Simultaneous determination of citalopram and its metabolites by high-performance liquid chromatography with column switching and fluorescence detection by direct plasma injection

High-performance liquid chromatography with a successive column-switching technique was developed for simultaneous determination of citalopram and its four metabolites in plasma. Plasma samples were injected directly, and the target compounds were purified and concentrated with an inexpensive commercial octadecyl guard column. Then, the six-port valve was switched, and the compounds retained in the column were eluted by the back-flush method using 20 mM phosphate buffer (pH 4.6)-acetonitrile (70:30, v/v) containing 0.1% diethylamine and separated with an ODS column. The compounds were assayed with a fluorescence detector at an excitation wavelength of 249 nm and an emission wavelength of 302 nm. At least 30 plasma samples could be treated with an octadecyl guard column. The limits of quantitation of this method were 2.0 ng/ml for citalopram, desmethylcitalopram, didesmethylcitalopram, citalopram propionic acid and citalopram N-oxide. This method was applied to a pharmacokinetic study in dogs and a toxicokinetic study in rats.     

Natural disturbance and tree species coexistence in an old-growth beech - dwarf bamboo forest,southwestern Japan

Abstract. The structure and composition of a cool-temperate old-growth beech (Fagus crenata) - dwarf bamboo (Sasa spp.) forest, partially affected by landslide disturbance, in the Daisen Forest Reserve of southwestern Japan, were investigated in relation to forest floor and canopy conditions. All stems ≥ 4 cm DBH were mapped on a 4-ha plot and analyses were made of population structure, spatial distribution and spatial association of major tree species. The dominant species, F. crenata, which had the maximum DBH among the species present, had the highest stem density. However, for other species, larger-sized species had lower stem density with few smaller stems or saplings, while smaller-sized species had higher stem density with many smaller stems or saplings. Canopy trees of F. crenata were distributed randomly in the plot, while its stems in the other layers and all other species were distributed patchily. Small patches represent gap-phase regeneration. Larger patches correlate with landslide disturbance, difference in soil age, or the presence/absence of Sasa. Cluster analysis for spatial associations among species and stems in the different layers revealed that the forest community consists of several groups. One main group was formed on sites not covered with Sasa. This group contained a successional subgroup (from Betula grossa to Acer mono and/or F. crenata) initiated by landslide disturbance and a subgroup of tree species that avoid Sasa. Another group was formed on sites with mature soils covered largely with Sasa. This contained associations of canopy trees of F. crenata and smaller-sized tree species such as Acanthopanax sciadophylloides and Acer japonicum. It is found that the community of this old-growth beech forest is largely organized by natural disturbance and heterogeneous conditions of the forest floor (difference in soil age and presence/absence of Sasa). The existence of these different factors and the different responses of species to them largely contribute to the maintenance of tree species diversity in this forest.; Keywords: Cluster analysis; Fagus crenata; Forest dynamics; Gap; Landslide; Spatial pattern.     

Sarcoplasmic reticulum calsequestrins: Structural and functional properties

Calsequestrin is the major Ca²⁺-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its -helical content and the intrinsic fluorescence upon binding of Ca²⁺. Calsequestrin binds Ca²⁺ with high-capacity and with moderate affinity and it functions as a Ca²⁺ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca²⁺ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsquestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

Synthesis of sulfated derivatives of curdlan and their anti-HIV activity

Sulfopropyl curdlan was synthesized, its structure was determined, and the anti-HIV activity was compared with that of standard curdlan sulfates obtained with piperidine N-sulfonic acid in dimethyl sulfoxide. It was shown that sulfopropyl curdlan exhibits weaker anti-HIV activity than curdlan sulfate. Curdlan sulfates were synthesized with a SO₃-pyridine complex in a heterogeneous phase. It was shown from ¹³C-NMR spectra of acetylated curdlan sulfates that they had a different substituent distribution from standard curdlan sulfate. The cytotoxicity of the curdlan sulfates was attributed to their heterogeneous structure.     

Fusarium poae and Fusarium crookwellense, Fungi Responsible for the Natural Occurrence of Nivalenol in Hokkaido

To determine the reasons for the natural occurrence of nivalenol in the northernmost area of Japan, scabby wheat was harvested from 19 crop fields in Hokkaido. Mycological surveys and analysis for mycotoxin contamination were performed. Among 13 wheat grain samples harvested in seven locations, 9, 2, and 6 samples were positive for deoxynivalenol, nivalenol, and zearalenone, respectively, at levels ranging from 0.03 to 1.28 μg/g, 0.04 to 1.22 μg/g, and 2 to 25 ng/g, respectively. The predominant Fusarium species of the scabby wheat examined were F. sporotrichioides, F. avenaceum, F. poae, and F. crookwellense. Fifteen of 48 F. poae isolates and all four F. crookwellense isolates were screened for the production of seven derivatives of trichothecenes and zearalenone respectively, on rice culture. One isolate of F. poae produced diacetoxyscirpenol alone (4.3 μg/g); seven produced nivalenol (1.3 to 23.8 μg/g), 4-acetylnivalenol (0.1 to 4.6 μg/g), and diacetoxyscirpenol (0.9 to 99.5 μg/g); and five produced nivalenol alone (0.4 to 3.5 μg/g). The remaining two isolates produced no trichothecenes. Zearalenone production was not found in any isolate of F. poae tested. All isolates of F. crookwellense produced nivalenol (0.9 to 22.5 μg/g), 4-acetylnivalenol (0.5 to 25.0 μg/g), and zearalenone (1.4 to 162.5 μg/g). From these results, it is apparent that deoxynivalenol and zearalenone, and occasionally nivalenol, occur naturally throughout Hokkaido, and it is suggested that nivalenol-producing F. poae and F. crookwellense strains are responsible for the natural contamination with nivalenol found in the northernmost area of Japan. Furthermore, it was found for the first time that several isolates of F. poae distributed in Hokkaido possessed the ability to produce both type A and type B trichothecenes.     

Correction to: Comparison of the usability of an automatic sleep staging program via portable 1-channel electroencephalograph and manual sleep staging with traditional polysomnography

Sleep and Biological Rhythms -