Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: isolation and pharmacological characterization of rOscp1

Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.     

Induction of a high-CO2-inducible, periplasmic protein, H43, and its application as a high-CO2-responsive marker for study of the high-CO2-sensing mechanism in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a broad range of environmental CO(2) concentrations. We observed that the cells synthesized a specific 43 kDa protein, H43, in the periplasmic space under photoautotrophic high-CO(2) conditions. Under low-CO(2) conditions, H43 disappeared. However, H43 mRNA expression was observed even under heterotrophic low-CO(2) conditions when the cells were grown with 17.4 mM acetate in darkness. When the cells were treated with 4,4'-dithiocyanatostilbene-2,2'-disulfonate (DIDS) and mersalyl to modify cell surface proteins, H43 mRNA expression was strongly affected under both heterotrophic and photoautotrophic conditions. The H43 induction pattern in a mitochondrial respiration-deficient mutant dum-1 that lacks cytochrome c oxidase was the same, but the level was much lower than that in the wild type. Even under illumination, the dissolved CO(2) concentration in the culture rapidly increased slightly following the addition of acetate and dramatically increased even further by the inhibition of photosynthesis with DCMU. Radiotracer experiments with [U-(14)C]acetate revealed that (14)CO(2) release from cells was greater in darkness than in the light due to the great stimulation of internal CO(2) evolution, resulting in an increase in external CO(2) concentration. Strong light inhibited H43 induction and DCMU promoted the induction under photoheterotrophic low-CO(2) conditions. The results demonstrate that H43 is strictly regulated by a concentration of CO(2) resulting from respiration and photosynthesis. Our results suggest that Chlamydomonas induces high-CO(2)-responsive protein H43 by sensing the concentration of ambient CO(2) with the contribution of cell surface protein.     

Inhibition of premature leaf abscission by a leafminer and its adaptive significance

We tested the possibility that a lepidopteran leafminer, Coptotriche japoniella Puplesis and Diskus, inhibits the host plant Eurya japonica Thunberg from abscising mined leaves prematurely to increase its survivorship in immature stage. We monitored abscission patterns of mined leaves with sacrificed larvae, mined leaves with living larvae, and unmined leaves from April to July 2004 and 2005 until leafminers emerged as adults. Unmined leaves rarely abscised before July. Mined leaves with sacrificed larvae fell at a constant rate after May, abscising significantly more than unmined leaves. In contrast, mined leaves with living larvae rarely fell before adult emergence; afterward they abscised rapidly. We also examined larval/pupal survivorship and mortality sources on the ground and trees after leafminers completed larval development. Leafminers on the ground suffered a higher mortality from predation than those on trees, and thus they emerged as adults on the ground less successfully. These findings suggest that the leafminer C. japoniella prevents the host plant from abscising mined leaves prematurely until adult emergence, thereby increasing their survivorship.     

Discovery of (-)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone--a potent NR2B-selective N-methyl D-aspartate (NMDA) antagonist for the treatment of pain

(-)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as an orally active NR2B-subunit selective N-methyl-d-aspartate (NMDA) receptor antagonist. It has very high selectivity for NR2B subunits containing NMDA receptors versus the HERG-channel inhibition (therapeutic index=4200 vs NR2B binding IC(50)). This compound has improved pharmacokinetic properties compared to the prototype CP-101,606.     

Bidirectional Ca2+ coupling of mitochondria with the endoplasmic reticulum and regulation of multimodal Ca2+ entries in rat brown adipocytes

How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca²⁺ entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca²⁺ or Mg²⁺ and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) produced by thapsigargin was abolished by removing extracellular Ca²⁺, depressed by depleting extracellular Na⁺, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca²⁺]_i, in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a Ca²⁺-free EGTA solution, and an Na⁺-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca²⁺ and fully blocked by oligomycin but not by thapsigargin or an Na⁺-deficient solution, was accompanied by a rise in cytoplasmic Mg²⁺ concentration, and was summated with a high pH-induced rise in [Ca²⁺]_i, whereas the extracellular Ca²⁺-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca²⁺ but not by thapsigargin, depressed by decreasing Na⁺, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca²⁺]_i in a Ca²⁺-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca²⁺ release, activating Ca²⁺ release from the ER and store-operated Ca²⁺ entry, and directly elicits a novel plasmalemmal Ca²⁺ entry, whereas Ca²⁺ release from the ER activates Ca²⁺ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. plasmalemmal calcium entry; calcium release; mitochondrial depolarization; FCCP     

Establishment and characterization of a cisplatin-resistant cell line (IGSK-1) from a poorly differentiated gastric adenocarcinoma

We successfully established a spontaneously cisplatin-resistant tumor cell line (designated as IGSK-1) derived from original gastric carcinoma. The patient was a 75-year-old Japanese woman. The histopathological diagnosis was gastric poorly differentiated adenocarcinoma accompanied with metastatic foci in lymph nodes, pT3, N2 M0, stage IIIB. The IGSK-1 cells grew as adhesive and monolayered cultures on the bottom of dishes. The susceptibility of the IGSK-1 cells to anti-cancer drugs was examined using oxygen electrode apparatus (Daikin, Tsukuba, JPN), and the results suggested TXL was effective, and CDDP, CPT-11 and 5-FU were not effective. Gastrin and somatostatin secretions were confirmed by immunohistochemical staining and also radioimmunoassay. Immunohistochemistry and radioimmunoassay for serotonin suggested the IGSK-1 cells might incorporate serotonin from the growth media. Spontaneously cisplatin-resistant gastric carcinoma cell line secreted gastrin and somatostatin is very important material for chemotherapy.     

Loss of cell wall alpha(1-3) glucan affects Cryptococcus neoformans from ultrastructure to virulence

Yeast cell walls are critical for maintaining cell integrity, particularly in the face of challenges such as growth in mammalian hosts. The pathogenic fungus Cryptococcus neoformans additionally anchors its polysaccharide capsule to the cell surface via alpha(1-3) glucan in the wall. Cryptococcal cells disrupted in their alpha glucan synthase gene were sensitive to stresses, including temperature, and showed difficulty dividing. These cells lacked surface capsule, although they continued to shed capsule material into the environment. