Bile acid-induced secretion in polarized monolayers of T84 colonic epithelial cells: Structure-activity relationships

Bile acid epimers and side-chain homologues are present in the human colon. To test whether such bile acids possess secretory activity, cultured T84 colonic epithelial cells were used to quantify the secretory properties of synthetic epimers and homologues of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). In our study, chloride secretion was measured as changes in short-circuit current (DeltaI(sc), in microA/cm2) with the use of voltage-clamped monolayers of T84 cells mounted in Ussing chambers. Bile acids were added at 0.5 mM, a concentration that did not alter transepithelial resistance. Data were expressed as peak DeltaI(sc) (means +/- SD). When added bilaterally, DCA stimulated a DeltaI(sc) response of 15.7 +/- 12.5 microA/cm2. The 12beta-OH epimer of DCA was less potent (DeltaI(sc) = 8.0 +/- 1.7 microA/cm2), whereas its 3beta-OH epimer had no effect. CDCA stimulated secretion (DeltaI(sc) = 8.2 +/- 5.5 microA/cm2), whereas both its 7beta-OH and 3beta-OH epimers were inactive, as was lithocholic acid. HomoDCA (1 additional side-chain carbon) was active (DeltaI(sc) = 7.8 +/- 4.8 microA/cm2), whereas norDCA (1 fewer carbon) and dinorDCA (2 fewer carbons) were not. Taurine conjugates of DCA and CDCA stimulated secretion (DeltaI(sc) = 12.3 +/- 7.5 and 8.8 +/- 4.8 microA/cm2, respectively) from the basolateral side but not the apical side. Uptake of taurine conjugates from the basolateral but not the apical side was shown by mass spectrometry. These studies indicate marked structural specificity for bile acid-induced chloride secretion and show that modification of bile acid structure by colonic bacteria modulates the secretory properties of these endogenous secretagogues.     

Human DNA polymerase kappa encircles DNA: implications for mismatch extension and lesion bypass

Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.     

Characterization of a multifunctional inositol phosphate kinase from rice and barley belonging to the ATP-grasp superfamily

OsIpk and HvIpk, inositol phosphate kinases, were cloned from rice (Oryza sativa L. var. indica, IR64) and barley (Hordeum vulgare) respectively. Sequence alignment showed that they belong to the ATP-grasp family, which includes inositol 1,3,4-trisphosphate 5/6-kinase from humans and Arabidopsis. Residues that are binding sites for ATP and coordinate magnesium in absence or presence of inositol phosphate are conserved and in total 23 residues are invariant among the twelve aligned inositol phosphate kinases. The genes were heterologously expressed in Escherichia coli and kinase activity assays with 17 different isomers of inositol mono-/di-/tri-/tetra-/pentaphosphate as well as phytate were performed. The strongest activity for both kinases was observed with Ins(3,4,5,6)P(4), which candidates as the primary substrate for these kinases in plants. Several species-specific differences between the two recombinant Ipks were observed. Rice OsIpk showed detectable kinase activity towards eight different substrates, whereas barley HvIpk showed kinase activity with all the substrates including inositol mono- and bisphosphates. HvIpk showed 3-kinase activity towards the Ins(1,4,5)P(3) substrate and it also interconverted the two substrates Ins(1,3,4,5)P(4) and Ins(1,3,4,6)P(4) by isomerase activity, which was not observed for the rice homologue. Both OsIpk and HvIpk had no detectable 2-kinase activity. Furthermore, the two Ipks showed phosphatase activity towards several inositol phosphates. Expression analysis by RT-PCR demonstrated that the Ipk gene was equally expressed in different tissues and developmental stages. Taken together, these results show that the Ipk kinase plays a significant role in the inositol phosphate interacting network in plants.     

CaMtw1, a member of the evolutionarily conserved Mis12 kinetochore protein family, is required for efficient inner kinetochore assembly in the pathogenic yeast Candida albicans

