NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.     

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

Background

Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods

We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results

We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion

The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.     

Newborn Body Fat: Associations with Maternal Metabolic State and Placental Size

Background

Neonatal body composition has implications for the health of the newborn both in short and long term perspective. The objective of the current study was first to explore the association between maternal BMI and metabolic parameters associated with BMI and neonatal percentage body fat and to determine to which extent any associations were modified if adjusting for placental weight. Secondly, we examined the relations between maternal metabolic parameters associated with BMI and placental weight.

Methods

The present work was performed in a subcohort (n = 207) of the STORK study, an observational, prospective study on the determinants of fetal growth and birthweight in healthy pregnancies at Oslo University Hospital, Norway. Fasting glucose, insulin, triglycerides, free fatty acids, HDL- and total cholesterol were measured at week 30–32. Newborn body composition was determined by Dual-Energy X-Ray Absorptiometry (DXA). Placenta was weighed at birth. Linear regression models were used with newborn fat percentage and placental weight as main outcomes.

Results

Maternal BMI, fasting glucose and gestational age were independently associated with neonatal fat percentage. However, if placental weight was introduced as a covariate, only placental weight and gestational age remained significant. In the univariate model, the determinants of placenta weight included BMI, insulin, triglycerides, total- and HDL-cholesterol (negatively), gestational weight gain and parity. In the multivariable model, BMI, total cholesterol HDL-cholesterol, gestational weight gain and parity remained independent covariates.

Conclusion

Maternal BMI and fasting glucose were independently associated with newborn percentage fat. This effect disappeared by introducing placental weight as a covariate. Several metabolic factors associated with maternal BMI were associated with placental weight, but not with neonatal body fat. Our findings are consistent with a concept that the effects of maternal BMI and a number of BMI-related metabolic factors on fetal fat accretion to a significant extent act by modifying placental weight.     

Regulated expression of inhibitor of apoptosis protein 3 in the rat corpus luteum

We sought to investigate the role inhibitor of apoptosis proteins (IAPs) play in the life cycle of the corpus luteum (CL) of the rat. We isolated two clones with amino acid homology to rat IAP2 (BIRC 3) and three to rat IAP3 (rIAP3; BIRC 4). The expression of rIAP3 mRNA was examined in the rat CL during and after pregnancy, in Day 8 pregnant rats after 24-h treatment of gonadotropin-releasing hormone-agonist (GnRH-Ag), and in a CL organ culture model of spontaneous apoptosis in the absence of tropic support with and without superoxide dismutase. We used real-time RT-PCR to quantitate rIAP3 mRNA expression. Interestingly, a significant reduction in rIAP3 levels was seen at the time of CL regression in the course of natural pregnancy and the GnRH-Ag model. Surprisingly, rIAP3 mRNA levels in the CL organ culture model of spontaneous apoptosis failed to show significant changes, although TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) reaction showed 30%-40% of the cells undergoing DNA fragmentation after 2 h in culture. In situ hybridization revealed that rIAP3 expression was localized to the cytoplasm of luteal and granulosa cells. These data clearly demonstrate both the presence of IAPs in the rat CL and the regulation of rIAP3 during in vivo apoptotic cell death, indicating a role for IAPs in the maintenance of CL function and demise.     

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

This study describes a method to rapidly screen hundreds of ion channel variants containing non-canonical amino acids. A proof-of-principle introducing photocrosslinking non-canonical amino acids into the human ion channel hASIC1a shows how this approach can provide insights into function and pharmacology.     

Magnoliid reproductive organs from the Cenomanian-Turonian of north-western Kazakhstan:Magnoliaceae andIlliciaceae

Cenomanian-Turonian sediments from the Sarbay locality in north-western Kazakhstan have yielded a rich assemblage of plant fossils including well preserved flowers, fruits, and seeds of angiosperms. This work describes fossil seeds assigned to theMagnoliaceae and theIlliciaceae. Three new species of the extinct magnoliaceous genusLiriodendroidea, L. asiatica, L. costata, andL. tenuitesta, are established and new information on the previously described species,L. alata, is provided. TheLiriodendroidea seeds are closely similar to seeds of extantLiriodendron, but are distinguished in being much smaller and winged. A new genus and species,Illiciospermum pusillum, is established based on seeds with close similarity to those of the extant genusIllicium. The seeds are small, anatropous and exotestal with outer epidermis of testa forming a palisade layer. The facets of the palisade cells have deeply undulate anticlinal walls. The micropyle area is seen on the outer integument as a transverse slit placed on a raised strophiole-like structure close to the hilum. TheIlliciospermum seeds represent the first unequivocal record of theIlliciaceae in the Cretaceous. Another seed of possible illiciaceous affinity is described as aff.Illiciospermum sp.     

Polymorphisms in COX-2, NSAID use and risk of basal cell carcinoma in a prospective study of Danes

We investigated the risk of basal cell carcinoma (BCC) in relation to a number of single nucleotide polymorphisms in genes involved in the inflammatory response. A case-control study including 322 BCC cases and a similar number of controls was nested in a population-based prospective study of 57,053 individuals (aged 50-64 at inclusion) in Denmark. NSAID use was associated with a slightly decreased risk of BCC (IRR=0.85, 95% CI=0.66-1.10). We found that two polymorphisms in COX-2, COX-2 A-1195G and T8473C were associated with risk of BCC. Carriers of the variant allele of COX-2 A-1195G had lower risk of BCC than homozygous wild type carriers (IRR=0.54, 95% CI=0.47-0.89). Homozygous carriers of the variant allele of COX-2 T8473C were at 2.27-fold higher risk of BCC (95% CI=1.31-3.92) than homozygous wild type allele carriers. The polymorphisms IL6 G-174C, IL8 T-251A, PPARgamma2 Pro(12)Ala, IL1beta T-31C, and IL10 C-592A were not associated with risk of BCC. We found no statistically significant interaction between polymorphisms and NSAID use in relation to risk of BCC. While it cannot be ruled out that the present findings are due to chance, the results indicate that high COX-2 expression may increase risk of BCC while NSAID use may be protective.     

Interaction of L Cells and Chlamydia psittaci: Entry of the Parasite and Host Responses to Its Development

The entry and development of Chlamydia psittaci in the L cell was studied by using purified, infectious parasites at high multiplicity. Entry of the parasite was accomplished by an act of phagocytosis by the host which was independent of an adsorption stage but was temperature-dependent. Kinetic studies of phagocytosis performed with (14)C-amino acid-labeled, purified parasites indicated that the rate of phagocytosis was directly proportional to the multiplicity of inoculation. Electron microscopy of cells infected at high multiplicity with purified infectious C. psittaci showed that phagocytosed chlamydiae were segregated in a host phagocytic vacuole throughout their developmental cycle which consisted of the transition of infecting elementary bodies to reticulate bodies dividing by binary fission, followed by the reemergence of a population of elementary bodies. The process of the transition was examined and a proposed sequence of intermediate bodies is presented. In isopycnic gradients of fractionated, infected L cells, chlamydial phagocytic vacuoles were apparent as a dense band distinct from lysosome and mitochondrion peaks, as indicated by acid phosphatase and cytochrome oxidase activities. Chlamydiae inactivated by heat or neutralized by antiserum were phagocytosed and appeared in lysosomes within 12 hr after infection according to electron microscopy; however, chlamydiae which were continuously inhibited in their development by chloramphenicol were retained intact in the cell for 24 hr without lysosomal response. The possibility of a lysosomal inhibitor on the native parasite is discussed.