Screening procedure for the analysis of distigmine bromide in serum by high-performance liquid chromatography-electrospray ionization mass spectrometry

A screening procedure was developed for the identification and quantification of distigmine bromide in serum samples by using liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS). In this method, distigmine bromide was analyzed in 0.5 mL serum by using pancuronium bromide as the internal standard, and gradient elution was performed using a reversed-phase column and a mixture of 10 mM-ammonium formate and methanol as the mobile phase. A highly sensitive assay could be performed with simple solid phase extraction using a cation exchange cartridge column by carrying out selected ion monitoring analysis in the positive ion detection mode. The procedure was validated in terms of linearity (0.9973 at 2.5 ng/mL). The inter- and intra-day precisions (coefficient of variation; CV%) were <8.5% and < 9.7%, respectively. The analytes were evaluated for stability and were found to be stable in serum for 1 week at 4 degrees C and 4 weeks at -30 degrees C, and successfully applied to in the analysis of two overdose cases. This method is sensitive and useful for the detection, quantification, and confirmation of distigmine bromide in serum.     

Application of real-time confocal microscopy for observation of living cells in tissue specimens.

Measurement of intracellular Ca2+ concentration ([Ca2+]i) has been a fundamental technique in cell biology. However, most investigations have used cultured or isolated cells as an experimental model, and consequently can provide only limited insight into the mechanisms that operate in tissue in situ. Useful information may be obtained by studying intact tissue specimens. High-speed confocal microscopes that can acquire digital images at video rate have recently been developed. These confocal microscopes which can acquire data in real-time enable [Ca2+]i dynamics of individual cells in intact tissue specimens to be observed. The present paper examines the use of fluorescent microscopy and confocal microscopy for [Ca2+]i imaging of living tissue. We analyzed the dynamics of the duodenal gland, lacrimal gland, intestinal smooth muscles, arterioles, myenteric plexus, and dorsal root ganglion. In these specimens, individual cells exhibited different [Ca2+]i dynamics, and the responses to transmitters/modulators were heterogeneous. In conclusion, real-time imaging provides a useful tool for observing dynamic changes in cells in situ, and it may lead to improve understanding tissue physiology.     

Isolation of a Periplasmic Molecular Chaperone-Like Protein of Rhodobacter sphaeroides f. sp. denitrificans That Is Homologous to the Dipeptide Transport Protein DppA of Escherichia coli

A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph Rhodobacter sphaeroides f. sp. denitrificans, in a manner similar to that of the Escherichia coli chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol. 37:333–339, 1996). The protein was isolated from the periplasm of the phototroph. It had a molecular mass of 58 kDa and had no subunits. The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of E. coli. The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein. The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution. The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding.     

Possible involvement of leaf gibberellins in the clock-controlled expression of XSP30, a gene encoding a xylem sap lectin, in cucumber roots

Root-produced organic compounds in xylem sap, such as hormones and amino acids, are known to be important in plant development. Recently, biochemical approaches have revealed the identities of several xylem sap proteins, but the biological functions and the regulation of the production of these proteins are not fully understood. XYLEM SAP PROTEIN 30 kD (XSP30), which is specifically expressed in the roots of cucumber (Cucumis sativus), encodes a lectin and is hypothesized as affecting the development of above-ground organs. In this report, we demonstrate that XSP30 gene expression and the level of XSP30 protein fluctuate in a diurnal rhythm in cucumber roots. The rhythmic gene expression continues for at least two or three cycles, even under continuous light or dark conditions, demonstrating that the expression of this gene is controlled by a circadian clock. Removal of mature leaves or treatment of shoots with uniconazole-P, an inhibitor of gibberellic acid (GA) biosynthesis, dampens the amplitude of the rhythmic expression; the application of GA negates these effects. These results suggest that light signals perceived by above-ground organs, as well as GA that is produced, possibly, in mature leaves, are important for the rhythmic expression of XSP30 in roots. This is the first demonstration of the regulation of the expression of a clock-controlled gene by GA.     

Degradation of the D1 protein of photosystem II under illumination in vivo: two different pathways involving cleavage or intermolecular cross-linking

The D1 protein of the photosystem II reaction center turns over the most rapidly of all the proteins of the thylakoid membrane under illumination in vivo. In vitro, the D1 protein sustained cleavage in a surface-exposed loop (DE loop) or cross-linking with another reaction center protein, the D2 protein or cytochrome b(559), under illumination. We found that the D1 protein was damaged in essentially the same way in vivo, although the resultant fragments and cross-linked adducts barely accumulated due to digestion by proteases. In vitro studies detected a novel stromal protease(s) that digested the adducts but not the monomeric D1 protein. These observations suggest that, in addition to cleavage, the cross-linking reactions themselves are processes involved in complete degradation of the D1 protein in vivo. Peptide mapping experiments located the cross-linking sites with the D2 protein among residues 226-244, which includes the cross-linking site with cytochrome b(559) [Barbato, R., et al. (1995) J. Biol. Chem. 270, 24032-24037], in the N-terminal part of the DE loop, while N-terminal amino acid sequencing of the fragment located the cleavage site around residue 260 in the C-terminal part of the loop. We propose a model explaining the occurrence of simultaneous cleavage and cross-linking and discuss the mechanisms of complete degradation of the D1 protein in vivo.     

Functional cooperation among Ras, STAT5, and phosphatidylinositol 3-kinase is required for full oncogenic activities of BCR/ABL in K562 cells.

BCR/ABL tyrosine kinase generated from the chromosomal translocation t(9;22) causes chronic myelogenous leukemia and acute lymphoblastic leukemia. To examine the roles of BCR/ABL-activated individual signaling molecules and their cooperation in leukemogenesis, we inducibly expressed a dominant negative (DN) form of Ras, phosphatidylinositol 3-kinase, and STAT5 alone or in combination in p210 BCR/ABL-positive K562 cells. The inducibly expressed DN Ras (N17), STAT5 (694F), and DN phosphatidylinositol 3-kinase (Delta p85) inhibited the growth by 90, 55, and 40%, respectively. During the growth inhibition, the expression of cyclin D2 and cyclin D3 was suppressed by N17, 694F, or Delta p85; that of cyclin E by N17; and that of cyclin A by Delta p85. In addition, N17 induced apoptosis in a small proportion of K562, whereas 694F and Delta p85 were hardly effective. In contrast, coexpression of two DN mutants in any combinations induced severe apoptosis. During these cultures, the expression of Bcl-2 was suppressed by N17, 694F, or Delta p85, and that of Bcl-XL by N17. Furthermore, although K562 was resistant to interferon-alpha- and dexamethasone-induced apoptosis, disruption of one pathway by N17, 694F, or Delta p85 sensitized K562 to these reagents. These results suggested that cooperation among these molecules is required for full leukemogenic activities of BCR/ABL.     

Decomposition of urea by size-fractionated planktonic community in a eutrophic reservoir in Japan

Free bacterial populations were separated from an intact planktonic community in water of a eutrophic reservoir in Japan by filtration through Whatman GF/ C glass fiber filters (mean porosity 1.2 µm). Urea decomposition by the free bacterial populations and the intact planktonic community was determined in six different months.The separated free bacteria apparently did not take part in urea-decomposition in waters of the reservoir through the year: the number of free heterotrophic bacteria increased during the urea-decomposition experiments, however, the concentration of urea did not decrease. Whereas, in five cases out of six, urea was decomposed by the intact planktonic community. Probably, phytoplankton were responsible for most of the urea-decomposition. On the assumption that the decomposition of urea obeyed first-order kinetics, rate constants were calculated to be 0.00–0.63 day^–1 with a mean value of 0.21 day^–1.     

Ultrastructural localization of acid phosphatase activity in the small intestinal absorptive cells of postnatal rats

Summary The ultrastructural localization of acid phosphatase activity was investigated in ultrathin (0.05 m) and semithin (0.5 m) sections of the small intestinal epithelial cells of postnatal rats. Until around the 15th day of neonatal life acid phosphatase activity in the duodenal and jejunal epithelial cells was observed on the microvillous membrane, the membrane of the tubulo-vacuolar system, the lateral cell membrane, the lysosomes, the Golgi apparatus and the GERL of Novikoff (1963). After about the 15th neonatal day, the tubulo-vacuolar system enzyme disappeared from both cells. Acid phosphatase activity then became localized on the microvillous membrane, the lateral cell membrane, the lysosomes, the Golgi apparatus, and the GERL, as in adult rats. During the suckling period, acid phosphatase in the ileal cells could be seen on the microvillous membrane, the lateral cell membrane, the Golgi apparatus, the GERL, the membrane of tubulo-vacuolar system and the supranuclear vacuole. At weaning, however, the tubulovacuolar system and the supranuclear vacuole enzyme disappeared, and only the lysosomes and the GERL of these cells showed acid phosphatase activity, as in the adult rat. It was concluded that the acid-phosphatase-containing tubulo-vacuolar system and the supranuclear vacuole in the epithelial cells of the distal intestine of suckling rats may possess a strong phagolysosomal function as well as having an absorptive capacity.     

D-Amino-acid-stimulated ethylene production in seed tissues

Ethylene production by axial and cotyledonary tissues excised from Xanthium pennsylvanicum Wallr. seeds was markedly (up to 5-fold) stimulated by the D-isomers of phenylalanine, valine, leucine, threonine, methionine and eithionine while the L-isomers caused no such effect. Responsiveness of these seed tissues to D-methionine appeared soon after the beginning of imbibition, reached a maximum after 6–12 and 12–24 h for the axial and cotyledonary tissues, respectively, and then decreased sharply. D-Phenylalanine and D-methionine also stimulated ethylene production in seed tissues of X. canadense Mill. and in cotyledonary segments from seeds of Helianthus annuus L., Cucurbita moschata Duch. and Vigna radiata (L.) Wilczek. The endogeneous ethylene production and the D-amino-acid-stimulated ethylene production by the seed segments was strongly inhibited by aminoethoxyvinyl glycine, a potent inhibitor of ethylene synthesis from L-methionine.