DKT1, a novel K+ channel from carrot, forms functional heteromeric channels with KDC1

We report the isolation and characterisation of DKT1, a new carrot K+ channel alpha-subunit belonging to the Shaker-like family. DKT1 is expressed in many tissues of the adult plant, suggesting that it may play important roles in both nutrition and other important physiological processes. During embryo development, DKT1 is expressed at later phases implying the involvement of K+ in embryo maturation. When co-expressed with KDC1 in Xenopus oocytes, DKT1 is able to form functional, heteromeric channels, suggesting that possible interactions between these two ion channels in plant tissues may modulate K+ uptake.     

Intermittent hypoxic training protects canine myocardium from infarction

This investigation examined cardiac protective effects of normobaric intermittent hypoxia training. Six dogs underwent intermittent hypoxic training for 20 consecutive days in a normobaric chamber ventilated intermittently with N2 to reduce fraction of inspired oxygen (FiO2) to 9.5%-10%. Hypoxic periods, initially 5 mins and increasing to 10 mins, were followed by 4-min normoxic periods. This hypoxia-normoxia protocol was repeated, initially 5 times and increasing to 8 times. The dogs showed no discomfort during intermittent hypoxic training. After 20 days of hypoxic training, the resistance of ventricular myocardium to infarction was assessed in an acute experiment. The left anterior descending (LAD) coronary artery was occluded for 60 mins and then reperfused for 5 hrs. At 30 mins of LAD occlusion, radioactive microspheres were injected through a left atrial catheter to assess coronary collateral blood flow into the ischemic region. After 5 hrs reperfusion, the heart was dyed to delineate the area at risk (AAR) of infarction and stained with triphenyl tetrazolium chloride to identify infarcted myocardium. During LAD occlusion and reperfusion, systemic hemodynamics and global left ventricular function were stable. Infarction was not detected in 4 hearts and was 1.6% of AAR in the other 2 hearts. In contrast, 6 dogs sham-trained in a chamber ventilated with compressed air and 5 untrained dogs subjected to the same LAD occlusion/reperfusion protocol had infarcts of 36.8% +/- 5.8% and 35.2% +/- 9.5% of the AAR, respectively. The reduction in infarct size of four of the six hypoxia-trained dogs could not be explained by enhanced collateral blood flow to the AAR. Hypoxia-trained dogs had no ventricular tachycardia or ventricular fibrillation. Three sham-trained dogs had ventricular tachycardia and two had ventricular fibrillation. Three untrained dogs had ventricular fibrillation. In conclusion, intermittent hypoxic training protects canine myocardium from infarction and life-threatening arrhythmias during coronary artery occlusion and reperfusion. The mechanism responsible for this potent cardioprotection merits further study.     

Augmented taurine release is not the mechanism of ischemic preconditioning’s cardioprotection

Summary. In ischemic preconditioning (IPC) a brief ischemic period protects the heart from a subsequent ischemic insult by an unknown mechanism. Osmotic swelling has been proposed to be a major cause of cell death when ischemic tissue is reperfused. The present study tests whether the preconditioned heart during reperfusion might release more taurine, an important osmolyte in the cardiac myocytes, to decrease cellular osmolarity, oppose swelling, and preserve viability. We collected the coronary effluent from isolated rabbit hearts for 10min before and 10min after preconditioning with 5min of global ischemia. The heart then experienced 15min of global ischemia and effluent was collected during reperfusion for 40min. A control group was studied similarly but without the preconditioning ischemia. Fifteen min of ischemia was chosen to avoid any taurine release caused by ischemic cell death. Taurine was measured with HPLC. In the IPC group there was a postischemic release over baseline of 5.09±1.51mol (approx 3.3% of the total taurine pool), whereas in the control group the release was not significantly different, 5.72±1.67mol. The percent of the taurine pool lost from each heart during reperfusion was calculated based on an assumption of a total content of 20M taurine/gm wet weight. Since the amount of taurine released by the isolated rabbit heart following ischemia was not different in preconditioned and non-preconditioned hearts, we conclude that reduced swelling through taurine release is not the mechanism of the cardioprotective effects of IPC.     

Synthesis and anticonvulsant evaluation of some new 2-substituted-3-arylpyrido[2,3-d]pyrimidinones

A series of 2-substituted-3-arylpyrido[2,3-d]pyrimidinones was prepared for evaluation as potential anticonvulsants. In murine screening, compounds 4a-c having a 2-oxo-2-(4-pyridyl)ethyl group in the 2-position and a 2-substituted phenyl moiety at the 3-position of the pyridopyrimidinone system displayed the most potent anti-seizure activity in both the maximal electroshock (MES) and pentylenetetrazol (scPTZ) tests at doses in the 3-10mg/kg range. Compound 4c showed no agonist activity at the GABA(A) receptor and was unable to block presynaptic sodium and calcium channels in vitro.     

