bluuebunny/crossref_metadata_2025 huggingface dataset:
Intramyocellular lipids and insulin resistance
Authors: Machann Jürgen , Häring Hans , Schick Fritz , Stumvoll Michael
Published: May 2004

Lipids are stored not only in adipocytes but also ‘ectopically’ in tissues such as muscle, liver, beta cells and others. From a metabolic perspective, intramyocellular lipids (IMCLs) have recently become a focus of interest. This review summarizes history, measurement techniques and interpretation of muscle lipid data. Problems in biopsies with the separation of those metabolically active lipid droplets in the cytoplasm of myocytes from further lipids in adipocytes are discussed as well as considerations important for analysis of correlations between IMCL content and insulin sensitivity under various circumstances. The relatively new approach to non‐invasive assessment of the IMCL content by magnetic resonance spectroscopy (MRS) is described in detail and exemplary spectra from different skeletal muscle types in humans are presented. The MRS technique allows human examinations of large cohorts for a detailed assessment of the interactions among metabolic parameters such as age, measures of adiposity, hormonal and ethnic factors and insulin resistance. IMCLs are generally positively correlated with measures of obesity and negatively with insulin sensitivity. Paradoxically, physical fitness (maximal aerobic capacity) increases both IMCL content and insulin sensitivity and therefore has to be taken into account as a confounding factor. Intervention studies with MRS further allowed to elucidate the regulation of IMCL. Molecular mechanisms and potential genetic factors on IMCL regulation are discussed as well as possible mechanisms of current treatment strategies for improving insulin sensitivity.
Plasma sex hormone‐binding globulin rather than corticosteroid‐binding globulin is a marker of insulin resistance in obese adult males
Authors: Lewis J. G. , Shand B. I. , Elder P. A. , Scott R. S.
Published: May 2004

Aim:  Plasma levels of corticosteroid‐binding globulin (CBG) and sex hormone‐binding globulin (SHBG) may be regulated by insulin. The aim of this study was to test the hypothesis that these steroid‐binding proteins are markers of insulin resistance and obesity in adult patients with the metabolic syndrome.

Methods:  Fasting blood samples were obtained from 108 male and 88 female overweight adult patients who had varying degrees of dyslipidaemia, adiposity and insulin resistance. We measured plasma levels of SHBG and CBG and investigated their correlation with insulin resistance [homeostasis model assessment (HOMA) % sensitivity] and anthropometric markers of adiposity.

Results:  In male patients, plasma SHBG correlated positively with HOMA (% sensitivity) and negatively with anthropometric measurements, including body mass index, waist circumference (cm) and percentage body fat. There was no correlation with CBG and any other parameter in the male patients. The female patients were treated as two groups, those not using oral contraceptives or hormone replacement therapy (n = 67) and those taking steroid medications (n = 21). Female patients using steroid medications had significantly higher SHBG levels but neither group showed any correlation between SHBG, insulin resistance and adiposity. Correlation studies of CBG with other parameters in the female subgroups did not reach statistical significance.

Conclusions:  We conclude that plasma SHBG is another surrogate marker for insulin resistance in obese males but not in obese females. It also appears that plasma CBG is not a useful marker of insulin resistance in patients with the metabolic syndrome.
Increased expression of resistin and tumour necrosis factor‐α in pig adipose tissue as well as effect of feeding treatment on resistin and cAMP pathway
Authors: Chen X. D. , Lei T. , Xia T. , Gan L. , Yang Z. Q.
Published: May 2004

Aim:  Resistin, a novel hormone, is expressed in mouse, rat and human adipose tissue. Its resistance to insulin is controversial. We hypothesized that resistin has the similar roles like those of tumour necrosis factor‐α (TNF‐α), a putative mediator of insulin resistance, and they are in common involved in regulating lipid metabolism in vivo.

Methods:  We demonstrated TNF‐α and resistin expression in different growth‐stage adipose tissue of genetically lean and obese pigs using semiquantitative reverse transcriptase‐polymerase chain reaction and Western blotting analysis. Moreover, we analysed the variations of resistin and some parameters associated with lipolysis pathway after body‐weight reduction in feeding‐restricted obese pigs. In addition, we localized resistin expression in the cell types from pig adipose tissue.

Results:  Overall, the expression of TNF‐α and resistin in obese pigs was statistically higher than their lean counterparts, although this difference in resistin mRNA expression tailed off with age. In individual pigs, their levels increased progressively with age and degree of obesity. After feeding restriction, resistin expression was gradually decreased. Simultaneously, feeding restriction also resulted in a significant decrease of serum triglyceride and plasma cAMP, free fatty acids and insulin as well as fat adenyl cyclase and cAMP. In addition, in isolated pig adipocytes, our data showed that resistin was localized not only to adipocytes but also to other unidentified stromal vascular cells.

