Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress

The NH2-terminal Jun kinases (JNKs) function in diverse roles through phosphorylation and activation of AP-1 components including ATF2 and c-Jun. However, the genes that mediate these processes are poorly understood. A model phenotype characterized by rapid activation of Jun kinase and enhanced DNA repair following cisplatin treatment was examined using chromatin immunoprecipitation with antibodies against ATF2 and c-Jun or their phosphorylated forms and hybridization to promoter arrays. Following genotoxic stress, we identified 269 genes whose promoters are bound upon phosphorylation of ATF2 and c-Jun. Binding did not occur following treatment with transplatin or the JNK inhibitor SP600125 or JNK-specific siRNA. Of 89 known DNA repair genes represented on the array, 23 are specifically activated by cisplatin treatment within 3-6 hr. Thus, the genotoxic stress response occurs at least partly via activation of ATF2 and c-Jun, leading to large-scale coordinate gene expression dominated by genes of DNA repair.     

Application of large-eddy simulation to the study of pulsatile flow in a modeled arterial stenosis

The technique of large-eddy simulation (LES) has been applied to the study of pulsatile flow through a modeled arterial stenosis. A simple stenosis model has been used that consists of a one-sided 50 percent semicircular constriction in a planar channel. The inlet volume flux is varied sinusoidally in time in a manner similar to the laminar flow simulations of Tutty (1992). LES is used to compute flow at a peak Reynolds number of 2000 and a Strouhal number of 0.024. At this Reynolds number, the flow downstream of the stenosis transitions to turbulence and exhibits all the classic features of post-stenotic flow as described by Khalifa and Giddens (1981) and Lieber and Giddens (1990). These include the periodic shedding of shear layer vortices and transition to turbulence downstream of the stenosis. Computed frequency spectra indicate that the vortex shedding occurs at a distinct high frequency, and the potential implication of this for noninvasive diagnosis of arterial stenoses is discussed. A variety of statistics have been also extracted and a number of other physical features of the flow are described in order to demonstrate the usefulness of LES for the study of post-stenotic flows.     

Distinct sites on G protein beta gamma subunits regulate different effector functions

G proteins interact with effectors at multiple sites and regulate their activity. The functional significance of multiple contact points is not well understood. We previously identified three residues on distinct surfaces of Gbetagamma that are crucial for G protein-coupled inward rectifier K(+) (GIRK) channel activation. Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K(+) currents. Each of the three Gbeta(1) mutants bound the channel indicating that the defects reflected mainly functional impairments. We tested these mutants for functional interactions with effectors other than K(+) channels. With N-type calcium channels, Gbetagamma wild type and mutants all inhibited basal currents. A depolarizing pre-pulse relieved Gbetagamma inhibition of Ca(2+) currents by the wild type and the S98T and T128F mutants but not the S67K mutant. Both wild type and mutant Gbetagamma subunits activated phospholipase C beta(2) with similar potencies; however, the S67K mutant showed reduced maximal activity. These data establish a pattern where mutations can alter the Gbetagamma regulation of a specific effector function without affecting other Gbetagamma-mediated functions. Moreover, Ser-67 showed this pattern in all three effectors tested, suggesting that this residue participates in a common functional domain on Gbeta(1) that regulates several effectors. These data show that distinct domains within Gbetagamma subserve specific functional roles.     

Relationship between esophageal muscle thickness and intraluminal pressure: an ultrasonographic study

A number of studies show a close temporal relationship between the rate of change in muscle thickness as detected by high-frequency intraluminal ultrasonography (HFIUS) and intraluminal pressure measured by manometry. There is a marked variability in esophageal contraction amplitude from one swallow to another at a given level in the esophagus and along the length of the esophagus. Furthermore, peristaltic pressures are higher in the distal compared with the proximal esophagus. The goal of this study was to evaluate the relationship between the baseline and peak muscle thickness and the contraction amplitude during swallow-induced contractions along the length of the esophagus. Fifteen normal subjects were studied using simultaneous esophageal pressures and HFIUS or HFIUS alone. Recordings were made during baseline and standardized swallows in the lower esophageal sphincter (LES) and at 2, 4, 6, 8, and 10 cm above the LES. HFIUS images were digitized, and esophageal muscle thickness and peak contraction amplitudes were measured. In the resting state, muscle thickness is higher in the LES compared with the rest of the esophagus. Baseline muscle thickness is also significantly higher at 2 cm vs. 10 cm above the LES. In a given subject and among different subjects, there is a good relationship between peak muscle thickness and peak peristaltic pressures (r = 0.55) at all sites along the length of the esophagus. The positive correlation between pressure and muscle thickness implies that the mean circumferential wall stress is fairly uniform from one swallow to another, irrespective of the contraction amplitude.     

Sustained esophageal contraction: a motor correlate of heartburn symptom

Heartburn occurs in the presence as well as the absence of acid reflux. We searched for a motor correlate of heartburn. Twelve subjects with heartburn were studied with 24-h synchronized pressure, pH, and high-frequency intraluminal ultrasound (HFIUS) imaging of the esophagus. The HFIUS images were analyzed every 2 s for a period of 2 min before and 30 s after the onset of heartburn during 20 acid reflux-positive and 20 acid reflux-negative heartburn episodes. The esophageal muscle thickness was measured as a marker of contraction. Esophageal pressure and HFIUS images were recorded during the Bernstein test in 15 subjects. Sustained esophageal contractions (SECs) were identified during 13 of 20 heartburn episodes associated with acid reflux and 15 of 20 heartburn episodes without acid reflux. SECs were detected during 2 of 40 matched control periods only (P < 0.05). The duration of SECs was 44.9 +/- 26.9 s. The Bernstein test reproduced heartburn symptoms in 8 of 15 subjects. SECs were identified during 6 of 8 (75%) Bernstein-positive and in 1 of 7 (14.3%) Bernstein-negative tests (P = 0.04). We conclude that a SEC precedes both spontaneous and induced heartburn symptoms and may be the cause of heartburn sensation.     

Adenosine modulates cell growth in baby hamster kidney (BHK) cells

Adenosine is known to modulate cell growth in a variety of mammalian cells either via the activation of receptors or through metabolism. We investigated the effect of adenosine on Baby Hamster Kidney (BHK) cell growth and attempted to determine its mechanism of modulation. In wild-type BHK cells, adenosine evoked a biphasic response in which a low concentration of adenosine (1-5 microM) produced an inhibition of colony formation but at higher concentrations (up to 50 microM) this inhibition was progressively reversed. However, no biphasic response was observed in an "adenosine kinase" deficient BHK mutant, "5a", which suggests that adenosine kinase plays an important role in the modulation of growth response to adenosine. Adenosine receptors did not appear to have a role in regulating cell growth of BHK cells. Specific A1 and A2 receptor antagonists were unable to reverse the effect of adenosine on cell growth. Even though a specific A3 adenosine receptor antagonist MRS-1220 partly reversed the inhibition in colony formation at 1 microM adenosine, it also affected the transport of adenosine. Thus adenosine transport and metabolism appears to play the major role in this modulation of cell growth as 5'-amino-5'-deoxyadenosine, an adenosine kinase inhibitor, reversed the inhibition of cell growth observed at 1 microM adenosine. These results, taken together, would suggest that adenosine modulates cell growth in BHK mainly through its transport and metabolism to adenine nucleotides.     

“They Know,They Agree,but They Don’t Do”- The Paradox of Tuberculosis Case Notification by Private Practitioners in Alappuzha District,Kerala, India

Background

Despite being a recognized standard of tuberculosis (TB) care internationally, mandatory TB case notification brings forth challenges from the private sector. Only three TB cases were notified in 2013 by private practitioners compared to 2000 TB cases notified yearly from the public sector in Alappuzha district. The study objective was to explore the knowledge, opinion and barriers regarding TB Notification among private practitioners offering TB services in Alappuzha, Kerala state, India.

Methods & Findings

This was a mixed-methods study with quantitative (survey) and qualitative components conducted between December 2013 and July 2014. The survey, using a structured questionnaire, among 169 private practitioners revealed that 88% were aware of mandatory notification. All patient-related details requested in the notification form (except government-issued identification number) were perceived to be important and easy to provide by more than 80% of practitioners. While more than 95% felt that notification should be mandatory, punitive action in case of failure to notify was considered unnecessary by almost two third. General practitioners (98%) were more likely to be aware of notification than specialists (84 %). (P<0.01). Qualitative purposive personal interviews (n=34) were carried out among private practitioners and public health providers. On thematic framework analysis of the responses, barriers to TB notification were grouped into three themes: ‘private provider misconceptions about notification’, ‘patient confidentiality, and stigma and discrimination ’and ‘lack of cohesion and coordination between public and private sector’. Private practitioners did not consider it necessary to notify TB cases treated with daily regimen.

Conclusion

Communication strategies like training, timely dissemination of information of policy changes and one-to-one dialogue with private practitioners to dispel misconceptions may enhance TB notification. Trust building strategies like providing feedback about referred cases from private sector, health personnel visit or a liaison private doctor may ensure compliance to public health activities.     

Insights into eukaryotic evolution from transmembrane domain lengths

Biological membranes, comprised of proteins anchored by their trans-membrane domains (TMDs) creating a semi-permeable phase with lipid constituents, serve as ‘checkposts’ for not only intracellular trafficking in eukaryotic cells but also for material transactions of all living cells with external environments. Hydropathy (or hydrophobicity) plots of ‘bitopic’ proteins (i.e. having single alpha-helical TMDs) are routinely utilized in biochemistry texts for predicting their TMDs. The number of amino acids (i.e. TMD length) embedded as alpha-helices may serve as indicators of thickness of biological membranes in which they reside under assumptions that are universally applied for fixing window sizes for identifying TMDs using hydropathy plots. In this work we explore variations in thickness of different eukaryotic biological membranes (reflected by TMD lengths of their resident proteins) over evolutionary time scales. Rigorous in silico analyses of over 23,000 non-redundant membrane proteins residing in different subcellular locations from over 200 genomes of fungi, plants, non-mammalian vertebrates and mammals, reveal that differences in plasma membrane and organellar TMD lengths have decreased over time (scales) of eukaryotic cellular evolution. While earlier work has indicated decreasing differences in TMD lengths with increasing ‘perceived’ organismal complexity, this work is the first report on TMD length variations as a function of evolutionary time of eukaryotic cellular systems. We report that differences in TMD lengths of bitopic proteins residing in plasma membranes and other intra-cellular locations have decreased with evolutionary time, suggesting better/more avenues of intracellular trafficking in the emergence of eukaryotic organisms.