bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of two different dosages of hydroxyzine for sedation in the paediatric dental patient
Authors: FAYTROUNY MAHMOUD , OKTE ZEYNEP , KUCUKYAVUZ ZUHAL
Published: June 2007

Aim.  This study compared the effect of two different dosages of hydroxyzine supported by 50% nitrous oxide inhalation sedation in child patients.

Subjects and methods.  Thirty uncooperative healthy children with an age range of 31–120 months were included in this study. Patients were randomly assigned into two groups. The patients in group 1 were given 20 mg of hydroxyzine (Atarax) 24 h preoperatively and on the operation day, 3.7 mg/kg hydroxyzine was administered orally. The patients in group 2 received 3.7 mg/kg hydroxyzine orally only on the operation day. All patients also received 50% nitrous oxide inhalation sedation. The child's behaviour was evaluated every 5 min by using Houpt Sedation Rating Scale. The oxygen saturation and heart rates were also followed.

Results.  The mean age of the children in the study was 61.9 months (SD 11.9) for group 1 and 53.7 months (SD 12.8) for group 2. Evaluation of the results showed that there were no significant differences (P < 0.05) between behavioural attitudes and sedation degree of the patients.

Conclusions.  Twenty milligrams of hydroxyzine administered 24 h preoperatively has no significant benefit on sedation of the child.
Bacterial diversity in aphthous ulcers
Authors: Marchini L. , Campos M. S. , Silva A. M. , Paulino L. C. , Nobrega F. G.
Published: June 2007

Introduction:  Recurrent aphthous ulcers are common lesions of the oral mucosa of which the etiology is unknown. This study aimed to estimate the bacterial diversity in the lesions and in control mucosa in pooled samples using a culture‐independent molecular approach.

Methods:  Samples were collected from ten healthy individuals and ten individuals with a clinical history of recurrent aphthous ulcers. After DNA extraction, the 16S ribosomal RNA bacterial gene was amplified by polymerase chain reaction with universal primers; amplicons were cloned, sequenced and matched to the GenBank database.

Results:  A total of 535 clones were analyzed, defining 95 bacterial species. We identified 62 putative novel phylotypes. In recurrent aphthous ulcer lesions 57 phylotypes were detected, of which 11 were known species. Control samples had 38 phylotypes, five of which were already known. Only three species or phylotypes were abundant and common to both groups (Gemella haemolysans, Streptococcus mitis strain 209 and Streptococcus pneumoniae R6). One genus was found only in recurrent aphthous ulcer samples (Prevotella) corresponding to 16% of all lesion‐derived clones.

Conclusion:  The microbiota found in recurrent aphthous ulcers and in the control groups diverged markedly and the rich variety of genera found can provide a new starting point for individual qualitative and quantitative analyses of bacteria associated with this oral condition.
Effect of cytokine and antigen stimulation on peripheral blood lymphocyte syndecan‐1 expression
Authors: Manakil J. F. , Seymour G. J. , Bartold P. M.
Published: June 2007

Introduction:  Cytokines are not only produced by activated lymphocytes but also interact with a number of cell‐surface molecules on the same cells. Syndecan‐1 is one such cell‐surface molecule, which has the capacity to bind a variety of growth factors as well as cytokines. The aim of this study was to examine the effects of transforming growth factor β (TGF‐β), interleukin‐1 (IL‐1), IL‐2, IL‐4, lipopolysaccharide (LPS) from Porphyromonas gingivalis and tetanus toxoid on syndecan‐1 expression by B and T lymphocytes.

Methods:  B and T lymphocytes were obtained from the peripheral blood of healthy donors. Following exposure to the above growth factors, cytokines and antigens, syndecan‐1 expression was determined by flow cytometry.

Results:  Subjects could be categorized as high or low expressors of syndecan‐1. In the high‐responder group TGF‐β1 alone resulted in a significant increase in syndecan‐1 expression by both B and T cells. None of the other cytokines and antigens produced a significant response. When analysed in combination, TGF‐β1 in combination with IL‐2, IL‐4, P. gingivalis LPS and tetanus toxoid all produced significant increases in syndecan‐1 expression by B cells. For T cells, combinations of TGF‐β1 with IL‐2 and tetanus toxoid resulted in increased syndecan‐1 expression.

Conclusions:  Both B and T lymphocytes synthesize the cell‐surface proteoglycan syndecan‐1 and its expression can be modulated by TGF‐β1, either alone or in combination with IL‐2, IL‐4 and LPS from P. gingivalis and tetanus toxoid. While these may reflect general responses under inflammatory conditions their biological significance requires further investigation.
Dysregulation of gene induction in corticostriatal circuits after repeated methylphenidate treatment in adolescent rats: differential effects on zif 268 and homer 1a
Authors: Cotterly Lindsay , Beverley Joel A. , Yano Motoyo , Steiner Heinz
Published: June 2007

Psychostimulants and other dopamine agonists produce molecular changes in neurons of cortico–basal ganglia–cortical circuits, and such neuronal changes are implicated in behavioural disorders. Methylphenidate, a psychostimulant that causes dopamine overflow (among other effects), alters gene regulation in neurons of the striatum. The present study compared the effects of acute and repeated methylphenidate treatment on cortical and striatal gene regulation in adolescent rats. Changes in the expression of the immediate–early genes zif 268 and homer 1a were mapped in 23 striatal sectors and 22 cortical areas that provide input to these striatal sectors, in order to determine whether specific corticostriatal circuits were affected by these treatments. Acute administration of methylphenidate (5 mg/kg, i.p.) produced modest zif 268 induction in cortical areas. These cortical zif 268 responses were correlated in magnitude with zif 268 induction in functionally related striatal sectors. In contrast, after repeated methylphenidate treatment (10 mg/kg, 7 days), cortical and striatal gene induction were dissociated. In these animals, the methylphenidate challenge (5 mg/kg) produced significantly greater gene induction (zif 268 and homer 1a) in the cortex. This enhanced response was widespread but regionally selective, as it occurred predominantly in premotor, motor and somatosensory cortical areas. At the same time, striatal gene induction was partly suppressed (zif 268) or unchanged (homer 1a). Thus, repeated methylphenidate treatment disrupted the normally coordinated gene activation patterns in cortical and striatal nodes of corticostriatal circuits. This drug‐induced dissociation in cortical and striatal functioning was associated with enhanced levels of behavioural stereotypies, suggesting disrupted motor switching function.
Sensitization to heat through G‐protein‐coupled receptor pathways in the isolated sciatic mouse nerve
Authors: Fischer Michael J. M. , Reeh Peter W.
Published: June 2007

We have previously shown that isolated rat sciatic nerve axons express capsaicin, heat and proton sensitivity and respond to stimulation with a Ca++‐dependent and graded calcitonin gene related peptide (CGRP) release. There is morphological evidence for stimulated vesicular exocytosis and for capsaicin receptor, transient receptor potential vanilloid type‐1 (TRPV1, formerly VR1) translocation in the axolemma of unmyelinated nerve fibres. In sensory nerve terminals CGRP release in response to noxious heat can be sensitized by activation of G‐protein‐coupled receptors and related protein kinases. We present evidence that also in isolated mouse sciatic nerve axons the intracellular protein kinase A (PKA)‐ and C (PKC)‐dependent transduction pathways modulate heat‐induced (45 °C) CGRP release. This is demonstrated using the direct activators, forskolin and phorbol 12‐myristate 13‐acetate (PMA), as well as prostaglandin E2 (PGE2) and bradykinin acting through G‐protein‐coupled receptors. Inhibition at rest of protein kinases A or C left heat‐induced CGRP release unchanged. In TRPV1 knockout animals no sensitization to heat was observed using a combined stimulation by prostaglandin E2 and bradykinin. To a surprising degree, peripheral nerve axons resemble peripheral sensory terminals in their common properties of sensory and signal transduction.
Identification of candidate transcriptional modulators involved in successful regeneration after nerve injury
Authors: Stam Floor J. , MacGillavry Harold D. , Armstrong Nicola J. , De Gunst Mathisca C. M. , Zhang Yi , Van Kesteren Ronald E. , Smit August B. , Verhaagen Joost
Published: June 2007

Successful regeneration of injured neurons requires a complex molecular response that involves the expression, modification and transport of a large number of proteins. The identity of neuronal proteins responsible for the initiation of regenerative neurite outgrowth is largely unknown. Dorsal root ganglion (DRG) neurons display robust and successful regeneration following lesion of their peripheral neurite, whereas outgrowth of central neurites is weak and does not lead to functional recovery. We have utilized this differential response to gain insight in the early transcriptional events associated with successful regeneration. Surprisingly, our study shows that peripheral and central nerve crushes elicit very distinct transcriptional activation, revealing a large set of novel genes that are differentially regulated within the first 24 h after the lesion. Here we show that Ankrd1, a gene known to act as a transcriptional modulator, is involved in neurite outgrowth of a DRG neuron‐derived cell line as well as in cultured adult DRG neurons. This gene, and others identified in this study, may be part of the transcriptional regulatory module that orchestrates the onset of successful regeneration.
Differential role of mGlu1 and mGlu5 receptors in rat hippocampal slice models of ischemic tolerance
Authors: Werner Claudia G. , Scartabelli Tania , Pancani Tristano , Landucci Elisa , Moroni Flavio , Pellegrini‐Giampietro Domenico E.
Published: June 2007

Activation of glutamate receptors has been proposed as a key factor in the induction of ischemic tolerance. We used organotypic rat hippocampal slices exposed to 30 min oxygen–glucose deprivation (OGD) to evaluate postischemic pyramidal cell death in the CA1 subregion. In this model, 10 min exposure to OGD 24 h before the exposure to toxic OGD was not lethal and reduced the subsequent OGD neurotoxicity by ∼ 53% (ischemic preconditioning). Similarly, a 30 min exposure to the group I mGlu receptor agonist DHPG (10 µM) significantly reduced OGD neurotoxicity 24 h later (pharmacological preconditioning). Ischemic tolerance did not develop when either the selective mGlu1 antagonists LY367385 and 3‐MATIDA or the AMPA/KA antagonist CNQX were present in the incubation medium during exposure to sublethal OGD. Neither the NMDA antagonist MK801 nor the mGlu5 antagonist MPEP affected the preconditioning process. On the other hand, pharmacological preconditioning was prevented not only by LY367385 or CNQX, but also by MPEP. In preconditioned slices, the toxic responses to AMPA or NMDA were reduced. The neurotoxicty of 100 µM DHPG in slices simultaneously exposed to a mild (20 min) OGD was differentially altered in the two preconditioning paradigms. After ischemic preconditioning, DHPG neurotoxicity was reduced in a manner that was sensitive to LY367385 but not to MPEP, whereas after pharmacological preconditioning it was enhanced in a manner that was sensitive to MPEP but not to LY367385. Our results show that mGlu1 and mGlu5 receptors are differentially involved in the induction and expression of ischemic tolerance following two diverse preconditioning stimuli.
Chronic interleukin‐6 alters the level of synaptic proteins in hippocampus in culture and in vivo
Authors: Vereyken Elly J. F. , Bajova Hilda , Chow Stephine , De Graan Pierre N. E. , Gruol Donna L.
Published: June 2007

There is now considerable evidence that the level of expression of the proinflammatory cytokine, interleukin‐6 (IL‐6), is increased in the central nervous system (CNS) during neuroinflammatory conditions such as occurs in neurological disorders and in disease and injury. However, our understanding of the consequences of increased expression of IL‐6 on the CNS is still limited, especially with respect to the developing nervous system, which is known to be particularly vulnerable to environmental factors. To address this issue, we investigated the properties of cultured hippocampal neurons exposed chronically to IL‐6 during the main period of morphological and physiological development, which occurs during the first 2 weeks of culture. IL‐6 was tested at 500 U/mL, considered to reflect a pathophysiologic concentration. The morphological features of neuronal development in the control and IL‐6‐treated cultures appeared similar. However, Western blot analysis showed a significant reduction in the level of Group‐II metabotropic receptors (mGluR2/3) and L‐type Ca2+ channels in the IL‐6‐treated cultures. A similar reduction in mGluR2/3 and L‐type Ca2+ channel protein was observed in transgenic mice that over‐express IL‐6 in the CNS through astrocyte production starting early in development. Analysis of Ca2+ signals produced by spontaneous synaptic network activity in the hippocampal cultures and effects of a mGluR2/3 agonist and antagonist showed that the reduced levels of mGluR2/3 impact on the functional properties of hippocampal synaptic network activity. These results have important implications relative to the mechanisms responsible for altered CNS function during conditions associated with increased levels of IL‐6 in the CNS.
Medicare Part D and Nursing Home Residents
Authors: Stevenson David G. , Huskamp Haiden A. , Keating Nancy L. , Newhouse Joseph P.
Published: June 2007

The objective of this study was to analyze national Part D formulary data to assess adequacy of coverage across seven drug classes commonly used by nursing home residents and older people, focusing on individuals dually eligible for Medicare and Medicaid and plans in which they enroll. Focusing at the molecule level, reasonably broad coverage across drug classes and minimal prior authorization overall was found. Of nonprotected classes, 69% of plans cover at least four of five Alzheimer's medications, 76% cover at least three of four bisphosphonates, 86% cover at least three of five proton pump inhibitors, and 61% cover at least four of six 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (statins). Nevertheless, a minority of plans are less generous, and some drug formulations important to nursing home residents are covered less well. For example, 11% of plans cover only one or two of the six statins. Of protected drug classes, plans generally cover all molecules, as expected. The majority of plans require no prior authorization for covered medications in six of seven classes reviewed (excepting bisphosphonates). A minority of plans once again are more stringent. For example, 22% and 9% of Part D Plans nationally require prior authorization for all covered Alzheimer's drugs and proton pump inhibitors, respectively. Random assignment of dually eligible patients to below‐benchmark plans means that some residents initially will be enrolled in these more‐restrictive plans. Part D allows nursing home residents to switch plans at any time, but there are important barriers to residents' self‐advocacy. Finally, it is unclear how well nursing homes and the pharmacies they work with will work across Part D plans, and vigilance will be required as the benefit proceeds.
Successful interim therapy with imatinib prior to allogeneic stem cell transplantation in Philadelphia chromosome‐positive acute myeloid leukemia
Authors: Cho Byung‐Sik , Kim Hee‐Je , Lee Seok , Eom Ki‐Sung , Min Woo‐Sung , Lee Jong‐Wook , Kim Chun‐Choo
Published: June 2007

Objectives:  Imatinib (Glivec, STI571) has been successfully used in patients with chronic myelogenous leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome (Ph). We used imatinib interim therapy for four consecutive patients with newly diagnosed Ph+ acute myeloid leukemia (AML). We monitored the patient status for minimal residual disease by real‐time quantitative polymerase chain reaction.

Methods and results:  Imatinib was administered on an interim schedule after each chemotherapy course. After the first imatinib cycle, all patients remained in sustained complete hematologic remission (CHR) with a decrease in the breakpoint cluster region of the Abelson oncogene locus transcript. All patients received a second imatinib cycle following consolidation and showed sustained CHR, including two cases with complete molecular remission. All cases underwent hematopoietic stem cell transplantation (HSCT) in favorable condition, and are still alive with a leukemia‐free status at 6, 6, 9, and 25 months after HSCT.

Conclusions:  As a first‐line interim therapy, imatinib appears to be a useful treatment strategy to provide a bridge to HSCT in patients with Ph+ AML. Further studies with a larger patient population and longer follow‐up are needed for accurate assessment of the impact of imatinib on the long‐term outcome of transplantation for patients with Ph+ AML.
Sources of Umami Taste in Cheddar and Swiss Cheeses
Authors: Drake S.L. , Carunchia Whetstine M.E. , Drake M.A. , Courtney P. , Fligner K. , Jenkins J. , Pruitt C.
Published: June 2007

ABSTRACT:  Umami plays an important role in the flavor of many cheese varieties. The purpose of this study was to identify the compound(s) responsible for umami taste in Cheddar and Swiss cheeses. Four Cheddar and 4 Swiss cheeses (two with low umami intensity and two with high umami intensity from each type) were selected using a trained sensory panel. Monosodium glutamate (MSG), disodium 5′‐inosine monophosphate (IMP), disodium 5′‐guanosine monophosphate (GMP), sodium chloride, lactic acid, propionic acid, and succinic acid were quantified in the cheeses instrumentally. Taste thresholds (best estimate thresholds, BETs) were determined for each compound in water. Subsequently, a trained descriptive sensory analysis panel evaluated each compound in odor‐free water across threshold concentrations to confirm that the thresholds were based on umami and not some other stimuli. Model system studies with trained panelists were then conducted with each compound individually or all compounds together. Comparison of analytical data and sensory thresholds indicated that IMP and GMP thresholds were 100‐fold higher than their concentrations in cheese. All other compounds contributed some umami taste within their concentration range in umami cheeses. Sensory analysis of model cheeses revealed that glutamic acid played the largest role in umami taste of both Cheddar and Swiss cheeses while succinic and propionic acids contributed to umami taste in Swiss cheeses. Knowledge of the key compounds associated with umami taste in cheeses will aid in the identification of procedures to enhance formation of this taste in cheese.
Effect of High Pressure on the Calpain–Calpastatin System in Fish Muscle
Authors: Chéret R. , Delbarre‐Ladrat C. , Verrez‐Bagnis V. , De Lamballerie M.
Published: June 2007

ABSTRACT:  Calpains (calcium‐activated neutral proteases) of sea bass (Dicentrarchus labrax L.) muscle may participate in the degradation of muscle tissue during postmortem storage. These enzymes are regulated by calpastatin, their endogenous specific inhibitor. The objective of this study was to evaluate the changes encountered by the calpain system during the postmortem storage of fish muscle after high‐pressure treatment. From 100 MPa, high‐pressure treatment of purified calpains results in a loss of their activity as well as in the dissociation of the heterodimeric form. In muscle, the high‐pressure processing decreases the initial activity of calpain. This loss in activity may be due to an inactivation by a change of structure. Initial calpastatin activity is not modified by the high‐pressure treatment, but it decreases during the storage from the beginning for a treatment at 300 MPa after which calpastatin is stable during 2 d. Therefore, this study also suggests that high‐pressure treatment could be a useful way to improve fish flesh quality.
Physicochemical and Volatiles Characterization of Trans‐Free Solid Fats Produced by Lipase‐Catalyzed Interesterification
Authors: Lee J.H. , Akoh C.C. , Lee K.‐T.
Published: June 2007

ABSTRACT:  Trans‐free solid fats were synthesized from fully hydrogenated soybean oil (FHSBO), olive oil (OO), and palm stearin (PS) at different substrate weight ratios (10:20:70, 10:40:50 and 10:50:40) via lipase‐catalyzed interesterification. The interesterified products contained mostly TAG (98.8% to 99.0%), and small amounts of MAG and DAG as by‐products. The major fatty acids were oleic acid, palmitic acid, and stearic acid in the interesterified products, and the melting points ranged from 39 to 45 °C. The amount of α‐tocopherol was reduced by 75% to 92%. Volatile analysis by solid‐phase microextraction indicated that OO and PS had distinct volatile profiles, in which 18 volatiles were retained in interesterified products. Furthermore, some volatiles disappeared or formed during processing. Electronic nose showed that the odors of substrates (OO and PS) were different from each other, and the odors of interesterified products were distinguishable from that of OO or PS. Among the interesterified products, the odor of blend FHSBO:OO:PS of 10:40:50 or 10:50:40 was different from that of blend FHSBO:OO:PS (10:20:70). However, no odor difference was observed between products blend FHSBO:OO:PS 10:40:50 and 10:50:40.
Rainfall‐Runoff Model for Typhoons Making Landfall in Taiwan1
Authors: Wu Ray‐Shyan , Shih Dong‐Sin , Chen Shih‐Wen
Published: June 2007

Abstract:  In this study, we examine a physical raster‐based distributed‐parameter flood simulation model combining one‐dimensional (1D) channel flow and two‐dimensional (2D) overland flow. Continuity equations based on the water budget concept and momentum equations based on Manning’s formula are included. The Shihmen reservoir watershed, situated in northern Taiwan, is selected as the study site, and data regarding the passage of Typhoons Xangsane [2000] and Nari [2001] are utilized to test the model. Comparative analysis shows that the description of the riverbed obtained by the gradient method is superior to that obtained by the GIS approach. This study suggests the Thiessen polygon method to interpolate spatial precipitation. The best calibrations are obtained at a spatial resolution of 160 m × 160 m, with a simulated time step of less than 5 s. The case study indicates that the proposed model has a good potential for inflow description, but does not accurately simulate the inflow amount. The proposed model is a flash reacting event‐based watershed model. To improve simulation accuracy, a good infiltration model and soil moisture model are needed, and their formulation could be our next task.
Automatic Calibration of Hydrologic Models With Multi‐Objective Evolutionary Algorithm and Pareto Optimization1
Authors: Confesor Remegio B. , Whittaker Gerald W.
Published: June 2007

Abstract:  In optimization problems with at least two conflicting objectives, a set of solutions rather than a unique one exists because of the trade‐offs between these objectives. A Pareto optimal solution set is achieved when a solution cannot be improved upon without degrading at least one of its objective criteria. This study investigated the application of multi‐objective evolutionary algorithm (MOEA) and Pareto ordering optimization in the automatic calibration of the Soil and Water Assessment Tool (SWAT), a process‐based, semi‐distributed, and continuous hydrologic model. The nondominated sorting genetic algorithm II (NSGA‐II), a fast and recent MOEA, and SWAT were called in FORTRAN from a parallel genetic algorithm library (PGAPACK) to determine the Pareto optimal set. A total of 139 parameter values were simultaneously and explicitly optimized in the calibration. The calibrated SWAT model simulated well the daily streamflow of the Calapooia watershed for a 3‐year period. The daily Nash‐Sutcliffe coefficients were 0.86 at calibration and 0.81 at validation. Automatic multi‐objective calibration of a complex watershed model was successfully implemented using Pareto ordering and MOEA. Future studies include simultaneous automatic calibration of water quality and quantity parameters and the application of Pareto optimization in decision and policy‐making problems related to conflicting objectives of economics and environmental quality.
Detection of Circulating Galactomannan in Serum Samples for Diagnosis ofPenicillium marneffeiInfection and Cryptococcosis among Patients Infected with Human Immunodeficiency Virus
Authors: Huang Yu-Tsung , Hung Chien-Ching , Liao Chun-Hsing , Sun Hsin-Yun , Chang Shan-Chwen , Chen Yee-Chun
Published: June 2007

Galactomannan (GM) is a heteropolysaccharide in the cell walls of mostAspergillusandPenicilliumspecies. Cross-reactivity ofCryptococcus neoformansgalactoxylomannan in anAspergillusGM test has also been reported. In this study, we used a PlateliaAspergillusenzyme immunoassay kit (Bio-Rad) to test serum samples obtained from 48 human immunodeficiency virus (HIV)-infected patients (15 with penicilliosis [7 with fungemia alone, 4 with cavitary lung lesions alone, 3 with both fungemia and cavitary lung lesions, and 1 with disseminated disease], 22 with cryptococcosis [11 with fungemia alone, 5 with cavitary lung lesions, 3 with both, and 3 with meningitis alone], and 11 without any invasive fungal infection [control]) for GM levels. None of the patients had aspergillosis or concurrent use of piperacillin-tazobactam or amoxicillin-clavulanate. The median time between diagnosis of fungal infection and collection of serum samples was 0 days for penicilliosis and 1.5 days for cryptococcosis. Of patients with penicilliosis, cryptococcosis, and controls, 73.3%, 13.6%, and 9%, respectively, had GM optical density (OD) indices of >0.5 (P= 0.0001). GM OD indices were higher for penicilliosis (median OD index, 4.419; range, 0.158 to >20) than for cryptococcosis (median, 0.247; range, 0.112 to 3.849) cases (P< 0.001). Patients with fungemic penicilliosis had higher OD indices (median, 10.628; range, 0.401 to >20) than patients with nonfungemic penicilliosis (median, 0.378; range, 0.158 to 4.419) and patients with cryptococcemia (median, 0.231; range, 0.112 to 1.168) (P< 0.001). Of the 15 patients with cavitary lung lesions, those with penicilliosis had higher antigen levels (median OD index, 1.641; range, 0.247 to >20) than those with cryptococcosis (median, 0.227; range, 0.112 to 3.849) (P= 0.011). This study showed that the GM OD index was significantly elevated for HIV patients with penicilliosis. The use of the GM antigen assay may facilitate earlier diagnosis ofPenicillium marneffeiinfection for HIV-infected patients in areas of endemicity.
PCR-Based Identification of Common Colonization Factor Antigens of Enterotoxigenic
Escherichia coli
Authors: Ghosal Abhisek , Bhowmick Rudra , Nandy Ranjan Kumar , Ramamurthy T. , Chatterjee Nabendu Sekhar
Published: June 2007

Colonization factor antigens (CFAs) of enterotoxigenic
Escherichia coli
(ETEC) have been classified into several groups based on their distinct antigenicity. We describe here a PCR-based method to detect common CFAs of ETEC, which were characterized using conventional serology. This PCR assay is simple and sensitive for the detection of expressed CFA genes.
Molecular Evidence of a New Strain of
Ehrlichia canis
from South America
Authors: Vinasco Javier , Li Olga , Alvarado Arnaldo , Diaz Diego , Hoyos Luis , Tabachi Luis , Sirigireddy Kamesh , Ferguson Carolyn , Moro Manuel H.
Published: June 2007

Blood samples from dogs with clinical signs compatible with ehrlichiosis were examined for infection of
Ehrlichia canis
using PCR, multiplex real-time PCR, and DNA sequencing analysis. Eleven of 25 samples were positive for a new strain of
E. canis
. This is the first molecular identification of
E. canis
infection in dogs from Peru.
Serotype 1 and 2 Bovine Noroviruses Are Endemic in Cattle in the United Kingdom and Germany
Authors: Oliver S. L. , Wood E. , Asobayire E. , Wathes D. C. , Brickell J. S. , Elschner M. , Otto P. , Lambden P. R. , Clarke I. N. , Bridger J. C.
Published: June 2007

The genomically and antigenically distinct bovine noroviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK have been associated with calf diarrhea. In the present seroprevalence study, both were found to be endemic in cattle from Germany and the United Kingdom, a finding in contrast to previous virus prevalence studies. They were less common than group A rotaviruses, particularly in calves, suggesting a different epidemiology.
Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA
Authors: Fradet-Turcotte Amélie , Vincent Caroline , Joubert Simon , Bullock Peter A. , Archambault Jacques
Published: June 2007

SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5′-GAGGC-3′ sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5′-GAGGC-3′ sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ∼10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.
A Rapid Progressor-Specific Variant Clone of Simian Immunodeficiency Virus Replicates Efficiently In Vivo Only in the Absence of Immune Reponses
Authors: Kuwata Takeo , Byrum Russell , Whitted Sonya , Goeken Robert , Buckler-White Alicia , Plishka Ronald , Iyengar Ranjini , Hirsch Vanessa M.
Published: June 2007

A subset of simian immunodeficiency virus (SIV)-infected macaques progresses rapidly to disease with transient SIV-specific immune responses and high viral loads. Unique SIV variants with convergent Env mutations evolve in these rapid progressor (RP) macaques. To address the pathogenic significance of RP-specific variants, we generated infectious molecular clones from the terminal-phase plasma of an RP macaque. Inoculation of macaques with a representative clone, SIVsmH635FC, resulted in a persistent viremia, comparable to that produced by pathogenic SIVsmE543-3, and a chronic disease with progressive loss of CD4
+
T cells. However, SIVsmH635FC did not reproduce the rapid-disease phenomenon. Molecular analyses of viruses from these macaques revealed rapid reversion to the wild-type SIVsmE543-3 sequence at two RP-specific sites and slower reversion at another three sites. SIVsmH635FC infection was not sufficient to cause rapid progression even following coinoculation with SIVsmE543-3, despite acute depletion of memory CD4
+
T cells. SIVsmH635FC competed efficiently during primary infection in the coinoculated macaques, but SIVsmE543-3 predominated after the development of SIV-specific immune responses. These data suggest that the replication fitness of the RP variant was similar to that of SIVsmE543-3 in a naïve host; however, SIVsmH635FC was at a disadvantage following the development of SIV-specific immune responses. Consistent with these findings, neutralization assays revealed that SIVsmH635FC was highly sensitive to neutralization but that the parental SIVsmE543-3 strain was highly resistant. This study suggests that the evolution of RP-specific variants is the result of replication in a severely immunocompromised host, rather than the direct cause of rapid progression.
Basic Residues in the Mason-Pfizer Monkey Virus Gag Matrix Domain Regulate Intracellular Trafficking and Capsid-Membrane Interactions
Authors: Stansell Elizabeth , Apkarian Robert , Haubova Sarka , Diehl William E. , Tytler Ewan M. , Hunter Eric
Published: June 2007

Mason-Pfizer monkey virus (M-PMV) capsids that have assembled in the cytoplasm must be transported to and associate with the plasma membrane prior to being enveloped by a lipid bilayer during viral release. Structural studies have identified a positive-charge density on the membrane-proximal surface of the matrix (MA) protein component of the Gag polyprotein. To investigate if basic amino acids in MA play a role in intracellular transport and capsid-membrane interactions, mutants were constructed in which lysine and arginine residues (R10, K16, K20, R22, K25, K27, K33, and K39) potentially exposed on the capsid surface were replaced singly and in pairs by alanine. A majority of the charge substitution mutants were released less efficiently than the wild type. Electron microscopy of mutant Gag-expressing cells revealed four distinct phenotypes: K16A and K20A immature capsids accumulated on and budded into intracellular vesicles; R10A, K27A, and R22A capsid transport was arrested at the cellular cortical actin network, while K25A immature capsids were dispersed throughout the cytoplasm and appeared to be defective at an earlier stage of intracellular transport; and the remaining mutant (K33A and K39A) capsids accumulated at the inner surface of the plasma membrane. All mutants that released virions exhibited near-wild-type infectivity in a single-round assay. Thus, basic amino acids in the M-PMV MA define both cellular location and efficiency of virus release.
Vaccine Platform for Prevention of Tuberculosis and Mother-to-Child Transmission of Human Immunodeficiency Virus Type 1 through Breastfeeding
Authors: Im Eung-Jun , Saubi Narcís , Virgili Goretti , Sander Clare , Teoh Denise , Gatell Jose M. , McShane Helen , Joseph Joan , Hanke Tomáš
Published: June 2007

Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born to human immunodeficiency virus type 1 (HIV-1)-positive mothers are at high risk of acquiring HIV-1 infection through breastfeeding in the first weeks of their lives. Thus, the development of a vaccine which would protect newborns against both of these major global killers is a logical yet highly scientifically, ethically, and practically challenging aim. Here, a recombinant lysine auxotroph ofMycobacterium bovisbacillus Calmette-Guérin (BCG), a BCG strain that is safer than those currently used and expresses an African HIV-1 clade-derived immunogen, was generated and shown to be stable and to induce durable, high-quality HIV-1-specific CD4+- and CD8+-T-cell responses. Furthermore, when the recombinant BCG vaccine was used in a priming-boosting regimen with heterologous components, the HIV-1-specific responses provided protection against surrogate virus challenge, and the recombinant BCG vaccine alone protected against aerosol challenge withM. tuberculosis. Thus, inserting an HIV-1-derived immunogen into the scheduled BCG vaccine delivered at or soon after birth may prime HIV-1-specific responses, which can be boosted by natural exposure to HIV-1 in the breast milk and/or by a heterologous vaccine such as recombinant modified vaccinia virus Ankara delivering the same immunogen, and decrease mother-to-child transmission of HIV-1 during breastfeeding.
Gag Influences Transformation by Abelson Murine Leukemia Virus and Suppresses Nuclear Localization of the v-Abl Protein
Authors: Yi Chae-Ryun , Rosenberg Naomi
Published: June 2007

Like the v-Onc proteins encoded by many transforming retroviruses, the v-Abl protein is expressed as a Gag-Onc fusion. Although the Gag-derived myristoylation signal targets the v-Abl protein to the plasma membrane, the protein contains the entire MA and p12 sequences and a small number of CA-derived residues. To understand the role of Gag sequences in transformation, mutants lacking portions of these sequences were examined for the effects of these deletions on v-Abl function and localization. Deletion of the N-terminal third of p12 or all of p12 enhanced the transformation of both pre-B cells and NIH 3T3 cells. In contrast, deletions in MA or a deletion removing all of Gag except the first 34 amino acids important for myristoylation highly compromised the ability to transform either cell type. Although all of the mutant proteins retained kinase activity, those defective in transformation were reduced in their ability to activate Erk, suggesting a role for Gag sequences in v-Abl signaling. Immunofluorescence analysis revealed that a v-Abl protein retaining only the first 34 amino acids of Gag localized to the nucleus. These data indicate that Gag sequences are important for normal v-Abl signaling and that they suppress nuclear localization of the molecule.
Crystal Structure of Lysine Sulfonamide Inhibitor Reveals the Displacement of the Conserved Flap Water Molecule in Human Immunodeficiency Virus Type 1 Protease
Authors: Nalam Madhavi N. L. , Peeters Anik , Jonckers Tim H. M. , Dierynck Inge , Schiffer Celia A.
Published: June 2007

Human immunodeficiency virus type 1 (HIV-1) protease has been continuously evolving and developing resistance to all of the protease inhibitors. This requires the development of new inhibitors that bind to the protease in a novel fashion. Most of the inhibitors that are on the market are peptidomimetics, where a conserved water molecule mediates hydrogen bonding interactions between the inhibitors and the flaps of the protease. Recently a new class of inhibitors, lysine sulfonamides, was developed to combat the resistant variants of HIV protease. Here we report the crystal structure of a lysine sulfonamide. This inhibitor binds to the active site of HIV-1 protease in a novel manner, displacing the conserved water and making extensive hydrogen bonds with every region of the active site.
Repeated social defeat increases the bactericidal activity of splenic macrophages through a Toll-like receptor-dependent pathway
Authors: Bailey Michael T. , Engler Harald , Powell Nicole D. , Padgett David A. , Sheridan John F.
Published: June 2007

Phagocytes of the innate immune system, such as monocytes/macrophages, represent a first line of defense against invading microorganisms. Psychological stress is often thought to suppress the functioning of these cells, in part due to the immunosuppressive activity of stress-induced glucocorticoid hormones. However, exposure to the stressor social disruption (SDR) has been shown to increase cytokine production by monocytes/macrophages and to reduce their sensitivity to corticosterone. Thus, it was hypothesized that splenic monocytes/macrophages from socially stressed mice would be primed to be more physiologically active than cells from nonstressed controls. Flow cytometry was used to demonstrate that exposure to SDR significantly increased the expression of Toll-like receptors (TLR) 2 and 4 on the surface of splenic macrophages. In a follow-up experiment, exposure to SDR also increased the ability of these macrophages to kill Escherichia coli ex vivo and in vivo. However, SDR failed to increase the bactericidal activity of splenic macrophages from C3H/HeJ mice, which lack functional TLR4. In mice with functional TLR4, the stress-induced increase in bactericidal activity was associated with a significant increase in macrophage gene expression for inducible nitric oxide synthase and subunits of the NADPH oxidase complex, which are responsible for generating reactive nitrogen and oxygen intermediates, respectively. This stress-induced increase in gene expression was not evident in the TLR4-deficient mice. These data indicate that SDR increases TLR expression, which in turn enhances the bactericidal activity of splenic macrophages, in part by increasing pathways responsible for reactive oxygen and nitrogen intermediate production.
Slc7a9knockout mouse is a good cystinuria model for antilithiasic pharmacological studies
Authors: Font-Llitjós Mariona , Feliubadaló Lídia , Espino Meritxell , Clèries Ramon , Mañas Sandra , Frey Isabelle M. , Puertas Sara , Colell Guillem , Palomo Sergio , Aranda Jessica , Visa Joana , Palacín Manuel , Nunes Virginia
Published: June 2007

Cystinuria is a hereditary disorder caused by a defect in the apical membrane transport system for cystine and dibasic amino acids in renal proximal tubules and intestine, resulting in recurrent urolithiasis. Mutations in SLC3A1 and SLC7A9 genes, that codify for rBAT/b0,+AT transporter subunits, cause type A and B cystinuria, respectively. In humans, cystinuria treatment is based on the prevention of calculi formation and its dissolution or breakage. Persistent calculi are treated with thiols [i.e., d-penicillamine (DP) and mercaptopropionylglycine (MPG)] for cystine solubilization. We have developed a new protocol with DP to validate our Slc7a9 knockout mouse model for the study of the therapeutic effect of drugs in the treatment of cystine lithiasis. We performed a 5-wk treatment of individually caged lithiasic mutant mice with a previously tested DP dose. To appraise the evolution of lithiasis throughout the treatment a noninvasive indirect method of calculi quantification was developed: calculi mass was quantified by densitometry of X-ray images from cystinuric mice before and after treatment. Urine was collected in metabolic cage experiments to quantify amino acids in DP-treated and nontreated, nonlithiasic mutant mice. We found significant differences between DP-treated and nontreated knockout mice in calculi size and in urinary cystine excretion. Histopathological analysis showed that globally nontreated mutant mice had more severe and diffuse urinary system damage than DP-treated mice. Our results validate the use of this mouse model for testing the efficacy of potential new drugs against cystinuria.
TGF-β signaling and its effect on glutaminase expression in LLC-PK1-FBPase+cells
Authors: Andratsch Manfred , Feifel Elisabeth , Taylor Lynn , O'Hayre Morgan , Schramek Herbert , Curthoys Norman P. , Gstraunthaler Gerhard
Published: June 2007

During systemic acidosis, renal proximal tubular cells exhibit enhanced rates of bicarbonate and ammonium ion synthesis and undergo extensive hypertrophy. The former adaptations are accomplished, in part, by increased expression of glutaminase (GA). LLC-PK1-FBPase+cells, a gluconeogenic line of porcine kidney cells, exhibit a rapid activation of the ERK1/2 and p38 MAPK pathways and a two- to threefold increase in GA mRNA when transferred to acidic medium (pH 6.9). Transforming growth factor-β (TGF-β), a potent activator of MAPK and Smad signaling cascades, also causes extensive renal hypertrophy. Thus the potential role of TGF-β in the renal response to metabolic acidosis was investigated. Western blot analyses established that in LLC-PK1-FBPase+cells, TGF-β activated the ERK1/2, p38 MAPK, and Smad1/5/8 pathways, but not the JNK and Smad2/3 pathways. Addition of TGF-β to cells cultured in normal medium (pH 7.4) produced a steady increase in GA mRNA, resulting in a twofold induction after 18 h. Western blot analysis indicated that treatment with either TGF-β or acidic medium resulted in an increased level of fibronectin. However, the effects of the two treatments on both GA mRNA and fibronectin levels occurred with different time courses and were additive. In addition, the rates of ammonia production were decreased slightly by addition of TGF-β. Finally, a GA-luciferase reporter construct, which is activated 3.5-fold by treatment with acidic medium, is not affected by TGF-β. Therefore, TGF-β and metabolic acidosis activate some of the same signaling pathways in LLC-PK1-FBPase+cells, but produce separate effects on GA expression.
Aldosterone induces epithelial-mesenchymal transition via ROS of mitochondrial origin
Authors: Zhang Aihua , Jia Zhanjun , Guo Xiaohua , Yang Tianxin
Published: June 2007

It has been well appreciated that aldosterone (Aldo) plays a direct profibrotic role in the kidney but the underlying mechanism is unclear. We examined the role of Aldo in epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Exposure of human renal proximal tubular cells to Aldo for 48 h dose dependently induced EMT as evidenced by conversion to the spindle-like morphology, loss of E-cadherin, and de novo expression of α-smooth muscle actin (SMA); the effect was noticeable at 50 nM and maximal at 100 nM. The EMT was completely blocked by the selective mineralocorticoid receptor (MR) antagonist eplerenone. Aldo time dependently increased intracellular reactive oxygen species (ROS) production that was detectable at 15 min and peaked (2.3-fold) at 60 min, as assessed by 2′,7′-dichlorofluorescin diacetate fluorescence. Aldo-induced oxidative stress and EMT were both abolished by the mitochondrial respiratory chain complex I inhibitor rotenone, but not the NADPH oxidase inhibitor apocynin. Aldo induced phosphorylation of ERK1/2 that was completely blocked by rotenone. Male 129-C57/BL6 mice were treated with deoxycorticosterone acetate (DOCA) salt (subcutaneous implantation of 50 mg of DOCA pellet plus 1% NaCl as drinking fluid) for 3 wk and animals were treated with vehicle or rotenone (600 ppm in diet) for the last week. DOCA salt induced a 2.5-fold increase in α-SMA and a 30% reduction of E-cadherin, as assessed by real-time RT-PCR, that were both restricted to renal epithelial cells, as determined by immunohistochemistry. In contrast, DOCA salt-induced changes in α-SMA and E-cadherin were completely blocked by treatment with rotenone. These observations suggest that Aldo induces EMT via MR-mediated, mitochondrial-originated, ROS-dependent ERK1/2 activation in renal tubular epithelial cells.
Anticipation in the Rodent Head Direction System Can Be Explained by an Interaction of Head Movements and Vestibular Firing Properties
Authors: van der Meer Matthijs A. A. , Knierim James J. , Yoganarasimha D. , Wood Emma R. , van Rossum Mark C. W.
Published: June 2007

The rodent head-direction (HD) system, which codes for the animal's head direction in the horizontal plane, is thought to be critically involved in spatial navigation. Electrophysiological recording studies have shown that HD cells can anticipate the animal's HD by up to 75–80 ms. The origin of this anticipation is poorly understood. In this modeling study, we provide a novel explanation for HD anticipation that relies on the firing properties of neurons afferent to the HD system. By incorporating spike rate adaptation and postinhibitory rebound as observed in medial vestibular nucleus neurons, our model produces realistic anticipation on a large corpus of rat movement data. In addition, HD anticipation varies between recording sessions of the same cell, between active and passive movement, and between different studies. Such differences do not appear to be correlated with behavioral variables and cannot be accounted for using earlier models. In the present model, anticipation depends on the power spectrum of the head movements. By direct comparison with recording data, we show that the model explains 60–80% of the observed anticipation variability. We conclude that HD afferent dynamics and the statistics of rat head movements are important in generating HD anticipation. This result contributes to understanding the functional circuitry of the HD system and has methodological implications for studies of HD anticipation.