Determination of duodenogastric reflux by quantification of bile acids in the gastric juice

The existence of duodeno-gastric reflux was evaluated in a group of 15 healthy subjects, in fasting and for 24 hours. The assessment of duodeno-gastric reflux was made by quantitation of the bile acids (BA) present in the gastric juice. The individual free and conjugated BA were separated and quantified by means of thin-layer chromatography and in situ spectrofluorometry. In 7 of the subjects studied no BA were detected, and in the other 8 subjects the BA levels were below 6 mumol reflux/hour. There were no free BA detected in any of the subjects. The levels of BA in gastric juice increased progressively with age, but there were no differences between sex. The chromatographic technique used is highly sensitive for the analysis of BA in biological samples. The study of BA in the gastric juice provides a quantitative and reliable assessment of the degree of duodeno-gastric reflux.     

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions

Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 M IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration percentage. High auxin concentrations (6.0 or 11.0 M) inhibited bud differentiation, especially when explants were cultured in darkness. On the other hand, low auxin levels and photoperiod improved shoot development. Excised shoots were induced to form roots by transfer to hormone-free MS medium with macronutrients at half strength. The obtained plantlets were ultimately grown in the greenhouse.Abbreviations BA benzyladenine - BM basal medium - IAA indoleacetic acid - MS Murashige & skoog - NAA -naphthaleneacetic acid     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Effect of prior ingestion of glucose or fructose on the performance of exercise of intermediate duration

The metabolic responses induced by the ingestion of a beverage containing glucose (G), fructose (F) or placebo (W) 30 min before exercise of high intensity and intermediate duration have been investigated; in these conditions the energy processes are mostly dependent on aerobic reactions. A group of 11 male recreational sportsmen ran on a treadmill, at an intensity corresponding to 82% of peak oxygen consumption, until exhaustion on three different occasions (after ingestion of a beverage containing 75 g of G, 75 g of F or W). Plasma glucose, insulin, and lactic acid concentrations were determined just prior to the ingestion of the beverages, 30 min afterwards and 10 and 30 min after completion of the exercise. The mean endurance time was 644 (SD 261) s after the ingestion of G, 611 (SD 227) s after the ingestion of F and 584 (SD 189) s after the ingestion of the W (P < 0.05 between G and W). No differences in the oxygen uptake, respiratory quotient or lactate concentrations between the three trials were observed. Both plasma glucose and insulin concentrations determined in samples obtained immediately before the onset of exercise were higher when G was ingested than when F (P < 0.05 andP < 0.05, respectively) or W (P < 0.001 and P < 0.005, respectively) were ingested. These findings would suggest that the ingestion of G prior to an effort of intermediate duration may improve physical performance.     

Purification and characterization of two 1,4-beta-xylan endohydrolases from the rumen fungus Neocallimastix frontalis.

Two beta-endoxylanases produced by Neocallimastix frontalis have been purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. Xylanase I is a nonglycosylated protein with an apparent molecular mass of 45 kDa. Xylanase II is a glycoprotein with an apparent molecular mass of 70 kDa. The pH optima of these enzymes were 5.5 and 6, respectively, and the temperature optimum was 55 degrees C for each enzyme. The endo mode of action of the enzymes was revealed by thin-layer chromatography of xylan hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, confirming the biochemical specificities of the enzymes. Both enzymes exhibited carboxymethyl cellulase activity, and xylanase I was absorbed on crystalline cellulose, indicating that these enzymes might belong to the F family of beta-1,4-glycanases.     

Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involvingcGMP and Ca2+ as second messengers

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane fractions. Agents that increase intracellularcAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system bycGMP. In fact, permeant analogs ofcGMP, dibutyrylcGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition ofcGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca²⁺ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca²⁺ ions.     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.