bluuebunny/crossref_metadata_2025 huggingface dataset:
Integration Targeting by Avian Sarcoma-Leukosis Virus and Human Immunodeficiency Virus in the Chicken Genome
Authors: Barr Stephen D. , Leipzig Jeremy , Shinn Paul , Ecker Joe R. , Bushman Frederic D.
Published: September 2005

We have analyzed the placement of sites of integration of avian sarcoma-leukosis virus (ASLV) and human immunodeficiency virus (HIV) DNA in the draft chicken genome sequence, with the goals of assessing species-specific effects on integration and allowing comparison to the distribution of chicken endogenous retroviruses (ERVs). We infected chicken embryo fibroblasts (CEF) with ASLV or HIV and sequenced 863 junctions between host and viral DNA. The relationship with cellular gene activity was analyzed by transcriptional profiling of uninfected or ASLV-infected CEF cells. ASLV weakly favored integration in active transcription units (TUs), and HIV strongly favored active TUs, trends seen previously for integration in human cells. The ERVs, in contrast, accumulated mostly outside TUs, including ERVs related to ASLV. The minority of ERVs present within TUs were mainly in the antisense orientation; consequently, the viral splicing and polyadenylation signals would not disrupt cellular mRNA synthesis. In contrast, de novo ASLV integration sites within TUs showed no orientation bias. Comparing the distribution of de novo ASLV integration sites to ERVs indicated that purifying selection against gene disruption, and not initial integration targeting, probably determined the ERV distribution. Further analysis indicated that ERVs in humans, mice, and rats showed similar distributions, suggesting purifying selection dictated their distributions as well.
Human Immunodeficiency Virus Mutagenesis during Antiviral Therapy: Impact of Drug-Resistant Reverse Transcriptase and Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors on Human Immunodeficiency Virus Type 1 Mutation Frequencies
Authors: Chen Renxiang , Yokoyama Masaru , Sato Hironori , Reilly Cavan , Mansky Louis M.
Published: September 2005

The development of antiviral drug resistance is an important problem in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Potent antiretroviral therapy is currently used for treatment, and typically consists of at least two reverse transcriptase (RT) inhibitors. We have previously reported that both drugs and drug-resistant RT mutants can increase virus mutation frequencies. To further assess the contributions of nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and drug-resistant RTs to HIV mutagenesis, a new high-throughput assay system was developed. This assay system was designed to specifically detect frameshift mutations in the luciferase gene in a single virus replication cycle. New drug-resistant RTs were identified that significantly altered virus mutation frequencies. Consistent with our previous observations of NRTIs, abacavir, stavudine, and zalcitabine increased HIV-1 mutation frequencies, supporting the general hypothesis that the NRTIs currently used in antiviral drug therapy increase virus mutation frequencies. Interestingly, similar observations were made with NNRTIs. This is the first report to show that NNRTIs can influence virus mutation frequencies. NNRTI combinations, NRTI-NNRTI combinations, and combinations of drug and drug-resistant RTs led to significant changes in the virus mutation frequencies compared to virus replication of drug-resistant virus in the absence of drug or wild-type virus in the presence of drug. This indicates that combinations of RT drugs or drugs and drug-resistant virus created during the evolution of drug resistance can act together to increase HIV-1 mutation frequencies, which would have important implications for drug therapy regimens. Finally, the influence of drug-resistant RT mutants from CRF01_AE viruses on HIV-1 mutation frequencies was analyzed and it was found that only a highly drug resistant RT led to altered virus mutation frequencies. The results further suggest that high-level drug-resistant RT can significantly influence virus mutation frequencies. A structural model that explains the mutation frequency data is discussed.
Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse Model
Authors: Li Zejun , Chen Hualan , Jiao Peirong , Deng Guohua , Tian Guobin , Li Yanbing , Hoffmann Erich , Webster Robert G. , Matsuoka Yumiko , Yu Kangzhen
Published: September 2005

We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans.
The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor
Authors: Tyagi Shweta , Surjit Milan , Lal Sunil K.
Published: September 2005

Hepatitis E virus (HEV), a human plus-stranded RNA virus, contains three open reading frames (ORF). Of these, ORF1 encodes the viral nonstructural polyprotein, ORF2 encodes the major capsid protein, and ORF3 codes for a phosphoprotein of undefined function. Recently, using the yeast two-hybrid system to screen a human cDNA liver library, we have isolated and characterized AMBP (α
1
-microglobulin/bikunin precursor), which specifically interacts with the ORF3 protein of HEV. The ORF3 protein expedites the processing and secretion of α
1
-microglobulin. When checked individually for interaction, the second processed protein from AMBP, bikunin, strongly interacted with the full-length ORF3 protein. This protein-protein interaction has been validated by immunoprecipitation in both COS-1 and Huh7 cells and by His
6
pull-down assays. In dual-labeling immunofluorescent staining, followed by fluorescence microscopy of transfected human liver cells, ORF3 colocalized with endogenously expressed bikunin. Finally, a 41-amino-acid C-terminal region of ORF3 has been found to be responsible for interacting with bikunin. The importance of this virus-host protein-protein interaction, with reference to the viral life cycle, has been discussed.
Human Immunodeficiency Virus Type 1 Virological Synapse Formation in T Cells Requires Lipid Raft Integrity
Authors: Jolly Clare , Sattentau Quentin J.
Published: September 2005

Human immunodeficiency virus type 1 (HIV-1) can spread directly between T cells by forming a supramolecular structure termed a virological synapse (VS). HIV-1 envelope glycoproteins (Env) are required for VS assembly, but their mode of recruitment is unclear. We investigated the distribution of GM1-rich lipid rafts in HIV-1-infected (effector) T cells and observed Env colocalization with polarized raft markers GM1 and CD59 but not with the transferrin receptor that is excluded from lipid rafts. In conjugates of effector T cells and target CD4
+
T cells, GM1, Env, and Gag relocated to the cell-cell interface. The depletion of cholesterol in the infected cell dispersed Env and GM1 within the plasma membrane, eliminated Gag clustering at the site of cell-cell contact, and abolished assembly of the VS. Raft integrity is therefore critical for Env and Gag coclustering and VS assembly in T-cell conjugates.
Identification and Characterization of Human Cytomegalovirus-Encoded MicroRNAs
Authors: Grey Finn , Antoniewicz Andy , Allen Edwards , Saugstad Julie , McShea Andy , Carrington James C. , Nelson Jay
Published: September 2005

MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.
Unique Acquisition of Cytotoxic T-Lymphocyte Escape Mutants in Infant Human Immunodeficiency Virus Type 1 Infection
Authors: Pillay Thillagavathie , Zhang Hua-Tang , Drijfhout Jan W. , Robinson Nicola , Brown Helen , Khan Munira , Moodley Jagadesa , Adhikari Miriam , Pfafferott Katja , Feeney Margaret E. , St. John Anne , Holmes Edward C. , Coovadia Hoosen M. , Klenerman Paul , Goulder Philip J. R. , Phillips Rodney E.
Published: September 2005

The role of cytotoxic T-lymphocyte (CTL) escape in rapidly progressive infant human immunodeficiency virus type 1 (HIV-1) infection is undefined. The data presented here demonstrate that infant HIV-1-specific CTL can select for viral escape variants very early in life. These variants, furthermore, may be selected specifically in the infant, despite the same CTL specificity being present in the mother. Additionally, pediatric CTL activity may be compromised both by the transmission of maternal escape variants and by mother-to-child transmission of escape variants that originally arose in the father. The unique acquisition of these CTL escape forms may help to explain the severe nature of some pediatric HIV infections.
Rift Valley Fever Virus NSs mRNA Is Transcribed from an Incoming Anti-Viral-Sense S RNA Segment
Authors: Ikegami Tetsuro , Won Sungyong , Peters C. J. , Makino Shinji
Published: September 2005

Analysis of purified Rift Valley fever virus (RVFV) particles demonstrated the presence of three negative-sense RNA genomes, plus three anti-viral-sense RNA segments. The virion-associated anti-viral-sense S segment served as a template for the synthesis of NSs mRNA immediately after infection. NSs protein synthesis also occurred early in infection, suggesting that NSs protein produced early in infection probably has biologically significant roles in virus replication and/or survival in the host. Translation inhibitor treatment of mammalian cells infected with viruses belonging to the
Bunyaviridae
family generally inhibits viral mRNA synthesis. However, RVFV NSs mRNA synthesis, but not N mRNA synthesis, was resistant to puromycin treatment during primary transcription, pointing to the uniqueness of RVFV NSs mRNA synthesis.
Sustained CD8
+
T-Cell Responses Induced after Acute Parvovirus B19 Infection in Humans
Authors: Norbeck Oscar , Isa Adiba , Pöhlmann Christoph , Broliden Kristina , Kasprowicz Victoria , Bowness Paul , Klenerman Paul , Tolfvenstam Thomas
Published: September 2005

Murine models have suggested that CD8
+
T-cell responses peak early in acute viral infections and are not sustained, but no evidence for humans has been available. To address this, we longitudinally analyzed the CD8
+
T-cell response to human parvovirus B19 in acutely infected individuals. We observed striking CD8
+
T-cell responses, which were sustained or even increased over many months after the resolution of acute disease, indicating that CD8
+
T cells may play a prominent role in the control of parvovirus B19 and other acute viral infections of humans, including potentially those generated by live vaccines.
BIZARRE INTERACTIONS AND ENDGAMES: Entomopathogenic Fungi and Their Arthropod Hosts
Authors: Roy H.E. , Steinkraus D.C. , Eilenberg J. , Hajek A.E. , Pell J.K.
Published: September 2005

▪ Abstract  Invertebrate pathogens and their hosts are taxonomically diverse. Despite this, there is one unifying concept relevant to all such parasitic associations: Both pathogen and host adapt to maximize their own reproductive output and ultimate fitness. The strategies adopted by pathogens and hosts to achieve this goal are almost as diverse as the organisms themselves, but studies examining such relationships have traditionally concentrated only on aspects of host physiology. Here we review examples of host-altered behavior and consider these within a broad ecological and evolutionary context. Research on pathogen-induced and host-mediated behavioral changes demonstrates the range of altered behaviors exhibited by invertebrates including behaviorally induced fever, elevation seeking, reduced or increased activity, reduced response to semiochemicals, and changes in reproductive behavior. These interactions are sometimes quite bizarre, intricate, and of great scientific interest.
Pharmacogenomics and Individualized Drug Therapy
Authors: Eichelbaum Michel , Ingelman-Sundberg Magnus , Evans William E.
Published: September 2005

Pharmacogenetics deals with inherited differences in the response to drugs. The best-recognized examples are genetic polymorphisms of drug-metabolizing enzymes, which affect about 30% of all drugs. Loss of function of thiopurine S-methyltransferase (TPMT) results in severe and life-threatening hematopoietic toxicity if patients receive standard doses of mercaptopurine and azathioprine. Gene duplication of cytochrome P4502D6 (CYP2D6), which metabolizes many antidepressants, has been identified as a mechanism of poor response in the treatment of depression. There is also a growing list of genetic polymorphisms in drug targets that have been shown to influence drug response. A major limitation that has heretofore moderated the use of pharmacogenetic testing in the clinical setting is the lack of prospective clinical trials demonstrating that such testing can improve the benefit/risk ratio of drug therapy.
West Nile Virus: Epidemiology and Clinical Features of an Emerging Epidemic in the United States
Authors: Hayes Edward B. , Gubler Duane J.
Published: September 2005

West Nile virus (WNV) was first detected in North America in 1999 during an outbreak of encephalitis in New York City. Since then the virus has spread across North America and into Canada, Latin America, and the Caribbean. The largest epidemics of neuroinvasive WNV disease ever reported occurred in the United States in 2002 and 2003. This paper reviews new information on the epidemiology and clinical aspects of WNV disease derived from greatly expanded surveillance and research on WNV during the past six years.
PERMEATION AND SELECTIVITY OF TRP CHANNELS
Authors: Owsianik Grzegorz , Talavera Karel , Voets Thomas , Nilius Bernd
Published: September 2005

▪ Abstract  Ion channels are pore-forming transmembrane proteins that allow ions to permeate biological membranes. Pore structure plays a crucial role in determining the ion permeation and selectivity properties of particular channels. In the past few decades, efforts have been undertaken to identify key elements of the pore regions of different classes of ion channels. In this review, we summarize current knowledge about permeation and selectivity of channel proteins from the transient receptor potential (TRP) superfamily. Whereas all TRP channels are permeable for cations, only two TRP channels are impermeable for Ca2+ (TRPM4, TRPM5), and two others are highly Ca2+ permeable (TRPV5, TRPV6). Despite the great advances in the TRP channel field during the past decade, only a limited number of reports have dealt with functional characterization of pore properties, biophysical aspects of cation permeation, or description of pore structures of TRP channels. This review gives an overview of available experimental and theoretical data and discusses the functional impact of pore-structure modifications on TRP channel properties.
Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes
Authors: Goldring Chris E. P. , Kitteringham Neil R. , Jenkins Rosalind , Lovatt Cerys A. , Randle Laura E. , Abdullah Azman , Owen Andrew , Liu Xiaoping , Butler Philip J. , Williams Dominic P. , Metcalfe Peter , Berens Christian , Hillen Wolfgang , Foster Brian , Simpson Alec , McLellan Lesley , Park B. Kevin
Published: September 2005

Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and on exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug-metabolizing enzymes, vary widely between species, among individual humans, and on exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. Here we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a second-generation doxycycline-inducible gene expression system and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely phase I (cytochrome P-450 2E1; CYP2E1) and phase II (glutathione S-transferase P1; GSTP1) drug metabolism, and three transcription factors that respond to chemical stress [nuclear factor erythroid 2 p45-related factors (NRF)1 and 2 and NFKB1 subunit of NF-κB]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.
DCEBIO stimulates Cl− secretion in the mouse jejunum
Authors: Hamilton Kirk L. , Kiessling Matt
Published: September 2005

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2 H-benzimidazol-2-one(DCEBIO) on the Cl− secretory response of the mouse jejunum using the Ussing short-circuit current ( Isc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in Isc (EC50 41 ± 1 μM). Pretreating tissues with 0.25 μM forskolin reduced the concentration-dependent increase in Isc by DCEBIO and increased the EC50 (53 ± 5 μM). Bumetanide blocked (82 ± 5%) the DCEBIO-stimulated Isc consistent with Cl− secretion. DCEBIO was a more potent stimulator of Cl− secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated Isc by >80% indicating the participation of CFTR in the DCEBIO-stimulated Isc response. Clotrimazole reduced DCEBIO-stimulated Isc by 67 ± 15%, suggesting the participation of the intermediate conductance Ca2+-activated K+ channel (IKCa) in the DCEBIO-activated Isc response. In the presence of maximum forskolin (10 μM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in Isc of 43 ± 5 μA/cm2 and then falling to a steady-state response of 17 ± 10 μA/cm2 compared with DCEBIO control tissues (61 ± 6 μA/cm2). The forskolin-stimulated Isc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated Isc, providing evidence that DCEBIO increased Cl− secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl− secretion of the mouse jejunum and that DCEBIO targets components of the Cl− secretory mechanism.
Spectral imaging microscopy demonstrates cytoplasmic pH oscillations in glial cells
Authors: Sánchez-Armáss Sergio , Sennoune Souad R. , Maiti Debasish , Ortega Filiberta , Martínez-Zaguilán Raul
Published: September 2005

Glial cells exhibit distinct cellular domains, somata, and filopodia. Thus the cytoplasmic pH (pHcyt) and/or the behavior of the fluorescent ion indicator might be different in these cellular domains because of distinct microenvironments. To address these issues, we loaded C6 glial cells with carboxyseminaphthorhodafluor (SNARF)-1 and evaluated pHcyt using spectral imaging microscopy. This approach allowed us to study pHcyt in discrete cellular domains with high temporal, spatial, and spectral resolution. Because there are differences in the cell microenvironment that may affect the behavior of SNARF-1, we performed in situ titrations in discrete cellular regions of single cells encompassing the somata and filopodia. The in situ titration parameters apparent acid-base dissociation constant (p K′a), maximum ratio (Rmax), and minimum ratio (Rmin) had a mean coefficient of variation approximately six times greater than those measured in vitro. Therefore, the individual in situ titration parameters obtained from specific cellular domains were used to estimate the pHcyt of each region. These studies indicated that glial cells exhibit pHcyt heterogeneities and pHcyt oscillations in both the absence and presence of physiological HCO3−. The amplitude and frequency of the pHcyt oscillations were affected by alkalosis, by acidosis, and by inhibitors of the ubiquitous Na+/H+ exchanger- and HCO3−-based H+-transporting mechanisms. Optical imaging approaches used in conjunction with BCECF as a pH probe corroborated the existence of pHcyt oscillations in glial cells.
Polarized trafficking of the aquaporin-3 water channel is mediated by an NH2-terminal sorting signal
Authors: Rai Tatemitsu , Sasaki Sei , Uchida Shinichi
Published: September 2005

Epithelial renal collecting duct cells express multiple types of aquaporin (AQP) water channels in a polarized fashion. AQP2 is specifically targeted to the apical cell domain, whereas AQP3 and AQP4 are expressed on the basolateral membrane. It is crucial that these AQP variants are sorted to their proper polarized membrane domains, because correct AQP sorting enables efficient water transport. However, the molecular mechanisms involved in the polarized targeting and membrane trafficking of AQPs remain largely unknown. In the present study, we have examined the polarized trafficking and surface expression of AQP3 in Madin-Darby canine kidney type II (MDCKII) cells in an effort to identify the molecular determinants of polarized targeting specificity. When expressed in MDCKII cells, the majority of the exogenous wild-type AQP3 was found to be targeted to the basolateral membrane, consistent with its localization pattern in vivo. A potential sorting signal consisting of tyrosine- and dileucine-based motifs was subsequently identified in the AQP3 NH2 terminus. When mutations were introduced into this signaling region, the basolateral targeting of the resulting mutant AQP3 was disrupted and the mutant protein remained in the cytoplasm. AQP2-AQP3 chimeras were then generated in which the entire NH2 terminus of AQP2 was replaced with the AQP3 NH2 terminus. This chimeric protein was observed to be mislocalized constitutively in the basolateral membrane, and mutations in the AQP3 NH2-terminal sorting signal abolished this effect. On the basis of these results, we conclude that an NH2-terminal sorting signal mediates the basolateral targeting of AQP3.
Macromolecule permeability of in situ and excised rodent skeletal muscle arterioles and venules
Authors: Sarelius Ingrid H. , Kuebel Julia M. , Wang Jianjie , Huxley Virginia H.
Published: August 2005

In microvessels, acute inflammation is typified by an increase in leukocyte-endothelial cell interactions, culminating in leukocyte transmigration into the tissue, and increased permeability to water and solutes, resulting in tissue edema. The goal of this study was to establish a method to quantify solute permeability ( Ps) changes in microvessels in intact predominantly blood-perfused networks in which leukocyte transmigratory behavior could be precisely described using established paradigms. We used intravital confocal microscopy to measure solute (BSA) flux across microvessel walls, hence Ps. A quantitative fluorescence approach (Huxley VH, Curry FE, and Adamson RH. Am J Physiol Heart Circ Physiol 252: H188–H197, 1987) was adapted to the imaged confocal tissue slice in which the fluorescent source volume and source surface area of the microvessel were restricted to the region of vessel that was contained within the imaged confocal tissue section. Ps measurements were made in intact cremaster muscle microvasculature of anesthetized mice and compared with measurements of Ps made in isolated rat skeletal muscle microvessels. Mouse arteriolar Ps was 9.9 ± 1.1 × 10−7 cm/s ( n = 16), which was not different from 8.4 ± 1.3 × 10−7 cm/s ( n = 6) in rat arterioles. Values in venules were significantly ( P < 0.05) higher: 44.4 ± 7.9 × 10−7 cm/s ( n = 14) in mice and 25.0 ± 3.7 × 10−7 cm/s in rats. Convective coupling was estimated to contribute <10% to the measured Ps in both microvessel types and both animal models. We conclude that this approach provides an appropriate quantification of Ps in the intact microvasculature and that arteriolar Ps, while lower than in venules, is nevertheless consistent with arterioles being a significant source of interstitial protein.
Use of simultaneous pressure and velocity measurements to estimate arterial wave speed at a single site in humans
Authors: Davies Justin E. , Whinnett Zachary I. , Francis Darrel P. , Willson Keith , Foale Rodney A. , Malik Iqbal S. , Hughes Alun D. , Parker Kim H. , Mayet Jamil
Published: August 2005

It has not been possible to measure wave speed in the human coronary artery, because the vessel is too short for the conventional two-point measurement technique used in the aorta. We present a new method derived from wave intensity analysis, which allows derivation of wave speed at a single point. We apply this method in the aorta and then use it to derive wave speed in the human coronary artery for the first time. We measured simultaneous pressure and Doppler velocity with intracoronary wires at the left main stem, left anterior descending and circumflex arteries, and aorta in 14 subjects after a normal coronary arteriogram. Then, in 10 subjects, serial measurements were made along the aorta before and after intracoronary isosorbide dinitrate. Wave speed was derived by two methods in the aorta: 1) the two-site distance/time method (foot-to-foot delay of pressure waveforms) and 2) a new single-point method using simultaneous pressure and velocity measurements. Coronary wave speed was derived by the single-point method. Wave speed derived by the two methods correlated well ( r = 0.72, P < 0.05). Coronary wave speed correlated with aortic wave speed ( r = 0.72, P = 0.002). After nitrate administration, coronary wave speed fell by 43%: from 16.4 m/s (95% confidence interval 12.6–20.1) to 9.3 m/s (95% confidence interval 6.5–12.0, P < 0.001). This single-point method allows determination of wave speed in the human coronary artery. Aortic wave speed is correlated to coronary wave speed. Finally, this technique detects the prompt fall in coronary artery wave speed with isosorbide dinitrate.
Calcium Dynamics and Compartmentalization in Leech Neurons
Authors: Andjelic Sofija , Torre Vincent
Published: September 2005

Calcium dynamics in leech neurons were studied using a fast CCD camera. Fluorescence changes (Δ F/ F) of the membrane impermeable calcium indicator Oregon Green were measured. The dye was pressure injected into the soma of neurons under investigation. Δ F/ F caused by a single action potential (AP) in mechanosensory neurons had approximately the same amplitude and time course in the soma and in distal processes. By contrast, in other neurons such as the Anterior Pagoda neuron, the Annulus Erector motoneuron, the L motoneuron, and other motoneurons, APs evoked by passing depolarizing current in the soma produced much larger fluorescence changes in distal processes than in the soma. When APs were evoked by stimulating one distal axon through the root, Δ F/ F was large in all distal processes but very small in the soma. Our results show a clear compartmentalization of calcium dynamics in most leech neurons in which the soma does not give propagating action potentials. In such cells, the soma, while not excitable, can affect information processing by modulating the sites of origin and conduction of AP propagation in distal excitable processes.
Interleukin 1 Beta Polymorphism (–511) and Risk of Stroke due to Small Vessel Disease
Authors: Dziedzic Tomasz , Slowik Agnieszka , Pera Joanna , Szczudlik Andrzej
Published: September 2005

Background: Interleukin 1 (IL-1) is a proinflammatory cytokine involved in atherogenesis and thrombosis. The aim of this study was to investigate the association between the IL-1β gene polymorphism (–511) and the risk of large vessel disease (LVD) stroke, small vessel disease (SVD) stroke and cardioembolic (CE) stroke. Methods: We genotyped 115 patients with LVD stroke and 194 matched controls, 122 patients with SVD stroke and 227 controls and 221 patients with CE stroke and 219 controls. The IL-1β polymorphism was analyzed by a polymerase chain reaction followed by a restriction enzyme digestion. Results: The allele T distribution did not change significantly between patients with LVD stroke and their controls nor between patients with CE stroke and their controls. The frequency of the T allele was higher in patients with SVD stroke than in the control group (37.7 vs. 30.4%, p = 0.05). After adjustment for vascular risk factor, the TT genotype was an independent risk factor for SVD stroke (OR: 2.40, 95% CI: 1.17–4.94). Conclusions: The TT genotype of the IL-1β gene may be associated with an increased risk of SVD stroke.
The AMPA Antagonist ZK 200775 in Patients with Acute Ischaemic Stroke: A Double-Blind, Multicentre, Placebo-Controlled Safety and Tolerability Study
Authors: Walters M.R. , Kaste Markku , Lees K.R. , Diener Hans C. , Hommel M. , De Keyser Jacques , Steiner Heike , Versavel Mark
Published: September 2005

Background and Purpose: ZK 200775 is a selective competitive AMPA receptor antagonist. It has demonstrated neuroprotective efficacy in experimental models of stroke and tolerability in healthy volunteers. We tested the safety and tolerability of ZK 200775 in patients with acute ischaemic stroke. Methods: In a multicentre double-blind, randomised, placebo-controlled phase II trial, 61 ischaemic stroke patients were treated with either placebo or active drug in a dose finding design. Twenty-five patients received placebo, 12 patients received a total dose of 262.5 mg in 48 h (group 1) and 13 patients received a total dose of 525 mg in 48 h (group 2), and 11 patients received a total dose of 105 mg over a period of 6 h (group 3). We studied the pharmacokinetics of the compound and the effect of the infusion on the neurologic and haemodynamic parameters of the patients. The study was not powered to detect neuroprotective efficacy. Results: In group 2 there was a significant transient worsening in the mean NIH stroke scale score 14– 18 h after the start of treatment. This was due to reduction of consciousness (stupor and coma) in 8 out of 13 patients. Level of consciousness improved approximately 6 h after cessation of infusion. No significant haemodynamic responses were observed. Even after reduction of the administered dose and duration of infusion to 6 h (group 3), 2 patients experienced a reduction in level of consciousness. The effect of ZK 200775 on level of consciousness gave cause for concern and the trial was stopped prematurely for safety reasons. Conclusions: The AMPA antagonist ZK 200775 reversibly worsened the neurological condition in patients with acute ischaemic stroke. Our results suggest that ZK 200775 exerts significant sedative effects in patients with acute stroke which preclude its further development as a neuroprotective agent in this indication.
TOAST Classification of Subtypes of Ischaemic Stroke: Diagnostic and Therapeutic Procedures in Stroke
Authors: Paradowski Bogusław , Maciejak Andrzej
Published: September 2005

Background: The purpose of this study was to classify ischaemic strokes according to TOAST in patients treated in the internal disease and neurological wards of a hospital in Lower Silesia, evaluate diagnostic and therapeutic methods, and compare their treatment effectiveness according to the Rankin scale. Methods: Two hundred and seventy-nine patients below 80 and 77 patients over 80 years of age suffering from ischaemic stroke were covered by the retrospective study. Results: Patients with large-artery atherosclerosis according to the TOAST classification and over 80 years old (p = 0.001) were more frequently hospitalised in the internal disease ward (p = 0.005). Strokes caused by small-vessel occlusion were more frequent in the neurological ward (p = 0.017). Conclusions: The authors suggest the general use of the TOAST classification and an active information campaign among doctors of various specialisations about standard procedures in stroke according to AHA and EUSI guidelines.
Benefit of Echocontrast-Enhanced Transcranial Arterial Color-Coded Duplex Ultrasound
Authors: Droste Dirk W. , Boehm Thomas , Ritter Martin A. , Dittrich Ralf , Ringelstein E. Bernd
Published: September 2005

Objectives: Proper assessment of the intracranial arteries by transcranial color-coded duplex sonography (TCCD) is occasionally made difficult by an insufficient temporal bone window, an unfavorable insonation angle, or low flow velocity or volume. In these cases, echocontrast could be helpful to increase the diagnostic confidence or to make the diagnosis at all. Material and Methods:We investigated 67 temporal windows of 47 patients with insufficient native transtemporal insonation conditions before and after the application of the second-generation (gas-filled) microbubble contrast agent Sonovue® (in 20 patients out of these 47, both temporal windows were insufficient, in the remaining 27 only one side). Results: As compared to the precontrast scans, echocontrast allowed for more segments to be evaluated by pulsed Doppler sonography (p < 0.0001) and for longer lumen segments to be displayed on color mode (p < 0.0001). With the help of contrast medium, flow velocity in the middle cerebral artery could be measured through 65 windows as compared to only 26 windows before contrast was applied (p < 0.0001). Conclusions: In patients with poor precontrast visualization of intracranial arteries, echocontrast-enhanced TCCD is very helpful.
Novel Magnetic Resonance Angiography Stage Grading for Moyamoya Disease
Authors: Houkin Kiyohiro , Nakayama Naoki , Kuroda Satoshi , Nonaka Tadashi , Shonai Takaharu , Yoshimoto Takashi
Published: September 2005

Background: Magnetic resonance angiography (MRA) has been acknowledged as a noninvasive diagnostic modality for moyamoya disease. However, in terms of staging of moyamoya disease, conventional angiography is still the gold standard. Therefore, the purpose of this study was to establish MRA grades for moyamoya disease as an alternative to conventional angiography. Methods: Twenty-two patients (44 sides) with moyamoya disease diagnosed by conventional angiography were evaluated by MRA during the past 5 years. MRA scores were assigned based on the severity of occlusive changes of the internal carotid artery, the horizontal portion of the middle cerebral artery, the anterior and the posterior cerebral arteries and the signals of the distal branches of these arteries. Total points ranged from 0 (normal) to 10 (most severe). Results: MRA scores (0–10) were significantly consistent with the conventional angiographic staging. Four grades based on this novel MRA scores correlated well with Suzuki’s stages, with high sensitivity and specificity. Conclusions: These novel MRA grades can be a reliable alternative to conventional staging. By employing these novel MRA grades, the use of conventional angiography can be avoided for the purpose of evaluation of the stages of moyamoya disease.
Hepatocyte Growth Factor Modulates H2O2-Induced Mesangial Cell Apoptosis through Induction of Heme Oxygenase-1
Authors: Radhakrishnan Neetu , Bhaskaran Madhu , Singhal Pravin C.
Published: September 2005

Oxidative stress plays an important role in the induction of mesangial cell (MC) injury. In the present study, we evaluated the molecular mechanism involved in hydrogen peroxide (H2O2)-induced MC apoptosis. In addition, we examined the role of heme oxygenase-1 (HO-1) in hepatocyte growth factor (HGF)-modulated, H2O2-induced MC injury. H2O2 promoted (p < 0.001) mouse MC (MMC) apoptosis. This effect of H2O2 was associated with translocation of cytochrome c from the mitochondrial to the cytosolic compartment. In addition, a caspase-9 inhibitor partially attenuated this effect of H2O2. These findings suggest that H2O2-induced MMC apoptosis is mediated through the mitochondrial pathway. HGF not only prevented H2O2-induced MMC apoptosis, but also inhibited H2O2-induced translocation of cytochrome c from the mitochondrial to the cytosolic compartment. HGF also promoted the expression of HO-1 by MMCs; interestingly, hemin inhibited (p < 0.001) H2O2-induced MMC apoptosis. On the other hand, zinc protoporphyrin inhibited the protective influence of HGF on H2O2-induced MMC apoptosis. These findings suggest that H2O2-induced apoptosis occurs through the mitochondrial pathway. HGF provides protection against H2O2-induced MMC apoptosis through induction of HO-1.
Establishment of Metanephros Transplantation in Mice Highlights Contributions by Both Nephrectomy and Pregnancy to Developmental Progression
Authors: Armstrong Shannon R. , Campbell Gordon R. , Campbell Julie H. , Little Melissa H.
Published: September 2005

Background: It has been demonstrated that embryonic kidneys (metanephroi) xenotransplanted into the omentum of adult recipients continue to develop and display immune protection due to their more naïve immune presentation. To date, this has been achieved using rat, pig and human metanephroi, with unilateral nephrectomy (UNX) of recipient rats a requisite of renal development. The aim of this study was to adapt this approach for use in mice and examine the parameters affecting successful onward development in this species. Methods: Metanephroi at embryonic age (E) 13.5 were transplanted either onto the body wall, abdominal fat pads or omentum of recipient isogenic C57/Bl6 mice using either sutures or polyglycolic acid mesh. Having established greatest success with polyglycolic acid mesh on the body wall, E12.5 and 15.5 days metanephroi from C57/Bl6 mice were then transplanted onto the body wall of control (non-pregnant non-UNX), UNX or 12.5 days post-coitum pregnant isogenic recipients. After 7 days, implanted tissue was harvested and examined using histology and immunohistochemistry for markers of renal maturation. The mean number of S-shaped bodies and glomeruli per section were recorded and statistically analysed for significant differences between all recipient groups and untransplanted metanephroi. The degree of development was scored qualitatively. Results: Transplanted E12.5 metanephroi developed S-shaped bodies and glomeruli in all recipient groups, although there were statistically higher numbers of S-shaped bodies in UNX (n = 2) and pregnant recipients (n = 9) than in control recipients (n = 4). Continued development, as indicated by mature vascularized glomeruli, was only observed in those E15.5 metanephroi transplanted into pregnant recipients (n = 11) with a 15.5-fold increase in S-shaped bodies and 4-fold increase in glomeruli compared with control transplants (n = 12). Conclusions: We have successfully established metanephros transplantation in mice and demonstrated enhancement of onward development of E12.5 metanephroi in response to both pregnancy and UNX. Using E15.5 metanephroi, continued development only occurred in pregnant recipients, implying pregnancy provides an environment conducive to continued organogenesis. This murine assay, when coupled with transgenically-tagged strains of mice, will allow the investigation of the relative contribution of donor and recipient cells to this process.
Potential Protective Effects of NSAIDs/ASA in Oxidatively Stressed Human Lens Epithelial Cells and Intact Mouse Lenses in Culture
Authors: Petersen A. , Zetterberg M. , Sjöstrand J. , Pålsson A.Z. , Karlsson J.-O.
Published: September 2005

Purpose: To study possible toxic effects of indomethacin, diclofenac, and celecoxib (NSAIDs) and acetylsalicylic acid (ASA) as well as potentially protective effects of these substances in oxidatively stressed human lens epithelial cells (HLEC) and in intact mouse lenses in culture. Methods: HLEC and mouse lenses were incubated with NSAIDs or ASA alone or in the presence of H2O2. To study apoptosis the cells were then either stained with Hoechst 33342 or assayed for caspase-3 activity. Mouse lenses were studied with respect to lens transparency. Results: Low concentrations of NSAIDs/ASA caused a significant protection against H2O2-induced apoptosis in HLEC whereas higher concentrations were toxic. Conclusion: The protective effects of NSAIDs/ASA against oxidative damage are confined to a relatively small therapeutic window.
Effect of Propolis in the Treatment of Experimental Staphylococcus aureus Keratitis in Rabbits
Authors: Oksuz H. , Duran N. , Tamer C. , Cetin M. , Silici S.
Published: September 2005

Purpose: The aim of this study was to investigate the antimicrobial and anti-inflammatory properties of an ethanolic extract of propolis, a natural resin produced by honeybees, and to determine synergistic activity between ciprofloxacin and propolis in the treatment of experimental Staphylococcus aureus keratitis. Methods: Sixteen young New Zealand white rabbits were given intrastromal injections of S. aureus strains. Twenty-four hours later, the rabbits were randomly divided into 4 groups: group 1 was treated with topical 0.3% ciprofloxacin drops along with the ethanolic extract of propolis drops; group 2 received topical 0.3% ciprofloxacin drops; group 3 was administered the ethanolic extract of propolis drops, and group 4, the control group, was treated with phosphate-buffered saline (PBS) drops. Drugs were instilled 8 times/day for 72 h. Twenty-four and 96 h after inoculation of bacteria, the eyes were examined by slit lamp to assess corneal opacity. Corneas were removed to count bacteria. Results: Slit lamp examination showed that the corneal opacity scores were significantly lower in eyes that received propolis plus ciprofloxacin than in those treated with ciprofloxacin (p = 0.041) or propolis (p = 0.006) or control eyes treated with PBS (p = 0.0001). There was no significant difference in eyes treated with ciprofloxacin and propolis (p = 1.00). There were significantly fewer bacteria in eyes that received propolis plus ciprofloxacin than in those treated with ciprofloxacin (p = 0.0001) or propolis (p = 0.0001) or control eyes treated with PBS (p = 0.0001). There was no significant difference in eyes treated with ciprofloxacin and propolis (p = 0.38). Conclusions: Taking these findings into consideration, we suggest that the ethanolic extract of propolis has antimicrobial and anti-inflammatory properties for S. aureus keratitis. The combination of ciprofloxacin and propolis had better therapeutic effects than either agent alone.
Local Resection of Rectal Tumors by Transanal Endoscopic Microsurgery: Experience with the First 70 Cases – with Invited Commentary
Authors: Røkke O. , Iversen K.B. , Øvrebø K. , Maartmann-Moe H. , Skarstein A. , Halvorsen J.F.
Published: September 2005

Background: Transanal endoscopic microsurgery (TEM) can access the whole rectum up to 20 cm from the anal verge. Due to its excellent view and accurate dissection, TEM is useful for the removal of adenoma and selected low risk cancers of the rectum. We report our experience with the first 70 patients presenting. Methods: A prospective descriptive study of 70 patients treated for rectal tumor with TEM from December 99 until October 2002 at Haukeland University Hospital. Results: TEM was performed in 37 men and 33 women, median age 70.5 (19–90) years, for anticipated adenoma (n = 64), adenocarcinoma (n = 3), rectal ulcer (n = 1), and re-resection after snare resection of rectal polyp with adenocarcinoma (n = 1) and carcinoid tumor (n = 1). The median observation time was 12 (1–33) months. The distance from the anal verge to the lower tumor border was 5.5 (2.5–14) cm. The median resected area was 15.4 (1.5–132) cm2. 56 of the 64 anticipated adenomas were true adenoma, resected without recurrences; 8 (12.5%) were unexpected adenocarcinoma. Three of these underwent a secondary rectal resection and 5 patients have been observed without recurrence. Of the 3 patients with known adenocarcinoma, there was one recurrence which was treated with a secondary curative rectal resection. Conclusions: TEM can access tumors in the whole rectum. Large tumors may be removed with low frequency of per- and postoperative complications and short hospital stay. TEM is highly useful for removal of rectal adenoma. A role for primary treatment of selected low-risk rectal cancers may emerge.
Locally Advanced Pancreatic Cancer Treated with Radiation and 5-Fluorouracil: A First Step to Neoadjuvant Treatment?
Authors: Smeenk Hans G. , de Castro Steve M.M. , Jeekel Johannes J. , Kazemier Geert , Busch Oliver R.C. , Incrocci Luca , Erdmann Joris , Hop W.C. , Gouma Dirk J. , van Eijck Casper H.J.
Published: September 2005

Aim of the Study: In two institutions, a retrospective analysis was performed on patients with histologically proven locally advanced pancreatic cancer without distant metastases. The aim of this analysis is to assess whether chemoradiotherapy provides survival benefit for patients with locally advanced pancreatic cancer. Methods: Forty-five patients from the Erasmus Medical Centre (Erasmus MC), Rotterdam, received 5-fluorouracil (5-FU) and radiotherapy and, 38 patients from the Academic Medical Centre Amsterdam (AMC) were offered the best supportive care. Radiotherapy consisted of 50 Gy external upper abdomen radiation in two courses, concomitant with intravenous 5-FU 25 mg/kg/ 24 h continuously on the first 4 days of each treatment course. Results: The treatment protocol was completed in 38 of 45 patients (84%) without complications. Radiological response was evaluated in 38 patients. Ten patients (26%) showed a partial response, stable disease was seen in 6 (16%) patients and progressive disease in 22 (58%) patients. A second-look operation was performed in 8 of 10 patients (72%) showing a radiological response, in 3 patients the tumour could be resected. Median overall survival time for the Erasmus MC group (n = 45) was 9.8 months compared to 7.6 months when the best supportive care was given (AMC group, p = 0.04). Conclusion: Although overall survival remains poor, treatment with 5-FU and radiotherapy might benefit some patients with locally advanced pancreatic cancer.