bluuebunny/crossref_metadata_2025 huggingface dataset:
Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle
Authors: Perkins Kelly J. , Basu Utpal , Budak Murat T. , Ketterer Caroline , Baby Santhosh M. , Lozynska Olga , Lunde John A. , Jasmin Bernard J. , Rubinstein Neal A. , Khurana Tejvir S.
Published: May 2007

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPα/β to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest “repressing repressors” as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.
Binding of CAP70 to Inducible Nitric Oxide Synthase and Implications for the Vectorial Release of Nitric Oxide in Polarized Cells
Authors: Navarro-Lérida Inmaculada , Martínez-Moreno Mónica , Ventoso Iván , Álvarez-Barrientos Alberto , Rodríguez-Crespo Ignacio
Published: May 2007

In this article we analyze the mechanisms by which the C-terminal four amino acids of inducible nitric oxide synthase (NOS2) interact with proteins that contain PDZ (PSD-95/DLG/ZO-1) domains resulting in the translocation of NOS2 to the cellular apical domain. It has been reported that human hepatic NOS2 associates to EBP50, a protein with two PDZ domains present in epithelial cells. We describe herein that NOS2 binds through its four carboxy-terminal residues to CAP70, a protein that contains four PDZ modules that is targeted to apical membranes. Interestingly, this interaction augments both the cytochrome c reductase and ·NO-synthase activities of NOS2. Binding of CAP70 to NOS2 also results in an increase in the population of active NOS2 dimers. In addition, CAP70 participates in the correct subcellular targeting of NOS2 in a process that is also dependent on the acylation state of the N-terminal end of NOS2. Hence, nonpalmitoylated NOS2 is unable to progress toward the apical side of the cell despite its interaction with either EBP50 or CAP70. Likewise, if we abrogate the interaction of NOS2 with either EBP50 or CAP70 by fusing the GFP reporter to the carboxy-terminal end of NOS2 palmitoylation is not sufficient to confer an apical targeting.
The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting
Authors: Bradshaw Niels , Walter Peter
Published: May 2007

The RNA component of the signal recognition particle (SRP) is universally required for cotranslational protein targeting. Biochemical studies have shown that SRP RNA participates in the central step of protein targeting by catalyzing the interaction of the SRP with the SRP receptor (SR). SRP RNA also accelerates GTP hydrolysis in the SRP·SR complex once formed. Using a reverse-genetic and biochemical analysis, we identified mutations in the E. coli SRP protein, Ffh, that abrogate the activity of the SRP RNA and cause corresponding targeting defects in vivo. The mutations in Ffh that disrupt SRP RNA activity map to regions that undergo dramatic conformational changes during the targeting reaction, suggesting that the activity of the SRP RNA is linked to the major conformational changes in the signal sequence-binding subunit of the SRP. In this way, the SRP RNA may coordinate the interaction of the SRP and the SR with ribosome recruitment and transfer to the translocon, explaining why the SRP RNA is an indispensable component of the protein targeting machinery.
Isolated ZP-N domains constitute the N-terminal extensions of Zona Pellucida proteins
Authors: Callebaut Isabelle , Mornon Jean-Paul , Monget Philippe
Published: May 2007

Zona Pellucida (ZP) domains have been found in a wide variety of extracellular proteins, in which they play essential role for polymerization. They are shared by the ZP proteins, which constitute the extracellular coat of animal eggs. Except from ZP3, constituting the primary sperm receptor, the ZP proteins possess, in addition to their C-terminal ZP domains, N-terminal extensions, which are thought to play an important role in the species-specific gamete recognition. Here, we show that these extensions are made of single or multiple copies of a small globular domain, which can be significantly related to the N-terminal region of ZP domains (ZP-N domains). This finding brings new insights into the molecular evolution of ZP proteins, which may have evolved around a common ZP-N architecture, and more generally into the noticeable sequence diversity of ZP-N domains, which can be found as isolated subunits or tightly associated with ZP-C domains to form complete, canonical ZP domains.

Contact:  isabelle.callebaut@impmc.jussieu.fr

Supplementary information: Supplementary data are available at Bioinformatics online.
Secondary structure based analysis and classification of biological interfaces: identification of binding motifs in protein–protein interactions
Authors: Guharoy Mainak , Chakrabarti Pinak
Published: May 2007

Motivation: The increasing amount of data on protein–protein interaction needs to be rationalized for deriving guidelines for the alteration or design of an interface between two proteins.

Results: We present a detaild structural analysis and comparison of homo- versus heterodimeric protein–protein interfaces. Regular secondary structures (helices and strands) are the main components of the former, whereas non-regular structures (turns, loops, etc.) frequently mediate interactions in the latter. Interface helices get longer with increasing interface area, but only in heterocomplexes. On average, the homodimers have longer helical segments and prominent helix–helix pairs. There is a surprising distinction in the relative orientation of interface helices, with a tendency for aligned packing in homodimers and a clear preference for packing at 90° in heterodimers. Arg and the aromatic residues have a higher preference to occur in all secondary structural elements (SSEs) in the interface. Based on the dominant SSE, the interfaces have been grouped into four classes: α, β, αβ and non-regular. Identity between protein and interface classes is the maximum for α proteins, but rather mediocre for the other protein classes. The interface classes of the two chains forming a heterodimer are often dissimilar. Eleven binding motifs can capture the prominent architectural features of most of the interfaces.

