Generation of Mouse Small Intestinal Epithelial Cell Lines That Allow the Analysis of Specific Innate Immune Functions

Cell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier.     

Biotransformation of 2,2-Dimethyl-1,3-Propanediol to 3-Hydroxypivalic Acid by Acetobacter Acetii DSMZ3508 and Related Bacteria

Acetobacter acetii DSMZ3508 and related bacteria converted 2,2-dimethyl-1,3-propanediol into 3-hydroxypivalic acid (2,2-dimethyl-3-hydroxypropionic acid; 3HP) during submerged cultivation in mineral salt medium. The maximum yield of 3-hydroxypivalic acid was 24.4% of the fed substrate after 18 days. Cultivation parameters, as pH, cell density, optimal substrate concentration, and oxygen supply for the bioconversion process were determined.     

Taxonomic distribution and origins of the extended LHC (light-harvesting complex) antenna protein superfamily

Background  

The extended light-harvesting complex (LHC) protein superfamily is a centerpiece of eukaryotic photosynthesis, comprising the LHC family and several families involved in photoprotection, like the LHC-like and the photosystem II subunit S (PSBS). The evolution of this complex superfamily has long remained elusive, partially due to previously missing families.     

Regulation of thyroid hormone metabolism in rat liver fractions

The nature of the conversion of thyroxine (T₄) to triiodothyronine (T₃) and reverse triiodothyronine (rT₃) was investigated in rat liver homogenate and microsomes. A 6-fold rise of T₃ and 2.5-fold rise of rT₃ levels determined by specific radioimmunoassays was observed over 6 h after the addition of T₄. An enzymic process is suggested that converts T₄ to T₃ and rT₃. For T₃ the optimal pH is 6 and for rT₃, 9.5. The converting activity for both T₃ and rT₃ is temperature dependent and can be suppressed by heat, H₂O₂, merthiolate and by 5-propyl-2-thiouracil. rT₃ and to a lesser degree iodide, were able to inhibit the production of T₃ in a dose related fashion. Therefore the pH dependendy, rT₃ and iodide may regulate the availability of T₃ or rT₃ depending on the metabolic requirements of thyroid hormones.     

Big Moose Basin: simulation of response to acidic deposition

The ILWAS model has been enhanced for application to multiple-lake hydrologic basins. This version of the model has been applied to the Big Moose basin, which includes Big Moose Lake and its tributary streams, lakes, and watersheds. The basin, as defined, includes an area of 96 km², with over 20 lakes and ponds, and 70 km of streams. Hydrologic and chemical calibrations have been made using data from seven sampling stations. When total atmospheric sulfur loading to the basin is halved, the model predicts, after four years of simulation, a decreasing sulfate concentration and to a lesser extent a rising alkalinity at Big Moose Lake outlet. At the end of four years, the results show an increase in pH of 0.1 to 0.5 pH units depending upon season.     

Renal handling of taurine,l-alanine,l-glutamate andd-glucose inOpsanus tau: studies on isolated brush border membrane vesicles

Summary Renal brush border membrane vesicles (bbmv) from the aglomerular toadfish (Opsanus tau), isolated by differential precipitation, were tested for their ability to actively translocate (i) taurine, known to be secreted by the kidney of several marine teleosts, and (ii)l-alanine,l-glutamic acid, andd-glucose, solutes that are normally reabsorbed in the filtering nephron. Vesicular taurine uptake displayed a Na⁺ dependence. Transport was greatest under conditions of an inward-directed Na⁺ gradient, but a significant stimulation by Na⁺ over K⁺ could also be observed in the absence of a salt gradient. At high extravesicular K⁺, the addition of valinomycin reduced taurine uptake. Na⁺-dependent³H-taurine flux was almost completely inhibited by non-labeled taurine (tracer replacement) or -alanine, but was unaffected byl-alanine. Replacement of medium chloride by SCN^– or NO ₃ ^– in the presence of Na⁺ resulted in significantly lower uptake rates under both anion gradient and anion equilibrium conditions, whereas Br^– could almost fully substitute for the stimulatory Cl^– action. These results indicate the presence of an electrogenic Na⁺-cotransport mechanism with specificity for -amino acids in the toadfish renal brush border. Whether the system under physiological conditions mediates reabsorption or secretion of taurine remains to be determined. Toadfish bbmv also translocatedl-alanine andl-glutamic acid in a Na⁺-dependent manner. Possible roles for these most likely reabsorptive transport systems in a non-filtering kidney are discussed.d-glucose uptake, however, appeared to occur via Na⁺-independent pathways, since it was not affected by phlorizin in the presence of Na⁺, or by Na⁺ replacement.Abbreviation bbmv brush border membrane vesicles     