Proper assembly of the kinetochore, a multi-protein complex that mediates attachment of centromere DNA to spindle microtubules on each chromosome, is required for faithful chromosome segregation. Each previously characterized member of the Mis12/Mtw1 protein family is part of an essential subcomplex in the kinetochore. In this work, we identify and characterize CaMTW1, which encodes the homologue of the human Mis12 protein in the pathogenic budding yeast Candida albicans. Subcellular localization and chromatin immunoprecipitation assays confirmed CaMtw1 is a kinetochore protein. CaMtw1 is essential for viability. CaMtw1-depleted cells and cells in which CaMtw1 was inactivated with a temperature-sensitive mutation had reduced viability, accumulated at the G2/M stage of the cell cycle, and exhibited increased chromosome missegregation. CaMtw1 depletion also affected spindle length and alignment. Interestingly, in C. albicans, CaMtw1 and the centromeric histone, CaCse4, influence each other for kinetochore localization. In addition, CaMtw1 is required for efficient kinetochore recruitment of another inner kinetochore protein, the CENP-C homologue, CaMif2. Mis12/Mtw1 proteins have well-established roles in the recruitment and maintenance of outer kinetochore proteins. We propose that Mis12/Mtw1 proteins also have important co-dependent interactions with inner kinetochore proteins and that these interactions may increase the fidelity of kinetochore formation.     

Cyclic presence and absence of conspecifics alters circadian clock phase but does not entrain the locomotor activity rhythm of the fruit fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms.     

Characterizing the epidemiology of the 2009 influenza A/H1N1 pandemic in Mexico

Background

Mexico''s local and national authorities initiated an intense public health response during the early stages of the 2009 A/H1N1 pandemic. In this study we analyzed the epidemiological patterns of the pandemic during April–December 2009 in Mexico and evaluated the impact of nonmedical interventions, school cycles, and demographic factors on influenza transmission.

Methods and Findings

We used influenza surveillance data compiled by the Mexican Institute for Social Security, representing 40% of the population, to study patterns in influenza-like illness (ILIs) hospitalizations, deaths, and case-fatality rate by pandemic wave and geographical region. We also estimated the reproduction number (R) on the basis of the growth rate of daily cases, and used a transmission model to evaluate the effectiveness of mitigation strategies initiated during the spring pandemic wave. A total of 117,626 ILI cases were identified during April–December 2009, of which 30.6% were tested for influenza, and 23.3% were positive for the influenza A/H1N1 pandemic virus. A three-wave pandemic profile was identified, with an initial wave in April–May (Mexico City area), a second wave in June–July (southeastern states), and a geographically widespread third wave in August–December. The median age of laboratory confirmed ILI cases was ∼18 years overall and increased to ∼31 years during autumn (p<0.0001). The case-fatality ratio among ILI cases was 1.2% overall, and highest (5.5%) among people over 60 years. The regional R estimates were 1.8–2.1, 1.6–1.9, and 1.2–1.3 for the spring, summer, and fall waves, respectively. We estimate that the 18-day period of mandatory school closures and other social distancing measures implemented in the greater Mexico City area was associated with a 29%–37% reduction in influenza transmission in spring 2009. In addition, an increase in R was observed in late May and early June in the southeast states, after mandatory school suspension resumed and before summer vacation started. State-specific fall pandemic waves began 2–5 weeks after school reopened for the fall term, coinciding with an age shift in influenza cases.

Conclusions

We documented three spatially heterogeneous waves of the 2009 A/H1N1 pandemic virus in Mexico, which were characterized by a relatively young age distribution of cases. Our study highlights the importance of school cycles on the transmission dynamics of this pandemic influenza strain and suggests that school closure and other mitigation measures could be useful to mitigate future influenza pandemics. Please see later in the article for the Editors'' Summary     

Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

Overexpression or expression of activating mutations of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. The present study employed Affymetrix oligonucleotide arrays to profile genes induced by ligand-activated EGFR with the receptor either moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes encoding proteins with functions in promoting cell proliferation, invasion, antiapoptosis, and angiogenesis featured prominently in the EGFRvIII- and EGFR-expressing cells. Surprisingly, it was found that ligand-activated EGFR induced the expression of a large group of genes known to be inducible by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1 and STAT3. The results thus demonstrate that ligand-activated EGFR at different expression levels results in different kinetics of signaling and induction of gene expression. In addition, the constitutively active variant EGFRvIII seems to activate only a subset of signal pathways and induce a subset of genes as compared to the ligand-activated EGFR.     

Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence in intronic DNA, the aim was to inhibit the amplification of genomic DNA without affecting the amplification of reverse-transcribed spliced mRNA. LNA was designed to bind within intron 5 in the x-box binding protein 1 (XBP1) gene. An irrelevant LNA oligonucleotide served as a negative control. In both PCR and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More than three DNA nucleotides reduced the LNA inhibition ability. The sequence specificity of the LNA was tested by investigating the number of LNA nucleotide mismatches permitted. The introduction of one mismatch maintained the inhibition of genomic amplification whereas two mismatches reduced the amplification. Our results show that LNA may be used to enhance the specificity of PCR by eliminating unwanted PCR products.