Characterization of Low pH-induced Catecholamine Secretion in the Rat Adrenal Medulla

Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O₂/5% CO₂. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ～6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca²⁺ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca²⁺ channel, but were completely inhibited in Ca²⁺-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH₄CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca²⁺ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca²⁺ release from intracellular stores, which is also induced by the Ca²⁺ influx.     

Plasmodesmal-mediated cell-to-cell transport in wheat roots is modulated by anaerobic stress

Summary Cell-to-cell transport of small molecules and ions occurs in plants through plasmodesmata. Plant roots are frequently subjected to localized anaerobic stress, with a resultant decrease in ATP. In order to determine the effect of this stress on plasmodesmal transport, fluorescent dyes of increasing molecular weight (0.46 to 10 kDa) were injected into epidermal and cortical cells of 3-day-old wheat roots, and their movement into neighboring cells was determined by fluorescence microscopy. Anaerobiosis was generated by N₂ gas or simulated by the presence of sodium azide, both of which reduced the ATP levels in the tissue by over 80%. In the absence of such stress, the upper limit for movement, or size exclusion limit (SEL), of cortical plasmodesmata was <1 kDa. The ATP analogue TNP-ADP (mw 681) moved across the plasmodesmata of unstressed roots, indicating that plasmodesmata may be conduits for nucleotide (ATP and ADP) exchange between cells. Upon imposition of stress, the SEL rose to between 5 and 10 kDa. This response of plasmodesmata to a decrease in the level of ATP suggests that they are constricted by an ATP-dependent process so as to maintain a restricted SEL. When roots are subjected to anaerobic stress, an increase in SEL may permit enhanced delivery of sugars to the affected cells of the root where anaerobic respiration could regenerate the needed ATP.Abbreviations F-dextran fluorescein-coupled dextran - LYCH Lucifer Yellow CH - SEL size exclusion limit - TNP-ADP 2-O-(trinitrophenyl)adenosine-5-diphosphate     

MOLECULAR PHYLOGENETIC ANALYSIS OF rbcL IN THE PRYMNESIOPHYTA1

The nucleotide sequences of rbcL genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were determined from six species of Prymnesiophyta to clarify their phylogenetic relationships. Molecular phylogenetic trees were constructed using PAUP (Phylogenetic Analysis Using Parsimony). These analyses suggest that the Prymnesiophyta, except for the Pavlovales, area relatively stable monophyletic group. Pleurochrysis carterae, included in the Isochrysidales, is a sister species of a monophyletic group consisting of other members of the Isochrysidales, Gephyrocapsa oceanica and Emiliania huxleyi, members of the Coccosphaerales, Calyptrosphaera sphaeroidea and Umbilicosphaera sibogae var. foliosa, and a member of the Prymnesiales, Chrysochromulina hirta. The nucleotide sequence of rbcL from G. oceanica was identical to that from E. huxleyi within the region examined. Our trees show that G. oceanica and E. huxleyi are more closely related to C. hirta than to U. sibogae, C. sphaeroidea, and P. carterae. These results suggest that orders in the Prymnesiophyceae, including the above-mentioned genera, should be redefined.     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of the nopaline synthase gene by double transformation: reactivation by segregation of the induced DNA

Tobacco plants were transformed with derivatives of a binary vector pMON505 and two kanamycin resistant lines that were nopaline positive were selected for second transformation. The plasmids used for the second transformation were derivatives of pMON850 which carries the nopaline synthase gene in addition to a gene for gentamicin resistance. Insertion of each transgene was confirmed by Southern hybridization. Surprisingly, we found that more than 50% of the doubly transformed tobacco plants were nopaline negative. Tobacco plants that were transformed only by the second vector exhibited nopaline accumulation. DNA methylation patterns at the HpaII site in the promoter region of the nopaline synthase gene did not correlate with the nopaline phenotype. In some plant lines, seedlings of the R1 generation which segregated out the second T-DNA insertion recovered the nop⁺ phenotype. These results indicate that nopaline accumulation was inhibited by the presence of the second T-DNA.Abbreviations T-DNA transferred DNA - NPTII neomycin phosphotransferase II - uidA -glucuronidase - Km kanamycin - Gm gentamicin - nop⁺ nopaline positive - nop^– nopaline negative - MS medium, Murashige-Skoog medium     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Cardiopulmonary function in bicycle racing over mountainous terrain at moderate altitude

To examine cardiopulmonary function during exercise in a mountainous region at moderate altitude, we measured cardiac frequency, oxygen consumption , and percentage arterial hemoglobin oxygen saturation (%SaO₂) before and after a bicycle race with a starting point at 638 m and finishing point at 1980 m. The time required to ascend an elevation of 10 m was prolonged with increasing altitude, and heart rate also increased with altitude. The %SaO₂ at the starting point and at the finishing point differed significantly (P<0.01). Faster cyclists exhibited higher %SaO₂ and lower , while slower cyclists exhibited a reduction in %SaO₂ and an increase in immediately after the race. The %SaO₂ recovery time was significantly correlated with the racing time (r=0.54,P<0.001). Therefore, the faster cyclists' oxygen debt upon completion of the race may be small and recovery of cardiopulmonary function may be fast, while the slower cyclists' oxygen debt may be large and recovery of cardiopulmonary function may be slow.