Stable expression of gastric proton pump activity at the cell surface

Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the alpha+beta-expressing cell line, K(+)-stimulated ATPase (K(+)-ATPase) activity and the acylphosphorylation of the alpha-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K(+)-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the alpha-subunit was retained in the intracellular compartment and was unstable in the absence of the beta-subunit, but it was stabilized and reached the cell surface in the presence of the beta-subunit. On the other hand, the beta-subunit was stable and able to travel to the cell surface in the absence of the alpha-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.     

Interactions of arsenic with human metallothionein-2

Arsenic is a toxic element that is found in the atmosphere, as well as in aquatic and terrestrial environments. We have demonstrated that As(3+) binds to human metallothionein-2 (hMT-2) by UV absorption spectroscopy, inductively coupled plasma-atomic emission spectrometry (ICP-AES), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-TOF-MS revealed that the structure of the adduct formed by arsenic and hMT-2 (As-hMT-2) was not homogeneous. The maximum molar ratio of arsenic to hMT-2 was found to be more than 6:1 on ICP-AES, UV absorption spectroscopy and MALDI-TOF-MS. The ratio of the number of sulfhydryl groups in hMT-2 that bound arsenic was 3:1, which is the same as the ratios reported previously for arsenic-glutathione and arsenic-phytochelatin complexes.     

Mutant luciferase enzymes from fireflies with increased resistance to benzalkonium chloride

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.     

Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata

CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.     

Allozyme analysis of cryptic species in the Asplenium niduscomplex from West Java,Indonesia

In various fern species, a large amount of rbcL sequence variation has been reported, and it is possible that these species contain several reproductively isolated cryptic species. In our previous study on Asplenium nidus L., it was suggested that the plants growing in Mt. Halimun National Park, West Java, Indonesia, consist of several cryptic species based on the results of crossing experiments among rbcL sequence types. In this study, we examined allozyme polymorphisms of five rbcL sequence types found in West Java in order to test the hypothesis that the assemblages of A. nidus delimited based on the rbcL sequences are separate Mendelian populations and gene flow is disrupted by reproductive isolation from one another. The calculated fixation indices suggested that the individuals in each rbcL type are randomly crossing at least in the investigated localities. Nevertheless, these rbcL-based assemblages were genetically differentiated in allozymes that are encoded in their nuclear genomes, and it is also suggested that gene flow is disrupted even between sympatrically distributed pairs of rbcL sequence types. Therefore, our findings support the view that the five rbcL sequence types in West Java are potential cryptic species. Electronic Publication     

Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.     

Rearing conditions required for behavioral compensation after unilateral cercal ablation in the cricket Gryllus bimaculatus

The rearing condition necessary for behavioral compensation after sensory deprivation was investigated in the cricket Gryllus bimaculatus. The right-cercus-ablated cricket was reared in a glass vial with a slightly larger diameter than the body length of the cricket. After two weeks of rearing in the vial, the air-puff-evoked escape behavior of the cricket was investigated. The response rate (relative occurrence of the escape behavior after a standard air puff) obtained was identical with that of crickets reared in a large cage. On the other hand, unlike crickets reared in a large cage, the distorted escape directional property of the cricket reared in the vial was not compensated at all. Control experiments proved that the restraint in the vial did not affect the motor system, and the air motion from environments was not essential for the compensational recovery of the escape direction. Therefore, the ablated crickets required spontaneous walking in order to compensate the directionality of their escape. A self-generated wind caused by spontaneous walking appears necessary for the crickets to realize the defect of their sensory system and to compensate the related escape behavior. A hypothesis for the compensation mechanism based on the efference copy signal is proposed.     

Molecular isolation and characterization of novel four subisoforms of ECE-2

Although the four polypeptides of blasticidin S (BS) deaminase (BSD) are packed rather tightly coordinated to the "structural and catalytic" zinc atom of each subunit, the C-terminal region of the enzyme was suggested to be somewhat molten and flexible [M. Kimura, S. Sekido, Y. Isogai, and I. Yamaguchi (2000) J. Biochem. 127, 955-963]. To understand roles of this flexible region, we constructed five C-terminal deletion variants of BSD (each successively deleted from the C-terminal end up to five residues) and analyzed their biochemical properties focusing on the structure and activity of the enzyme. BSD and all of the deletion mutants showed the unique rigid conformation (e.g., characterized by their stabilities in SDS solution) and high levels of resistance against protease digestions. Furthermore, both the wild-type and deletion apoenzymes exhibited similar physical properties in thermodynamic refolding into the stable tetramer conformation. However, these small C-terminal deletions exerted deleterious effects on the catalytic efficiency of the enzyme as indicated by their strongly reduced k(cat)/K(m) value. Judging from the altered kinetic parameters and unaltered structural properties of the deletion variants, these C-terminal residues appear to be directly involved in enzyme-substrate interaction. In this short flexible region, Tyr-126, Trp-128, and Gly-130 were the key residues. Most notably, removal of Gly-130 markedly increased K(m) for BS without affecting its k(cat) value. These results indicate that the flexible C-terminal region is important for catalytic function and that a single Gly residue at the C-terminal end of BSD contributes significantly in facilitating access of a substrate to the active site.     

Arg tyrosine kinase is involved in homologous recombinational DNA repair

c-Abl plays important roles in cellular response to DNA damage. However, possible roles for Arg (Abl-related gene) in DNA damage response are unknown. Here, we show that ionizing radiation (IR)-induced Rad51 focus formation is reduced in Arg-deficient cells generated from a chicken B cell line by targeted disruption. This is consistent with the findings that Arg-deficient cells display hypersensitivity to IR, elevated frequencies of IR-induced chromosomal aberrations, and reduced targeted integration frequencies. All of these abnormalities in DNA damage repair are also observed in ATM-deficient cells but not in c-Abl-deficient cells. Finally, we show that Arg interacts with and phosphorylates Rad51 in 293T cells. These results suggest that Arg plays a role in homologous recombinational (HR) DNA repair by phosphorylating Rad51.     

Identification of genes responsive to sodium butyrate in colonic epithelial cells

We identified genes responsive to sodium butyrate (SB) in colonic epithelial cells using cDNA microarrays. Treatment with 2 mM SB of colonic epithelial cells (MCE301), which was derived from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen, arrested cell growth and showed a differentiated phenotype accompanying an increase in alkaline phosphatase activity. Of the approximately 900 genes analyzed, SB down-regulated 25 genes and up-regulated 88 genes by a factor of 2.0 or greater. Northern blot or TaqMan and Western blot analyses confirmed that the mRNA and protein levels of cyclin D1 and the level of proliferating cell nuclear antigen decreased, whereas the levels of integrin beta1 and osteopontin increased. The present results regarding the changes in gene expression, arrived at using microarrays, will provide a basis for a further understanding of the molecular mechanisms of cell growth arrest and differentiation in response to SB in colonic epithelial cells.