KDC1, a novel carrot root hair K+ channel. Cloning, characterization, and expression in mammalian cells

Potassium is an essential nutrient which plays an important role in many aspects of plant growth and development. Plants have developed a number of highly specific mechanisms to take up potassium from the soil; these include the expression of K(+) transporters and potassium channels in root cells. Despite the fact that root epidermal and hair cells are in direct contact with the soil, the role of these tissues in K(+) uptake is not well understood. Here we report the molecular cloning and functional characterization of a novel potassium channel KDC1 which forms part of a new subfamily of plant K(in) channels. Kdc1 was isolated from carrot root RNA and in situ hybridization experiments show Kdc1 to be highly expressed in root hair cells. Expressing the KDC1 protein in Chinese hamster ovary cells identified it as a voltage and pH-dependent inwardly rectifying potassium channel. An electrophysiological analysis of carrot root hair protoplasts confirmed the biophysical properties of the Kdc1 gene product (KDC1) in the heterologous expression system. KDC1 thus represents a major K(+) uptake channel in carrot root hair cells.     

A role for both ecology and geography as mechanisms of genetic differentiation in specialized butterflies

An important mechanism of speciation for specialized phytophagous insects is host-associated differentiation, in which natural selection drives the evolution of reproductive isolation. Here we use molecular population genetics data to test the hypothesis that regional and local specialization on three alternate hosts restricts gene flow in the Mitoura gryneus species complex of butterflies. Over half of the variation in mitochondrial DNA sequences is explained by host plant association (?_CT = 0.57, P = 0.002) consistent with the hypothesis of host-associated divergence on the three hosts. AFLP analyses revealed the number of clusters of individuals was K = 2, with all individuals associated with one host grouping separately from all other host-associated individuals. Combined with previous experimental results, these findings present evidence of varying levels of differentiation among host associations and identify a role for both isolation in allopatry as well as ecological factors in limiting gene exchange. The Mitoura species complex includes multiple, differentiated lineages at varying stages of divergence, providing an opportunity to examine the multifarious mechanisms that generate biodiversity in phytophagous insects.     

A prospective study of human papillomavirus (HPV) testing to resolve uncertainty in colposcopy

J. Bowring, R. Albrow, A. Fisher, G. Downey, J. Cullimore, J. Patnick, P.G. Walker and H.C. Kitchener
A prospective study of human papillomavirus (HPV) testing to resolve uncertainty in colposcopy Objective: UK colposcopy services are seeing increased workloads, a large proportion of which are follow‐up appointments. The English Cervical Screening Programme HPV Special Interest Group identified five subcategories of colposcopy clinic patients who often require prolonged follow‐up regimes for low‐grade abnormalities. Human papillomavirus (HPV) testing has a high negative predictive value, meaning that HPV‐negative women are at very low risk of underlying disease. Our objectives were to quantify the number of HPV‐negative women in each study subcategory and to evaluate the number who could potentially be discharged from colposcopy on the basis of their results. Methods: Four colposcopy clinics prospectively identified women according to five categories over 12 months. All women underwent cytological testing and high‐risk HPV (hrHPV) testing using the Hybrid Capture 2 test. Management outcomes and decisions based on a knowledge of the HPV status were recorded. Results: Data available on 755 women showed that 422/755 (55.9%) and 260/755 (34.4%) had persistent cervical intraepithelial neoplasia grade 1 (CIN1) (Category 1) or a minor abnormality following treatment (Category 2), respectively. In Categories 1 and 2, 51.7% and 60.2%, respectively, were hrHPV negative. The rates with biopsies of CIN2 or worse (CIN2+) across the two categories were 3/355 (0.8%) and 21/291 (7.0%) for hrHPV‐negative and hrHPV‐positive women, respectively. Conclusion: The incorporation of hrHPV testing within organized cervical screening programmes has been widely accepted. hrHPV testing for the clinical scenarios outlined in this study detects women who are hrHPV negative and therefore at low risk of underlying disease, potentially reducing anxiety and inconvenience for women and costs to colposcopy services.     

A novel approach for using dielectric spectroscopy to predict viable cell volume (VCV) in early process development

Online monitoring of viable cell volume (VCV) is essential to the development, monitoring, and control of bioprocesses. The commercial availability of steam‐sterilizable dielectric‐spectroscopy probes has enabled successful adoption of this technology as a key noninvasive method to measure VCV for cell‐culture processes. Technological challenges still exist, however. For some cell lines, the technique's accuracy in predicting the VCV from probe‐permittivity measurements declines as the viability of the cell culture decreases. To investigate the cause of this decrease in accuracy, divergences in predicted vs. actual VCV measurements were directly related to the shape of dielectric frequency scans collected during a cell culture. The changes in the shape of the beta dispersion, which are associated with changes in cell state, are quantified by applying a novel “area ratio” (AR) metric to frequency‐scanning data from the dielectric‐spectroscopy probes. The AR metric is then used to relate the shape of the beta dispersion to single‐frequency permittivity measurements to accurately predict the offline VCV throughout an entire fed‐batch run, regardless of cell state. This work demonstrates the possible feasibility of quantifying the shape of the beta dispersion, determined from frequency‐scanning data, for enhanced measurement of VCV in mammalian cell cultures by applying a novel shape‐characterization technique. In addition, this work demonstrates the utility of using changes in the shape of the beta dispersion to quantify cell health. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:479–487, 2014     

Interactions of the Protein-tyrosine Phosphatase-α with the Focal Adhesion Targeting Domain of Focal Adhesion Kinase Are Involved in Interleukin-1 Signaling in Fibroblasts

Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca²⁺ release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα.