, augmented affinity for heparin-Sepharose, and a marked increase in specific infectivity for SW13 cells relative to the respective parental viruses, while the specific infectivity for Vero cells was not altered. Therefore, host cell adaptation of passage variants was most likely a consequence of altered receptor usage for virus attachment-entry with the involvement of cell surface glycosaminoglycans (GAG) in this process. In vivo blood clearance kinetics of the passage variants was markedly faster and viremia was reduced relative to the parental viruses, suggesting that affinity for GAG (ubiquitously present on cell surfaces and extracellular matrices) is a key determinant for the neuroinvasiveness of encephalitic flaviviruses. A difference in pathogenesis between WNV and JEV, which was reflected in more efficient growth in the spleen and liver of the WNV parent and passage variants, accounted for a less pronounced loss of neuroinvasiveness of GAG binding variants of WNV than JEV. Single gain-of-net-positive-charge amino acid changes at E protein residue 49, 138, 306, or 389/390, putatively positioned in two clusters on the virion surface, define molecular determinants for GAG binding and concomitant virulence attenuation that are shared by the JEV serotype flaviviruses.
Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle
Authors: Mir M. A. , Panganiban A. T.
Published: July 2004

Hantaviruses are tripartite negative-sense RNA viruses and members of the
Bunyaviridae
family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3′ and 5′ ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.
Itinerary of Hepatitis B Viruses: Delineation of Restriction Points Critical for Infectious Entry
Authors: Funk Anneke , Mhamdi Mouna , Lin Li , Will Hans , Sirma Hüseyin
Published: July 2004

Little is known about cellular determinants essential for human hepatitis B virus infection. Using the duck hepatitis B virus as a model, we first established a sensitive binding assay for both virions and subviral particles and subsequently elucidated the characteristics of the early viral entry steps. The infection itinerary was found to initiate with the attachment of viral particles to a low number of binding sites on hepatocytes (about 10
4
per cell). Virus internalization was fully accomplished in less than 3 h but was then followed by a period of unprecedented length, about 14 h, until completion of nuclear import of the viral genome. Steps subsequent to virus entry depended on both intact microtubules and their dynamic turnover but not on actin cytoskeleton. Notably, cytoplasmic trafficking of viral particles and emergence of nuclear covalently closed circular DNA requires microtubules during entry only at and for specific time periods. Taken together, these data disclose for the first time a series of steps and their kinetics that are essential for the entry of hepatitis B viruses into hepatocytes and are different from those of any other virus reported so far.
Coiled-Coil Domains in Glycoproteins B and H Are Involved in Human Cytomegalovirus Membrane Fusion
Authors: Lopper Matthew , Compton Teresa
Published: July 2004

Human cytomegalovirus (CMV) utilizes a complex route of entry into cells that involves multiple interactions between viral envelope proteins and cellular receptors. Three conserved viral glycoproteins, gB, gH, and gL, are required for CMV-mediated membrane fusion, but little is known of how these proteins cooperate during entry (E. R. Kinzler and T. Compton, submitted for publication). The goal of this study was to begin defining the molecular mechanisms that underlie membrane fusion mediated by herpesviruses. We identified heptad repeat sequences predicted to form alpha-helical coiled coils in two glycoproteins required for fusion, gB and gH. Peptides derived from gB and gH containing the heptad repeat sequences inhibited virus entry when introduced coincident with virus inoculation onto cells or when mixed with virus prior to inoculation. Neither peptide affected binding of CMV to fibroblasts, suggesting that the peptides inhibit membrane fusion. Both gB and gH coiled-coil peptides blocked entry of several laboratory-adapted and clinical strains of human CMV, but neither peptide affected entry of murine CMV or herpes simplex virus type 1 (HSV-1). Although murine CMV and HSV-1 gB and gH have heptad repeat regions, the ability of human CMV gB and gH peptides to inhibit virus entry correlates with the specific residues that comprise the heptad repeat region. The ability of gB and gH coiled-coil peptides to inhibit virus entry independently of cell contact suggests that the coiled-coil regions of gB and gH function differently from those of class I, single-component fusion proteins. Taken together, these data support a critical role for alpha-helical coiled coils in gB and gH in the entry pathway of CMV.
Effect of Vaccine Use in the Evolution of Mexican Lineage H5N2 Avian Influenza Virus
Authors: Lee Chang-Won , Senne Dennis A. , Suarez David L.
Published: July 2004

An outbreak of avian influenza (AI) caused by a low-pathogenic H5N2 type A influenza virus began in Mexico in 1993 and several highly pathogenic strains of the virus emerged in 1994-1995. The highly pathogenic virus has not been reported since 1996, but the low-pathogenic virus remains endemic in Mexico and has spread to two adjacent countries, Guatemala and El Salvador. Measures implemented to control the outbreak and eradicate the virus in Mexico have included a widespread vaccination program in effect since 1995. Because this is the first case of long-term use of AI vaccines in poultry, the Mexican lineage virus presented us with a unique opportunity to examine the evolution of type A influenza virus circulating in poultry populations where there was elevated herd immunity due to maternal and active immunity. We analyzed the coding sequence of the HA1 subunit and the NS gene of 52 Mexican lineage viruses that were isolated between 1993 and 2002. Phylogenetic analysis indicated the presence of multiple sublineages of Mexican lineage isolates at the time vaccine was introduced. Further, most of the viruses isolated after the introduction of vaccine belonged to sublineages separate from the vaccine's sublineage. Serologic analysis using hemagglutination inhibition and virus neutralization tests showed major antigenic differences among isolates belonging to the different sublineages. Vaccine protection studies further confirmed the in vitro serologic results indicating that commercial vaccine was not able to prevent virus shedding when chickens were challenged with antigenically different isolates. These findings indicate that multilineage antigenic drift, which has not been observed in AI virus, is occurring in the Mexican lineage AI viruses and the persistence of the virus in the field is likely aided by its large antigenic difference from the vaccine strain.
Human Cytomegalovirus-Encoded G Protein-Coupled Receptor US28 Mediates Smooth Muscle Cell Migration through Gα12
Authors: Melnychuk Ryan M. , Streblow Daniel N. , Smith Patricia P. , Hirsch Alec J. , Pancheva Dora , Nelson Jay A.
Published: July 2004

Coupling of G proteins to ligand-engaged chemokine receptors is the paramount event in G-protein-coupled receptor signal transduction. Previously, we have demonstrated that the human cytomegalovirus-encoded chemokine receptor US28 mediates human vascular smooth muscle cell (SMC) migration in response to either RANTES or monocyte chemoattractant protein 1. In this report, we identify the G proteins that couple with US28 to promote vascular SMC migration and identify other signaling molecules that play critical roles in this process. US28-mediated cellular migration was enhanced with the expression of the G-protein subunits Gα12 and Gα13, suggesting that US28 may functionally couple to these G proteins. In correlation with this observation, US28 was able to activate RhoA, a downstream effector of Gα12 and Gα13 in cell types with these G proteins but not in those without them and activation of RhoA was dependent on US28 stimulation with RANTES. In addition, inactivation of RhoA or the RhoA-associated kinase p160ROCK with a dominant-negative mutant of RhoA or the small molecule inhibitor Y27632, respectively, abrogated US28-induced SMC migration. The data presented here suggest that US28 functionally signals through Gα12 family G proteins and RhoA in a ligand-dependent manner and these signaling molecules are important for the ability of US28 to induce cellular migration.
Is virtual bronchoscopy useful for physicians practising in a district general hospital?
Authors: Dheda K , Roberts C M , Partridge M R , Mootoosamy I
Published: July 2004

Background: Virtual bronchoscopy software is now available to district general hospitals (DGHs). There is limited information on the clinical utility of virtual bronchoscopy and whether it offers any additional information over conventional axial computed tomography in the setting of a busy DGH chest unit.

Methods: Virtual bronchoscopy and computed tomography findings were compared in all patients who had a virtual bronchoscopy study over a 12 month period.

Results: Eighteen consecutive patients had virtual bronchoscopy for a specific clinical indication over the study period. Additional information was conveyed by virtual bronchoscopy in five patients (in four patients the airways distal to an obstruction were better visualised thereby influencing decisions about airway stenting and in one patient the virtual bronchoscopy study showed an endobronchial lesion missed on computed tomography). In nine patients who were unfit for fibreoptic bronchoscopy (FOB) the radiologist was more confident in excluding an obstructive airway lesion. The main indication for performing a virtual bronchoscopy study was to rule out an obstructive airway lesion in patients who were unfit for FOB (n = 11).

Conclusion: Virtual bronchoscopy is feasible and useful in the management of a few selected patients in a DGH chest unit. Virtual bronchoscopy may convey additional information over computed tomography when the distal airways need to be visualised and for discrete endoluminal lesions.
Iron deficiency anaemia in general practice: clinical outcomes over three years and factors influencing diagnostic investigations
Authors: Yates J M , Logan E C M , Stewart R M
Published: July 2004

Background: Iron deficiency anaemia (IDA) may be a sign of significant gastrointestinal disease, and delayed diagnosis may result in chronic morbidity. Studies in patients referred to hospital for investigation of their anaemia have shown that 5%–15% have a gastrointestinal cancer but there are few studies of patients presenting to primary care. Factors influencing further investigation in these patients have not previously been identified.

Patients and methods: A cohort of patients presenting to their general practitioners (GPs) with IDA was identified and clinical outcomes recorded. Logistic regression was used to determine which factors influenced GPs to investigate the anaemia.

Results: 43% of patients had investigations within three months and serious pathology was found in 30% of these; 13% of patients were considered unfit for further investigation and 8% refused to have any. Independent predictors of non-investigation were a mild anaemia (odds ratio (OR) 0.38, confidence interval (CI) 0.23 to 0.61, p<0.001), female gender (OR 0.49, CI 0.3 to 0.8, p = 0.004), a previous history of anaemia (OR 0.39, CI 0.24 to 0.64, p<0.001), and age <65 years (OR 0.44, CI 0.26 to 0.74, p = 0.002). During the entire study period gastrointestinal cancer was diagnosed in 48 patients (11%); 17% of men had colorectal cancer. Of 263 patients alive at 12 months without a confirmed diagnosis, 113 (43%) had recurrent or persistent anaemia during the study period.

Conclusion: Although the overall prevalence of gastrointestinal cancer in patients presenting to primary care is similar to that seen in secondary care, the diagnosis may be delayed due to lack of appropriate investigations resulting in significant morbidity.
Clinical guidelines online: do they improve compliance?
Authors: Williams J G , Cheung W Y , Price D E , Tansey R , Russell I T , Duane P D , Al-Ismail S A , Wani M A
Published: July 2004

Background: The introduction of intranet services in a district general hospital provided an opportunity to put evidence based national guidelines online to facilitate access and promote application of best practice in acute medical care. This study evaluated the effectiveness of this approach.

Method: Local guidelines were made available online at ward terminals after they had been distributed in paper form. An interrupted time series design was used to evaluate the impact on compliance with three preselected guidelines, which addressed the management of suspected deep vein thrombosis, upper gastrointestinal bleeding, and stroke. This was supplemented by a qualitative assessment of the views of medical staff.

Results: There was a significant increase in the adherence to the guidelines for stroke when they were made available online, but this was not demonstrable for deep vein thrombosis or upper gastrointestinal bleeding.

Qualitative interviews with junior medical staff and consultants after the study was completed revealed that there was confusion regarding the application of the guidelines for deep vein thrombosis and little active support from the gastroenterologists for the guidelines for upper gastrointestinal bleeding. The stroke guidelines were actively promoted by their author and widely supported.

Conclusion: Making guidelines available online will not be effective unless they are actively promoted and represent a consensus view.
An approach to drug induced delirium in the elderly
Authors: Alagiakrishnan K , Wiens C A
Published: July 2004

Drugs have been associated with the development of delirium in the elderly. Successful treatment of delirium depends on identifying the reversible contributing factors, and drugs are the most common reversible cause of delirium. Anticholinergic medications, benzodiazepines, and narcotics in high doses are common causes of drug induced delirium. This article provides an approach for clinicians to prevent, recognise, and manage drug induced delirium. It also reviews the mechanisms for this condition, especially the neurotransmitter imbalances involving acetylcholine, dopamine, and gamma aminobutyric acid and discusses the age related changes that may contribute to altered pharmacological effects which have a role in delirium. Specific interventions for high risk elderly with the goal of preventing drug induced delirium are discussed.
Severe acute respiratory syndrome (SARS): epidemiology and clinical features
Authors: Hui D S C , Chan M C H , Wu A K , Ng P C
Published: July 2004

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease with a significant morbidity and mortality. The major clinical features include persistent fever, chills/rigor, myalgia, malaise, dry cough, headache, and dyspnoea. Older subjects may present without the typical febrile response. Common laboratory features include lymphopenia, thrombocytopenia, raised alanine transaminases, lactate dehydrogenase, and creatine kinase. The constellation of compatible clinical and laboratory findings, together with certain characteristic radiological features and lack of clinical response to broad spectrum antibiotics, should arouse suspicion of SARS. Measurement of serum RNA by real time reverse transcriptase-polymerase chain reaction technique has a detection rate of 75%–80% in the first week of the illness.
Ectomycorrhizal colonization of greenhouse-grown Douglas-fir (Pseudotsuga menziesii) seedlings by inoculum associated with the roots of refuge plants sampled from a Douglas-fir forest in the southern interior of British Columbia
Authors: Hagerman Shannon M , Durall Daniel M
Published: July 2004

Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings were grown in the greenhouse in a sterilized mixture of forest soil and vermiculite, which had been inoculated with root fragments from one of six different ectomycorrhizal under story plant species (Arctostaphylos uva-ursi (L.) Spreng, P. menziessi, Salix bebbiana Bebb, Alnus viridis subsp. sinuata (Regel) Ä. Löve & and D. Löve (alder), Betula papyrifera Marsh. (paper birch), Populus tremuloides Michx.) and arbuscular mycorrhizal Calamagrostis rubescens Buckl. (pinegrass) sampled from a dry Douglas-fir forest in the southern interior of British Columbia. The overall objective of the present study was to investigate the inoculum potential of these ectomycorrhizal roots for colonizing Douglas-fir seedlings. A total of seven ectomycorrhizal morphotypes formed on the bioassay seedlings, which were colonized by all treatments except the control. Seedlings growing in soil inoculated with root fragments of Douglas-fir, Arctostaphylos, and paper birch had greater ectomycorrhizal richness and a higher percentage of colonized fine roots relative to the pinegrass and alder treatments. The community of ectomycorrhizal fungi that colonized the bioassay seedlings differed from that associated with some of the same refuge plants assessed in a previously reported field-based study at this site. Different ectomy corrhizal fungal colonization strategies and the retention of refuge plants are discussed in relation to the colonization of outplanted seedlings following clearcutting.Key words: ectomycorrhizae, refuge plants, inoculum potential, soil bioassay, Pseudotsuga menziesii.
Macrofungus communities correlate with moisture and nitrogen abundance in two old-growth conifer forests, Olympic National Park, Washington, USA
Authors: Trudell Steven A , Edmonds Robert L
Published: July 2004

We characterized the epigeous macrofungus communities in two old-growth conifer forests by collecting sporocarps. Despite the similarity in dominant tree species in the two forests, the macrofungus communities were very different. At the drier, nitrogen-poor Deer Park area, the macrofungi were dominated by ectomycorrhizal species in the genera Cortinarius, Tricholoma, Hydnellum, Suillus, and Sarcodon. At the wetter, higher nitrogen Hoh Valley, the macrofungi were characterized by ectomycorrhizal species in different genera, such as Inocybe, Russula, Amanita, Boletus, and Phaeocollybia, and saprotrophic fungi accounted for a greater proportion of the community. Species richness was similar at the two areas, but sporocarp production was much higher at Deer Park. We propose that (i) these community differences developed over a long time; (ii) they are largely related to differences in ecosystem moisture and nitrogen abundance; and (iii) within the ectomycorrhizal fungi, possible causal mechanisms involve mycelial morphology and carbon allocation within the symbioses. The apparent response to relatively small but presumably long-term differences in nitrogen abundance suggests that sporocarp production by macrofungi could be an effective bioindicator and should be considered in determination of critical loads for atmospheric nitrogen deposition to temperate and boreal forests.Key words: critical nitrogen loads, ectomycorrhizal fungi, macrofungi, macrofungus communities, nitrogen, old-growth conifer forests.
Ultrastructure of theAspergillus nidulans hypA1restrictive phenotype shows defects in endomembrane arrays and polarized wall deposition
Authors: Kaminskyj Susan GW , Boire Melissa R
Published: July 2004

Aspergillus nidulans Eidam (G. Wint.) wild-type hyphal morphogenesis requires the hypA gene product. Like its homolog in Saccharomyces cerevisiae Meyen ex E.C. Hansen, TRS120, hypA encodes a cytoplasmic protein likely associated with endomem branes. hypA is not essential, but hypA1 temperature-sensitive strains grow poorly at restrictive temperatures. In younger cells, endomembrane arrays were aberrant, only sometimes resembling wild type. In older cells, Golgi equivalents were swollen, impacted with electron-dense granules. In hypA1 strains grown at 42 °C, the poorly polarized hyphae lack recognizable Spitzenkörper and have walls at least four-fold thicker than those of wild-type or hypA1 strains grown at 28 °C. At restrictive temperatures, both hyphal width and wall thickness increase markedly in basal regions, suggesting wall deposition is impaired. Septa are thicker than in wild type, but have medial pores and Woronin bodies. Individual nuclei and mitochondria are smaller at 42 °C than at 28 °C, but each collectively occupies similar proportions of the cytoplasm. Mitochondrial cristae are reduced in number and width at 42 °C, possibly compromising metabolic efficiency; in older cells, cristae are widely spaced and randomly inserted. If hypA1 cells grown at 42 °C are shifted to 28 °C, the thickened wall is precisely degraded for growth of wild-type branches, which form within 1 h, suggesting areas of nascent polarity formed at 42 °C require the hypA product for wild-type function.Key words: endomembrane, filamentous fungus, electron microscopy, cell wall, secretion, Saccharomyces TRS120.
Ectomycorrhizal fungal communities in an Abies firma forest, with special reference to ectomycorrhizal associations between seedlings and mature trees
Authors: Matsuda Yosuke , Hijii Naoki
Published: July 2004

Ectomycorrhizal fungal communities of both seedlings and mature trees of Abies firma Sieb. et Zucc. were studied based on morphological and molecular analyses of ectomycorrhizal (ECM) roots. A total of 142 seedlings and 28 soil cores were collected from a 10 m × 30 m plot, and 37 morphotypes were recorded from seedlings and 23 from mature trees. A morphotype, type 21, formed by an unidentified fungus that is likely a Russula species, was dominant on both seedlings (30% of the total number of the ECM roots detected) and mature trees (56%) and overlapped them spatially, suggesting a hyphal linkage between mature trees and seedlings in the plot. A total of 12 morphotypes were recorded from soil blocks underneath fruiting bodies of undescribed Russula sp. 1, the dominant aboveground fruiting body in the plot, among which type 21 was the most abundant. The pattern of restriction fragment length polymorphism of this type was identical to that of the fruiting body of Russula sp. 1. These results indicate that Russula sp. 1 was the dominant ECM fungus associated with both mature trees and seedlings and would play an important role in the dynamics and maintenance of this Abies-dominated forest site.Key words: Abies firma, ectomycorrhizal fungal community, morphotype, PCR-RFLP analysis, tree age.
A simple description of the spectra of confined hydrogen, helium, and lithium
Authors: Patil S H , Varshni Y P
Published: July 2004

A simple description of the spectrum of confined hydrogen atom is obtained in terms of an scheme involving interpolation between the expressions for large and small values of the confining radius. Satisfactory agreement is shown to exist between the eigen energies thus calculated and exact values. The scheme is extended for helium and lithium. With the simulation of interaction between the electrons by a screened nuclear charge, some energy levels are deduced for the confined helium and lithium.PACS Nos.: 03.65.Ge, 73.21.La, 78.67.Hc
Advances in Quantitative Electroencephalogram Analysis Methods
Authors: Thakor Nitish V. , Tong Shanbao
Published: July 2004

▪ Abstract  Quantitative electroencephalogram (qEEG) plays a significant role in EEG-based clinical diagnosis and studies of brain function. In past decades, various qEEG methods have been extensively studied. This article provides a detailed review of the advances in this field. qEEG methods are generally classified into linear and nonlinear approaches. The traditional qEEG approach is based on spectrum analysis, which hypothesizes that the EEG is a stationary process. EEG signals are nonstationary and nonlinear, especially in some pathological conditions. Various time-frequency representations and time-dependent measures have been proposed to address those transient and irregular events in EEG. With regard to the nonlinearity of EEG, higher order statistics and chaotic measures have been put forward. In characterizing the interactions across the cerebral cortex, an information theory-based measure such as mutual information is applied. To improve the spatial resolution, qEEG analysis has also been combined with medical imaging technology (e.g., CT, MR, and PET). With these advances, qEEG plays a very important role in basic research and clinical studies of brain injury, neurological disorders, epilepsy, sleep studies and consciousness, and brain function.
Tissue Engineering Applications of Therapeutic Cloning
Authors: Atala Anthony , Koh Chester J.
Published: July 2004

▪ Abstract  Few treatment options are available for patients suffering from diseased and injured organs because of a severe shortage of donor organs available for transplantation. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for replacement therapy. Scientists in the field of tissue engineering apply the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. The present chapter reviews recent advances that have occurred in therapeutic cloning and tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure.
Advances in High-Field Magnetic Resonance Imaging
Authors: Hu Xiaoping , Norris David G.
Published: July 2004

▪ Abstract  Among advances in magnetic resonance imaging (MRI), the increase of the magnetic field strength is perhaps one of the most significant. The use of high magnetic fields for in vivo magnetic resonance is motivated by a number of considerations. Advantages are increases in signal-to-noise ratio, blood-oxygenation level–dependent contrast, and spectral resolution, while disadvantages include potential reduction of contrast in anatomic imaging owing to lengthening of T1 and effects of susceptibility of high fields. To address these challenges, technical advances have been made in various aspects of MRI, allowing high-field MRI to provide exquisite morphological and functional details in clinical and research settings. This review provides an overview of technical issues and applications of high-field MRI.
Tissue Engineering of Ligaments
Authors: Vunjak-Novakovic G. , Altman Gregory , Horan Rebecca , Kaplan David L.
Published: July 2004

▪ Abstract  Tissue engineering is emerging as a significant clinical option to address tissue and organ failure by implanting biological substitutes for the compromised tissues. As compared to the transplantation of cells alone, engineered tissues offer the potential advantage of immediate functionality. Engineered tissues can also serve as physiologically relevant models for controlled studies of cells and tissues designed to distinguish the effects of specific signals from the complex milieu of factors present in vivo. A high number of ligament failures and the lack of adequate options to fully restore joint functions have prompted the need to develop new tissue engineering strategies. We discuss the requirements for ligament reconstruction, the available treatment options and their limitations, and then focus on the tissue engineering of ligaments. One representative tissue engineering system involving the integrated use of adult human stem cells, custom-designed scaffolds, and advanced bioreactors with dynamic loading is described.