Contact:  pinak@boseinst.ernet.in

Supplementary information: A separate file is provided with 3 tables and 2 figures, which are referred to with a prefix ‘S’ in text.
Dominican Children with HIV Not Receiving Antiretrovirals: Massage Therapy Influences their Behavior and Development
Authors: Hernandez-Reif Maria , Shor-Posner Gail , Baez Jeannette , Soto Solange , Mendoza Rosangela , Castillo Raquel , Quintero Noaris , Perez Eddy , Zhang Guoyan
Published: May 2007

Forty‐eight children (M age = 4.8 years) infected with HIV/AIDS and living in the Dominican Republic were randomly assigned to a massage therapy or a play session control group. The children in the massage therapy group received two weekly 20‐min massages for 12 weeks; the children in the control group participated in a play session (coloring, playing with blocks) for the same duration and length as the massage therapy group. Overall, the children in the massage therapy group improved in self‐help abilities and communication, suggesting that massage therapy may enhance daily functioning for children with HIV/AIDS. Moreover, the HIV infected children who were six or older also showed a decrease in internalizing behaviors; specifically depressive/anxious behaviors and negative thoughts were reduced. Additionally, baseline assessments revealed IQ equivalence below normal functioning for 70% of the HIV infected children and very high incidences of mood problems (depression, withdrawn) for 40% of the children and anxiety problems for 20% of the children, suggesting the need for better monitoring and alternative interventions in countries with limited resources to improve cognition and the mental health status of children infected with HIV/AIDS.
A Robust Transformation Procedure for Interpreting Political Text
Authors: Martin Lanny W. , Vanberg Georg
Published: May 2007

In a recent article in theAmerican Political Science Review, Laver, Benoit, and Garry (2003, “Extracting policy positions from political texts using words as data,” 97:311—331) propose a new method for conducting content analysis. TheirWordscoresapproach, by automating text-coding procedures, represents an advance in content analysis that will potentially have a large long-term impact on research across the discipline. To allow substantive interpretation, the scores produced by theWordscoresprocedure require transformation. In this note, we address several shortcomings in the transformation procedure introduced in the original program. We demonstrate that the original transformation distorts the metric on which content scores are placed—hindering the ability of scholars to make meaningful comparisons across texts—and that it is very sensitive to the texts that are scored—opening up the possibility that researchers may generate, inadvertently or not, results that depend on the texts they choose to include in their analyses. We propose a transformation procedure that solves these problems.
Incidence of Chest Tube Malposition in the Critically Ill
Authors: Remérand Francis , Luce Virginie , Badachi Yasmina , Lu Qin , Bouhemad Belaïd , Rouby Jean-Jacques
Published: May 2007

Background

Malposition of percutaneously inserted chest tubes is considered as a rare complication in critically ill patients. Its incidence, however, remains uncertain. The aims of the study were to assess the true incidence of chest tube malposition in critically ill patients and to identify predicting factors.

Methods

The authors prospectively studied 122 chest tubes percutaneously inserted in 75 consecutive critically ill patients. For clinical reasons independent of the study, thoracic computed tomography scanning was performed in 63 patients, allowing direct visualization of 106 chest tubes. Based on these findings, chest tube position was classified as intrapleural, intrafissural, or intraparenchymal. Factors predicting chest tube malposition were analyzed by univariate and multivariate analysis.

Results

The mean delay between chest tube placement and thoracic scan was 3.5+/-2.9 days. Twenty-two chest tubes were diagnosed as being intrafissural (21%), and 10 were diagnosed as being intraparenchymal (9%). The only predicting factor associated with the risk of malposition was the use of a trocar for the percutaneous insertion of the chest tube (P=0.032).

Conclusions

Malposition was detected in 30% of percutaneously inserted chest tubes, a higher incidence than previously reported. Avoiding the use of a trocar may reduce significantly the incidence of chest tube malposition.
Effects of Xenon Anesthesia on Cerebral Blood Flow in Humans
Authors: Laitio Ruut M. , Kaisti Kaike K. , Låangsjö Jaakko W. , Aalto Sargo , Salmi Elina , Maksimow Anu , Aantaa Riku , Oikonen Vesa , Sipilä Hannu , Parkkola Riitta , Scheinin Harry
Published: May 2007

Background

Animal studies have demonstrated a strong neuroprotective property of xenon. Its usefulness in patients with cerebral pathology could be compromised by deleterious effects on regional cerebral blood flow (rCBF).

Methods

15O-labeled water was used to determine rCBF in nine healthy male subjects at baseline and during 1 minimum alveolar concentration (MAC) of xenon (63%). Anesthesia was based solely on xenon. Absolute changes in rCBF were quantified using region-of-interest analysis and voxel-based analysis.

Results

Mean arterial blood pressure and arterial partial pressure for carbon dioxide remained unchanged. The mean (+/-SD) xenon concentration during anesthesia was 65.2+/-2.3%. Xenon anesthesia decreased absolute rCBF by 34.7+/-9.8% in the cerebellum (P<0.001), by 22.8+/-10.4% in the thalamus (P=0.001), and by 16.2+/-6.2% in the parietal cortex (P<0.001). On average, xenon anesthesia decreased absolute rCBF by 11.2+/-8.6% in the gray matter (P=0.008). A 22.1+/-13.6% increase in rCBF was detected in the white matter (P=0.001). Whole-brain voxel-based analysis revealed widespread cortical reductions and increases in rCBF in the precentral and postcentral gyri.

Conclusions

One MAC of xenon decreased rCBF in several areas studied. The greatest decreases were detected in the cerebellum, the thalamus and the cortical areas. Increases in rCBF were observed in the white matter and in the pre- and postcentral gyri. These results are in clear contradiction with ketamine, another N-methyl-D-aspartate antagonist and neuroprotectant, which induces a general increase in cerebral blood flow at anesthetic concentrations.
Effects of Vasopressin on Microcirculatory Blood Flow in the Gastrointestinal Tract in Anesthetized Pigs in Septic Shock
Authors: Hiltebrand Luzius B. , Krejci Vladimir , Jakob Stephan M. , Takala Jukka , Sigurdsson Gisli H.
Published: May 2007

Background

Vasopressin increases arterial pressure in septic shock even when alpha-adrenergic agonists fail. The authors studied the effects of vasopressin on microcirculatory blood flow in the entire gastrointestinal tract in anesthetized pigs during early septic shock.

Methods

Thirty-two pigs were intravenously anesthetized, mechanically ventilated, and randomly assigned to one of four groups (n=8 in each; full factorial design). Group S (sepsis) and group SV (sepsis-vasopressin) were made septic by fecal peritonitis. Group C and group V were nonseptic control groups. After 300 min, group V and group SV received intravenous infusion of 0.06 U.kg.h vasopressin. In all groups, cardiac index and superior mesenteric artery flow were measured. Microcirculatory blood flow was recorded with laser Doppler flowmetry in both mucosa and muscularis of the stomach, jejunum, and colon.

Results

While vasopressin significantly increased arterial pressure in group SV (P<0.05), superior mesenteric artery flow decreased by 51+/-16% (P<0.05). Systemic and mesenteric oxygen delivery and consumption decreased and oxygen extraction increased in the SV group. Effects on the microcirculation were very heterogeneous; flow decreased in the stomach mucosa (by 23+/-10%; P<0.05), in the stomach muscularis (by 48+/-16%; P<0.05), and in the jejunal mucosa (by 27+/-9%; P<0.05), whereas no significant changes were seen in the colon.

Conclusion

Vasopressin decreased regional flow in the superior mesenteric artery and microcirculatory blood flow in the upper gastrointestinal tract. This reduction in flow and a concomitant increase in the jejunal mucosa-to-arterial carbon dioxide gap suggest compromised mucosal blood flow in the upper gastrointestinal tract in septic pigs receiving low-dose vasopressin.
Roles of Amino Acids and Subunits in Determining the Inhibition of Nicotinic Acetylcholine Receptors by Competitive Antagonists
Authors: Dilger James P. , Vidal Ana Maria , Liu Man , Mettewie Claire , Suzuki Takahiro , Pham Anh , Demazumder Deeptankar
Published: May 2007

Background

Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the alpha-epsilon interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors.

Methods

HEK293 cells were transfected with wild-type or mutant (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates.

Results

Effects of mutations were antagonist specific. alphaY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates.

Conclusion

Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.
Antihyperalgesic Effect of a Recombinant Herpes Virus Encoding Antisense for Calcitonin Gene–related Peptide
Authors: Tzabazis Alexander Z. , Pirc Geanine , Votta-Velis Effrossyni , Wilson Steven P. , Laurito Charles E. , Yeomans David C.
Published: May 2007

Background

Calcitonin gene-related peptide (CGRP) is contained in and released by small-diameter, nociceptive primary afferent sensory neurons. Upon spinal release, one of the effects of CGRP seems to be to sensitize dorsal horn neurons to subsequent input from nociceptive afferents and, consequently, to induce a behavioral hyperalgesia. Therefore, attenuating evoked release of CGRP from central terminals of nociceptors should have an antihyperalgesic effect.

Methods

The authors applied a recombinant herpes vector, encoding an antisense sequence to the whole CGRP gene, to the dorsal surface of the hind paw of mice to knock down expression of the peptide selectively in primary afferents innervating this tissue.

Results

Herpes virus-based vector encoding an antisense sequence for the whole CGRP clearly reduced CGRP immunoreactivity in the infected spinal dorsal horn levels as well as in cultured dorsal root ganglia neurons. Selective knockdown of CGRP in primary afferents significantly attenuated the thermal, C-fiber hyperalgesia normally observed after topical application of capsaicin. The effect of viral vector-mediated knockdown of CGRP was comparable to the effect of intrathecal application of the CGRP antagonist CGRP8-37, but lasted for 14 weeks after one single application.

Conclusion

Viral vector-mediated knockdown of CGRP in primary afferent neurons provides a promising tool for treatment of chronic pain states as well as for studies investigating the pathophysiology underlying these conditions.
Postoperative Analgesic Effects of Continuous Wound Infiltration with Diclofenac after Elective Cesarean Delivery
Authors: Lavand'homme Patricia M. , Roelants Fabienne , Waterloos Hilde , De Kock Marc F.
Published: May 2007

Background

Postoperative pain mostly results from sensitization of afferent fibers at injury sites driving central sensitization. Recently, peripheral processes have gained attention as mechanisms of hyperalgesia, and prostaglandins are among highly sensitizing agents. To date, perioperative administration of a single local dose of nonsteroidal antiinflammatory drugs has shown inconclusive efficacy. Rather than a single bolus, the current study evaluates the postoperative analgesic effect of diclofenac continuous intrawound infusion after elective cesarean delivery.

Methods

Ninety-two parturients were randomly allocated to receive a 48-h continuous intrawound infusion with 240 ml containing 300 mg diclofenac, 0.2% ropivacaine, or saline. In the ropivacaine and saline groups, patients also received 75 mg intravenous diclofenac every 12 h for 48 h. Postoperative evaluation included intravenous morphine consumption by patient-controlled analgesia and visual analog pain scores. Punctate mechanical hyperalgesia surrounding the wound and presence of residual pain after 1 and 6 months were also assessed.

Results

Continuous diclofenac infusion significantly reduced postoperative morphine consumption (18 mg; 95% confidence interval, 12.7-22.2) in comparison with saline infusion and systemic diclofenac (38 mg; 95% confidence interval, 28.8-43.7) (P=0.0009) without unique adverse effects. Postoperative analgesia produced by local diclofenac infusion was as effective as local ropivacaine infusion with systemic diclofenac.

Conclusions

After elective cesarean delivery, continuous intrawound infusion of diclofenac demonstrates a greater opioid-sparing effect and better postoperative analgesia than the same dose administered as an intermittent intravenous bolus.
What Tidal Volumes Should Be Used in Patients without Acute Lung Injury?
Authors: Schultz Marcus J. , Haitsma Jack J. , Slutsky Arthur S. , Gajic Ognjen , Warltier David C.
Published: May 2007

Mechanical ventilation practice has changed over the past few decades, with tidal volumes (VT) decreasing significantly, especially in patients with acute lung injury (ALI). Patients without acute lung injury are still ventilated with large--and perhaps too large--VT. Studies of ventilator-associated lung injury in subjects without ALI demonstrate inconsistent results. Retrospective clinical studies, however, suggest that the use of large VT favors the development of lung injury in these patients. Side effects associated with the use of lower VT in patients with ALI seem to be minimal. Assuming that this will be the case in patients without ALI/acute respiratory distress syndrome too, the authors suggest that the use of lower VT should be considered in all mechanically ventilated patients whether they have ALI or not. Prospective studies should be performed to evaluate optimal ventilator management strategies for patients without ALI.
Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprinting
Authors: Thies Frank L. , König Wolfgang , König Brigitte
Published: May 2007

Bacterial vaginosis (BV) is a prevalent infection in women of reproductive age associated with numerous sequelae, including preterm delivery, amniotic fluid infections and an increased risk of acquiring sexually transmitted diseases. The vaginal microbiota in BV patients is characterized by a shift from lactobacilli to a diverse spectrum of mostly anaerobic bacteria. In this study, terminal restriction fragment length polymorphism (T-RFLP) was used to characterize the vaginal bacterial communities from 50 women with BV and 20 healthy subjects. In the BV samples, 23 species or phylotypes from 17 genera could be identified, including Atopobium vaginae, Megasphaera sp., Lactobacillus iners, Gardnerella vaginalis and three recently described phylotypes from the order Clostridiales. The number of detected species or phylotypes was on average 6.3 per sample (range 2–14). In contrast, in normal samples, only Lactobacillus species could be identified. In conclusion, T-RFLP provides a rapid and reliable technique to investigate the diversity of the predominant vaginal microbiota and allows differentiation of the flora of BV and healthy women. As such, T-RFLP may be helpful both in the diagnosis of BV from vaginal fluids and in a better understanding of the bacterial succession involved in the aetiology of BV.
Evaluation of Albicans ID2 and Biggy agar for the isolation and direct identification of vaginal yeast isolates
Authors: Ilkit Macit , Hilmioglu Suleyha , Tasbakan Meltem , Aydemir Sohret
Published: May 2007

In this study, 250 vaginal samples from patients with vulvovaginal candidosis were inoculated onto two chromogenic media, Albicans ID2 and Biggy agar, as well as onto Sabouraud chloramphenicol agar, yielding a total of 63 yeast (25.2 %) on all three media. These strains were identified as Candida glabrata in 20 (31.8 %) samples, Candida albicans in 15 samples (23.8 %), Candida tropicalis in 10 samples (15.9 %), Candida krusei in five samples (7.9 %), Candida kefyr in five samples (7.9 %), Candida dubliniensis in four samples (6.3 %), Candida parapsilosis in two samples (3.2 %) and Candida guilliermondii in two samples (3.2 %). Mixed fungal cultures and bacterial growth or filamentous fungi were not detected on any of the selected media. The sensitivity and specificity of the Albicans ID2 and Biggy agar with regard to the identification of C. albicans were 80.0 and 64.6 %, and 86.7 and 56.3 %, respectively. This study showed these two chromogenic media to be as effective as Sabouraud chloramphenicol agar with respect to fungal detection. However, neither Albicans ID2 nor Biggy agar was sufficient for reliable differentiation of yeasts to the species level.
Use of a colorimetric assay to measure differences in cytotoxicity of Mycobacterium tuberculosis strains
Authors: Castro-Garza Jorge , Barrios-García Hugo B. , Cruz-Vega Delia Elva , Said-Fernández Salvador , Carranza-Rosales Pilar , Molina-Torres Carmen A. , Vera-Cabrera Lucio
Published: May 2007

Several techniques have been used to quantify the cytotoxicity produced by Mycobacterium tuberculosis bacilli on cell monolayers; however, they are semi-quantitative or time consuming. Herein, a method based on crystal violet (CV) uptake by THP-1 cell monolayers is described. This colorimetric method quantifies the cytotoxic effect as a function of the number of remaining cells after the infection with M. tuberculosis. Since this micro-organism is not stained by the dye, it does not produce a background that affects absorbance readings. As determined by CV assay (CVA), M. tuberculosis strain H37Rv destroyed 10.5 % of THP-1 cell monolayers at 24 h and 50.52 % at 72 h, while M. tuberculosis strains lacking the complete phospholipase C locus produced a reduced cytotoxic effect. The damage estimated by microscopy corresponded to the effect quantified by CVA. The results show that the use of CVA is a rapid, sensitive and reliable quantitative assay to measure the cytotoxicity of different M. tuberculosis strains.
Molecular characterization and analysis of a gene encoding the acidic repeat protein (Arp) of Treponema pallidum
Authors: Liu Hsi , Rodes Berta , George Robert , Steiner Bret
Published: May 2007

The acidic repeat protein (arp) genes from three subspecies of the treponemeTreponema pallidum(T. pallidumsubsp.pallidum, Nichols strain;T. pallidumsubsp.pertenue, CDC-1 and CDC-2 strains; andT. pallidumsubsp.endemicum, Bosnia A strain) were cloned and sequenced. The predicted protein sequence contained a high percentage of glutamic acid, hence the name acidic repeat protein, or Arp. The protein had a potential membrane-spanning domain and a signal peptidase I site. The gene from the Nichols strain ofT. pallidumsubsp.pallidumcontained a set of 14 nearly identical repeats of a 60 bp sequence, which occupied ∼51 % of the length of the gene. Analyses ofarpfrom laboratory strains showed that the 5′ and 3′ ends of the genes were conserved, but there was considerable heterogeneity in the number of repeats of this 60 bp sequence. Based on amino acid variations, the 14 sequence repeats could be classified into three types, which were named type I, type II and type III repeats. The type II repeat was the most common in the strains examined. Thearpgene of the Nichols strain was subsequently cloned into the expression vector pBAD/TOPO ThioFusion. The expressed protein was detected in a Western blot assay using rabbit immune sera produced againstT. pallidum, or synthetic peptides derived from the repeat sequences. Using an ELISA, rapid plasma reagin (RPR) test-positive sera reacted with synthetic peptides derived from the repeat region but not with peptides derived from N and C termini of the Arp protein. These results show that the Arp protein is immunogenic and could prove to be a useful target for serological diagnosis ofT. palliduminfection.
Phenotypic and genotypic traits and epidemiological implication of Vibrio cholerae O1 and O139 strains in India during 2003
Authors: Chatterjee Souvik , Ghosh Kausik , Raychoudhuri Amit , Pan Arpita , Bhattacharya Mihir Kumar , Mukhopadhyay Asish Kumar , Ramamurthy T. , Bhattacharya Sujit Kumar , Nandy Ranjan Kumar
Published: May 2007

During 2003,Vibrio choleraeO1 Ogawa was the predominant serotype among diarrhoeal patients admitted to different hospitals in India. With the exception of 3 strains from Kolkata, none of 172 strains examined exhibited resistance to tetracycline, but 45.7 % showed reduced susceptibility to ciprofloxacin. Extensive molecular characterization using randomly amplified polymorphic DNA analysis, ribotyping and PFGE revealed that almost all the strains within a serogroup were clonally related. Along with the H pulsotype, a newly described L pulsotype of recently emerged O1 Inaba strains was detected among the O1 Ogawa strains from 2003. The striking similarity in their molecular properties and antibiograms indicated that at least certain clones of recently emerged Inaba strains from 2004 may have evolved from O1 Ogawa strains. This view was further supported by the detection of a nearly identicalwbeTregion among the O1 Ogawa and recently emerged Inaba strains, the latter differing only by a single point mutation. Since 2003, a hiatus in the isolation of serogroup O139 was observed and these strains share the same PFGE profiles as those isolated during 2000. Organization of tandemly arranged CTXEl, CTXCaland truncated CTXCal(devoid ofctxAB) prophages was unique among the majority of these O139 strains.
CodY-affected transcriptional gene expression of Streptococcus pyogenes during growth in human blood
Authors: Malke Horst , Ferretti Joseph J.
Published: May 2007

In an attempt to expand the available knowledge of pathogen–host interactions during ex vivo growth of Streptococcus pyogenes (GAS) in nonimmune whole human blood, the extents to which the expression of 51 genes including regulators with known targets, established virulence factors, physiologically important transporters and metabolic enzyme genes was differentially affected in the presence or absence of a functional codY gene were determined. The results obtained by quantitative real-time PCR using the M49 strain NZ131 showed that CodY influenced GAS gene activity in a dynamic fashion, with differential responses detected for 26 genes and occasionally characterized by discordance in the blood environment compared to laboratory medium. Degenerate derivatives of the recently discovered CodY box potentially serving as a cis-regulatory element for CodY action were identified in the upstream regions of 15 genes of the NZ131 genome, and these genes featured sequence motifs identical to the NZ131 CodY box in all completely sequenced S. pyogenes genomes. As none of these genes represented a genuine virulence factor, it seems likely, therefore, that the observed differential transcription of the majority of virulence genes was caused by indirect actions of CodY as part of a regulatory network.
Pneumococci responsible for invasive disease and discharging ears in children in Sydney, Australia
Authors: Watson Michael , Brett Maggie , Brown Mitchell , Stewart Marianne G. , Warren Shirley ,   for the New South Wales Pneumococcal Network
Published: May 2007

The serotypes and molecular clones of penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) responsible for invasive pneumococcal disease (IPD) and discharging ears in metropolitan New South Wales were characterized to form a baseline prior to introduction of the heptavalent conjugate pneumococcal vaccine in Australia. Pneumococci isolated between 1 July 2000 and 30 June 2003 in Sydney from children <15 years were tested for antibiotic susceptibilities and serotyped. Penicillin-nonsusceptible pneumococci were typed by multilocus sequence typing and BOX PCR. During this period, 97 (13.9 %) of 698 pneumococci from IPD that were serotyped were penicillin-nonsusceptible. Of 607 pneumococci from discharging ears, 157 (26.1 %) were penicillin-nonsusceptible. Serotype 14 was the predominant serotype responsible for IPD and serotype 19F predominated from discharging ears. The heptavalent vaccine serotypes accounted for 613 (87.8 %) of all invasive isolates and 420 (69.8 %) of all isolates from discharging ears. Representatives of the major international clones were present among the PNSP. The majority of serotypes and clones that showed penicillin-nonsusceptibility are present within the vaccine. Serotype switching was also noted to have occurred prior to introduction of the vaccine. This study provides a characterization of the pneumococcal serotypes associated with IPD and discharging ears that will be useful for detecting potential selective effects of the vaccine. This surveillance should be continued, as it will be important to monitor the frequency and distribution of serotypes in the post-vaccine era.
Typing of Histoplasma capsulatum strains by fatty acid profile analysis
Authors: Zarnowski Robert , Miyazaki Makoto , Dobrzyn Agnieszka , Ntambi James M. , Woods Jon P.
Published: May 2007

The performance of fatty acid profiling for strain differentiation ofHistoplasma capsulatumwas assessed. Total fatty acids were isolated from the yeast-phase cells of seven stock and two previously unreported clinical strains ofH. capsulatumvar.capsulatum, as well as from one unreported clinical strain and one stock strain ofH. capsulatumvar.duboisii, and one strain of each of three other dimorphic zoopathogenic fungal species,Blastomyces dermatitidis,Paracoccidioides brasiliensisandSporothrix schenckii. Different colony morphology and pigmentation types of theH. capsulatumstrains were also included. The most frequently occurring fatty acids were oleic, palmitic, stearic and linoleic acids. There were variations in the relative percentage fatty acid contents ofH. capsulatumstrains that could be used for strain identification and discrimination. Differentiation betweenH. capsulatumstrains was achieved by the comparison of detected fatty acids accompanied by principal component analysis using calculated Varimax-rotated principal component loadings. Statistical analysis yielded three major principal components that explained over 94 % of total variance in the data. All the strains ofH. capsulatumvar.capsulatumRFLP classes II and III were grouped into two distinct clusters: the heterogenic RFLP class I formed a large, but also well-defined group, whereas the outgroup strains ofH. capsulatumvar.duboisii,B. dermatitidis,P. brasiliensisandS. schenckiiwere shifted away. These data suggest that fatty acid profiling can be used inH. capsulatumstrain classification and epidemiological studies that require strain differentiation at the intraspecies level.
Species identification and strain differentiation of clinical Candida isolates using the DiversiLab system of automated repetitive sequence-based PCR
Authors: Wise Mark G. , Healy Mimi , Reece Kristy , Smith Rebecca , Walton Dobbie , Dutch Wendy , Renwick Alex , Huong Joe , Young Steve , Tarrand Jeffrey , Kontoyiannis Dimitrios P.
Published: May 2007

The DiversiLab system, which uses repetitive sequence-based PCR (rep-PCR) to genotype micro-organisms, was evaluated as a molecular typing tool for members of the genus Candida. Initially, 41 clinical Candida spp. (7 Candida krusei, 10 Candida parapsilosis, 7 Candida albicans, 10 Candida tropicalis and 7 Candida glabrata), previously identified at the species level by morphological and biochemical analysis, were analysed with the DiversiLab system. Species identification was confirmed by DNA sequence analysis of the contiguous internal transcribed spacer (ITS) region (ITS1–5.8S–ITS2). On the basis of an 80 % similarity threshold, rep-PCR consistently clustered like species and this set of isolates, along with five ATCC reference strains, was used to create a DNA fingerprint library with the DiversiLab software. Subsequently, an additional set of 115 clinical Candida isolates, identified biochemically as C. albicans (n=94), C. glabrata (n=8), C. parapsilosis (n=5), C. tropicalis (n=3), C. krusei (n=3) and Candida lusitaniae (n=2), isolated at a regional reference laboratory, were typed using DiversiLab. One hundred and six of these isolates clustered with members of the Candida library at >80 % similarity and thus could be assigned species identification, and initial calculations showed that identification via rep-PCR fingerprinting was 95 % concordant (101/106) with the biochemical/morphological identification. However, ITS region sequencing of the five discrepant samples, as well as the nine isolates that were <80 % similar to the database samples, showed that nine were misidentified with traditional biochemical/morphological methods. For the misidentified isolates, the sequence-based identification was in agreement with the DiversiLab clustering, yielding an actual correlation of >99 %. As traditional techniques can take several days to provide information about Candida at the genus/species level, genotyping with the DiversiLab system holds promise for more-rapid speciation of members of this genus. This system may also be useful for epidemiological studies such as source tracking that require Candida subspecies discrimination.
In vitro activity of piperacillin/tazobactam and ertapenem against Bacteroides fragilis and Escherichia coli in pure and mixed cultures
Authors: Valéria dos Santos Kênia , Diniz Cláudio Galuppo , Coutinho Simone Cristina , Apolônio Ana Carolina Morais , Geralda de Sousa-Gaia Luciana , Nicoli Jacques Robert , Farias Luiz de Macêdo , Roque de Carvalho Maria Auxiliadora
Published: May 2007

Ertapenem and piperacillin/tazobactam are β-lactam antibiotics with a broad spectrum of activity used for the treatment of mixed infections in which Bacteroides fragilis and Escherichia coli play an important aetiological role. In this study, the activities of piperacillin/tazobactam and ertapenem (MIC and time–kill kinetics) against these bacteria were compared. MICs were determined by the agar dilution method, and the time and slope of time–kill curves were analysed. In the in vitro pharmacodynamic assays, pure and mixed cultures of E. coli and B. fragilis were exposed to peak concentrations of ertapenem (8.0 μg ml−1) and piperacillin/tazobactam (64.0/8.0 μg ml−1) for 48 h. Treatment with ertapenem reduced the viability of E. coli and/or B. fragilis by 3 logs in all experiments, whereas piperacillin/tazobactam only affected the viability of B. fragilis. Both drugs exhibited their fastest rates of killing when bacteria were grown in mixed cultures. According to the results, ertapenem exhibited activity similar to that of piperacillin/tazobactam against B. fragilis alone or in mixed culture. However, ertapenem exhibited a markedly higher activity against E. coli alone or in combination with B. fragilis relative to piperacillin/tazobactam.
Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources
Authors: Coote J. G. , Stewart-Tull D. E. S. , Owen R. J. , Bolton F. J. , Siemer Berit L. , Candlish Denise , Thompson D. H. , Wardlaw A. C. , On S. L. W. , Candlish A. , Billcliffe Bronwen , Jordan Penelope J. , Kristiansen K. , Borman Pauline
Published: May 2007

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.
Rapid quantification of hepatitis B virus DNA by direct real-time PCR from serum without DNA extraction
Authors: Cheng Zheng-Jiang , Hu Li-Hua , Fu Wen-Rong , Li Yi-Rong
Published: May 2007

The purpose of this study was to quantify hepatitis B virus DNA by direct real-time PCR from serum without the need for DNA extraction. Crossing point (Cp) values were determined automatically using the second derivative maximum mode. Since serum samples from patients are inevitably haemolysed, lipaemic or icteric, the interference of endogenous substances from the serum in real-time PCR was evaluated. The result showed that, although serum protein quenched the intensity of fluorescence, the Cp value adopted to calculate the quantity of DNA copies remained unchanged. Importantly, real-time PCR from serum with or without DNA extraction reached a high level of concordance. This direct serum PCR method without the DNA extraction and gel electrophoresis allows for substantial labour and cost savings. In addition, it is also suitable for rapid DNA quantification during clinical diagnosis.
Autophagy suppresses tumor progression by limiting chromosomal instability
Authors: Mathew Robin , Kongara Sameera , Beaudoin Brian , Karp Cristina M. , Bray Kevin , Degenhardt Kurt , Chen Guanghua , Jin Shengkan , White Eileen
Published: May 2007

Autophagy is a bulk degradation process that promotes survival under metabolic stress, but it can also be a means of cell death if executed to completion. Monoallelic loss of the essential autophagy gene beclin1 causes susceptibility to metabolic stress, but also promotes tumorigenesis. This raises the paradox that the loss of a survival pathway enhances tumor growth, where the exact mechanism is not known. Here, we show that compromised autophagy promoted chromosome instability. Failure to sustain metabolism through autophagy was associated with increased DNA damage, gene amplification, and aneuploidy, and this genomic instability may promote tumorigenesis. Thus, autophagy maintains metabolism and survival during metabolic stress that serves to protect the genome, providing an explanation for how the loss of a survival pathway leads to tumor progression. Identification of this novel role of autophagy may be important for rational chemotherapy and therapeutic exploitation of autophagy inducers as potential chemopreventive agents.
Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis
Authors: Larsen Rachel A. , Cusumano Christina , Fujioka Akina , Lim-Fong Grace , Patterson Paula , Pogliano Joe
Published: May 2007

Prokaryotes rely on a distant tubulin homolog, FtsZ, for assembling the cytokinetic ring essential for cell division, but are otherwise generally thought to lack tubulin-like polymers that participate in processes such as DNA segregation. Here we characterize a protein (TubZ) from the Bacillus thuringiensis virulence plasmid pBtoxis, which is a member of the tubulin/FtsZ GTPase superfamily but is only distantly related to both FtsZ and tubulin. TubZ assembles dynamic, linear polymers that exhibit directional polymerization with plus and minus ends, movement by treadmilling, and a critical concentration for assembly. A point mutation (D269A) that alters a highly conserved catalytic residue within the T7 loop completely eliminates treadmilling and allows the formation of stable polymers at a much lower protein concentration than the wild-type protein. When expressed in trans, TubZ(D269A) coassembles with wild-type TubZ and significantly reduces the stability of pBtoxis, demonstrating a direct correlation between TubZ dynamics and plasmid maintenance. The tubZ gene is in an operon with tubR, which encodes a putative DNA-binding protein that regulates TubZ levels. Our results suggest that TubZ is representative of a novel class of prokaryotic cytoskeletal proteins important for plasmid stability that diverged long ago from the ancient tubulin/FtsZ ancestor.
Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFκB
Authors: Yang Jinbo , Liao Xudong , Agarwal Mukesh K. , Barnes Laura , Auron Philip E. , Stark George R.
Published: May 2007

gp130-linked cytokines such as interleukin-6 (IL-6) stimulate the formation of tyrosine-phosphorylated signal transducer and activator of transcription 3 (P-STAT3), which activates many genes, including the STAT3 gene itself. The resulting increase in the concentration of unphosphorylated STAT3 (U-STAT3) drives a second wave of expression of genes such asRANTES,IL6,IL8,MET, andMRASthat do not respond directly to P-STAT3. Thus, U-STAT3 sustains cytokine-dependent signaling at late times through a mechanism completely distinct from that used by P-STAT3. Many U-STAT3-responsive genes have κB elements that are activated by a novel transcription factor complex formed when U-STAT3 binds to unphosphorylated NFκB (U-NFκB), in competition with IκB. The U-STAT3/U-NFκB complex accumulates in the nucleus with help from the nuclear localization signal of STAT3, activating a subset of κB-dependent genes. Additional genes respond to U-STAT3 through an NFκB-independent mechanism. The role of signal-dependent increases in U-STAT3 expression in regulating gene expression is likely to be important in physiological responses to gp130-linked cytokines and growth factors that activate STAT3, and in cancers that have constitutively active P-STAT3.