bluuebunny/crossref_metadata_2025 huggingface dataset:
Sumoylation of the P Protein at K254 Plays an Important Role in Growth of Parainfluenza Virus 5
Authors: Sun Dengyun , Xu Pei , He Biao
Published: July 2011

The P protein of parainfluenza virus 5 (PIV5) is an essential cofactor of the viral RNA-dependent RNA polymerase. Phosphorylation of the P protein can positively or negatively regulate viral gene expression, depending on the precise phosphorylation sites. Sumoylation, a process of adding small ubiquitin-like modifier (SUMO) to proteins posttranslationally, plays an important role in regulating protein function. In this study, we have found that the P protein of PIV5 was sumoylated with SUMO1 in both transfected and infected cells. The K254 residue of the P protein is within a consensus sumoylation motif. Mutation of the P protein at K254 to arginine (P-K254R) reduced PIV5 minigenome activity, as well as the sumoylation level of the P protein. Incorporation of K254R into a recombinant PIV5 (rPIV5-P-K254R) resulted in a virus that grew to a lower titer and had lower levels of viral RNA synthesis and protein expression than wild-type PIV5, suggesting that sumoylation of the P protein at K254 is important for PIV5 growth. Biochemical studies did not reveal any defect of P-K254R in its interactions with viral proteins NP and L or formation of homotetramers. We propose that sumoylation of the P protein at K254 regulates PIV5 gene expression through a host protein.
Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location
Authors: Swedan Samer , Andrews Joel , Majumdar Tanmay , Musiyenko Alla , Barik Sailen
Published: July 2011

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression.
Escape from Transcriptional Shutoff during Poliovirus Infection: NF-κB-Responsive Genes IκBa and A20
Authors: Doukas Tammy , Sarnow Peter
Published: July 2011

It has been known for a long time that infection of cultured cells with poliovirus results in the overall inhibition of transcription of most host genes. We examined whether selected host genes can escape transcriptional inhibition by thiouridine marking newly synthesized host mRNAs during viral infection. Using cDNA microarrays hybridized to cDNAs made from thiolated mRNAs, a small set of host transcripts was identified and their expression verified by quantitative PCR and Northern and Western blot analyses. These transcripts were synthesized from genes that displayed enrichment for NF-κB binding sites in their promoter regions, suggesting that some NF-κB-regulated promoters can escape the virus-induced inhibition of transcription. In particular, two negative regulators of NF-κB, IκBa and A20, were upregulated during viral infection. Depletion of A20 enhanced viral RNA abundance and viral yield, arguing that cells respond to virus infection by counteracting NF-κB-induced proviral effects.
Enterovirus 71 and Coxsackievirus A16 3C Proteases: Binding to Rupintrivir and Their Substrates and Anti-Hand, Foot, and Mouth Disease Virus Drug Design
Authors: Lu Guangwen , Qi Jianxun , Chen Zhujun , Xu Xiang , Gao Feng , Lin Daizong , Qian Wangke , Liu Hong , Jiang Hualiang , Yan Jinghua , Gao George F.
Published: July 2011

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of hand, foot, and mouth disease (HFMD), which is prevalent in Asia. Thus far, there are no prophylactic or therapeutic measures against HFMD. The 3C proteases from EV71 and CVA16 play important roles in viral replication and are therefore ideal drug targets. By using biochemical, mutational, and structural approaches, we broadly characterized both proteases. A series of high-resolution structures of the free or substrate-bound enzymes were solved. These structures, together with our cleavage specificity assay, well explain the marked substrate preferences of both proteases for particular P4, P1, and P1′ residue types, as well as the relative malleability of the P2 amino acid. More importantly, the complex structures of EV71 and CVA16 3Cs with rupintrivir, a specific human rhinovirus (HRV) 3C protease inhibitor, were solved. These structures reveal a half-closed S2 subsite and a size-reduced S1′ subsite that limit the access of the P1′ group of rupintrivir to both enzymes, explaining the reported low inhibition activity of the compound toward EV71 and CVA16. In conclusion, the detailed characterization of both proteases in this study could direct us to a proposal for rational design of EV71/CVA16 3C inhibitors.
Characterization of the Interaction between Human Respiratory Syncytial Virus and the Cell Cycle in Continuous Cell Culture and Primary Human Airway Epithelial Cells
Authors: Wu Weining , Munday Diane C. , Howell Gareth , Platt Gareth , Barr John N. , Hiscox Julian A.
Published: July 2011

Viruses can modify conditions inside cells to make them more favorable for replication and progeny virus production. One way of doing this is through manipulation of the cell cycle, a process that describes the ordered growth and division of cells. Analysis of model cell lines, such as A549 cells and primary airway epithelial cells, infected with human respiratory syncytial virus (HRSV) has shown alteration of the cell cycle during infection, although the signaling events were not clearly understood. In this study, targeted transcriptomic analysis of HRSV-infected primary airway epithelial cells revealed alterations in the abundances of many mRNAs encoding cell cycle-regulatory molecules, including decreases in the D-type cyclins and corresponding cyclin-dependent kinases (CDK4 and CDK6 [CDK4/6]). These alterations were reflected in changes in protein abundance and/or relocalization in HRSV-infected cells; taken together, they were predicted to result in G
0
/G
1
phase arrest. In contrast, there was no change in the abundances of D-type cyclins in A549 cells infected with HRSV. However, the abundance of the G
1
/S phase progression inhibitor p21
WAF1/CIP1
was increased over that in mock-treated cells, and this, again, was predicted to result in G
0
/G
1
phase arrest. The G
0
/G
1
phase arrest in both HRSV-infected primary cells and A549 cells was confirmed using dual-label flow cytometry that accurately measured the different stages of the cell cycle. Comparison of progeny virus production in primary and A549 cells enriched in G
0
/G
1
using a specific CDK4/6 kinase inhibitor with asynchronously replicating cells indicated that this phase of the cell cycle was more efficient for virus production.
Epstein-Barr Virus Nuclear Antigen 1 Replication and Segregation Functions in Nasopharyngeal Carcinoma Cell Lines
Authors: Sivachandran Nirojini , Thawe Natalia N. , Frappier Lori
Published: July 2011

Nasopharyngeal carcinomas (NPC) are usually Epstein-Barr virus (EBV) positive, but, with the exception of C666-1 cells, these cells lose the EBV genomes when grown in culture. Maintenance of EBV requires the viral EBV nuclear antigen 1 (EBNA1) protein, which ensures the replication and mitotic segregation of the genomes through interactions with OriP. Here we compare the abilities of C666-1 and NPC cells that have lost EBV genomes to replicate and segregate OriP plasmids. We found that either cell line can replicate and maintain OriP plasmids for extended periods under conditions where low levels of EBNA1 are expressed but that high EBNA1 levels selectively interfered with mitotic segregation.
Estimating person-based injury incidence: accuracy of an algorithm to identify readmissions from hospital discharge data
Authors: Davie Gabrielle , Samaranayaka Ari , Langley John D , Barson Dave
Published: July 2011

Background

Effective use of routinely collected hospital discharge data (HDD) to estimate injury incidence requires a separate identification of new injuries from readmissions for a previous injury. The aim was to determine the accuracy of a computerised algorithm to identify injury readmissions in HDD.

Methods

A random sample of 2000 events (‘key events’) were selected from the 2006 injury subset of New Zealand's HDD. Discharge histories from 1989 to 2007 were extracted for individuals and manually reviewed by at least two people to determine the ‘gold standard’ readmission status of each key event. The algorithm relies on four variables: unique national person identifier, dates of injury, admission and discharge. Reviewers were provided with these variables as well as additional discharge information (eg, discharge type and external cause code narrative) recorded in the HDD. Results of the manual review were compared to those obtained from the algorithm.

Results

The algorithm assigned 1811 (90.6%) as incident admissions compared to 1800 (90.0%) classified by the gold standard. Agreement was 97.9%, and accuracy measures (sensitivity, specificity, negative predictive value and positive predictive value) ranged from 87% to 99%. No statistically significant differences between readmission assignation by the algorithm and the gold standard were observed by age, nature of injury, external cause of injury or body region.

Conclusions

Any country with electronic HDD could readily identify readmissions and, thus, accurately estimate injury incidence from HDD, providing that a unique person identifier and the date of injury were included in addition to the obligatory dates of admission and discharge.
Regulation breach on farm
Authors: Steele Mike
Published: July 2011

A farmer client, known to be sceptical of all things veterinary, requests a visit. Examination of a pregnant suckler cow reveals an infected, ulcerated growth on its third eyelid. You provisionally diagnose squamous cell carcinoma of the third eyelid, and suggest removal of the mass under local anaesthetic, with the caveat that by the time the cow had calved and reared its calf, the growth would probably have returned and culling would then be recommended. However, the client insists on palliative treatment with a time limit, without removal of the mass. A courtesy visit a week later reveals that the cow is showing signs of pain from the growth. On arrival at the farm, however, you notice several untagged dead calves and two dead sheep in a ditch on the farmer's land by the entrance. How do you approach the farmer to remind him of his responsibilities to both the cow's welfare and the dead animals?
Understanding viruses
Authors: Erles Kerstin
Published: July 2011

Viruses are important pathogens that are frequently encountered in veterinary practice. The diagnosis of viral infections has become more readily available owing to the development of rapid molecular methods. This article explains the basic properties of viruses, how they interact with their host and how this knowledge aids the diagnosis and control of infection. It also briefly discusses different diagnostic techniques and the challenges posed by viral evolution.
Treatment of high risk symptomatic intracranial atherosclerosis with balloon mounted coronary stents and Wingspan stents: single center experience over a 10 year period
Authors: Tarlov Nicholas , Jahan Reza , Saver Jeffrey L , Sayre James W , Ali Latisha K , Kim Doojin , Duckwiler Gary R , Tateshima Satoshi , Viñuela Fernando , Liebeskind David S
Published: July 2011

Background

Stenting of symptomatic intracranial atherosclerosis remains under investigation, yet this option to potentially avert subsequent stroke has been offered at select centers under humanitarian device exemption and off-label use for several years.

Methods

Retrospective case series of consecutive patients undergoing stenting with Wingspan and balloon mounted coronary stents for symptomatic intracranial atherosclerosis at a single institution. Recurrent symptomatic ischemia in the territory of the stented artery was ascertained. Rates of recurrent ischemic stroke were calculated per patient-year of follow-up and were compared with medically treated patients in the Warfarin–Aspirin Symptomatic Intracranial Disease (WASID) trial.

Results

During the 10 year study period, 41 cases of intracranial stenting were identified. Stenoses were severe (>70%) in 88% of patients. Stenting procedures occurred a median of 14 days from the most recent symptomatic event. 19 Wingspan stents and 22 balloon mounted coronary stents were deployed. Four strokes occurred within 24 h of stenting, seven within 1 month and eight within 3 months. By 3 months after stenting, no further strokes occurred during up to 2 years of follow-up. Patients had 0.194 ischemic strokes per person-year of follow-up, compared with 0.083 ischemic strokes per person-year of follow-up in the aspirin arm of WASID and 0.065 ischemic strokes per person-year of follow-up in the warfarin arm of WASID.

Conclusions

Stenting of symptomatic intracranial atherosclerosis in a high risk subset of cases with advanced degree of luminal stenosis may be associated with an increased early risk of recurrent ischemic stroke.
Farming, growing up on a farm, and haematological cancer mortality
Authors: Mannetje Andrea 't , Eng Amanda , Pearce Neil
Published: July 2011

Objectives

Occupation as a farmer has been associated with increased risks of haematological cancers in adults. This study aimed to examine whether farm exposures in childhood contribute to these risks, by using parental occupation in farming as a proxy for growing up on a farm.

Methods

New Zealand death records (1998-2003) of persons aged 35-85 were extracted (n=114 289). For 82.3% usual occupation and the occupation of at least one of the parents could be coded (n=94 054). Unconditional logistic regression analyses included 3119 haematological cancer deaths (cases) and 90 935 deaths from other causes (controls). ORs for farming and growing up on a farm were adjusted for each other, year of birth, age at death, socio-economic status, Māori ethnicity, immigration status and sex.

Results

Growing up on a livestock farm was positively associated with haematological cancer (OR 1.22, 95% CI 1.05 to 1.41), particularly for poultry farms (OR 2.99, 95% CI 1.44 to 6.21), while growing up on a crop farm was not (OR 0.81, 95% CI 0.64 to 1.03). Crop farming in adulthood was associated with an increased haematological cancer risk (OR 1.49, 95% CI 1.13 to 1.96), while livestock farming was not (OR 0.80, 95% CI 0.63 to 1.00), except for beef cattle farming (OR 2.99, 95% CI 1.28 to 7.00). These results did not change appreciably when different control groups with different causes of death were used.

Conclusions

These results could suggest a role for early life biological exposures in the development of haematological cancers.
Home‐based subcutaneous continuous glucose monitoring in 10 diabetic dogs
Authors: Affenzeller N. , Thalhammer J. G. , Willmann M.
Published: July 2011

A subcutaneous continuous glucose monitoring system (GlucoDay; Menarini Diagnostics) based on microdialysis was investigated for its clinical applicability in veterinary medicine. Ten diabetic dogs, referred as clinically stable, were equipped with this system and sent home for a maximum observation period of 48 hours. Time of insulin administration, feeding and other events were written in a diary and plotted afterwards in the glucose graph. Implantation of the microdialysis fibre, acceptance of the device and evaluation of individual canine glucose profiles were without complication. Based on the monitoring data, recommended treatment adjustments were given to the referring veterinarians in all 10 dogs; hypoglycaemic or prolonged hyperglycaemic episodes were detected in six dogs.
Haematological analysis of calves with bovine neonatal pancytopenia
Authors: Witt K. , Weber C. N. , Meyer J. , Buchheit‐Renko S. , Müller K. E.
Published: July 2011

Blood was obtained from 61 neonatal Holstein calves originating from a farm in Germany with a high incidence of bovine neonatal pancytopenia (BNP). In order to detect alterations that might be related to BNP, selected haematological analytes were determined. Haematological examinations demonstrated alterations in at least two of the three cell lineages in 10 calves (16.39 per cent). Six animals (9.84 per cent) developed a bleeding disorder indicative of BNP at approximately two weeks of age. None of these animals showed alterations in complete blood cell count at sampling in the first week of life. In weeks when calves with BNP were born, an increase in the number of apparently healthy calves demonstrating decreases in blood cell counts was observed.
Evidence-based risk assessment and recommendations for physical activity clearance: stroke and spinal cord injury1This paper is one of a selection of papers published in this Special Issue, entitled Evidence-based risk assessment and recommendations for physical activity clearance, and has undergone the Journal’s usual peer review process.
Authors: Zehr E. Paul
Published: July 2011

Physical activity (PA) has potential benefits after stroke or spinal cord injury (SCI), especially in improving efficiency and functional capacity in activities of daily living. Currently, many who could benefit from PA may be routinely excluded from participation because of myths related to functional capacity and the concern for harm. The purpose of this review was to evaluate the literature for reports of adverse events during exercise after stroke or SCI, and to provide recommendations regarding exercise participation in supervised and unsupervised environments. Studies were evaluated for quality, and the summary level and quality of evidence were evaluated using the AGREE rubric, modified to address the main outcome measure of adverse events. Levels of exercise stress were evaluated for aerobic activities, using an established rubric. Included in the current analysis were 32 studies for stroke and 4 for SCI. In aggregate, this yielded a total of 730 experimental participants with stroke and 143 with SCI. It should be noted that almost all studies were not designed to examine naturally occurring adverse events from PA. Significant contraindications to unsupervised exercise include manifestation of autonomic dysreflexia in SCI and cardiovascular comorbidity after stroke. There are clear benefits of exercise training on physiological outcomes in stroke and SCI, but the relation between outcomes and safety remains unclear. However, taken on balance, the risk-to-benefit ratio favors the recommendation of exercise. This recommendation is based on studies in which participants were almost universally screened for participation in supervised environments. Thus, the grading of evidence for finding adverse events to support this conclusion is inadequate.
STATE OWNERSHIP, CORPORATE TOURNAMENT AND EXECUTIVE COMPENSATION: EVIDENCE FROM PUBLIC LISTED FIRMS IN CHINA
Authors: SU DONGWEI
Published: July 2011

This article tests several predictions of tournament theory on executive compensation in the context of a transition economy. Using an unbalanced panel of 34,701 executives in 1,386 public listed firms in China during 1999 and 2006, the paper finds that (1) pay increases as executives move up the corporate hierarchy into higher ranks; (2) pay gap is the largest between the first- and second-tier executives; (3) pay dispersion increases with the number of tournament participants and the risk of the business environment; (4) state ownership of shares reduces pay, pay gap and the sensitivities of the contestant pool and business risk to pay dispersion; (5) board composition and independence, CEO duality and the independence of the supervisory committee all affect pay and pay dispersion. Overall, this paper shows that listed firms in China, as they become more and more market-oriented, have adopted a pay structure that is largely consistent with the predictions of tournament theory, and that it is important to consider both state ownership and corporate governance in analyzing executive compensation structure.
Structural investigations of hexadecafluoro(phthalocyaninato)ruthenium(II) F16PcRu with EXAFS spectroscopy
Authors: Bertagnolli Helmut , Krishnan Venkata , Youssef Tamer Ezzat , Hanack Michael
Published: July 2011

Amorphous hexadecafluoro(phthalocyaninato)ruthenium(II) F16PcRu (1) has been studied with EXAFS spectroscopy. It has been shown that F16PcRu has a dimeric structure in the solid state.
Porphyrin hexamer with a triphenylene core unit: Spectroscopy, electrochemistry and controllable supramolecular formation
Authors: Sakai Hayato , Rabbani Mohammad Gulam , Hasobe Taku
Published: July 2011

A series of free-base and zinc porphyrin hexamers, where six porphyrin units are linked to a triphenylene core through amide or ester linkage, was designed and synthesized in an effort to study their spectroscopic and electrochemical properties, and aggregate formation. The distances between triphenylene core and porphyrin moieties are tuned by changing the lengths of spacer alkyl chains. The absorption spectral studies indicates that the porphyrin units in a hexamer behave like monomeric porphyrins, while a strong fluorescence quenching of triphenylene core was observed suggesting the deactivation of the excited state of triphenylene. The redox potentials of porphyrin units in a hexamer were estimated by differential pulse voltammetry (DPV). These values are very similar to those of the corresponding monomer. Consequently, absorption and electrochemical data demonstrate that six porphyrin units of (H2PAC5)6TPh behave like monomers independently in the ground state. The hexamers undergo supramolecular aggregation spontaneously in different fashions based on the solvent systems as well as spacer lengths. Highly ordered and aligned monolayer-based patterns are formed when hexamers are allowed to aggregate from toluene solution onto carbon-coated copper films. In contrast, hexamers undergo aggregation to nanoparticles in toluene/acetonitrile mixed solvent system and the particle sizes increase with increasing the spacer alkyl chain lengths.
Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor
Authors: Murphy Jane E. , Roosterman Dirk , Cottrell Graeme S. , Padilla Benjamin E. , Feld Micha , Brand Eva , Cedron Wendy J. , Bunnett Nigel W. , Steinhoff Martin
Published: July 2011

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.
Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells
Authors: Chou Feng-Pai , Xu Han , Lee Ming-Shyue , Chen Ya-Wen , Durand Richards O. X. , Swanson Richard , Olson Steven T. , Johnson Michael D. , Lin Chen-Yong
Published: July 2011

Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptase-antithrombin complexes were not detected in all but two of a panel of seven breast, prostate, and ovarian cancer cell lines. Using purified active matriptase, we further characterize the formation of matriptase-antithrombin complex and show that heparin can significantly potentiate the inhibitory potency of antithrombin against matriptase. Second-order rate constants for the inhibition were determined to be 3.9 × 103 M−1s−1 in the absence of heparin and 1.2 × 105 M−1s−1 in the presence of heparin, a 30-fold increase, consistent with the established role of heparin in activating antithrombin function. Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells.
Alanyl-glutamine promotes intestinal epithelial cell homeostasis in vitro and in a murine model of weanling undernutrition
Authors: Ueno Priscilla M. , Oriá Reinaldo B. , Maier Elizabeth A. , Guedes Marjorie , de Azevedo Orleancio G. , Wu David , Willson Tara , Hogan Simon P. , Lima Aldo A. M. , Guerrant Richard L. , Polk D. Brent , Denson Lee A. , Moore Sean R.
Published: July 2011

Alanyl-glutamine (Ala-Gln) has recently been shown to enhance catch-up growth and gut integrity in undernourished children from Northeast Brazil. We hypothesized that the intestinal epithelial effects of Ala-Gln in malnourished weanling mice and mouse small intestinal epithelial (MSIE) cells would include modulation of barrier function, proliferation, and apoptosis. Dams of 10-day-old suckling C57BL/6 pups were randomized to a standard diet or an isocaloric Northeast Brazil “regional basic diet,” moderately deficient in protein, fat, and minerals. Upon weaning to their dam's diet on day of life 21, pups were randomized to Ala-Gln solution or water. At 6 wk of age, mice were killed, and jejunal tissue was collected for morphology, immunohistochemistry, and Ussing chamber analysis of transmucosal resistance and permeability. Proliferation of MSIE cells in the presence or absence of Ala-Gln was measured by MTS and bromodeoxyuridine assays. MSIE apoptosis was assessed by annexin and 7-amino-actinomycin D staining. Pups of regional basic diet-fed dams exhibited failure to thrive. Jejunal specimens from undernourished weanlings showed decreased villous height and crypt depth, decreased transmucosal resistance, increased permeability to FITC-dextran, increased claudin-3 expression, and decreased epithelial proliferation and increased epithelial apoptosis (as measured by bromodeoxyuridine and cleaved caspase-3 staining, respectively). Undernourished weanlings supplemented with Ala-Gln showed improvements in weight velocity, villous height, crypt depth, transmucosal resistance, and epithelial proliferation/apoptosis compared with unsupplemented controls. Similarly, Ala-Gln increased proliferation and reduced apoptosis in MSIE cells. In summary, Ala-Gln promotes intestinal epithelial homeostasis in a mouse model of malnutrition-associated enteropathy, mimicking key features of the human disease.
RUNX transcription factors: association with pediatric asthma and modulated by maternal smoking
Authors: Haley Kathleen J. , Lasky-Su Jessica , Manoli Sara E. , Smith Lacey A. , Shahsafaei Aliakbar , Weiss Scott T. , Tantisira Kelan
Published: July 2011

Intrauterine smoke exposure (IUS) is a strong risk factor for development of airways responsiveness and asthma in childhood. Runt-related transcription factors (RUNX1–3) have critical roles in immune system development and function. We hypothesized that genetic variations in RUNX1 would be associated with airway responsiveness in asthmatic children and that this association would be modified by IUS. Family-based association testing analysis in the Childhood Asthma Management Program genome-wide genotype data showed that 17 of 100 RUNX1 single-nucleotide polymorphisms (SNPs) were significantly ( P < 0.03–0.04) associated with methacholine responsiveness. The association between methacholine responsiveness and one of the SNPs was significantly modified by a history of IUS exposure. Quantitative PCR analysis of immature human lung tissue with and without IUS suggested that IUS increased RUNX1 expression at the pseudoglandular stage of lung development. We examined these associations by subjecting murine neonatal lung tissue with and without IUS to quantitative PCR ( N = 4–14 per group). Our murine model showed that IUS decreased RUNX expression at postnatal days (P)3 and P5 ( P < 0.05). We conclude that 1) SNPs in RUNX1 are associated with airway responsiveness in asthmatic children and these associations are modified by IUS exposure, 2) IUS tended to increase the expression of RUNX1 in early human development, and 3) a murine IUS model showed that the effects of developmental cigarette smoke exposure persisted for at least 2 wk after birth. We speculate that IUS exposure-altered expression of RUNX transcription factors increases the risk of asthma in children with IUS exposure.
Intrinsic contractile properties of the crucian carp (Carassius carassius) heart during anoxic and acidotic stress
Authors: Stecyk Jonathan A. W. , Larsen Bent C. , Nilsson Göran E.
Published: July 2011

The crucian carp ( Carassius carassius ) seems unique among vertebrates in its ability to maintain cardiac performance during prolonged anoxia. We investigated whether this phenomenon arises in part from a myocardium tolerant to severe acidosis or because the anoxic crucian carp heart may not experience a severe extracellular acidosis due to the fish's ability to convert lactate to ethanol. Spontaneously contracting heart preparations from cold-acclimated (6–8°C) carp were exposed (at 6.5°C) to graded or ungraded levels of acidosis under normoxic or anoxic conditions and intrinsic contractile performance was assessed. Our results clearly show that the carp heart is tolerant of acidosis as long as oxygen is available. However, heart rate and contraction kinetics of anoxic hearts were severely impaired when extracellular pH was decreased below 7.4. Nevertheless, the crucian carp heart was capable of recovering intrinsic contractile performance upon reoxygenation regardless of the severity of the anoxic + acidotic insult. Finally, we show that increased adrenergic stimulation can ameliorate, to a degree, the negative effects of severe acidosis on the intrinsic contractile properties of the anoxic crucian carp heart. Combined, these findings indicate an avoidance of severe extracellular acidosis and adrenergic stimulation are two important factors protecting the intrinsic contractile properties of the crucian carp heart during prolonged anoxia, and thus likely facilitate the ability of the anoxic crucian carp to maintain cardiac pumping.
Peripheral microvascular response to muscle contraction is unaltered by early diabetes but decreases with age
Authors: Slade Jill M. , Towse Theodore F. , Gossain Ved V. , Meyer Ronald A.
Published: July 2011

Long-term or untreated diabetes leads to micro- and macrovascular complications. However, there are few tests to evaluate microvascular function. A postcontraction blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) technique was exploited to measure peripheral microvascular function in diabetics and healthy controls matched with respect to age, body mass index, and physical activity. Postcontraction BOLD microvascular response was measured following 1-s maximal isometric ankle dorsiflexion in individuals with diabetes mellitus type I [DMI, n = 15, age 33 ± 3 yr (means ± SE), median diabetes duration = 5.5 yr] and type II (DMII, n = 16, age 45 ± 2 yr, median duration = 2.4 yr); responses were compared with controls (CONI and CONII). Peripheral macrovascular function of the popliteal and tibial arteries was assessed during exercise hyperemia with phase contrast magnetic resonance angiography following repetitive exercise. There were no group differences as a result of diabetes in peripheral microvascular function (peak BOLD response: DMI = 2.04 ± 0.38% vs. CONI = 2.08 ± 0.48%; DMII = 0.93 ± 0.24% vs. CONII = 1.13 ± 0.24%; mean ± SE), but the BOLD response was significantly influenced by age (partial r = −0.384, P = 0.003), supporting its sensitivity as a measure of microvascular function. Eleven individuals had no microvascular BOLD response, including three diabetics with neuropathy and four controls with a family history of diabetes. There were no differences in peripheral macrovascular function between groups when assessing exercise hyperemia or the pulsitility and resistive indexes. Although the BOLD microvascular response was not impaired in early diabetes, these results encourage further investigation of muscle BOLD as it relates to peripheral microvascular health.
Effect of inspired air conditions on exercise-induced bronchoconstriction and urinary CC16 levels in athletes
Authors: Bolger C. , Tufvesson E. , Anderson S. D. , Devereux G. , Ayres J. G. , Bjermer L. , Sue-Chu M. , Kippelen P.
Published: July 2011

Injury to the airway epithelium has been proposed as a key susceptibility factor for exercise-induced bronchoconstriction (EIB). Our goals were to establish whether airway epithelial cell injury occurs during EIB in athletes and whether inhalation of warm humid air inhibits this injury. Twenty-one young male athletes (10 with a history of EIB) performed two 8-min exercise tests near maximal aerobic capacity in cold dry (4°C, 37% relative humidity) and warm humid (25°C, 94% relative humidity) air on separate days. Postexercise changes in urinary CC16 were used as a biomarker of airway epithelial cell perturbation and injury. Bronchoconstriction occurred in eight athletes in the cold dry environment and was completely blocked by inhalation of warm humid air [maximal fall in forced expiratory volume in 1 s = 18.1 ± 2.1% (SD) in cold dry air and 1.7 ± 0.8% in warm humid air, P < 0.01]. Exercise caused an increase in urinary excretion of CC16 in all subjects ( P < 0.001), but this rise in CC16 was blunted following inhalation of warm humid air [median CC16 increase pre- to postchallenge = 1.91 and 0.35 ng/μmol in cold dry and warm humid air, respectively, in athletes with EIB ( P = 0.017) and 1.68 and 0.48 ng/μmol in cold dry and warm humid air, respectively, in athletes without EIB ( P = 0.002)]. The results indicate that exercise hyperpnea transiently disrupts the airway epithelium of all athletes (not only in those with EIB) and that inhalation of warm moist air limits airway epithelial cell perturbation and injury.
Progressive adaptation in regional parenchyma mechanics following extensive lung resection assessed by functional computed tomography
Authors: Yilmaz Cuneyt , Tustison Nicholas J. , Dane D. Merrill , Ravikumar Priya , Takahashi Masaya , Gee James C. , Hsia Connie C. W.
Published: July 2011

In adult canines following major lung resection, the remaining lobes expand asymmetrically, associated with alveolar tissue regrowth, remodeling, and progressive functional compensation over many months. To permit noninvasive longitudinal assessment of regional growth and function, we performed serial high-resolution computed tomography (HRCT) on six male dogs (∼9 mo old, 25.0 ± 4.5 kg, ±SD) at 15 and 30 cmH2O transpulmonary pressure (Ptp) before resection (PRE) and 3 and 15 mo postresection (POST3 and POST15, respectively) of 65–70% of lung units. At POST3, lobar air volume increased 83–148% and tissue (including microvascular blood) volume 120–234% above PRE values without further changes at POST15. Lobar-specific compliance (Cs) increased 52–137% from PRE to POST3 and 28–79% from POST3 to POST15. Inflation-related parenchyma strain and shear were estimated by detailed registration of corresponding anatomical features at each Ptp. Within each lobe, regional displacement was most pronounced at the caudal region, whereas strain was pronounced in the periphery. Regional three-dimensional strain magnitudes increased heterogeneously from PRE to POST3, with further medial-lateral increases from POST3 to POST15. Lobar principal strains (PSs) were unchanged or modestly elevated postresection; changes in lobar maximum PS correlated inversely with changes in lobar air and tissue volumes. Lobar shear distortion increased in coronal and transverse planes at POST3 without further changes thereafter. These results establish a novel use of functional HRCT to map heterogeneous regional deformation during compensatory lung growth and illustrate a stimulus-response feedback loop whereby postresection mechanical stress initiates differential lobar regrowth and sustained remodeling, which in turn, relieves parenchyma stress and strain, resulting in progressive increases in lobar Cs and a delayed increase in whole lung Cs.
Lung injury caused by high tidal volume mechanical ventilation and hyperoxia is dependent on oxidant-mediated c-Jun NH2-terminal kinase activation
Authors: Makena Patrudu S. , Gorantla Vijay K. , Ghosh Manik C. , Bezawada Lavanya , Balazs Louisa , Luellen Charlean , Parthasarathi Kuashik , Waters Christopher M. , Sinclair Scott E.
Published: July 2011

Both prolonged exposure to hyperoxia and large tidal volume mechanical ventilation can each independently cause lung injury. However, the combined impact of these insults is poorly understood. We recently reported that preexposure to hyperoxia for 12 h, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone (Makena et al. Am J Physiol Lung Cell Mol Physiol 299: L711–L719, 2010). The upstream mechanisms of this lung injury and apoptosis have not been clearly elucidated. We hypothesized that lung injury in this model was dependent on oxidative signaling via the c-Jun NH2-terminal kinases (JNK). We, therefore, evaluated lung injury and apoptosis in the presence of N-acetyl-cysteine (NAC) in both mouse and cell culture models, and we provide evidence that NAC significantly inhibited lung injury and apoptosis by reducing the production of ROS, activation of JNK, and apoptosis. To confirm JNK involvement in apoptosis, cells treated with a specific JNK inhibitor, SP600125, and subjected to preexposure to hyperoxia, followed by mechanical stretch, exhibited significantly reduced evidence of apoptosis. In conclusion, lung injury and apoptosis caused by preexposure to hyperoxia, followed by high tidal volume mechanical ventilation, induces ROS-mediated activation of JNK and mitochondrial-mediated apoptosis. NAC protects lung injury and apoptosis by inhibiting ROS-mediated activation of JNK and downstream proapoptotic signaling.
Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABAA receptors
Authors: Meera Pratap , Wallner Martin , Otis Thomas S.
Published: July 2011

Extrasynaptic GABAA receptors (eGABARs) allow ambient GABA to tonically regulate neuronal excitability and are implicated as targets for ethanol and anesthetics. These receptors are thought to be heteropentameric proteins made up of two α subunits—either α4 or α6—two β2 or β3 subunits, and one δ subunit. The GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4- c)pyridin-3(-ol) (THIP) has been proposed as a selective ligand for eGABARs. Behavioral and in vitro studies suggest that eGABARs have nanomolar affinity for THIP; however, all published studies on recombinant versions of eGABARs report micromolar affinities. Here, we examine THIP sensitivity of native eGABARs on cerebellar neurons and on reconstituted GABARs in heterologous systems. Concentration-response data for THIP, obtained from cerebellar granule cells and molecular layer interneurons in wild-type and δ subunit knockout slices, confirm that submicromolar THIP sensitivity requires δ subunits. In recombinant experiments, we find that δ subunit coexpression leads to receptors activated by nanomolar THIP concentrations (EC50 of 30–50 nM for α4β3δ and α6β3δ), a sensitivity almost 1,000-fold higher than receptors formed by α4/6 and β3 subunits. In contrast, γ2 subunit expression significantly reduces THIP sensitivity. Even when δ subunit cDNA or cRNA was supplied in excess, high- and low-sensitivity THIP responses were often apparent, indicative of variable mixtures of low-affinity αβ and high-affinity αβδ receptors. We conclude that δ subunit incorporation into GABARs leads to a dramatic increase in THIP sensitivity, a defining feature that accounts for the unique behavioral and neurophysiological properties of THIP.
Representation of limb kinematics in Purkinje cell simple spike discharge is conserved across multiple tasks
Authors: Hewitt Angela L. , Popa Laurentiu S. , Pasalar Siavash , Hendrix Claudia M. , Ebner Timothy J.
Published: July 2011

Encoding of movement kinematics in Purkinje cell simple spike discharge has important implications for hypotheses of cerebellar cortical function. Several outstanding questions remain regarding representation of these kinematic signals. It is uncertain whether kinematic encoding occurs in unpredictable, feedback-dependent tasks or kinematic signals are conserved across tasks. Additionally, there is a need to understand the signals encoded in the instantaneous discharge of single cells without averaging across trials or time. To address these questions, this study recorded Purkinje cell firing in monkeys trained to perform a manual random tracking task in addition to circular tracking and center-out reach. Random tracking provides for extensive coverage of kinematic workspaces. Direction and speed errors are significantly greater during random than circular tracking. Cross-correlation analyses comparing hand and target velocity profiles show that hand velocity lags target velocity during random tracking. Correlations between simple spike firing from 120 Purkinje cells and hand position, velocity, and speed were evaluated with linear regression models including a time constant, τ, as a measure of the firing lead/lag relative to the kinematic parameters. Across the population, velocity accounts for the majority of simple spike firing variability (63 ± 30% of Radj2), followed by position (28 ± 24% of Radj2) and speed (11 ± 19% of Radj2). Simple spike firing often leads hand kinematics. Comparison of regression models based on averaged vs. nonaveraged firing and kinematics reveals lower Radj2values for nonaveraged data; however, regression coefficients and τ values are highly similar. Finally, for most cells, model coefficients generated from random tracking accurately estimate simple spike firing in either circular tracking or center-out reach. These findings imply that the cerebellum controls movement kinematics, consistent with a forward internal model that predicts upcoming limb kinematics.
Functional magnetic resonance adaptation reveals the involvement of the dorsomedial stream in hand orientation for grasping
Authors: Monaco Simona , Cavina-Pratesi Cristiana , Sedda Anna , Fattori Patrizia , Galletti Claudio , Culham Jody C.
Published: July 2011

Reach-to-grasp actions require coordination of different segments of the upper limbs. Previous studies have examined the neural substrates of arm transport and hand grip components of such actions; however, a third component has been largely neglected: the orientation of the wrist and hand appropriately for the object. Here we used functional magnetic resonance imaging adaptation (fMRA) to investigate human brain areas involved in processing hand orientation during grasping movements. Participants used the dominant right hand to grasp a rod with the four fingers opposing the thumb or to reach and touch the rod with the knuckles without visual feedback. In a control condition, participants passively viewed the rod. Trials in a slow event-related design consisted of two sequential stimuli in which the rod orientation changed (requiring a change in wrist posture while grasping but not reaching or looking) or remained the same. We found reduced activation, that is, adaptation, in superior parieto-occipital cortex (SPOC) when the object was repeatedly grasped with the same orientation. In contrast, there was no adaptation when reaching or looking at an object in the same orientation, suggesting that hand orientation, rather than object orientation, was the critical factor. These results agree with recent neurophysiological research showing that a parieto-occipital area of macaque (V6A) is modulated by hand orientation during reach-to-grasp movements. We suggest that the human dorsomedial stream, like that in the macaque, plays a key role in processing hand orientation in reach-to-grasp movements.