Conclusions:  TNF‐α and resistin are increased in obesity. Weight loss results in the decrease of resistin and lipolysis‐associated parameters, suggesting the possible association between resistin and lipolysis pathway. Cell localization of resistin expression implies the possibility of other biological actions of resistin.
Physicochemical factors affecting the rapid bactericidal efficacy of the phenolic antibacterial triclosan
Authors: Taylor T. J. , Seitz E. P. , Fox P. , Fischler G. E. , Fuls J. L. , Weidner P. L.
Published: May 2004

Synopsis

The antimicrobial activity of triclosan (TCS; 2,4,4′‐trichloro‐2′‐hydroxydiphenyl ether) in aqueous solutions is shown to directly depend upon two key physicochemical parameters: % saturation and saturation solubility. Saturated solutions of TCS in water, water–propylene glycol (PG) mixtures, and aqueous surfactant systems are shown to effect rapid, potent bacterial reductions (e.g. >4log10 reduction of Staphylococcus aureus in 15 s contact time in a time kill suspension test). In surfactant solutions, increasing the surfactant: TCS ratio causes a decrease in antibacterial efficacy, consistent with a model for micellar solubilization where the micelle binding constant, K (=X/cw) increases with decreasing TCS concentration in the micelles (X), resulting in decreased concentration of bioavailable TCS in the water (continuous) phase (cw). The rapid and potent reductions of bacteria reported here were surprising and support the existence of a non‐specific mode of action for TCS, such as gross membrane disruption, in addition to the specific modes of action reported by others.
Assessment of visibility of facial wrinkle reduction by various types of observers
Authors: Westerink J. H. D. M.
Published: May 2004

The prime objective of many facial wrinkle‐reduction treatments is to achieve visible improvement. The visibility of before/after treatment differences is therefore often part of the efficacy assessment. This paper investigates whether the background knowledge of the people acting as observers in such assessments has an impact on the results, e.g., the subjects themselves are familiar with their faces, skin professionals have much experience in judging skin quality, and thus both might be more sensitive to small changes.

In a clinical study, 44 female subjects were regularly treated during a period of 12 weeks with one of three wrinkle‐reduction treatments: K, L and M (placebo). Photographs were taken before treatment and after 6 and 12 weeks. Three different types of observers judged the photographs:

Observer type I: Twenty‐four lay observers were given the 0‐ and 6‐week and the 0‐ and 12‐week pairs of photographs of all subjects to indicate the one with the least wrinkles in a two‐alternative forced‐choice procedure;

Observer type II: The subjects themselves were given the 0‐ and 6‐week and the 0‐ and 12‐week pairs of their own photographs (eight replications) to indicate the photograph with the least wrinkles (two‐alternative forced‐choice);

Observer type III: A trained panel of skin professionals (n = 3) each gave a 9‐point Fitzpatrick wrinkle‐severity score for all individual 0‐ and 12‐week photographs.

It was found that the lay observers perceived the same differences as the subjects themselves: significant improvements after 12 weeks for treatment K (P < 0.0005 and P = 0.005, respectively). No visible effects were seen for treatments L and M, but, most importantly, a significant difference between treatments K and M (placebo) (P = 0.015 and P = 0.01 for independent observers and the subjects themselves, respectively). The trained panel also identified this difference between K and M (P = 0.013) in favor of K, but here it was due to a significant deterioration over time of the ‘placebo‐treated’ wrinkles (M, P = 0.03).

Thus, in conclusion, no indications were found that extra knowledge – in the form of familiarity with the own face or in the form of professional training – results in the identification of more treatments that show significantly visible wrinkle reduction.
A Nineteenth‐century Revival: Romanesque Revisited
Authors: Bullen J. B.
Published: May 2004

Book reviewed:

Kathleen Curran, The Romanesque Revival: Religion, Politics, and Transnational Exchange
Love, Actually…
Authors: Shaw‐Miller Lindsey
Published: May 2004

Books reviewed:

Klaske Muizelaar and Derek Phillips, Picturing Men and Women in the Dutch Golden Age

H. Rodney Nevitt, Jr., Art and the Culture of Love in Seventeenth‐century Holland
Different cytokine levels in thrombolysis patients as predictors for clinical outcome
Authors: Mazzotta G. , Sarchielli P. , Caso V. , Paciaroni M. , Floridi A. , Floridi A. , Gallai V.
Published: May 2004

Thrombolytic therapy not always improves clinical outcome in ischemic stroke patients. This could cause lymphomonocyte accumulation in the infarcted brain area. These produce an excessive amount of proinflammatory cytokines, such as IL‐1beta, IL‐6 and TNF‐alfa. The aim of our study was to determine ILs levels in fibrinolytic therapy treated patients, compared with healthy controls and to evaluate if the varying levels can predictors of neurological outcome. Eighteen patients underwent thrombolytic treatment with t‐PA within 3 h. Plasma levels of IL‐1beta, IL‐6, TNF‐alfa and IL‐10 were determined by ELISA method before and within 24 h after t‐PA infusion and compared with controls. Significantly higher levels of IL‐1beta and Il‐6 emerged in stroke patients before treatment compared with the control group (P < 0.05 and 0.04, respectively). Slightly higher plasma levels of TNF‐alfa and lower plasma levels of IL‐10 were also found at base line in stroke patients. After thrombolytic treatment no significant variations were observed in the levels of TNF‐alfa and IL‐6, whereas a trend toward lower values for IL‐1beta and higher levels for IL‐10 was observed. Positive correlations among the values of IL‐6, TNF‐alfa and National Institute of Health Stroke Scale (NIHSS) at discharges were observed. A similar correlation with modified Rankin scale score at 3 month was found. Pre‐treatment cytokine status seems to influence pre‐and long‐term clinical outcome. Therefore an investigation into the possible predictor of cytokines seem worthy.
Primary headaches in an outpatient neurology headache clinic in East Hungary
Authors: Gesztelyi G. , Bereczki D.
Published: May 2004

