bluuebunny/crossref_metadata_2025 huggingface dataset:
Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells
Authors: Gingrich Kevin J. , Tran Son , Nikonorov Igor M. , Blanck Thomas J.
Published: November 2005

Background

Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation.

Methods

HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy.

Results

Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels.

Conclusions

The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.
Effect of Labor Epidural Analgesia with and without Fentanyl on Infant Breast-feeding
Authors: Beilin Yaakov , Bodian Carol A. , Weiser Jane , Hossain Sabera , Arnold Ittamar , Feierman Dennis E. , Martin Gregory , Holzman Ian
Published: November 2005

Background

The influence of labor epidural fentanyl on the neonate is controversial. The purpose of this study was to determine whether epidural fentanyl has an impact on breast-feeding.

Methods

Women who previously breast-fed a child and who requested labor epidural analgesia were randomly assigned in a double-blinded manner to one of three groups: (1) no fentanyl group, (2) intermediate-dose fentanyl group (intent to administer between 1 and 150 microg epidural fentanyl), or (3) high-dose epidural fentanyl group (intent to administer > 150 microg epidural fentanyl). On postpartum day 1, the mother and a lactation consultant separately assessed whether the infant was experiencing difficulty breast-feeding, and a pediatrician assessed infant neurobehavior. All women were contacted 6 weeks postpartum to determine whether they were still breast-feeding.

Results

Sixty women were randomly assigned to receive no fentanyl, 59 were randomly assigned to receive an intermediate dose, and 58 were randomly assigned to receive high-dose fentanyl. On postpartum day 1, women who were randomly assigned to receive high-dose fentanyl reported difficulty breast-feeding (n = 12, 21%) more often than women who were randomly assigned to receive an intermediate fentanyl dose (n = 6, 10%), or no fentanyl (n = 6, 10%), although this did not reach statistical significance (P = 0.09). There was also no significant difference among groups in breast-feeding difficulty based on the lactation consultant's evaluation (40% difficulty in each group; P = 1.0). Neurobehavior scores were lowest in the infants of women who were randomly assigned to receive more than 150 microg fentanyl (P = 0.03). At 6 weeks postpartum, more women who were randomly assigned to high-dose epidural fentanyl were not breast-feeding (n = 10, 17%) than women who were randomly assigned to receive either an intermediate fentanyl dose (n = 3, 5%) or no fentanyl (n = 1, 2%) (P = 0.005).

Conclusions

Among women who breast-fed previously, those who were randomly assigned to receive high-dose labor epidural fentanyl were more likely to have stopped breast-feeding 6 weeks postpartum than woman who were randomly assigned to receive less fentanyl or no fentanyl.
Frontal Slab Composite Magnetic Resonance Neurography of the Brachial Plexus
Authors: Raphael David T. , McIntee Diane , Tsuruda Jay S. , Colletti Patrick , Tatevossian Ray
Published: November 2005

Background

Magnetic resonance neurography (MRN) is an imaging method by which nerves can be selectively highlighted. Using commercial software, the authors explored a variety of approaches to develop a three-dimensional volume-rendered MRN image of the entire brachial plexus and used it to evaluate the accuracy of infraclavicular block approaches.

Methods

With institutional review board approval, MRN of the brachial plexus was performed in 10 volunteer subjects. MRN imaging was performed on a GE 1.5-tesla magnetic resonance scanner (General Electric Healthcare Technologies, Waukesha, WI) using a phased array torso coil. Coronal STIR and T1 oblique sagittal sequences of the brachial plexus were obtained. Multiple software programs were explored for enhanced display and manipulation of the composite magnetic resonance images. The authors developed a frontal slab composite approach that allows single-frame reconstruction of a three-dimensional volume-rendered image of the entire brachial plexus. Automatic segmentation was supplemented by manual segmentation in nearly all cases. For each of three infraclavicular approaches (posteriorly directed needle below midclavicle, infracoracoid, or caudomedial to coracoid), the targeting error was measured as the distance from the MRN plexus midpoint to the approach-targeted site.

Results

Composite frontal slabs (coronal views), which are single-frame three-dimensional volume renderings from image-enhanced two-dimensional frontal view projections of the underlying coronal slices, were created. The targeting errors (mean +/- SD) for the approaches-midclavicle, infracoracoid, caudomedial to coracoid-were 0.43 +/- 0.67, 0.99 +/- 1.22, and 0.65 +/- 1.14 cm, respectively.

Conclusion

Image-processed three-dimensional volume-rendered MNR scans, which allow visualization of the entire brachial plexus within a single composite image, have educational value in illustrating the complexity and individual variation of the plexus. Suggestions for improved guidance during infraclavicular block procedures are presented.
A New Inguinal Approach for the Obturator Nerve Block
Authors: Choquet Olivier , Capdevila Xavier , Bennourine Khaled , Feugeas Jean-Louis , Bringuier-Branchereau Sophie , Manelli Jean-Claude
Published: November 2005

Background

Obturator nerve block is highly recommended for knee surgery in addition to a femoral nerve block. The main disadvantage of the classic approach at the pubic tubercle is low patient acceptance due to pain and discomfort. The authors hypothesized that the use of a new inguinal obturator nerve block technique would reduce pain and discomfort in patients.

Methods

The inguinal approach was simulated in five fresh cadavers. Injection of latex was performed in two cadavers. The location of the needle and the extent of latex solution were analyzed. Fifty patients scheduled to undergo arthroscopic knee surgery were randomly assigned to receive obturator nerve block using either the inguinal (n = 25) or the pubic tubercle approach (n = 25).

Results

In all cadavers, the needle was close to the obturator nerve branches, which were surrounded by the latex solution. In the clinical study, visual analog scale pain scores and discomfort of block placement were significantly lower in the inguinal group compared with the pubic tubercle group (P < 0.01). In the inguinal group, there was a significant decrease in block performance time (P < 0.05) and in bolus of propofol and fentanyl used for the procedure (P < 0.01). Twenty minutes after application of the block, adductor strength decrease, occurrence, and location of cutaneous distribution of the obturator nerve were not significantly different between the groups. The incidence of minor complications was significantly increased in the pubic tubercle group (P < 0.05). No major complications were observed.

Conclusions

The new inguinal approach decreases patient discomfort and pain of block placement as well as the time and sedation and analgesics required for a similar quality of sensory and motor block compared with the pubic tubercle approach.
Bayesian Prediction Bounds and Comparisons of Operating Room Times Even for Procedures with Few or No Historic Data
Authors: Dexter Franklin , Ledolter Johannes
Published: November 2005

Background

Lower prediction bounds (e.g., for fasting), upper prediction bounds (e.g., to schedule delays between sequential surgeons), comparisons of operating room (OR) times (e.g., when sequencing cases among ORs), and quantification of case uncertainty (e.g., for sequencing a surgeon's list of cases) can be done accurately for combinations of surgeon and scheduled procedure(s) by using historic OR times. The authors propose that when there are few or no historic data, the predictive distribution of the OR time of a future case be centered at the scheduled OR time, and its proportional uncertainty be based on that of other surgeons and procedures. When there are a moderate or large number of historic data, the historic data alone are used in the prediction. When there are a small number of historic data, a weighted combination is used.

Methods

This Bayesian method was tested with all 65,661 cases from a hospital.

Results

Bayesian prediction bounds were accurate to within 2% (e.g., the 5% lower bounds exceeded 4.9% of the actual OR times). The predicted probability of one case taking longer than another was estimated to within 0.7%. When sequencing a surgeon's list of cases to reduce patient waiting past scheduled start times, both the scheduled OR time and the variability in historic OR times should be used together when assessing which cases should be done first.

Conclusions

The authors validated a practical way to calculate prediction bounds and compare the OR times of all cases, even those with few or no historic data for the surgeon and the scheduled procedure(s).
Pushing the envelope: microinjection of Minute virus of mice into Xenopus oocytes causes damage to the nuclear envelope
Authors: Cohen Sarah , Panté Nelly
Published: November 2005

Parvoviruses are small DNA viruses that replicate in the nucleus of their host cells. It has been largely assumed that parvoviruses enter the nucleus through the nuclear pore complex (NPC). However, the details of this mechanism remain undefined. To study this problem, the parvovirus Minute virus of mice (MVM) was microinjected into the cytoplasm of Xenopus oocytes and a transmission electron microscope was used to visualize the effect of the virus on the host cell. It was found that MVM caused damage to the nuclear envelope (NE) in a time- and concentration-dependent manner. Damage was predominantly to the outer nuclear membrane and was often near the NPCs. However, microinjection experiments in which the NPCs were blocked showed that NE damage induced by MVM was independent of the NPC. To address the question of whether this effect of MVM is specific to the NE, purified organelles were incubated with MVM. Visualization by electron microscopy revealed that MVM did not affect all intracellular membranes. These data represent a novel form of virus-induced damage to host cell nuclear structure and suggest that MVM is imported into the nucleus using a unique mechanism that is independent of the NPC, and involves disruption of the NE and import through the resulting breaks.
Structural proteins of Hepatitis C virus induce interleukin 8 production and apoptosis in human endothelial cells
Authors: Balasubramanian Anuradha , Munshi Neru , Koziel Margaret J. , Hu Zongyi , Liang T. Jake , Groopman Jerome E. , Ganju Ramesh K.
Published: November 2005

Hepatitis C virus (HCV) infection is associated with inflammation of liver endothelium, which contributes to the pathogenesis of chronic hepatitis. The mechanism of this endothelitis is not understood, since the virus does not appear to infect endothelial cells productively. Here, an ‘innocent bystander’ mechanism related to HCV proteins was hypothesized and it was investigated whether the binding of HCV particles to human endothelium induced functional changes in the cells. Exposure of human umbilical vein endothelial cells (HUVECs) to HCV-like particles (HCV-LPs) resulted in increased interleukin 8 (IL8) production and induction of apoptosis. The IL8 supernatants collected after stimulation of HUVECs with HCV-LPs, BV-GUS (control baculovirus containingβ-glucuronidase) and appropriate controls were used to assay the transendothelial migration of neutrophils. This assay confirmed that HCV-LP-induced IL8 was functionally active. Using specific NF-κB inhibitors, it was also shown that HCV-LP-induced NF-κB activity mediated IL8 production in HUVECs. Apoptosis appeared to be mediated by the Fas/Fas-L pathway, as neutralizing antibodies for Fas and Fas-L significantly protected HUVECs against HCV-LP-induced apoptosis. Treatment of HUVECs with HCV-LPs also enhanced cellular Fas-L expression and augmented caspase-3 activation. This was confirmed by using a specific caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethyl ketone. As shown by blocking of specific chemokine receptors for IL8 on HUVECs, the induction of IL8 did not appear to contribute to HCV-LP-induced apoptosis. These results suggest that HCV proteins can trigger the release of inflammatory chemokines such as IL8 and cause endothelial apoptosis, thereby facilitating endothelitis.
Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores
Authors: Koschinski Andreas , Wengler Gerd , Wengler Gisela , Repp Holger
Published: November 2005

Recently, class II fusion proteins have been identified on the surface of alpha- and flaviviruses. These proteins have two functions besides membrane fusion: they generate an isometric lattice on the viral surface and they form ion-permeable pores at low pH. An attempt was made to identify inhibitors for the ion pores generated by the fusion proteins of the alphaviruses Semliki Forest virus and Sindbis virus. These pores can be detected and analysed in three situations: (i) in the target membrane during virus entry, by performing patch-clamp measurements of membrane currents; (ii) in the virus particle, by studying the entry of propidium iodide; and (iii) in the plasma membrane of infected cells, by Fura-2 fluorescence imaging of Ca2+ entry into infected cells. It is shown here that, at a concentration of 0·1 mM, rare earth ions block the ion permeability of alphavirus ion pores in all three situations. Even at a concentration of 0·5 mM, these ions do not block formation of the viral fusion pore, as they do not inhibit entry or multiplication of alphaviruses. The data indicate that ions flow through the ion pores into the virus particle in the endosome and from the endosome into the cytoplasm after fusion of the viral envelope with the endosomal membrane. These ion flows, however, are not necessary for productive infection. The possibility that the ability of class II fusion proteins to form ion-permeable pores reflects their origin from protein toxins that form ion-permeable pores, and that entry via class II fusion proteins may resemble the entry of non-enveloped viruses, is discussed.
In vivo detection of scrapie cases from blood by infrared spectroscopy
Authors: Carmona Pedro , Monzón Marta , Monleón Eva , Badiola Juan José , Monreal Jaime
Published: November 2005

In the present study, infrared spectroscopy was shown to be able to distinguish healthy and scrapie-infected animals by analysis of the white-cell membranous fraction from blood. Infrared spectroscopy was able to detect not only clinical cases, but also animals at a preclinical stage of the disease. These findings suggest this technique as an accurate in vivo diagnostic tool that could be applied to animal as well as human samples. In addition to possibly avoiding the slaughter of a huge number of animals with the socio-economic consequences that this poses, the test could be expected to become useful in the prevention of human transmission by blood transfusion.
Rotaviruses interact with α4β7 and α4β1 integrins by binding the same integrin domains as natural ligands
Authors: Graham Kate L. , Fleming Fiona E. , Halasz Peter , Hewish Marilyn J. , Nagesha Hadya S. , Holmes Ian H. , Takada Yoshikazu , Coulson Barbara S.
Published: November 2005

Group A rotaviruses are major intestinal pathogens that express potentialα4β1 andα4β7 integrin ligand sequences Leu–Asp–Val and Leu–Asp–Ile in their outer capsid protein VP7, and Ile–Asp–Ala in their spike protein VP4. Monkey rotavirus SA11 can use recombinantα4β1 as a cellular receptor. In this study a new potentialα4β1,α4β7 andα9β1 integrin ligand sequence, Tyr–Gly–Leu, was identified in VP4. It was shown that several human and monkey rotaviruses boundα4β1 andα4β7, but notα9β1. Binding toα4β1 mediated the infectivity and growth of monkey rotaviruses, and binding toα4β7 mediated their infectivity. A porcine rotavirus interacted withα4 integrins at a post-binding stage to facilitate infection. Activation ofα4β1 increased rotavirus infectivity. Cellular treatment with peptides containing theα4 integrin ligand sequences Tyr–Gly–Leu and Ile–Asp–Ala eliminated virus binding toα4 integrins and infectivity. In contrast, rotavirus recognition ofα4 integrins was unaffected by a peptide containing the sequence Leu–Asp–Val or by a mutation in the VP7 Leu–Asp–Val sequence. VP4 involvement in rotavirus recognition ofα4β1 was demonstrated with rotavirus reassortants. Swapping and point mutagenesis ofα4 surface loops showed that rotaviruses required the sameα4 residues and domains for binding as the naturalα4 integrin ligands: mucosal addressin cell adhesion molecule-1, fibronectin and vascular cell adhesion molecule-1. Several rotaviruses are able to useα4β7 andα4β1 for cell binding or entry, through the recognition of the sameα4-subunit domains as naturalα4 ligands.
Effective treatment of retrovirus-induced suppression of antibody responses with CpG oligodeoxynucleotides
Authors: Kraft Anke R. M. , Arndt Tanja , Hasenkrug Kim J. , Dittmer Ulf
Published: November 2005

Most retroviruses induce severe immunosuppression during acute infection. We have used the Friend retrovirus mouse model to demonstrate that immunostimulatory B-type CpG oligodeoxynucleotides (ODN) have a protective effect against retrovirus-induced suppression of antibody responses to potent B-cell antigens. CD8+ T cells were critical for effective treatment with CpG-ODN, since in vivo depletion of these cells from treated mice impaired protection from retrovirus-induced immunosuppression. Protection also required IFN-γ, as neutralization of this cytokine abolished the therapeutic effect of CpG-ODN. These findings may have implications for the treatment of immunosuppressive virus infections.
Membrane deformations induced by the matrix protein of vesicular stomatitis virus in a minimal system
Authors: Solon Jérôme , Gareil Olivier , Bassereau Patricia , Gaudin Yves
Published: November 2005

The matrix (M) protein of vesicular stomatitis virus plays a key role in both assembly and budding of progeny virions. In vitro experiments have shown a strong propensity of M protein to bind to vesicles containing negatively charged phospholipids. In vivo, it has also been demonstrated that recruitment of some cellular proteins by M protein is required for efficient virus budding and release of newly synthesized virions in the extracellular medium. The ability of M protein to deform target membranes in vitro was investigated in this study. It was shown that incubation of purified M protein with giant unilamellar vesicles results in the formation of patches of M protein at their surface, followed by deformations of the membrane toward the inside of the vesicle, which could be observed in phase-contrast microscopy. This provides the first evidence that M protein alone is able to impose the correct budding curvature on the membrane. Using confocal microscopy, patches of M protein that colocalized with negatively charged lipid domains a few minutes after vesicle injection were observed. After a longer incubation period, membrane deformations appeared in these domains. At this time, a strict colocalization of M protein, negatively charged lipids and membrane deformation was observed. The influence on this process of the basic N-terminal part of the protein and of the previously identified hydrophobic loop has also been investigated. Interestingly, the final fission event has never been observed in our experimental system, indicating that other partners are required for this step.
The majority of human immunodeficiency virus type 1 particles present within splenic germinal centres are produced locally
Authors: Dumaurier Marie-Jeanne , Gratton Sophie , Wain-Hobson Simon , Cheynier Rémi
Published: November 2005

In most stages of human immunodeficiency virus (HIV) infection, cell-free viral particles can be detected in germinal centres (GCs) that are principally retained, in the form of immune complexes, on the surface of follicular dendritic cells (FDCs). The source of this virus remains unknown, although it is agreed that the FDCs themselves are not infected productively. By sequencing HIV viral DNA, genomic RNA and spliced mRNA isolated from individual splenic white pulps, it was shown here that the majority of HIV-1 viral particles are produced locally within the supporting lymphoid structure and do not result from trapping of circulating viruses or immune complexes. These findings underline the exquisite spatial organization of HIV-1 replication in vivo, suggesting a local origin for viruses trapped in splenic GCs.
Evaluation of CD8+ T-cell and antibody responses following transient increased viraemia in rhesus macaques infected with live, attenuated simian immunodeficiency virus
Authors: Metzner Karin J. , Moretto Walter J. , Donahoe Sean M. , Jin Xia , Gettie Agegnehu , Montefiori David C. , Marx Preston A. , Binley James M. , Nixon Douglas F. , Connor Ruth I.
Published: November 2005

In vivodepletion of CD8+T cells results in an increase in viral load in macaques chronically infected with simian immunodeficiency virus (SIVmac239Δnef). Here, the cellular and humoral immune responses associated with this transient period of enhanced viraemia in macaques infected with SIVmac239Δnef were characterized. Fourteen days afterin vivoCD8+T-cell depletion, two of six macaques experienced a 1–2 log10increase in anti-gp130 and p27 antibody titres and a three- to fivefold increase in gamma interferon-secreting SIV-specific CD8+T cells. Three other macaques had modest or no increase in anti-gp130 antibodies and significantly lower titres of anti-p27 antibodies, with minimal induction of functional CD8+T cells. Four of the five CD8-depleted macaques experienced an increase in neutralizing antibody titres to SIVmac239. Induction of SIV-specific immune responses was associated with increases in CD8+T-cell proliferation and fluctuations in the levels of signal-joint T-cell receptor excision circles in peripheral blood cells. Five months after CD8+T-cell depletion, only the two high-responding macaques were protected from intravenous challenge with pathogenic SIV, whilst the remaining animals were unable to control replication of the challenge virus. Together, these findings suggest that a transient period of enhanced antigenaemia during chronic SIV infection may serve to augment virus-specific immunity in some, but not all, macaques. These findings have relevance for induction of human immunodeficiency virus (HIV)-specific immune responses during prophylactic and therapeutic vaccination and for immunological evaluation of structured treatment interruptions in patients chronically infected with HIV-1.
Co-evolutionary patterns of variation in small and large RNA segments of Crimean-Congo hemorrhagic fever virus
Authors: Chamberlain John , Cook Nicola , Lloyd Graham , Mioulet Valerie , Tolley Howard , Hewson Roger
Published: November 2005

The genus Nairovirus of the family Bunyaviridae includes the Crimean-Congo haemorrhagic fever (CCHF) species group. The species is predominated by the hazard-group 4 pathogens, from which the name and majority of strain entries are derived. Additionally, the species embraces hazard-group 2 viruses that are classified as members by antigenic cross-reactivity. CCHF viruses have a tripartite RNA genome consisting of large (L), medium (M) and small (S) segments. Here, the sequence characterization of previously undescribed L and S segments from novel strains originating in the Middle East and Africa is reported. Further scrutiny of this data with phylogenetic tools, in the context of other publicly available sequence information, reveals analogous grouping patterns between the L and S segments. These groups correlate with the geographical distribution of strain isolation and indicate that the L and S segments of CCHF viruses have evolved together.
Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrovirus (family Baculoviridae)
Authors: Young Vivienne L. , Simpson Robert M. , Ward Vernon K.
Published: November 2005

Baculovirus chitinases and other family 18 glycohydrolases have been shown to possess both exo- and endochitinase activities when assayed against fluorescent chito-oligosaccharides. Homology modelling of the chitinase ofEpiphyas postvittana nucleopolyhedrovirus(EppoNPV) againstSerratia marcescenschitinase A indicated that the enzyme possesses an N-terminal polycystic kidney 1 (PKD1) domain for chitin-substrate feeding and anα/βTIM barrel catalytic domain characteristic of a family 18 glycohydrolase. EppoNPV chitinase has many features in common with other baculovirus chitinases, including high amino acid identity, an N-terminal secretion signal and a functional C-terminal endoplasmic reticulum-retention sequence. EppoNPV chitinase displayed exo- and endochitinolytic activity against fluorescent chito-oligosaccharides, withKmvalues of 270±60 and 240±40 μM against 4MU-(GlcNAc)2and 20±6 and 14±7 μM against 4MU-(GlcNAc)3for native and recombinant versions of the enzyme, respectively. In contrast, digestion and thin-layer chromatography analysis of short-chain (GlcNAc)2–6chito-oligosaccharides without the fluorescent 4-methylumbelliferone (4MU) moiety produced predominantly (GlcNAc)2, indicating an exochitinase, although low-level endochitinase activity was detected. Digestion of long-chain colloidalβ-chitin and analysis by mass spectrometry identified a single 447 Da peak, representing a singly charged (GlcNAc)2complexed with a sodium adduct ion, confirming the enzyme as an exochitinase with no detectable endochitinolytic activity. Furthermore, (GlcNAc)3–6substrates, but not (GlcNAc)2, acted as inhibitors of EppoNPV chitinase. Short-chain substrates are unlikely to interact with the aromatic residues of the PKD1 substrate-feeding mechanism and hence may not accurately reflect the activity of these enzymes against native substrates. Based upon these results, the chitinase of the baculovirus EppoNPV is an exochitinase.
Recombination in circulating Human enterovirus B: independent evolution of structural and non-structural genome regions
Authors: Lukashev Alexander N. , Lashkevich Vasilii A. , Ivanova Olga E. , Koroleva Galina A. , Hinkkanen Ari E. , Ilonen Jorma
Published: November 2005

The complete nucleotide sequences of eight Human enterovirus B (HEV-B) strains were determined, representing five serotypes, E6, E7, E11, CVB3 and CVB5, which were isolated in the former Soviet Union between 1998 and 2002. All strains were mosaic recombinants and only the VP2–VP3–VP1 genome region was similar to that of the corresponding prototype HEV-B strains. In seven of the eight strains studied, the 2C–3D genome region was most similar to the prototype E30, EV74 and EV75 strains, whilst the remaining strain was most similar to the prototype E1 and E9 strains in the non-structural protein genome region. Most viruses also bore marks of additional recombination events in this part of the genome. In the 5′ non-translated region, all strains were more similar to the prototype E9 than to other enteroviruses. In most cases, recombination mapped to the VP4 and 2ABC genome regions. This, together with the star-like topology of the phylogenetic trees for these genome regions, identified these genome parts as recombination hot spots. These findings further support the concept of independent evolution of enterovirus genome fragments and indicate a requirement for more advanced typing approaches. A range of available phylogenetic methods was also compared for efficient detection of recombination in enteroviruses.
Mutational analysis of the avian pneumovirus conserved transcriptional gene start sequence identifying critical residues
Authors: Edworthy Nicole L. , Easton Andrew J.
Published: November 2005

Seven of the eight genes in the avian pneumovirus (APV) genome contain a conserved 9 nt transcriptional start sequence with the virus large (L) polymerase gene differing from the consensus at three positions. The sequence requirements of the APV transcriptional gene start sequence were investigated by generating a series of mutations in which each of the nine conserved bases was mutated to each of the other three possible nucleotides in a minigenome containing two reporter genes. The effect of each mutation was assessed by measuring the relative levels of expression from the altered and unaltered gene start sequences. Mutations at positions 2, 7 and 9 significantly reduced transcription levels while alterations to position 5 had little effect. The L gene start sequence directed transcription at levels approximately 50 % below that of the consensus gene start sequence. These data suggest that there are common features in pneumovirus transcriptional control sequences.
Inhibition of host peripheral blood mononuclear cell proliferation ex vivo by Rinderpest virus
Authors: Heaney J. , Cosby S. L. , Barrett T.
Published: November 2005

Rinderpest, or cattle plague, is caused by Rinderpest virus (RPV), which is related most closely to human Measles virus (MV), both being members of the genus Morbillivirus, a group of viruses known to have strong immunosuppressive effects in vitro and in vivo. Here, it was shown that peripheral blood mononuclear cells (PBMCs) isolated from cattle experimentally infected with either wild-type or vaccine strains of RPV impaired the proliferation of PBMCs derived from uninfected animals; however, in contrast to either mild or virulent strains of wild-type virus, the inhibition induced by the vaccine was both weak and transient. Flow-cytometric analysis of PBMCs obtained from cattle infected with different strains of RPV showed that the proportion of infected cells was virus dose-dependent and correlated with lymphoproliferative suppression.
Both soluble and membrane-anchored forms of Felid herpesvirus 1 glycoprotein G function as a broad-spectrum chemokine-binding protein
Authors: Costes B. , Ruiz-Argüello M. B. , Bryant N. A. , Alcami A. , Vanderplasschen A.
Published: November 2005

Recently, glycoprotein G (gG) of several alphaherpesviruses infecting large herbivores was shown to belong to a new family of chemokine-binding proteins (vCKBPs). In the present study, the function of Felid herpesvirus 1 (FeHV-1) gG as a vCKBP was investigated and the following conclusions were reached: (i) FeHV-1 secreted gG is a high-affinity broad-spectrum vCKBP that binds CC, CXC and C chemokines; (ii) gG is the only vCKBP expressed by FeHV-1 that binds CCL3 and CXCL1; (iii) secreted gG blocks chemokine activity by preventing their interaction with high-affinity cellular receptors; (iv) the membrane-anchored form of gG expressed on the surface of infected cells is also able to bind chemokines; and (v) the vCKBP activity is conserved among different field isolates of FeHV-1. Altogether, these data demonstrate that FeHV-1 gG is a new member of the vCKBP-4 family. Moreover, this study is the first to demonstrate that gG expressed at the surface of FeHV-1-infected cells can also bind chemokines.
Expression of the high-risk human papillomavirus type 18 and 45 E7 oncoproteins in cervical carcinoma biopsies
Authors: Fiedler Marc , Ressler Sigrun , Campo-Fernández Beatriz , Laich Andreas , Jansen Lars , Widschwendter Andreas , Viertler Hans-Peter , Bacher Nicole , Morandell Dieter , Müller-Holzner Elisabeth , Dürst Matthias , Jansen-Dürr Pidder , Zwerschke Werner
Published: November 2005

E7 proteins are major oncoproteins of high-risk human papillomaviruses (HPVs), which play a key role in cervical carcinogenesis. These proteins have been shown to immortalize primary human cells. Due to the absence of antibodies with suitable sensitivity and specificity, little is known about expression of the E7 oncoproteins in naturally infected tissues. Recently, high-level expression of the E7 protein of HPV-16, the most prevalent oncogenic HPV type, was demonstrated in cervical carcinomas by immunohistochemistry; however, approximately 15 additional high-risk HPV types are known to be associated with cervical carcinoma. It is unknown whether the E7 oncoproteins of HPV-18 and -45, the second and third most prevalent HPV types, are expressed in cervical cancers. Using antibodies against HPV-18 and -45 E7 proteins, it is shown here for the first time that the HPV-18 and -45 E7 proteins can be detected in cervical carcinoma biopsies. Together with anti-HPV-16 E7 antibodies, this could create the possibility of detecting E7 oncoproteins in approximately 80 % of all cervical cancers.
RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)
Authors: Yue Constanze , Genersch Elke
Published: November 2005

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.
Assembly of functional hepatitis C virus glycoproteins on infectious pseudoparticles occurs intracellularly and requires concomitant incorporation of E1 and E2 glycoproteins
Authors: Sandrin Virginie , Boulanger Pierre , Penin Francois , Granier Christelle , Cosset François-Loïc , Bartosch Birke
Published: November 2005

Hepatitis C virus (HCV) E1 and E2 envelope glycoproteins (GPs) displayed on retroviral cores (HCVpp) are a powerful and highly versatile model system to investigate wild-type HCV entry. To further characterize this model system, the cellular site of HCVpp assembly and the respective roles of the HCV GPs in this process were investigated. By using a combination of biochemical methods with confocal and electron microscopic techniques, it was shown that, in cells producing HCVpp, both E1 and E2 colocalized with retroviral core proteins intracellularly, presumably in multivesicular bodies, but not at the cell surface. When E1 and E2 were expressed individually with retroviral core proteins, only E2 colocalized with and was incorporated on retroviral cores. Conversely, the colocalization of E1 with retroviral core proteins and its efficient incorporation occurred only upon co-expression of E2. Moreover, HCVpp infectivity correlated strictly with the presence of both E1 and E2 on retroviral cores. Altogether, these results confirm that the E1E2 heterodimer constitutes the prebudding form of functional HCV GPs and, more specifically, show that dimerization with E2 is a prerequisite for efficient E1 incorporation onto particles.
Synchrotron X-ray diffraction study of the tetragonal–cubic phase boundary of nanocrystalline ZrO2–CeO2synthesized by a gel-combustion process
Authors: Lamas D. G. , Fuentes R. O. , Fábregas I. O. , Fernández de Rapp M. E. , Lascalea G. E. , Casanova J. R. , Walsöe de Reca N. E. , Craievich A. F.
Published: November 2005

The crystal structures of a number of nanocrystalline ZrO2–CeO2solid solutions, synthesized by a pH-controlled nitrate-glycine gel-combustion process, were studied. By using a synchrotron X-ray diffractometer, small peaks of the tetragonal phase, which correspond to forbidden reflections in the case of a perfect cubic fluorite structure, were clearly detected. By monitoring the most intense of these reflections, 112, as a function of the CeO2content, the tetragonal–cubic phase boundary was found to be at 85 (5) mol% CeO2. For a CeO2content up to 68 mol%, a tetragonal phase withc/a> 1 (known as thet′ form) was detected, whereas, between 68 and 85 mol% CeO2, the existence of a tetragonal phase withc/a= 1 and oxygen anions displaced from their ideal positions in the cubic phase (thet′′ form) was verified. Finally, solid solutions with higher CeO2contents exhibit the cubic fluorite-type phase.
A global multi-technique approach to study low-resolution solution structures
Authors: Nöllmann Marcelo , Stark W. Marshall , Byron Olwyn
Published: November 2005

Finding the conformation of large macromolecular complexes has become an important problem in structural biology, which is not always soluble by high-resolution techniques such as X-ray crystallography and NMR spectroscopy. Solution biophysical properties can provide direct or indirect structural information on these large complexes. A general systematic approach to the construction of a structural model of the macromolecule consistent with all the experimental solution properties is currently lacking. In this paper, such an approach is presented, where generalized rigid-body modelling is combined with a Monte Carlo/simulated-annealing optimization method, to search over a large range of possible conformations for the structure that best fits solution experimental properties derived from small-angle scattering, fluorescence resonance energy transfer and analytical ultracentrifugation.
Crystalline microstructure of sepiolite influenced by grinding
Authors: Kojdecki Marek Andrzej , Bastida Joaquín , Pardo Pablo , Amorós Pedro
Published: November 2005

The crystalline microstructure of ground sepiolite has been investigated. A reference sample of sepiolite and products of its comminution by dry grinding were studied through X-ray diffraction pattern analysis, specific surface measurements by nitrogen adsorption and complementary analysis of field emission scanning electron microscope images. A statistical model of polycrystals was applied to describe and determine the crystalline microstructure of the studied specimens. The model parameters characterizing the microstructure were prevalent crystallite shape, volume-weighted crystallite size distribution and second-order crystalline lattice strain distribution, and they were determined for each sample by modelling a selected part of the X-ray diffraction pattern and fitting the simulated pattern to a measured one. A strict correlation of microstructure parameters with grinding time and with specific surface magnitudes was observed. A parallelepiped with edge-length ratios almost independent of grinding time (for longer times) was found to be the predominant crystallite shape. The crystallite size distributions were found to be close to logarithmic normal ones, with the mean values decreasing with increasing grinding time and the standard-deviation-to-mean-value ratios approximately constant. The second-order crystalline lattice strain distributions were found to be close to some simple function with the mean value equal to zero, the mean deviation increasing with increasing grinding time and the standard-to-mean-deviation ratios approximately constant. It was demonstrated that the specific surface can be calculated on the basis of the microstructure characteristics. Some details of the relation between crystallites and crystalline grains were explained by comparing the results of analysesviaX-ray diffraction and scanning electron microscopy.
A possible additional role of mineral oil in successful flash cooling
Authors: Gakhar Lokesh , Wiencek John M.
Published: November 2005

Traditional explanations for the efficacy of mineral oil in successful flash cooling of a protein crystal correctly point to the removal of excess liquid around the crystal to prevent external ice formation. Based on the physics of the well known Leidenfrost phenomenon, an additional role that mineral oil possibly plays in aiding vitrification is suggested: that of improving cooling rates for a protein crystal plunged into liquid nitrogen. The full potential of liquid heat transfer when using liquid nitrogen is not realized in conventional cooling techniques due to film boiling that occurs around larger protein crystals. However, a thin layer of an insulating material, such as a mineral oil, around the protein crystal can prevent this vapor film from forming by raising the Leidenfrost temperature. Surface cooling then occurs in the more efficient nucleate boiling regime where liquid nitrogen is in contact with the crystal throughout the quench. Using bare and coated thermocouples, the validity of a predictive Leidenfrost temperature equation for use in liquid-nitrogen plunge cooling of protein crystals is demonstrated.
Pressure acid leaching of nickel laterite ores: anin situdiffraction study of the mechanism and rate of reaction
Authors: Madsen Ian C. , Scarlett Nicola V. Y. , Whittington Barry I.
Published: November 2005

This paper outlines the use of an innovative system for thein situinvestigation of hydrothermal reactions by X-ray diffraction. The key features are the use of: (i) a purpose-built capillary reaction vessel which allows close emulation of the conditions present in mineral processing plants; (ii) MoKα radiation, to ensure that the X-ray beam penetrates through the capillary, and (iii) an Inel CPS120 position-sensitive detector, to enable simultaneous collection of a wide range (120° 2θ) of diffraction data. The pressure acid leaching (PAL) of nickel laterite ores is used to illustrate the capabilities of this system, with a particular focus on the PAL of saprolite in strong H2SO4at 493 K. Saprolitic ore, which largely consists of serpentine mineral phases [(Mg,Fe,Ni,Al)3(Si,Al)2O5(OH)4], undergoes a number of mineralogical changes during both the acid leaching reaction and subsequent cooling, thus making it difficult to examine accurately using traditional post-reactionex situtechniques. In particular, kieserite (MgSO4.H2O), which forms during leaching, has a negative temperature coefficient of solubility, causing it to dissolve on cooling. Thein situtechnique described in this paper allows the direct observation of kieserite formation during the saprolite PAL at 493 K and its dissolution upon cooling to ambient temperature.
Time-of-flight neutron transmission of mosaic crystals
Authors: Santisteban Javier R.
Published: November 2005

The energy-dispersive neutron transmission of mosaic crystals presents a series of dips in intensity as a result of reflection in the crystal planes. The positions of these dips can be exploited for the definition of the crystal orientation with a resolution of 1 min of arc. The widths of these dips depend on crystal orientation, on the reflection index, on the mosaicity, and on the incident-beam divergence. The capability of the technique to define the orientation and mosaicity of a Cu crystal has been assessed through time-of-flight experiments. A Cu monochromator has been plastically deformed by uniaxial tensionin situ, and the evolution of crystal orientation and mosaicity was tracked using the technique. Several crystal reflections at different locations of the sample were simultaneously studied during the experiment. A linear increase of mosaicity on deformation at an average rate of ∼5 min per percentage of shear strain was observed. The reorientation of the crystal as a result of the applied load showed variations across the specimen.
UV microscopy at 280 nm is effective in screening for the growth of protein microcrystals
Authors: Lunde Christopher S. , Rouhani Shahab , Remis Jonathan P. , Ruzin Steven E. , Ernst James A. , Glaeser Robert M.
Published: November 2005

This paper describes the relatively simple modification of a light microscope to operate with ultraviolet-based optics. Using a 280 nm UV illumination source, protein microcrystals are readily visualized in gels made from hydrated bilayers of phospholipids. Non-colored proteins stand out as clearly as colored proteins in this system, the imaging of which is based on UV absorption by tryptophan residues. In addition, protein crystals are easily distinguished from salt crystals. Artifacts from the lipid-based crystallization medium, which are frequently seen in brightfield microscopy, are greatly reduced when viewed in this UV-based microscope.
Microstructure characterization of nanocrystalline ZrSiO4synthesized by ball-milling and high-temperature annealing
Authors: Pradhan S. K. , Sinha M.
Published: November 2005

Microstructure characterization and phase transformation kinetics of a high-energy ball-milled and post-annealed stoichiometric (1:1 mol%)m-ZrO2and amorphous (a-) SiO2powder mixture have been investigated by Rietveld analysis of X-ray powder diffraction data. The experimental results reveal that the ball-milling of stoichiometric powder results in the formation ofc-ZrO2/t-PSZ phases only. The ZrSiO4phase was not found to form even after 30 h of milling. However, an almost stoichiometric ZrSiO4(commercially known as zircon) phase is found to form even when a sample that was ball-milled for 5 min was post-annealed at 1473 K for 1 h. It appears that the intermediatet-PSZ phase plays an important role in zircon phase formation. The content of ZrSiO4phase increases very sharply within 15 min of milling and remains almost unchanged after 30 h of ball-milling. It is also found that nanocrystalline zircon particles are almost free from lattice strain. Contamination by an α-Fe2O3phase from the milling media results in a colour gradient (white to reddish) in the nanocrystalline ZrSiO4phase.