Oxygen transfer properties and dimensions of red blood cells in high-altitude camelids,dromedary camel and goat

Summary To estimate the advantage of the small red blood cells (RBC) of high-altitude camelids for O₂ transfer, the kinetics of O₂ uptake into and release from the RBC obtained from llama, vicuña and alpaca were investigated at 37°C with a stopped-flow technique. O₂ transfer conductance of RBC (G) was estimated from the rate of O₂ saturation change and the corresponding O₂ pressure difference between medium and hemoglobin. For comparison, O₂ kinetics for the RBC of a lowaltitude camelid (dromedary camel) and the pygmy goat were determined and previously measured values for human RBC were used. O₂ transfer of RBC was found to be strongly influenced by extracellular diffusion, except with O₂ release into dithionite solutions of sufficiently high concentration (>30 mM). TheG values measured in these standard conditions,G _st (in mmol · min^–1 · Torr^–1 · (ml RBC)^–1) were: high-altitude camelids, 0.58 (averaged for llama, alpaca and vicuña since there were no significant interspecific differences); camel 0.42; goat, 0.42; man, 0.39. The differences can in part be attributed to expected effects of the size and shape of the RBC (volume, surface area, mean thickness), as well as to the intracellular O₂ diffusivity which depends on the concentration of cellular hemoglobin. The highG _st of RBC of highaltitude camelids may be considered to enhance O₂ transfer in lungs and tissues. But the O₂ transfer conductance of blood, , equal toG _st multiplied by hematocrit (in mmol · min^–1 · Torr^–1 · (ml blood)^–1), was only slightly higher as compared to other species: 0.20 (llama, alpaca, vicuña), 0.14 (camel), 0.18 (goat), 0.17 (man).Abbreviations DPG 2,3-diphosphoglycerate - G conductance - Hb hemoglobin - RBC red blood cells - percent saturation of hemoglobin     

Implications for cytoplasmic pH,protonmotice force,and amino-acid transport across the plasmalemma of Riccia fluitans

By means of pH-sensitive microelectrodes, cytoplasmic pH has been monitored continuously during amino-acid transport across the plasmalemma of Riccia fluitans rhizoid cells under various experimental conditions. (i) Contrary to the general assumption that import of amino acids (or hexoses) together with protons should lead to cytoplasmic acidification, an alkalinization of 0.1–0.3 pH_c units was found for all amino acids tested. Similar alkalinizations were recorded in the presence of hexoses and methylamine. No alkalinization occurred when the substrates were added in the depolarized state or in the presence of cyanide, where the electrogenic H⁺-pump is inhibited. (ii) After acidification of the cytoplasm by means of various concentrations of acetic acid, amino-acid transport is massively altered, although the protonmotive force remained essentially constant. It is suggested that H⁺-cotransport is energetically interconnected with the proton-export pump which is stimulated by the amino-acid-induced depolarization, thus causing proton depletion of the cytoplasm. It is concluded that, in order to investigate H⁺-dependent cotransport processes, the cytoplasmic pH must be measured and be under continuous experimental control; secondly, neither pH nor the protonmotive force across a membrane are reliable quantities for analysing a proton-dependent process.Abbreviations 3-OMG 3-oxymethylglucose - pH_c cytoplasmic pH - _m electrical potential difference across the respective membrane, i.e. membrane potential - _H+/F (=pmf) electrochemical proton gradient     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol