Determination of piretanide and furosemide in pharmaceuticals and human urine by high-performance liquid chromatography with amperometric detection

A high-performance liquid chromatographic method with electrochemical detection (ED) has been developed for the determination of two diuretics: 4-phenoxy-3-(1-pyrrolidinyl)-5-sulfamoylbenzoic acid (piretanide) and 4-chloro-2-furfurylamino-5-sulfamoylbenzoic acid (furosemide). The chromatographic separation was performed on a μBondapak C₁₈ column with a mobile phase of acetonitrile-water (40:60) containing 5 mM KH₂PO₄/K₂HPO₄ and with a flow-rate of 1 ml/min (69 bar). The temperature was optimized at 30 ± 0.2°C. The amperometric detector equipped with a glassy carbon electrode was operated at + 1200 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds in two concentration ranges (ppm and ppb), obtaining a reproducibility in terms of relative standard deviations lower than 1% for within-day and 4% for day-to-day and determination limits of 15 ppb for both compounds. Recoveries greater than 90% were obtained for spiked urine samples, using a liquid-liquid extraction method in the sample clean-up procedure. The LC-ED method was applied to commercially available pharmaceuticals (Seguril, furosemide 40 mg, and Perbilén, piretanide 6 mg) and urine samples obtained from healthy volunteers and hypertensive patients.     

The channel-forming protein proaerolysin remains a dimer at low concentrations in solution

Proaerolysin, the proform of the channel-forming protein aerolysin, is secreted as a dimer by Aeromonas sp. The protein also exists as a dimer in the crystal, as well as in solution, at least at concentrations in the region of 500 microg/ml. Recently it has been argued that proaerolysin becomes monomeric at concentrations below 100 microg/ml and that only the monomeric form of the protoxin can bind to cell surface receptors (Fivaz, M., Velluz, M.-C., and van der Goot, F. G. (1999) J. Biol. Chem. 274, 37705-37708). Here we show, using non-denaturing polyacrylamide electrophoresis, chemical cross-linking, and analytical ultracentrifugation, that proaerolysin remains dimeric at the lowest concentrations of the protein that we measured (less than 5 microg/ml) and that the dimeric protoxin is quite capable of receptor binding.     

Cytochemical Localization of Calcium in Prefusion Myoblasts from the Chick Embryo Myotome

Myoblast fusion is a Ca²⁺-dependent process. The aim of this report was to study the localization of Ca²⁺ in prefusion myoblasts from the brachial somites of chick embryos (51–108h of incubation), using the potassium pyroantimonate cytochemical method. When observed under a transmission electron microscope, electron-dense precipitates of Ca²⁺-antimonate were found in the basement membrane of the myotome, which separates the myotome from the adjacent mesenchyma. Within myoblasts, triads and sarcoplasmic reticulum associated with the first newly formed sarcomeres were observed, but a T-tubule network was not found. Moreover, Ca²⁺-antimonate precipitates were not observed in structures resembling T-tubules or sarcoplasmic reticulum. The results suggest that sarcomerogenesis and sarcoplasmic reticulum development occur simultaneously and that prefusion myoblasts have neither a T-tubule network nor Ca²⁺ deposits on sarcoplasmic reticulum. Small Ca²⁺ pools were found in the myoblast nuclei, cytoplasmic vesicles and mitochondrias. Ca²⁺-antimonate precipitates periodically distributed at the cell periphery, close to the cell membrane, were observed. These precipitates could represent internal Ca²⁺ stores located in the peripheral couplings and it is proposed that these pools of Ca²⁺ could be mobilized before fusion, leading to the increase in free intracellular Ca²⁺ that precedes myoblast fusion.     

Mechanisms involved in the cellular calcium homeostasis in vascular smooth muscle: calcium pumps

The regulation of cytosolic Ca2+ homeostasis is essential for cells, and particularly for vascular smooth muscle cells. In this regulation, there is a participation of different factors and mechanisms situated at different levels in the cell, among them Ca2+ pumps play an important role. Thus, Ca2+ pump, to extrude Ca2+; Na+/Ca2+ exchanger; and different Ca2+ channels for Ca2+ entry are placed in the plasma membrane. In addition, the inner and outer surfaces of the plasmalemma possess the ability to bind Ca2+ that can be released by different agonists. The sarcoplasmic reticulum has an active role in this Ca2+ regulation; its membrane has a Ca2+ pump that facilitates luminal Ca2+ accumulation, thus reducing the cytosolic free Ca2+ concentration. This pump can be inhibited by different agents. Physiologically, its activity is regulated by the protein phospholamban; thus, when it is in its unphosphorylated state such a Ca2+ pump is inhibited. The sarcoplasmic reticulum membrane also possesses receptors for 1,4,5-inositol trisphosphate and ryanodine, which upon activation facilitates Ca2+ release from this store. The sarcoplasmic reticulum and the plasmalemma form the superficial buffer barrier that is considered as an effective barrier for Ca2+ influx. The cytosol possesses different proteins and several inorganic compounds with a Ca2+ buffering capacity. The hypothesis of capacitative Ca2+ entry into smooth muscle across the plasma membrane after intracellular store depletion and its mechanisms of inhibition and activation is also commented.     

Early blooming's challenges: extended flowering season, diverse pollinator assemblage and the reproductive success of Gynodioecious Daphne laureola

BACKGROUND AND AIMS: The scarcity and unpredictability of active pollinators during late winter in temperate areas tends to favour extended flowering seasons and increased floral longevity in early blooming species, which are usually pollinated by diverse sets of insects. Daphne laureola is a gynodioecious woody perennial that flowers from January to April in southern Spain, a period characterized by cold temperatures, frequent rains and irregular snowfalls. METHODS: Pollinators were excluded at four different times during the flowering season in order to determine the effect of decreased exposure to pollinators on fruit set in female and hermaphrodite individuals. The role of nocturnal and diurnal pollination on reproductive success in each gender was simultaneously evaluated by selective exclusion. KEY RESULTS: A 50 % reduction in the flowering period decreased fruit set of females by 50 %, whereas the corresponding decrease in self-compatible hermaphrodites was only approx. 25 %. Day-active hymenopterans and lepidopterans were infrequent visitors, and nocturnal pollinators were inefficient, suggesting that pollen beetles, Meligethes elongatus, were the main pollinators of D. laureola in the study region. CONCLUSIONS: Beetles were less abundant in pollenless females, although discrimination did not apparently result in pollination limitation of female reproduction. A preference of beetles for sunny locations emphasized the relevance of abiotic conditions for pollination of this early blooming shrub.     

Genetic admixture of European FRDA genes is the cause of Friedreich ataxia in the Mexican population

Friedreich ataxia accounts for approximately 75% of European recessive ataxia patients. Approximately 98% of pathogenic chromosomes have large expansions of a GAA triplet repeat in the FRDA gene (E alleles), and strong linkage disequilibrium among polymorphisms spanning the FRDA locus indicates a common origin for all European E alleles. In contrast, we found that only 14 of 151 (9.3%) Mexican Mestizo patients with recessive ataxia were homozygous for E alleles. Analysis of polymorphisms spanning the FRDA locus revealed that all Mestizo E alleles had the common European haplotype, indicating that they share a single origin. Genetic admixture levels were determined, which revealed that the relative contributions to the Mestizo FRDA gene pool by Native American and European genes were 76-87% and 13-24%, respectively, commensurate with the observed low prevalence of Friedreich ataxia in Mestizos. This indicates that Friedreich ataxia in Mexican Mestizos is due to genetic admixture of European mutant FRDA genes in the Native American gene pool that existed prior to contact with Europeans.