bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparative study of the efficacy of AphthealTM in the management of recurrent minor aphthous ulceration
Authors: Murray Brenda , McGuinness Niall , Biagioni Paul , Hyland Paula , Lamey Philip‐John
Published: July 2005

Background:  Recurrent minor aphthous ulceration (RAU) is a common condition which is multifactorial in origin.

Methods:  This study, firstly, aimed to treat the prodromal stage of RAU with AphthealTM (5% amlexanox paste) to determine if ulcer development could be prevented. A second arm of the study investigated treatment of RAU with AphthealTM once ulceration had developed. Ulcer duration, ulcer size and associated pain were measured. Both groups of subjects had previously undergone a no‐treatment run‐in period to establish these parameters over an untreated episode of ulceration.

Results:  By day 3, only 35% of the prodromal group had developed an ulcer compared with 97% of the ulcer group (P < 0.001). In the treated ulcer group only 66% had an ulcer present by day 3. Treatment at the onset of prodromal symptoms reduced the maximum ulcer size score by 84% (P < 0.01), extent of ulceration by 88% (P < 0.01), maximum pain score by 69% (P < 0.01) and extent of pain by 85% (P < 0.01) compared to no treatment.

Conclusion:  Treatment with AphthealTM at the onset of prodromal RAU symptoms can prevent progression to ulcer development and significantly reduced symptoms if ulcers do develop.
Hypoxia‐inducible factor 1α in oral cancer
Authors: Brennan P. A. , Mackenzie N. , Quintero M.
Published: July 2005

Background:  Hypoxia is a common feature of many cancers. It contributes to local and systemic tumour progression as well as potentially compromising radiotherapy and chemotherapy. Hypoxia‐inducible factor 1α (HIF‐1α) is an essential component in changing the transcriptional response of tumours under hypoxia. It targets the transcription of over 60 genes involved in many aspects of cancer biology including cell survival, glucose metabolism, cell invasion and angiogenesis.

Methods:  In this review, we discuss the relevant literature on HIF‐1α with specific emphasis on oral cancer. We also present some of our preliminary data on HIF‐1α in oral cancer.

Results:  Although there are a few conflicting reports of its prognostic significance, over expression of HIF‐1α seems to play an adverse role in the malignant progression of head and neck cancer by facilitating the adaptation of cells to hypoxia as well as contributing to the invasive properties and angiogenesis in these tumours.

Conclusions:  HIF‐1α has an important role to play in pathophysiology of oral cancer, both under normal and hypoxic conditions. The pharmacological manipulation of HIF‐1α has marked effects on tumour growth, and it could prove to be an important target for drug therapy, both in oral cancer and in other hypoxia‐dependent disease states.
Comparative evaporimetry in man
Authors: Shah Jigna H. , Zhai Hongbo , Maibach Howard I.
Published: July 2005

Background/aims: Transepidermal water loss (TEWL) measurements are frequently used; the most widely utilized instrument has been the open chamber device. Traditional open‐chamber systems for measuring TEWL have limitations related to ambient and body‐induced airflows near the probe, probe size, measurement site and angles. Recent technology provides a portable and battery‐operated closed chamber Evaporimeter. It is presumably less affected by external factors such as room‐ or body‐induced airflows, breathing or heating of the probe by the operators' hand. This study compares this commercial instrument, a closed chamber device, with a traditional open‐chamber device.

Methods: Two open chamber devices and one closed chamber device were used in such a way that each instrument could simultaneously measure TEWL in an identical experimental environment with known relative humidity and temperature values. TEWL values of seven locations on healthy volunteers were measured simultaneously with each instrument in the same room according to the guidelines of the standardization group of the European society of contact dermatitis.

Results: For TEWL values of all forearm locations, there was no statistically significant difference between the mean values measured by all three instruments. For TEWL values of forehead locations, there was a significant difference between the mean values of open chamber device and closed chamber device. For the forehead, values from closed chamber device were higher than the open one.

Conclusion: The TEWL values measured by all instruments were constant with small standard deviations. These instruments appear sufficiently robust that a standardization group could document similarities and differences with in vitro models. We suspect that both techniques will continue to be used and that the closed method will find increasing use because of its ease of use.
Detection of asymmetric blotches (asymmetric structureless areas) in dermoscopy images of malignant melanoma using relative color
Authors: Stoecker William V. , Gupta Kapil , Stanley R. Joe , Moss Randy H. , Shrestha Bijaya
Published: July 2005

Background: Dermoscopy, also known as dermatoscopy or epiluminescence microscopy (ELM), is a non‐invasive, in vivo technique, which permits visualization of features of pigmented melanocytic neoplasms that are not discernable by examination with the naked eye. One prominent feature useful for melanoma detection in dermoscopy images is the asymmetric blotch (asymmetric structureless area).

Method: Using both relative and absolute colors, blotches are detected in this research automatically by using thresholds in the red and green color planes. Several blotch indices are computed, including the scaled distance between the largest blotch centroid and the lesion centroid, ratio of total blotch areas to lesion area, ratio of largest blotch area to lesion area, total number of blotches, size of largest blotch, and irregularity of largest blotch.

Results: The effectiveness of the absolute and relative color blotch features was examined for melanoma/benign lesion discrimination over a dermoscopy image set containing 165 melanomas (151 invasive melanomas and 14 melanomas in situ) and 347 benign lesions (124 nevocellular nevi without dysplasia and 223 dysplastic nevi) using a leave‐one‐out neural network approach. Receiver operating characteristic curve results are shown, highlighting the sensitivity and specificity of melanoma detection. Statistical analysis of the blotch features are also presented.

Conclusion: Neural network and statistical analysis showed that the blotch detection method was somewhat more effective using relative color than using absolute color. The relative‐color blotch detection method gave a diagnostic accuracy of about 77%.
Effect of hormonal replacement therapy on skin biophysical properties of menopausal women
Authors: Guinot Christiane , Malvy Denis , Ambroisine Laurence , Latreille Julie , Mauger Emmanuelle , Guéhenneux Sabine , Morizot Frédérique , Tschachler Erwin
Published: July 2005

Background/purpose: Hormonal replacement therapy (HRT) has been shown in the past to influence well being as well as several somatic features in menopausal women. The aim of this analysis was to study the effect of HRT on various biophysical properties of the skin of menopausal women. Two sub‐samples were built to test the effect (1) of ‘short‐term’ HRT in recently menopausal women (n=15), and (2) the effect of ‘medium‐ and long‐term’ HRT in menopausal women (n=78).

Methods: The analysis was performed on data from a study of 106 menopausal Caucasian women with apparent healthy skin. Self‐report data on the menopausal status, the duration of the menopause, whether or not HRT has been taken and the duration of the HRT were collected. A series of biophysical measurements on the cheek, the forehead and the inner forearm were performed under controlled environmental conditions. Twenty women were menopausal for less than 5 years (eight with HRT for at least 1 year and seven who had never taken HRT) and 86 women for at least 5 years (35 with HRT for at least 5 years and 43 who had never taken HRT). Analyses of covariance with adjustment for age were performed.

Results: With regard to skin colour, the measurements in women treated for at least 1 year were significantly higher for red intensity (a*) and lower for brightness (L*) on the forearm as compared with the non‐treated women. Furthermore, yellow intensity (b*) mean values were higher on the forehead and the forearm in women treated for at least 5 years compared with the non‐treated women. Concerning sebum casual level, the mean values were significantly higher on the forehead and the cheek in women treated for at least 5 years. Regarding skin surface parameters reflecting hydration, the mean values for capacitance on the forehead and the cheek were significantly higher in women treated for at least 5 years. Finally, as regards with skin relief, parameters mean values for amplitude and roughness on the forehead were significantly higher in women treated for at least 5 years.

Conclusion: The skin colour parameters showed a higher red intensity value in menopausal women who had been treated for at least 1 year. In menopausal women who had been treated for at least 5 years, the biophysical measurements were significantly higher for the parameters reflecting hydration and sebum secretion, which generally decrease after the menopause. These features were associated with higher values for the yellow intensity parameter and the skin relief parameters on the forehead. Our results support the subjective impression and the clinical evaluation concerning the impact of HRT on the development and the evolution of some skin properties after menopause.
Vasodilator Effect of Endothelin in Cutaneous Microcirculation of Heart Failure Patients
Authors: Andersson Sven E. , Edvinsson Marie‐Louise , Alving Kjell , Edvinsson Lars
Published: July 2005

Abstract: The heart failure syndrome is associated with a reduced vasodilatory capacity in cutaneous microvessels. The aim of this study was to investigate the hypothesis that an altered activity of the endothelin system contributes to this reduction. The skin blood flow was recorded by laser Doppler flowmetry in patients with congestive heart failure and in age‐ and gender‐matched controls without clinical signs of heart failure. The vessels were stimulated by iontophoretic administration of endothelin‐1. The involvement of the endothelinA receptor was studied by co‐administration of a specific antagonist; the role of the endothelinB receptor was studied by the administration of the selective agonist sarafotoxin 6c. The plasma levels of endothelin‐1, C‐reactive peptide and N‐terminal‐pro‐Brain Natriuretic Peptide were elevated in heart failure patients. Unexpected, endothelin‐1 induced an endothelinA mediated vasodilation. In the heart failure group the dilation was reduced to less than half as compared to control. The response to local warming was reduced in parallel indicating that the attenuation of the response in the heart failure group can be explained by the general decline in vascular reactivity. The response to endothelinB receptor stimulation did not differ between the groups. The reduction in endothelin‐1 responsiveness is paralleled by a general reduction in microvascular vasodilatory capacity, a phenomenon of increased vascular stiffness in the of heart failure subjects.
Involvement of Different Calcium Channels in the Depolarization‐Evoked Release of Noradrenaline from Sympathetic Neurones in Rabbit Carotid Artery*
Authors: Uhrenholt Torben R. , Nedergaard Ove A.
Published: July 2005

Abstract: The calcium channels coupled to noradrenaline release from sympathetic neurones in the rabbit isolated carotid artery were examined. Rings of carotid artery were preloaded with (‐)‐[3H]noradrenaline and the fractional 3H overflow evoked by electrical‐field stimulation was determined by liquid scintillation spectrometry. The N‐type Ca2+ channel blocking agent ω‐conotoxin GVIA (3×10−9‐6×10−8 M) reduced the stimulation‐evoked 3H overflow. The maximal inhibition was seen with 3×10−8 M. The maximal reduction was more marked at a low (2 Hz) stimulation frequency than at a high one (30 Hz). Mibefradil (10−6 M) irreversibly reduced the 3H overflow evoked by field stimulation (2 Hz). At 30 Hz, the reduction was more marked than at 2 Hz. Mibefradil (3×10−6–10−5 M) enhanced the passive 3H outflow. The reduction of the stimulation (30 Hz)‐evoked 3H overflow seen with ω‐conotoxin GVIA (3×10−8 M) was enhanced by mibefradil (10−6 M) and unaffected by nimodipine (10−5 M) and ω‐agatoxin IVA (10−8 M). We conclude that the stimulation‐evoked release of noradrenaline from sympathetic neurones in rabbit carotid artery at a low frequency (2 Hz) is mediated mainly by the N‐type calcium channels. At a high frequency (30 Hz), T‐type Ca2+ channels are also involved.
The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano  ‐electrical transducer channels
Authors: Marcotti Walter , Van Netten Sietse M. , Kros Corné J.
Published: July 2005

The most serious side‐effect of the widely used aminoglycoside antibiotics is irreversible intracellular damage to the auditory and vestibular hair cells of the inner ear. The mechanism of entry into the hair cells has not been unequivocally resolved. Here we report that extracellular dihydrostreptomycin not only blocks the mechano‐electrical transducer channels of mouse outer hair cells at negative membrane potentials, as previously shown, but also enters the cells through these channels, which are located in the cells' mechanosensory hair bundles. The voltage‐dependent blocking kinetics indicate an open‐channel block mechanism, which can be well described by a two barrier–one binding site model, quantifying the antibiotic's block of the channel as well as its permeation in terms of the associated rate constants. The results identify the open transducer channels as the main route for aminoglycoside entry. Intracellularly applied dihydrostreptomycin also blocks the transducer channels, but at positive membrane potentials. However, the potency of the block was two orders of magnitude lower than that due to extracellular dihydrostreptomycin. Extracellular Ca2+ increases the free energy of the barrier nearest the extracellular side and of the binding site for dihydrostreptomycin. This reduces both the entry of dihydrostreptomycin into the channel and the channel's affinity for the drug. In vivo, where the extracellular Ca2+ concentration in the endolymph surrounding the hair bundles is < 100 μm, we predict that some 9000 dihydrostreptomycin molecules per second enter each hair cell at therapeutic drug concentrations.
Long-Term Monitoring of Bacteria Undergoing Programmed Population Control in a Microchemostat
Authors: Balagaddé Frederick K. , You Lingchong , Hansen Carl L. , Arnold Frances H. , Quake Stephen R.
Published: July 2005

Using an active approach to preventing biofilm formation, we implemented a microfluidic bioreactor that enables long-term culture and monitoring of extremely small populations of bacteria with single-cell resolution. We used this device to observe the dynamics of
Escherichia coli
carrying a synthetic “population control” circuit that regulates cell density through a feedback mechanism based on quorum sensing. The microfluidic bioreactor enabled long-term monitoring of unnatural behavior programmed by the synthetic circuit, which included sustained oscillations in cell density and associated morphological changes, over hundreds of hours.
Atlantic Ocean Forcing of North American and European Summer Climate
Authors: Sutton Rowan T. , Hodson Daniel L. R.
Published: July 2005

Recent extreme events such as the devastating 2003 European summer heat wave raise important questions about the possible causes of any underlying trends, or low-frequency variations, in regional climates. Here, we present new evidence that basin-scale changes in the Atlantic Ocean, probably related to the thermohaline circulation, have been an important driver of multidecadal variations in the summertime climate of both North America and western Europe. Our findings advance understanding of past climate changes and also have implications for decadal climate predictions.
Genome of the Host-Cell Transforming Parasite
Theileria annulata
Compared with
T. parva
Authors: Pain Arnab , Renauld Hubert , Berriman Matthew , Murphy Lee , Yeats Corin A. , Weir William , Kerhornou Arnaud , Aslett Martin , Bishop Richard , Bouchier Christiane , Cochet Madeleine , Coulson Richard M. R. , Cronin Ann , de Villiers Etienne P. , Fraser Audrey , Fosker Nigel , Gardner Malcolm , Goble Arlette , Griffiths-Jones Sam , Harris David E. , Katzer Frank , Larke Natasha , Lord Angela , Maser Pascal , McKellar Sue , Mooney Paul , Morton Fraser , Nene Vishvanath , O'Neil Susan , Price Claire , Quail Michael A. , Rabbinowitsch Ester , Rawlings Neil D. , Rutter Simon , Saunders David , Seeger Kathy , Shah Trushar , Squares Robert , Squares Steven , Tivey Adrian , Walker Alan R. , Woodward John , Dobbelaere Dirk A. E. , Langsley Gordon , Rajandream Marie-Adele , McKeever Declan , Shiels Brian , Tait Andrew , Barrell Bart , Hall Neil
Published: July 2005

Theileria annulata
and
T. parva
are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of
T. annulata
and compared it with that of
T. parva
to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a
Theileria
-specific protein domain [frequently associated in
Theileria
(FAINT)] present in a large number of secreted proteins.
Genome Analysis Reveals Pili in Group B
Streptococcus
Authors: Lauer Peter , Rinaudo Cira D. , Soriani Marco , Margarit Immaculada , Maione Domenico , Rosini Roberto , Taddei Anna Rita , Mora Marirosa , Rappuoli Rino , Grandi Guido , Telford John L.
Published: July 2005

Pili are essential virulence factors in many Gram-negative bacteria; however, they have not been described in most important Gram-positive pathogens. While screening the sequence of multiple genomes of Group B
Streptococcus
, we identified protective antigens that formed high molecular weight polymers. Immunogold electron microscopy revealed that the structures have a pilus-like form. These large structures have gone unrecognized in decades of studies of Group B
Streptococcus
.
Variable Control of Ets-1 DNA Binding by Multiple Phosphates in an Unstructured Region
Authors: Pufall Miles A. , Lee Gregory M. , Nelson Mary L. , Kang Hyun-Seo , Velyvis Algirdas , Kay Lewis E. , McIntosh Lawrence P. , Graves Barbara J.
Published: July 2005

Cell signaling that culminates in posttranslational modifications directs protein activity. Here we report how multiple Ca
2+
-dependent phosphorylation sites within the transcription activator Ets-1 act additively to produce graded DNA binding affinity. Nuclear magnetic resonance spectroscopic analyses show that phosphorylation shifts Ets-1 from a dynamic conformation poised to bind DNA to a well-folded inhibited state. These phosphates lie in an unstructured flexible region that functions as the allosteric effector of autoinhibition. Variable phosphorylation thus serves as a “rheostat” for cell signaling to fine-tune transcription at the level of DNA binding.
A High-Pressure Structure in Curium Linked to Magnetism
Authors: Heathman S. , Haire R. G. , Le Bihan T. , Lindbaum A. , Idiri M. , Normile P. , Li S. , Ahuja R. , Johansson B. , Lander G. H.
Published: July 2005

Curium lies at the center of the actinide series and has a half-filled shell with seven 5
f
electrons spatially residing inside its radon core. As a function of pressure, curium exhibits five different crystallographic phases up to 100 gigapascals, of which all but one are also found in the preceding element, americium. We describe here a structure in curium, Cm III, with monoclinic symmetry, space group
C2/c
, found at intermediate pressures (between 37 and 56 gigapascals). Ab initio electronic structure calculations agree with the observed sequence of structures and establish that it is the spin polarization of curium's 5
f
electrons that stabilizes Cm III. The results reveal that curium is one of a few elements that has a lattice structure stabilized by magnetism.
Sulfolobus tengchongensis
Spindle-Shaped Virus STSV1: Virus-Host Interactions and Genomic Features
Authors: Xiang Xiaoyu , Chen Lanming , Huang Xiaoxing , Luo Yuanmin , She Qunxin , Huang Li
Published: July 2005

A virus infecting the hyperthermophilic archaeon
Sulfolobus tengchongensis
has been isolated from a field sample from Tengchong, China, and characterized. The virus, denoted STSV1 (
Sulfolobus tengchongensis
spindle-shaped virus 1), has the morphology of a spindle (230 by 107 nm) with a tail of variable length (68 nm on average) at one end and is the largest of the known spindle-shaped viruses. After infecting its host, the virus multiplied rapidly to high titers (>10
10
PFU/ml). Replication of the virus retarded host growth but did not cause lysis of the host cells. STSV1 did not integrate into the host chromosome and existed in a carrier state. The STSV1 DNA was modified in an unusual fashion, presumably by virally encoded modification systems. STSV1 harbors a double-stranded DNA genome of 75,294 bp, which shares no significant sequence similarity to those of fuselloviruses. The viral genome contains a total of 74 open reading frames (ORFs), among which 14 have a putative function. Five ORFs encode viral structural proteins, including a putative coat protein of high abundance. The products of the other nine ORFs are probably involved in polysaccharide biosynthesis, nucleotide metabolism, and DNA modification. The viral genome divides into two nearly equal halves of opposite gene orientation. This observation as well as a GC-skew analysis point to the presence of a putative viral origin of replication in the 1.4-kb intergenic region between ORF1 and ORF74. Both morphological and genomic features identify STSV1 as a novel virus infecting the genus
Sulfolobus
.
Stimulation of Hepatitis C Virus (HCV) Nonstructural Protein 3 (NS3) Helicase Activity by the NS3 Protease Domain and by HCV RNA-Dependent RNA Polymerase
Authors: Zhang Chen , Cai Zhaohui , Kim Young-Chan , Kumar Ranjith , Yuan Fenghua , Shi Pei-Yong , Kao Cheng , Luo Guangxiang
Published: July 2005

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione
S
-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.
Foamy Virus Bet Proteins Function as Novel Inhibitors of the APOBEC3 Family of Innate Antiretroviral Defense Factors
Authors: Russell Rebecca A. , Wiegand Heather L. , Moore Michael D. , Schäfer Alexandra , McClure Myra O. , Cullen Bryan R.
Published: July 2005

Foamy viruses are a family of complex retroviruses that establish common, productive infections in a wide range of nonhuman primates. In contrast, humans appear nonpermissive for foamy virus replication, although zoonotic infections do occur. Here we have analyzed the ability of primate and mouse APOBEC3G proteins to inhibit the infectivity of primate foamy virus (PFV) virions produced in their presence. We demonstrate that several APOBEC3 proteins can potently inhibit the infectivity of a PFV-based viral vector. This inhibition correlated with the packaging of inhibitory APOBEC3 proteins into PFV virions, due to a specific PFV Gag/APOBEC3 interaction, and resulted in the G to A hypermutation of PFV reverse transcripts. While inhibition of PFV virion infectivity by primate APOBEC3 proteins was largely relieved by coexpression of the PFV Bet protein, a cytoplasmic auxiliary protein of previously uncertain function, Bet failed to relieve inhibition caused by murine APOBEC3. PFV Bet bound to human, but not mouse, APOBEC3 proteins in coexpressing cells, and this binding correlated with the specific inhibition of their incorporation into PFV virions. Of note, both PFV Bet and a second Bet protein, derived from an African green monkey foamy virus, rescued the infectivity of Vif-deficient human immunodeficiency virus type 1 (HIV-1) virions produced in the presence of African green monkey APOBEC3G and blocked the incorporation of this host factor into HIV-1 virion particles. However, neither foamy virus Bet protein reduced APOBEC3 protein expression levels in virion producer cells. While these data identify the foamy virus Bet protein as a functional ortholog of the HIV-1 Vif auxiliary protein, they also indicate that Vif and Bet block APOBEC3 protein function by distinct mechanisms.
Activation of the N-Ras–PI3K–Akt-mTOR Pathway by Hepatitis C Virus: Control of Cell Survival and Viral Replication
Authors: Mannová Petra , Beretta Laura
Published: July 2005

The hepatitis C virus (HCV) replication complex is localized within detergent-resistant membranes or lipid rafts. We analyzed the protein contents of detergent-resistant fractions isolated from Huh7 cells expressing a self-replicating full-length HCV-1b genome. Using two-dimensional gel electrophoresis followed by mass spectrometry, we identified N-Ras as one of the proteins in which expression was increased in the detergent-resistant fractions from HCV genomic replicon clones compared to control cells. N-Ras is an activator of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We found that the activities of PI3K and Akt, as well as the activity of their downstream target, mTOR, in the HCV-replicating cells were increased. Both PI3K-Akt- and mTOR-dependent pathways have been shown to promote cell survival. In agreement with this, HCV replicon cells were resistant to serum starvation-induced apoptosis. We also characterized the role of this pathway in HCV replication. Reduction of N-Ras expression by transfection of N-Ras small interfering RNA (siRNA) resulted in increased replication of HCV. We observed a similar increase in HCV replication in cells treated with the PI3K inhibitor LY294002 and in cells transfected with mTOR siRNA. Taken together, these data suggest that increased N-Ras levels in subcellular sites of HCV replication and stimulation of the prosurvival PI3K-Akt pathway and mTOR by HCV not only protect cells against apoptosis but also contribute to the maintenance of steady-state levels of HCV replication. These effects may contribute to the establishment of persistent infection by HCV.
RING-H2 Protein WSSV249 from White Spot Syndrome Virus Sequesters a Shrimp Ubiquitin-Conjugating Enzyme, PvUbc, for Viral Pathogenesis
Authors: Wang Zhilong , Chua Hui Kheng , Gusti Ayu A. R. A. , He Fang , Fenner Beau , Manopo Ivanus , Wang Hai , Kwang Jimmy
Published: July 2005

Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of
WSSV249
and
PvUbc
increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that
WSSV249
and
PvUbc
display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.
Involvement of Nuclear Export in Human Papillomavirus Type 18 E6-Mediated Ubiquitination and Degradation of p53
Authors: Stewart Deborah , Ghosh Anirban , Matlashewski Greg
Published: July 2005

The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes.
Influenza Virus Infection Increases p53 Activity: Role of p53 in Cell Death and Viral Replication
Authors: Turpin Elizabeth , Luke Kimberly , Jones Jeremy , Tumpey Terrence , Konan Kouacou , Schultz-Cherry Stacey
Published: July 2005

The induction of apoptotic cell death is a hallmark of influenza virus infection. Although a variety of cellular and viral proteins have been implicated in this process, to date no conserved cellular pathway has been identified. In this study, we report that the tumor suppressor protein p53 is essential for the induction of cell death in influenza virus-infected cells. In primary human lung cells, influenza virus increased p53 protein levels. This was also noted in the human lung cell line A549, along with the up-regulation of p53-dependent gene transcription. Reduction of p53 activity in A549 cells inhibited influenza virus-induced cell death as measured by trypan blue exclusion and caspase activity. These findings were not cell type specific. Influenza virus-induced cell death was absent in mouse embryo fibroblasts isolated from p53 knockout mice, which was not the case in wild-type mouse embryo fibroblasts, suggesting that p53 is a common cellular pathway leading to influenza virus-induced cell death. Surprisingly, inhibiting p53 activity led to elevated virus replication. Mechanistically, this may be due to the decrease in interferon signaling in p53-deficient cells, suggesting that functional p53 is involved in the interferon response to influenza infection. To our knowledge, these are the first studies demonstrating that p53 is involved in influenza virus-induced cell death and that inhibiting p53 leads to increased viral titers, potentially through modulation of the interferon response.
Herpes Simplex Virus Type 1 Capsids Transit by the
trans
-Golgi Network, Where Viral Glycoproteins Accumulate Independently of Capsid Egress
Authors: Turcotte Sophie , Letellier Josée , Lippé Roger
Published: July 2005

Egress of herpes capsids from the nucleus to the plasma membrane is a complex multistep transport event that is poorly understood. The current model proposes an initial envelopment at the inner nuclear membrane of capsids newly assembled in the nucleus. The capsids are then released in cytosol by fusion with the outer nuclear membrane. They are finally reenveloped at a downstream organelle before traveling to the plasma membrane for their extracellular release. Although the
trans
-Golgi network (TGN) is often cited as a potential site of reenvelopment, other organelles have also been proposed, including the Golgi, endoplasmic reticulum-Golgi intermediate compartment, aggresomes, tegusomes, and early or late endosomes. To clarify this important issue, we followed herpes simplex virus type 1 egress by immunofluorescence under conditions that slowed intracellular transport and promoted the accumulation of the otherwise transient reenvelopment intermediate. The data show that the capsids transit by the TGN and point to this compartment as the main reenvelopment site, although a contribution by endosomes cannot formally be excluded. Given that viral glycoproteins are expected to accumulate where capsids acquire their envelope, we examined this prediction and found that all tested could indeed be detected at the TGN. Moreover, this accumulation occurred independently of capsid egress. Surprisingly, capsids were often found immediately adjacent to the viral glycoproteins at the TGN.
A Specific Region of 37 Amino Acid Residues in the SPRY (B30.2) Domain of African Green Monkey TRIM5α Determines Species-Specific Restriction of Simian Immunodeficiency Virus SIVmac Infection
Authors: Nakayama Emi E. , Miyoshi Hiroyuki , Nagai Yoshiyuki , Shioda Tatsuo
Published: July 2005

Human immunodeficiency virus type 1 (HIV-1) efficiently enters cells of Old World monkeys but encounters a block before reverse transcription. This restriction is mediated by a dominant repressive factor. Recently, a member of the tripartite motif (TRIM) family proteins, TRIM5α, was identified as a blocking factor in a rhesus macaque cDNA library. Among Old World monkey cell lines, the African green monkey kidney cell line CV1 is highly resistant to not only HIV-1 but also simian immunodeficiency virus SIVmac infection. We analyzed TRIM5α of CV1 cells and HSC-F cells, a T-cell line from a cynomolgus monkey, and found that both CV1- and HSC-F-TRIM5αs could inhibit CD4-dependent HIV-1 infection, as well as vesicular stomatitis virus glycoprotein-mediated infection. CV1-TRIM5α could also inhibit SIVmac infection, whereas HSC-F-TRIM5α could not. In the SPRY (B30.2) domain of CV1-TRIM5α, there was a 20-amino-acid duplication that was not present in HSC-F-TRIM5α. A chimeric TRIM5α containing 37 amino acid residues from CV1-TRIM5α, which spanned the 20-amino-acid duplication, in the background of HSC-F-TRIM5α fully gained the ability to inhibit SIVmac infection. Conversely, the mutant CV1-TRIM5α lacking the 20-amino-acid duplication completely lost the ability to restrict SIVmac infection. These findings clearly indicated that a specific region of 37 amino acid residues in the SPRY domain of CV1-TRIM5α contained a determinant of species-specific restriction of SIVmac.
High Incidence of Scrapie Induced by Repeated Injections of Subinfectious Prion Doses
Authors: Jacquemot Catherine , Cuche Céline , Dormont Dominique , Lazarini Françoise
Published: July 2005

To clarify the mechanisms leading to the development of Creutzfeldt-Jakob disease in some recipients of pituitary-derived human growth hormone (hGH), we investigated the effects of repeated injections of low prion doses in mice. The injections were performed, as in hGH-treated children, by a peripheral route at short intervals and for an extended period. Twelve groups of 24 mice were intraperitoneally inoculated one, two, or five times per week for 200 days with 2 × 10
−5
to 2 × 10
−8
dilutions of brain homogenate containing the mouse-adapted C506M3 scrapie strain. Sixteen control mice were injected once a week for 200 days with a 2 × 10
−4
dilution of normal brain homogenate. Of mice injected in a single challenge with a scrapie inoculum of a 2 × 10
−4
, 2 × 10
−5
, or 2 × 10
−6
dilution, 2/10, 1/10, and 0/10 animals developed scrapie, respectively. Control mice remained healthy. One hundred thirty-five of 135 mice injected with repeated prion doses of a 2 × 10
−5
or 2 × 10
−6
dilution succumbed to scrapie. Of mice injected with repeated scrapie doses of a 2 × 10
−7
or 2 × 10
−8
dilution, 52/59 and 38/67 animals died of scrapie, respectively. A high incidence of scrapie was observed in mice receiving repeated doses at low infectivity, whereas there was no disease in mice that were injected once with the same doses. Repeated injections of low prion doses thus constitute a risk for development of prion disease even if the same total dose inoculated in a single challenge does not induce the disease.
In Vivo Self-Interaction of Nodavirus RNA Replicase Protein A Revealed by Fluorescence Resonance Energy Transfer
Authors: Dye Billy T. , Miller David J. , Ahlquist Paul
Published: July 2005

Flock house virus (FHV) is the best-characterized member of the
Nodaviridae
, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.
Respiratory Syncytial Virus Influences NF-κB-Dependent Gene Expression through a Novel Pathway Involving MAP3K14/NIK Expression and Nuclear Complex Formation with NF-κB2
Authors: Choudhary Sanjeev , Boldogh Steve , Garofalo Roberto , Jamaluddin Mohammad , Brasier Allan R.
Published: July 2005

A member of the
Paramyxoviridae
family of RNA viruses, respiratory syncytial virus (RSV), is a leading cause of epidemic respiratory tract infection in children. In children, RSV primarily replicates in the airway mucosa, a process that alters epithelial cell chemokine expression, thereby inducing airway inflammation. We investigated the role of the mitogen-activated protein kinase kinase kinase 14/NF-κB-inducing kinase (NIK) in the activation of NF-κB-dependent genes in alveolus-like A549 cells. RSV infection induces a time dependent increase of NIK mRNA and protein expression that peaks 12 to 24 h after viral exposure. Immunoprecipitation kinase assays indicate that NIK kinase activity is activated even more rapidly (within 6 h of RSV adsorption) associated with an endogenous ∼50-kDa NF-κB2 substrate. Because NIK associates with IKKα to mediate processing of the 100-kDa NF-κB2 precursor into its 52-kDa DNA binding isoform (“p52”), the effects of RSV on NIK complex formation with IKKα and NF-κB2 were determined by coimmunoprecipitation assay. We find that NIK, IKKα, and both 100 kDa- and 52-kDa NF-κB2 isoforms strongly complex 15 h after exposure to RSV at times subsequent to NIK kinase activation. Western immunoblot and microaffinity DNA pull-down assays showed a parallel increase in nuclear translocation and DNA binding of the NF-κB2-Rel B complex. Interestingly, we make the novel observations that NIK also transiently translocates into the nucleus complexed with 52-kDa NF-κB2. Small interfering RNA-mediated NIK “knock-down” blocked RSV-inducible 52-kDa NF-κB2 processing and interfered with the early activation of a subset of NF-κB-dependent genes, indicating the importance of this activation pathway in the genomic NF-κB response to RSV. Together, these data indicate that RSV infection rapidly activates the noncanonical NF-κB activation pathway prior to the more potent canonical pathway activation. This appears to be through a novel mechanism involving induction of NIK kinase activity, expression, and nuclear translocation of a ternary complex with IKKα and processed NF-κB2.
Novel Human Monoclonal Antibody Combination Effectively Neutralizing Natural Rabies Virus Variants and Individual In Vitro Escape Mutants
Authors: Bakker Alexander B. H. , Marissen Wilfred E. , Kramer R. Arjen , Rice Amy B. , Weldon William C. , Niezgoda Michael , Hanlon Cathleen A. , Thijsse Sandra , Backus Harold H. J. , de Kruif John , Dietzschold Bernhard , Rupprecht Charles E. , Goudsmit Jaap
Published: July 2005

The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity, breadth of neutralization, and coverage of natural rabies virus (RV) isolates and in vitro escape mutants were set for each individual antibody, and the complementarities of the two MAbs were defined at the onset. First, we identified and characterized one human MAb (CR57) with high in vitro and in vivo neutralizing potency and a broad neutralization spectrum. The linear antibody binding site was mapped on the RV glycoprotein as antigenic site I by characterizing CR57 escape mutants. Secondly, we selected using phage display a complementing antibody (CR4098) that recognized a distinct, nonoverlapping epitope (antigenic site III), showed similar neutralizing potency and breadth as CR57, and neutralized CR57 escape mutants. Reciprocally, CR57 neutralized RV variants escaping CR4098. Analysis of glycoprotein sequences of natural RV isolates revealed that the majority of strains contain both intact epitopes, and the few remaining strains contain at least one of the two. In vitro exposure of RV to the combination of CR57 and CR4098 yielded no escape mutants. In conclusion, a novel combination of human MAbs was discovered suitable to replace RIG.
Contributions of the Viral Genetic Background and a Single Amino Acid Substitution in an Immunodominant CD8+T-Cell Epitope to Murine Coronavirus Neurovirulence
Authors: MacNamara Katherine C. , Chua Ming Ming , Phillips Joanna J. , Weiss Susan R.
Published: July 2005

The immunodominant CD8+T-cell epitope of a highly neurovirulent strain of mouse hepatitis virus (MHV), JHM, is thought to be essential for protection against virus persistence within the central nervous system. To test whether abrogation of this H-2Db-restricted epitope, located within the spike glycoprotein at residues S510 to 518 (S510), resulted in delayed virus clearance and/or virus persistence we selected isogenic recombinants which express either the wild-type JHM spike protein (RJHM) or spike containing the N514S mutation (RJHMN514S), which abrogates the response to S510. In contrast to observations in suckling mice in which viruses encoding inactivating mutations within the S510 epitope (epitope escape mutants) were associated with persistent virus and increased neurovirulence (Pewe et al., J Virol. 72:5912-5918, 1998), RJHMN514Swas not more virulent than the parental, RJHM, in 4-week-old C57BL/6 (H-2b) mice after intracranial injection. Recombinant viruses expressing the JHM spike, wild type or encoding the N514S substitution, were also selected in which background genes were derived from the neuroattenuated A59 strain of MHV. Whereas recombinants expressing the wild-type JHM spike (SJHM/RA59) were highly neurovirulent, A59 recombinants containing the N514S mutation (SJHMN514S/RA59) were attenuated, replicated less efficiently, and exhibited reduced virus spread in the brain at 5 days postinfection (peak of infectious virus titers in the central nervous system) compared to parental virus encoding wild-type spike. Virulence assays in BALB/c mice (H-2d), which do not recognize the S510 epitope, revealed that attenuation of the epitope escape mutants was not due to the loss of a pathogenic immune response directed against the S510 epitope. Thus, an intact immunodominant S510 epitope is not essential for virus clearance from the CNS, the S510 inactivating mutation results in decreased virulence in weanling mice but not in suckling mice, suggesting that specific host conditions are required for epitope escape mutants to display increased virulence, and the N514S mutation causes increased attenuation in the context of A59 background genes, demonstrating that genes other than that for the spike are also important in determining neurovirulence.
Suppressors of a Host Range Mutation in the Rabbitpox Virus Serpin SPI-1 Map to Proteins Essential for Viral DNA Replication
Authors: Luttge Benjamin G. , Moyer Richard W.
Published: July 2005

The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (
hr
) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1
hr
mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted
hr
phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R
hr
mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism.