Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.     

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Regulation of voltage-gated Ca channels by lipids

Great skepticism has surrounded the question of whether modulation of voltage-gated Ca²⁺ channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acid's carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in Ca_V2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP₂), a palmitoylated accessory subunit (β_2a) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M₁ muscarinic receptors may stimulate multiple lipases to break down the PIP₂ associated with VGCCs and leave PIP₂'s freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs.     

Utilization of Serine, Leucine, Isoleucine, and Valine byThermoanaerobacter brockiiin the Presence of Thiosulfate orMethanobacteriumsp. as Electron Acceptors

Thermoanaerobacter brockiifermented serine to acetate and ethanol. It oxidized leucine to isovalerate, isoleucine to 2-methylbutyrate, and valine to isobutyrate only in the presence of thiosulfate, or when co-cultured withMethanobacteriumsp. This oxidative deamination was rendered thermodynamically possible by the ability ofT. brockiito reduce thiosulfate to sulfide or the transfer of reducing equivalents to the hydrogenotrophic methanogen. The results suggest thatT. brockiimay be of ecological significance in thermal environments in the turnover of amino acids, especially with thiosulfate or H₂-utilizing methanogens are present.     

The occurrence and frequency of 2n pollen in 2x, 4x,and 6x wild,tuber-bearing Solanum species from Mexico,and Central and South America

Summary The occurrence of 2n pollen-producing plants was investigated in 187 plant introductions (PIs) of 38 wild species of tuber-bearing Solanum. These 2x, 4x, and 6x species are from Mexico, and Central and South America. The determination of 2n pollen-producing plants was conducted using acetocarmine glycerol. Plants with more than 1% large-size pollen were regarded as 2n pollen-producing plants. 2n pollen-producing plants were identified in the following species: 10 out of 12 Mexican 2x species, seven of nine South American 2x species, seven of seven Mexican and Central American 4x species, five of five South American 4x species, and five of five Mexican 6x species. The frequency of 2n pollen-producing plants varied among species at the same ploidy level, but the range of frequency, generally between 2 and 10% among species, was similar over different ploidy levels. The general occurrence of 2n pollen in both 2x and polyploid species, which are evolutionarily related, is evidence that the mode of polyploidization in tuber-bearing Solanums is sexual polyploidization. Furthermore, the frequencies of 2n pollen-producing plants in autogamous disomic polyploid species were not markably different from those of their related diploid species. It is thought that the frequent occurrence of 2n gametes with autogamy tends to disturb the fertility and consequently reduce fitness of polyploids. Thus, we propose that the breeding behavior of polyploids and the occurrence of 2n gametes may be genetically balanced in order to conserve high fitness in polyploid species in tuberbearing Solanum.Paper No. 3114 from the Laboratory of Genetics. Research supported by the College of Agriculture and Life Sciences; International Potato Center; USDA, SEA, CGRO 84-CRCR-1-1389; and Frito Lay, Inc.     

Oxygen: a master regulator of pancreatic development?

Beyond its role as an electron acceptor in aerobic respiration, oxygen is also a key effector of many developmental events. The oxygen‐sensing machinery and the very fabric of cell identity and function have been shown to be deeply intertwined. Here we take a first look at how oxygen might lie at the crossroads of at least two of the major molecular pathways that shape pancreatic development. Based on recent evidence and a thorough review of the literature, we present a theoretical model whereby evolving oxygen tensions might choreograph to a large extent the sequence of molecular events resulting in the development of the organ. In particular, we propose that lower oxygenation prior to the expansion of the vasculature may favour HIF (hypoxia inducible factor)‐mediated activation of Notch and repression of Wnt/β‐catenin signalling, limiting endocrine cell differentiation. With the development of vasculature and improved oxygen delivery to the developing organ, HIF‐mediated support for Notch signalling may decline while the β‐catenin‐directed Wnt signalling is favoured, which would support endocrine cell differentiation and perhaps exocrine cell proliferation/differentiation.     

MUC4 involvement in ErbB2/ErbB3 phosphorylation and signaling in response to airway cell mechanical injury

The receptor tyrosine kinases ErbB2 and ErbB3 are phosphorylated in response to injury of the airway epithelium. Since we have shown that the membrane mucin MUC4 can act as a ligand/modulator for ErbB2, affecting its localization in polarized epithelial cells and its phosphorylation, we questioned whether Muc4 was involved, along with ErbB2 and ErbB3, in the damage response of airway epithelia. To test this hypothesis, we first examined the localization of MUC4 in human airway samples. Both immunocytochemistry and immunofluorescence showed a co‐localization of MUC4 and ErbB2 at the airway luminal surface. Sequential immunoprecipitation and immunoblotting from airway cells demonstrated that the MUC4 and ErbB2 are present as a complex in airway epithelial cells. To assess the participation of MUC4 in the damage response, cultures of NCI‐H292 or airway cells were scratch‐wounded, then analyzed for association of phospho‐ErbB2 and ‐ErbB3 with MUC4 by sequential immunoprecipitation and immunoblotting. Wounded cultures exhibited increased phosphorylation of both receptors in complex with MUC4. Scratch wounding also increased activation of the downstream pathway through Akt, as predicted from our previous studies on Muc4 effects on ErbB2 and ErbB3. The participation of MUC4 in the phosphorylation response was also indicated by siRNA repression of MUC4 expression, which resulted in diminution of the phosphorylation of ErbB2 and ErbB3. These studies provide a new model for the airway epithelial damage response, in which the MUC4–ErbB2 complex is a key element in the sensor mechanism and phosphorylation of the receptors. J. Cell. Biochem. 107: 112–122, 2009. © 2009 Wiley‐Liss, Inc.