Alternative splicing variants of human arsenic (+3 oxidation state) methyltransferase

Arsenic (+3 oxidation state) methyltransferase (As3MT) catalyzes the methylation of trivalent arsenic (As(III)) to monomethylarsonate (MMA(V)) and dimethylarsinic acid (DMA(V)), and plays an important role in the detoxification of arsenicals. Here, we report the identification of two splicing variants of the human As3MT gene. One splicing variant was an exon-3 skipping (Δ3) form which produced a premature stop codon, and the other was an exon-4 and -5 skipping (Δ4,5) form which produced a 31.1 kDa As3MT protein. In addition to the full-length mRNA of As3MT, Δ4,5 mRNAs were detected in HepG2, A549, HL60, K562, and HEK293 cells. The methyltransferase activity of the recombinant Δ4,5 As3MT and wild-type As3MT proteins purified from Escherichia coli was determined. Speciation analysis by HPLC–ICP-MS showed a clear peak of MMA(V) after incubation of As(III) with the wild-type As3MT protein, but not with the Δ4,5 As3MT protein. In addition, COS-7 cells transfected with Δ4,5 As3MT cDNA did not convert As(III) to MMA(V) or DMA(V). The lack of methyltransferase activity of Δ4,5 As3MT seems to be related to the deletion of an S-adenosylmethionine-binding site and a critical cysteine residue. These data suggest that the expression pattern of splicing variants of the As3MT gene may affect the capacity for arsenic methylation in cells.     

Organization of the replication control region of plasmid ColIb-P9.

We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair IncI alpha group ColIb-P9 plasmid. Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ and inc. The repZ gene encodes a protein with a molecular weight of 39,000, which probably functions as an initiator for the ColIb-P9 replicon. The inc gene that phenotypically governs the incompatibility encodes an RNA with a size of about 70 bases. This small RNA acts in trans to repress the expression of repZ, thereby functioning to maintain a constant copy number of the ColIb-P9 replicon in host cells.     

Tool Use Specific Adult Neurogenesis and Synaptogenesis in Rodent (Octodon degus) Hippocampus

We previously demonstrated that degus (Octodon degus), which are a species of small caviomorph rodents, could be trained to use a T-shaped rake as a hand tool to expand accessible spaces. To elucidate the neurobiological underpinnings of this higher brain function, we compared this tool use learning task with a simple spatial (radial maze) memory task and investigated the changes that were induced in the hippocampal neural circuits known to subserve spatial perception and learning. With the exposure to an enriched environment in home cage, adult neurogenesis in the dentate gyrus of the hippocampus was augmented by tool use learning, but not radial maze learning, when compared to control conditions. Furthermore, the proportion of new synapses formed in the CA3 region of the hippocampus, the target area for projections of mossy fiber axons emanating from newborn neurons, was specifically increased by tool use learning. Thus, active tool use behavior by rodents, learned through multiple training sessions, requires the hippocampus to generate more novel neurons and synapses than spatial information processing in radial maze learning.     

Day-night changes in production of carbohydrate and protein by natural phytoplankton population from Lake Biwa, Japan

The ¹³C and gas chromatography-mass spectrometry (¹³C-GC-MS)method was applied to determine the day-night changes in thecomposition of photosynthetic products of the natural phytoplanktonpopulation from Lake Biwa, Japan. Glucose is the most abundantmonosaccharide in acid-hydrolyzable carbohydrate. The contributionof glucose was high in incubatesd samples in daytime and decreasedduring the night. Other monosaccharides (rhamnose, fucose, ribose,arabinose, xylose, mannose and galactose) and amino acids tendedto be produced throughout both day- and night-time. These resultssuggested that the carbon flows from glucose, which might constitutereserve glucan, to other monosaccharides and amino acids duringnight-time. The disproportionate production of glucose (reservedglucan) during daytime was thus partly cancelled out at night.     

Suppressor T lymphocyte induction by a factor released from cultured blastocysts

For the analysis of immunologic escape mechanisms of embryos during the implantation period in mice, the effects of culture supernatant of blastocysts on in vitro responsiveness to alloantigen of mice was investigated. Blastocyst-cultured conditioned medium was prepared by culturing late blastocysts of outbred ICR mice for 5 days. The addition of culture supernatant containing four or eight blastocysts to allogeneic mixed lymphocyte culture inhibited both the MLR responses and the generation of cytotoxic T lymphocytes (CTL). Preincubation of the culture supernatant with lymphocytes syngeneic to the responder cells of MLR induced potent suppressor cell activity in the MLR. The supernatant did not inhibit the activity of CTL at the effector phase, but preinduced suppressor cells obtained by incubation of splenocytes with the supernatant showed almost complete suppression of CTL activity at the effector phase. Both of the suppressor cells, active on MLR and at the generation phase of CTL as well as active at the effector phase, had a surface phenotype of Thy-1+ and Ig-. The suppressive material could be extracted from the eight-cell stage of fertilized ova or blastocysts but not from unfertilized ova, indicating that the production of the factor(s) is dependent on the stages of early embryogenesis. These results suggest that the active induction of suppressor T lymphocytes by the factor(s) released from implanted embryos is one of the protective mechanisms from maternal immunologic attack.     

Impact of the Distance from the Stent Edge to the Residual Plaque on Edge Restenosis following Everolimus-Eluting Stent Implantation

Objectives

This study aimed to assess the relation between stent edge restenosis (SER) and the distance from the stent edge to the residual plaque using quantitative intravascular ultrasound.

Background

Although percutaneous coronary intervention with drug-eluting stents has improved SER rates, determining an appropriate stent edge landing zone can be challenging in cases of diffuse plaque lesions. It is known that edge vascular response can occur within 2 mm from the edge of a bare metal stent, but the distance to the adjacent plaque has not been evaluated for drug-eluting stents.

Methods

A total of 97 proximal residual plaque lesions (plaque burden [PB] >40%) treated with everolimus-eluting stents were retrospectively evaluated to determine the distance from the stent edge to the residual plaque.

Results

The SER group had significantly higher PB (59.1 ± 6.1% vs. 51.9 ± 9.1% for non-SER; P = 0.04). Higher PB was associated with SER, with the cutoff value of 54.74% determined using receiver operating characteristic (ROC) curve analysis. At this cutoff value of PB, the distance from the stent edge to the lesion was significantly associated with SER (odds ratio = 2.05, P = 0.035). The corresponding area under the ROC curve was 0.725, and the cutoff distance value for predicting SER was 1.0 mm.

Conclusion

An interval less than 1 mm from the proximal stent edge to the nearest point with the determined PB cutoff value of 54.74% was significantly associated with SER in patients with residual plaque lesions.     

Local and chemical distribution of phlorotannins in brown algae

The local and chemical distribution of phlorotannins among the Japanese Laminariaceae, Eisenia bicyclis, Ecklonia cava and Ecklonia kurome, was investigated. As a result of light microscopy observations with vanillin-HCl staining, phlorotannins were found to be accumulated within the vegetative cells of the outer cortical layer of the thalli, regardless of the species, stage of growth or organ. Crude phlorotannins comprised about 3.0% of the algal powder for each of the algae. High-performance liquid chromatography (HPLC) showed that the phlorotannins of E. bicyclis were composed of phloroglucinol (0.9%), phloroglucinol tetramer (4.4%), eckol (7.5%), phlorofucofuroeckol A (21.9%), dieckol (23.4%), and 8,8'-bieckol (24.6%), plus some other unknown phenolic compounds (17.3%). The composition of the phlorotannins differed little among the Laminariaceae, except for a significantly larger amount of the tetramer, MW 478, in E. bicyclis.     

Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein

The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 542-547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracycline-inducible ("Tet-On") system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress.     

Studies on the oligomeric structure of yeast aldehyde dehydrogenase by cross-linking with bifunctional reagents.

The molecular w:ight of yeast aldehyde dehydrogenase determined by sucrose density gradient centrifugation was 207,000 +/- 13,000. The enzyme activity was proportional to the enzyme concentration in the range of 2 X 10(-11) M to 1 X 10(-7) M. Cross-linking patterns obtained with yeast aldehyde dehydrogenase after treatment with a series of diimidoesters of increasing chain lengths with different reaction times resulted in the appearance of tetramers as the largest cross-linked product of the enzyme subunits. The molecular weights of its monomer, dimer, trimer, and tetramer were, 57,000, 114,000, 171,000, and 228,000, respectively, as estimated from their mobilities on SDS-electrophoresis. In tetramers monomers are probably assembled in a heterologous square arrangement.     

Surfactant-modified yeast whole-cell biocatalyst displaying lipase on cell surface for enzymatic production of structured lipids in organic media

The cell surface engineering system, in which functional proteins are genetically displayed on microbial cell surfaces, has recently become a powerful tool for applied biotechnology. Here, we report on the surfactant modification of surface-displayed lipase to improve its performance for enzymatic synthesis reactions. The lipase activities of the surfactant-modified yeast displaying Rhizopus oryzae lipase (ROL) were evaluated in both aqueous and nonaqueous systems. Despite the similar lipase activities of control and surfactant-modified cells in aqueous media, the treatment with nonionic surfactants increased the specific lipase activity of the ROL-displaying yeast in n-hexane. In particular, the Tween 20-modified cells increased the cell surface hydrophobicity significantly among a series of Tween surfactants tested, resulting in 8–30 times higher specific activity in organic solvents with relatively high log P values. The developed cells were successfully used for the enzymatic synthesis of phospholipids and fatty acid methyl esters in n-hexane, whereas the nontreated cells produced a significantly low yield. Our results thus indicate that surfactant modification of the cell surface can enhance the potential of the surface-displayed lipase for bioconversion.