The majority of patients with primary headache do not seek medical help. In the present study we analyse the characteristics of those patients with primary headaches who consulted a single headache outpatient service in East Hungary. All consecutive patients with primary headaches referred to the headache center between February 2002 and April 2003 were asked to fill in a questionnaire on headache characteristics and the migraine disability score (MIDAS) form. A total of 327 patients had some form of primary headache: 42% migraine, 31% tension‐type headache, 1% cluster headache and 26% had combination headache. Of the patients 95% were younger than 60 years of age. Only 16% of the patients were male. Chronic daily headache was found in 41% of patients. Although headache was the least severe in tension‐type headache, reported disability occurred on much more days in this group than in migraine patients. The majority of patients with primary headache were in the active age group. Patients with tension‐type headache need more attention, as disability is more severe in this patient group than generally assumed. More attention to the headaches of men and the elderly could improve the quality of life in these groups of the Hungarian population.
A controlled prospective inception cohort study on the post‐concussion syndrome outside the medicolegal context
Authors: Mickevičiene D. , Schrader H. , Obelieniene D. , Surkiene D. , Kunickas R. , Stovner L. J. , Sand T.
Published: May 2004

In an earlier historical cohort study on the post‐concussion syndrome (PCS) in Lithuania, a country in which there are few confounding factors, the validity of this condition as a disease entity could not be confirmed. In order to register the post‐traumatic symptoms, the influence of sociodemographic factors, and the effect of expectation on these symptoms more reliably, we performed a controlled prospective study. Three hundred subjects with concussion were followed up with repeated questionnaires for 1 year. For each study subject, a sex‐ and age‐matched control person with minor non‐head injury was identified. These controls received similar questionnaires. Headache both after 3 months and after 1 year did not differ significantly between the head‐injured participants and the non‐head‐injured controls. Several other symptoms attributed to PCS did not differ significantly between the groups after 3 months. After 1 year the vast majority of symptoms did not differ significantly. Exceptions were slightly significant differences concerning memory problems, concentration problems, dizziness and tiredness. These differences were insignificant when analysing symptoms in unmarried and/or people with lower education, separately. No relationship between cognitive dysfunction and the severity of concussion was found. Although the possibility of a mild organic brain injury cannot be completely ruled out, our results cast doubt on the validity of PCS as a useful clinical entity, at least for head injuries with loss of consciousness for <15 min. Sociodemographic factors and expectation seem to influence reporting of symptoms after concussion.
On Probation: Pickled and Nothing to Say
Authors: Mantle Greg , Moore Stephen
Published: May 2004

Abstract: The probation service is at risk of serious harm from the discourses of toughness and cure. People subject to community rehabilitation orders are categorised first and foremost as ‘offenders’ by probation officers, their managers and governors. Not surprisingly, people on probation are increasingly viewed as bad or as misfits, either way they have nothing intrinsically valuable to contribute to their supervision nor to the work of the probation service more generally. As a counter to this exclusion and disenfranchisement, two replacement discourses are commended: first, the language of userism/consumerism; and, second, an integration of strain and rational choice crime theories, as an alternative to the ‘psychic prisons’ of conservatism and individual positivism fast enveloping the service.
The Provenance of Red Figure Vases From Locri Epizephiri (Southern Italy): New Evidence by Chemical Analysis
Authors: Mirti P. , Gulmini M. , Pace M. , Elia D.
Published: May 2004

Thirty‐nine samples of red figure pottery found at Locri Epizephiri were analysed for 18 chemical elements by inductively coupled plasma emission spectroscopy. The finds, dated from the end of the sixth to the fourth century bc, were assigned to Attic, Sicilian and Locrian workshops due to their style of execution. Prior to facing the archaeological questions, possible biases on sample classification due to variable firing conditions, or to alteration during burial, were investigated: the results supported the opportunity of a refiring pre‐treatment. Multivariate statistical treatment of the analytical data confirms the separation between Attic and non‐Attic samples; comparison with the composition of black gloss pottery suggests that all the non‐Attic red figure vases could be the output of Locrian workshops.
Isothermal Time‐Series Determination of the Rate of Diffusion of Water in Pachuca Obsidian*
Authors: Anovitz L. M. , Elam J. M. , Riciputi L. R. , Cole D. R.
Published: May 2004

Friedman and Smith's (1960) article introducing an exciting, potentially precise and inexpensive method of dating obsidian artefacts has thus far failed to reach its potential. Numerous efforts to refine, improve and even redevelop the method since that time have similarly failed to achieve the original promise. Only within the last eight years have significant improvements been made, due to both improved analytical techniques and a better understanding of the hydration process. However, most of our mechanistic understanding of the interaction of water with rhyolitic glass is based on experiments performed on melts and glasses at temperatures above their glass transitions, conditions inappropriate for investigation of near‐surface environmental conditions. Unfortunately, studies detailing the temporal evolution of the diffusion profile at low temperatures are rare, and few useful data are available on the low‐temperature diffusive hydration of silicate glasses.

This paper presents data on the experimental hydration of obsidian from the Pachuca source (a.k.a. Sierra de las Navajas, Basin of Mexico) at 75°C for times ranging from 3 to 562 days, and compares these results with data for samples obtained from a stratigraphic excavation of the Chalco site in the Basin of Mexico. Samples have been analysed using secondary ion mass spectrometry (SIMS) to provide concentration/depth data. While 75°C is still significantly above the temperatures at which archaeological obsidians hydrate, it is well below the glass transition temperature (approx. 400°C) and thus processes are likely to be similar to those that occur in nature, but fast enough to be observed over a laboratory timescale. The results demonstrate that a simple square‐root‐of‐time model of the evolution of the diffusion profile is not adequate to describe the diffusion process, as measured diffusion profiles exhibit the effects of concentration‐ and time‐dependent, non‐Fickian diffusion. With progressive hydration, characteristic diffusion coefficients first decrease, then increase with time. Surface concentration increases with time, but an intermediate plateau is observed in its time evolution that is consistent with results obtained from the suite of Chalco samples. Both of these effects have been observed during diffusion in glassy polymer systems and are associated with the build‐up and relaxation of self‐stress caused by the influx of diffusing material.
Career Story: Financial Services Statistician
Authors: Mohamed Omar
Published: May 2004

Have you ever been direct mailed by Marks & Spencer Money? If so, the chances are it is because you were interested in the product, Omar Mohamed tells Helen Joyce. A statistician with M&S Money's Marketing Department, he believes that the fast-growing field of financial services is unjustly overlooked by job-seeking statisticians.
Interleukin-13 Overexpression by Tax Transactivation: a Potential Autocrine Stimulus in Human T-Cell Leukemia Virus-Infected Lymphocytes
Authors: Wäldele Katja , Schneider Grit , Ruckes Tobias , Grassmann Ralph
Published: May 2004

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein induces growth transformation and is critical for the pathogenesis of the HTLV-1-induced adult T-cell leukemia (ATL). It stimulates the cell cycle and transactivates cellular genes. Here we show that the expression of interleukin-13 (IL-13) is upregulated as a consequence of Tax in HTLV-1-transformed T cells and ATL-derived cultures. IL-13 exerts proliferative and antiapoptotic functions and is linked to leukemogenesis, since it stimulates Hodgkin lymphoma cells by an autocrine mechanism. Overexpression of IL-13 RNA and protein was confirmed in HTLV-1-positive and Tax-transformed cells. Induction of endogenous IL-13 levels intax-transfected Jurkat cells and in conditional Tax-expressing transformed T lymphocytes suggested that Tax can replace signals required for IL-13 synthesis. For functional analysis, the IL-13 promoter and deletion variants were cloned into luciferase reporter plasmids. Experiments with transfected human T lymphocytes revealed a 16-fold stimulation of the IL-13 promoter by Tax. Experiments with Tax mutants indicated that none of the classical transactivation pathways (SRF, CREB, and NF-κB) is sufficient for the transactivation; at least two different Tax functions are required for full transactivation. The IL-13 promoter is stimulated via two elements; one is a NF-AT binding P element, and the other is a putative AP-1 site. The following observations suggest that IL-13 may stimulate HTLV-1-transformed cells by an autocrine mechanism: (i) the HTLV-1-transformed cells express the IL-13 receptor on their surface, and (ii) STAT6, a downstream effector of IL-13 signaling, is constitutively activated. Thus, in summary, Tax, by transactivating the promoter, induces IL-13 overexpression that possibly leads to an autocrine stimulation of HTLV-1-infected cells.
Inhibition of Reovirus by Mycophenolic Acid Is Associated with the M1 Genome Segment
Authors: Hermann Laura L. , Coombs Kevin M.
Published: May 2004

Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, inhibits reovirus replication and viral RNA and protein production. In mouse L929 cells, antiviral effects were greatest at 30 μg of MPA/ml. At this dosage, MPA inhibited replication of reovirus strain T3D more than 1,000-fold and inhibited replication of reovirus strain T1L nearly 100-fold, compared to non-drug-treated controls. Genetic reassortant analysis indicated the primary determinant of strain-specific differences in sensitivity to MPA mapped to the viral M1 genome segment, which encodes the minor core protein μ2. MPA also inhibited replication of both strains of reovirus in a variety of other cell lines, including Vero monkey kidney and U373 human astrocytoma cells. Addition of exogenous guanosine to MPA-treated reovirus-infected cells restored viral replicative capacity to nearly normal levels. These results suggest the μ2 protein is involved in the uptake and processing of GTP in viral transcription in infected cells and strengthens the evidence that the μ2 protein can function as an NTPase and is likely a transcriptase cofactor.
Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches
Authors: Murakami Pete , Havenga Menzo , Fawaz Farah , Vogels Ronald , Marzio Giuseppe , Pungor Erno , Files Jim , Do Linh , Goudsmit Jaap , McCaman Michael
Published: May 2004

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 × 10
12
vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.
Molecular Analysis of the Protease-Resistant Prion Protein in Scrapie and Bovine Spongiform Encephalopathy Transmitted to Ovine Transgenic and Wild-Type Mice
Authors: Baron Thierry , Crozet Carole , Biacabe Anne-Gaëlle , Philippe Sandrine , Verchere Jérémie , Bencsik Anna , Madec Jean-Yves , Calavas Didier , Samarut Jacques
Published: May 2004

The existence of different strains of infectious agents involved in scrapie, a transmissible spongiform encephalopathy (TSE) of sheep and goats, remains poorly explained. These strains can, however, be differentiated by characteristics of the disease in mice and also by the molecular features of the protease-resistant prion protein (PrP
res
) that accumulates into the infected tissues. For further analysis, we first transmitted the disease from brain samples of TSE-infected sheep to ovine transgenic [Tg(OvPrP4)] and to wild-type (C57BL/6) mice. We show that, as in sheep, molecular differences of PrP
res
detected by Western blotting can differentiate, in both ovine transgenic and wild-type mice, infection by the bovine spongiform encephalopathy (BSE) agent from most scrapie sources. Similarities of an experimental scrapie isolate (CH1641) with BSE were also likewise found following transmission in ovine transgenic mice. Secondly, we transmitted the disease to ovine transgenic mice by inoculation of brain samples of wild-type mice infected with different experimental scrapie strains (C506M3, 87V, 79A, and Chandler) or with BSE. Features of these strains in ovine transgenic mice were reminiscent of those previously described for wild-type mice, by both ratios and by molecular masses of the different PrP
res
glycoforms. Moreover, these studies revealed the diversity of scrapie strains and their differences with BSE according to labeling by a monoclonal antibody (P4). These data, in an experimental model expressing the prion protein of the host of natural scrapie, further suggest a genuine diversity of TSE infectious agents and emphasize its linkage to the molecular features of the abnormal prion protein.
Nef Stimulates Human Immunodeficiency Virus Type 1 Replication in Primary T Cells by Enhancing Virion-Associated gp120 Levels: Coreceptor-Dependent Requirement for Nef in Viral Replication
Authors: Lundquist Christopher A. , Zhou Jing , Aiken Christopher
Published: May 2004

The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) replication through an unknown mechanism. We and others have previously reported that efficient HIV-1 replication in activated primary CD4
+
T cells depends on the ability of Nef to downregulate CD4 from the cell surface. Here we demonstrate that Nef greatly enhances the infectivity of HIV-1 particles produced in primary T cells. Nef-defective HIV-1 particles contained significantly reduced quantities of gp120 on their surface; however, Nef did not affect the levels of virion-associated gp41, indicating that Nef indirectly stabilizes the association of gp120 with gp41. Surprisingly, Nef was not required for efficient replication of viruses that use CCR5 for entry, nor did Nef influence the infectivity or gp120 content of these virions. Nef also inhibited the incorporation of CD4 into HIV-1 particles released from primary T cells. We propose that Nef, by downregulating cell surface CD4, enhances HIV-1 replication by inhibiting CD4-induced dissociation of gp120 from gp41. The preferential requirement for Nef in the replication of X4-tropic HIV-1 suggests that the ability of Nef to downregulate CD4 may be most important at later stages of disease when X4-tropic viruses emerge.
Proteolytic Cleavage of the Catalytic Subunit of DNA-Dependent Protein Kinase during Poliovirus Infection
Authors: Graham Kareem L. , Gustin Kurt E. , Rivera Carlos , Kuyumcu-Martinez N. Muge , Choe Sunny S. , Lloyd Richard E. , Sarnow Peter , Utz Paul J.
Published: May 2004

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PKCS. Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PKCSis cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PKCSenzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PKCSis a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PKCSis directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PKCSis cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PKCSduring poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or “immunocryptic,” protein fragments.
Novel Nuclear Herniations Induced by Nuclear Localization of a Viral Protein
Authors: Hoyt Cristen C. , Bouchard Ron J. , Tyler Kenneth L.
Published: May 2004

A common consequence of viral infection is perturbation of host cell nuclear functions. For cytoplasmically replicating viruses, this process may require regulated transport of specific viral proteins into the nucleus. Here, we describe a novel form of virus-induced perturbation of host cell nuclear structures. Active signal-mediated nuclear import of the reovirus σ1s protein results in redistribution of nuclear pore complexes and nuclear lamins and formation of nuclear herniations. These herniations represent a previously undescribed mechanism by which cytoplasmic viral infection can perturb nuclear architecture and induce cytopathic effects, which ultimately lead to disease pathogenesis in the infected host.
Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein K8.1 Is Dispensable for Virus Entry
Authors: Luna Rafael E. , Zhou Fuchun , Baghian Abolgashem , Chouljenko Vladimir , Forghani Bagher , Gao Shou-Jiang , Kousoulas Konstantin G.
Published: May 2004

Kaposi's sarcoma-associated herpesvirus (KSHV) is considered the etiologic agent of Kaposi's sarcoma and several lymphoproliferative disorders. Recently, the KSHV genome was cloned into a bacterial artificial chromosome and used to construct a recombinant KSHV carrying a deletion of the viral interferon regulatory factor gene (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol.
76:
6185-6196, 2002). The K8.1 glycoprotein is a structural component of the KSHV particle and is thought to facilitate virus entry by binding to heparan sulfate moieties on cell surfaces. To further address the role of K8.1 in virus infectivity, a K8.1-null recombinant virus (BAC36ΔK8.1) was constructed by deletion of most of the K8.1 open reading frame and insertion of a kanamycin resistance gene cassette within the K8.1 gene. Southern blotting and diagnostic PCR confirmed the presence of the engineered K8.1 gene deletion. Transfection of the wild-type genome (BAC36) and mutant genome (BAC36ΔK8.1) DNAs into 293 cells in the presence or absence of the complementing plasmid (pCDNAK8.1A), transiently expressing the K8.1A gene, produced infectious virions in the supernatants of transfected cells. These results demonstrated that the K8.1 glycoprotein is not required for KSHV entry into 293 cells.
Direct Binding of Hepatitis C Virus Core to gC1qR on CD4+and CD8+T Cells Leads to Impaired Activation of Lck and Akt
Authors: Yao Zhi Qiang , Eisen-Vandervelde Audrey , Waggoner Stephen N. , Cale Evan M. , Hahn Young S.
Published: May 2004

Complement plays a pivotal role in the regulation of innate and adaptive immunity. It has been shown that the binding of C1q, a natural ligand of gC1qR, on T cells inhibits their proliferation. Here, we demonstrate that direct binding of the hepatitis C virus (HCV) core to gC1qR on T cells leads to impaired Lck/Akt activation and T-cell function. The HCV core associates with the surface of T cells specifically via gC1qR, as this binding is inhibited by the addition of either anti-gC1qR antibody or soluble gC1qR. The binding affinity constant of core protein for gC1qR, as determined by BIAcore analysis, is 3.8 × 10−7M. The specificity of the HCV core-gC1qR interaction is confirmed by reduced core binding on Molt-4 T cells treated with gC1qR-silencing small interfering RNA and enhanced core binding on GPC-16 guinea pig cells transfected with human gC1qR. Interestingly, gC1qR is expressed at higher levels on CD8+than on CD4+T cells, resulting in more severe core-induced suppression of the CD8+-T-cell population. Importantly, T-cell receptor-mediated activation of the Src kinases Lck and ZAP-70 but not Fyn and the phosphorylation of Akt are impaired by the HCV core, suggesting that it inhibits the very early events of T-cell activation.
Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth
Authors: Das Subash C. , Pattnaik Asit K.
Published: May 2004

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase (RdRp) complex. It is phosphorylated at two different domains. Using defective interfering (DI) RNA or minigenomic RNA templates, we previously demonstrated that phosphorylation within the amino-terminal domain I is essential for transcription, whereas phosphorylation within the carboxy-terminal domain II is necessary for replication. For the present study, we examined the role of the phosphorylation of residues in these domains in the life cycle of VSV. Various mutant P coding sequences were inserted into a full-length cDNA clone of VSV, and the virus recovery, kinetics of growth, and mRNA and protein synthesis were examined. We observed that virus recovery was completely abolished when all three phosphate acceptor sites in domain I or both sites in domain II were replaced with alanine. Single or double mutations in domain I (with the exception of P60/64) or single mutations in domain II had no adverse effect on virus recovery. VSVP227, carrying alanine at position 227, showed reduced kinetics of virus growth but increased kinetics of viral mRNA synthesis in infected cells. More interestingly, this particular virus exhibited a significantly reduced cytopathic effects and apoptosis in infected cells, implying that P may be involved in these processes. Furthermore, we found that DI RNAs of different sizes were generated by high-multiplicity passaging of various mutant VSVs, indicating that the viral RdRp may play a significant role in the process of DI particle generation. Taken together, our results suggest that the phosphorylation of residues in domains I and II of VSV P is indispensable for virus growth.
Biodistribution of Radioiodinated Adenovirus Fiber Protein Knob Domain after Intravenous Injection in Mice
Authors: Awasthi Vibhudutta , Meinken George , Springer Karen , Srivastava Suresh C. , Freimuth Paul
Published: May 2004

The knob domains from the fiber proteins of adenovirus serotypes 2 and 12 were labeled with radioiodine and then injected into the bloodstreams of mice. Knob proteins with functional binding sites for the coxsackie and adenovirus receptor (CAR) were cleared rapidly from the circulation, with radioactivity appearing predominantly in the stomach, while knob mutants unable to bind to CAR remained in the blood circulation for a prolonged period. The clearance of radiolabeled wild-type knob from the blood was slowed by coinjecting an excess of unlabeled wild-type knob protein. An earlier study showed that
99m
Tc-labeled knob protein with intact CAR-binding activity also cleared rapidly from the blood circulation of mice, with radioactivity accumulating predominantly in the liver (K. R. Zinn et al., Gene Ther. 5:798-808, 1998). Together these results suggest that rapid clearance of knob protein from the blood results from specific binding to CAR in the liver and that the bound knob then enters a degradative pathway. The elevated levels of radioiodine in the stomach observed in our experiments are consistent with deiodination of labeled knob by dehalogenases in hepatocyte microsomes and uptake of the resultant free radioiodine by Na/I symporters in the gastric mucosa. Although CAR has been shown to localize in tight junctions of polarized epithelial cells, where it functions in intercellular adhesion, the results of our study suggest that a subset of CAR molecules in the liver is highly accessible to ligands in the blood and able to rapidly deliver bound ligand to an intracellular degradative compartment.
Characterization of Two
Autographa californica
Nucleopolyhedrovirus Proteins, Ac145 and Ac150, Which Affect Oral Infectivity in a Host-Dependent Manner
Authors: Lapointe Renee , Popham Holly J. R. , Straschil Ursula , Goulding David , O'Reilly David R. , Olszewski Julie A.
Published: May 2004

The genome of the baculovirus
Autographa californica
nuclear polyhedrosis virus (Ac
M
NPV) contains two homologues,
orf145
and
orf150
, of the
Heliothis armigera
Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both
orf145
and
orf150
were deleted from the Ac
M
NPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in
Trichoplusia ni
and
Heliothis virescens
larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) Ac
M
NPV for
T. ni
but not for
H. virescens
. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for
H. virescens
. Intrahemocoelic injection of budded virus from the double-deletion virus into
H. virescens
larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of Ac
M
NPV, the extent of which is host dependent.
Ability of the Human Cytomegalovirus IE1 Protein To Modulate Sumoylation of PML Correlates with Its Functional Activities in Transcriptional Regulation and Infectivity in Cultured Fibroblast Cells
Authors: Lee Hye-Ra , Kim Do-Jun , Lee Jang-Mi , Choi Cheol Yong , Ahn Byung-Yoon , Hayward Gary S. , Ahn Jin-Hyun
Published: May 2004

In one of the earliest events in human cytomegalovirus (HCMV)-infected cells, the major immediate-early (IE) protein IE1 initially targets to and then disrupts the nuclear structures known as PML oncogenic domains (PODs) or nuclear domain 10. Recent studies have suggested that modification of PML by SUMO is essential to form PODs and that IE1 both binds to PML and may disrupt PODs by preventing or removing SUMO adducts on PML. In this study, we showed that in contrast to herpes simplex virus type 1 (HSV-1) IE110 (ICP0), the loss of sumoylated forms of PML by cotransfected IE1 was resistant to the proteasome inhibitor MG132 and that IE1 did not reduce the level of unmodified PML. Reduced sumoylation of PML was also observed in U373 cells after infection with wild-type HCMV and proved to require IE1 protein expression. Mutational analysis revealed that the central hydrophobic domain of IE1, including Leu174, is required for both PML binding and loss of PML sumoylation and confirmed that all IE1 mutants tested that were deficient in these functions also failed both to target to PODs and to disrupt PODs. These same mutants were also inactive in several reporter gene transactivation assays and in inhibition of PML-mediated repression. Importantly, a viral DNA genome containing an IE1 gene with a deletion [IE1(Δ290-320)] that was defective in these activities was not infectious when transfected into permissive fibroblast cells, but the mutant IE1(K450R), which is defective in IE1 sumoylation, remained infectious. Our mutational analysis strengthens the idea that interference by IE1 with both the sumoylation of PML and its repressor activity requires a physical interaction with PML that also leads to disruption of PODs. These activities of IE1 also correlate with several unusual transcriptional transactivation functions of IE1 and may be requirements for efficient initiation of the lytic cycle in vivo.
Lyssavirus Matrix Protein Induces Apoptosis by a TRAIL-Dependent Mechanism Involving Caspase-8 Activation
Authors: Kassis Raïd , Larrous Florence , Estaquier Jérôme , Bourhy Hervé
Published: May 2004

Lyssaviruses, which are members of theRhabdoviridaefamily, induce apoptosis, which plays an important role in the neuropathogenesis of rabies. However, the mechanisms by which these viruses mediate neuronal apoptosis have not been elucidated. Here we demonstrate that the early induction of apoptosis in a model of lyssavirus-infected neuroblastoma cells involves a TRAIL-dependent pathway requiring the activation of caspase-8 but not of caspase-9 or caspase-10. The activation of caspase-8 results in the activation of caspase-3 and caspase-6, as shown by an increase in the cleavage of the specific caspase substrate in lyssavirus-infected cells. However, neither caspase-1 nor caspase-2 activity was detected during the early phase of infection. Lyssavirus-mediated cell death involves an interaction between TRAIL receptors and TRAIL, as demonstrated by experiments using neutralizing antibodies and soluble decoy TRAIL-R1/R2 receptors. We also demonstrated that the decapsidation and replication of lyssavirus are essential for inducing apoptosis, as supported by UV inactivation, cycloheximide treatment, and the use of bafilomycin A1 to inhibit endosomal acidification. Transfection of cells with the matrix protein induced apoptosis using pathways similar to those described in the context of viral infection. Furthermore, our data suggest that the matrix protein of lyssaviruses plays a major role in the early induction of TRAIL-mediated apoptosis by the release of a soluble, active form of TRAIL. In our model, Fas ligand (CD95L) appears to play a limited role in lyssavirus-mediated neuroblastoma cell death. Similarly, tumor necrosis factor alpha does not appear to play an important role.
Target Specificity of Human Immunodeficiency Virus Type 1 NCp7 Requires an Intact Conformation of Its CCHC N-Terminal Zinc Finger
Authors: Ramboarina S. , Druillennec S. , Morellet N. , Bouaziz S. , Roques B. P.
Published: May 2004

The modification of zinc-binding residues inside the conserved CCHC motif of human immunodeficiency virus type 1 NCp7, in particular into CCHH, induces a complete loss of infectivity. Since the mutant His28NCp7 has been shown to be devoid of infectivity in vivo, the structure-function relationships of the mutant His28(12-53)NCp7 were investigated by nuclear magnetic resonance and surface plasmonic resonance. Although the Cys28→His mutation modifies drastically the structure of the core domain (residues 12 to 53) of NCp7, His28(12-53)NCp7 still interacts with a 10-fold-lower affinity to specific nucleic acid targets, such as SL3, a stem-loop critically involved in viral RNA packaging, and without affinity change with the nonspecific, single-stranded nucleic acid poly(T). Moreover, His28(12-53)NCp7 and native (12-53)NCp7 displayed the same affinity with reverse transcriptase, but the natures of the complexes are probably different, accounting for the drastic reduction in the amount of RNA packaged in the mutated virus. We propose a structural model of His28(12-53)NCp7 that provides insights into the NCp7 structural features necessary for target recognition and that shows that the specific native structure of the zinc finger domain is strictly required for the optimal target selectivity of NCp7.
Engagement of ICAM-3 Provides a Costimulatory Signal for Human Immunodeficiency Virus Type 1 Replication in both Activated and Quiescent CD4+T Lymphocytes: Implications for Virus Pathogenesis
Authors: Barat Corinne , Gervais Philippe , Tremblay Michel J.
Published: May 2004

Human immunodeficiency virus type 1 (HIV-1) replication is regulated by several extracellular signals. We demonstrate that intercellular adhesion molecule 3 (ICAM-3) acts as a costimulating molecule to increase HIV-1 transcription and viral production, a process allowing productive infection of quiescent CD4+T lymphocytes. The present work suggests an important role for ICAM-3 in HIV-1 replication.
Ultrastructure and immunocytochemistry of the hostpathogen interface in poinsettia leaves infected with powdery mildew
Authors: Celio G J , Mims. C W , Richardson E A
Published: May 2004

Transmission electron microscopy of high-pressure frozen and freeze-substituted samples was used to examine details of the hostpathogen interface in poinsettia leaf epidermal cells infected by the powdery mildew fungus Oidium sp. Cryofixation provided excellent preservation of both host cells and fungal haustoria. Each haustorium possessed a slender neck and an expanded body with numerous slender, aseptate, coiled lobes. The extrahaustorial membrane that separated each haustorium from the cytoplasm of an invaded host cell appeared thicker than other cellular membranes and was highly convoluted, particularly near the distal end of the haustorium neck. The neck of each haustorium was encased by a papilla that formed in response to the development of a haustorium. Immunogold labeling was used to elucidate the chemical nature of these papillae. Epitopes of callose, xyloglucan, and arabinogalactan proteins were localized in three separate regions of papillae. The localization sites for these compounds in poinsettia pa pillae differed somewhat from their reported locations in papillae of various other plants infected by either true fungi or oomycetes. In the current study, no labeling with the antibodies used was observed on the extrahaustorial matrix, the extrahaustorial membrane, the fungal plasma membrane, or the host plasma membrane.Key words: Oidium sp., Euphorbia pulcherrima, hostpathogen interactions, papillae, haustoria.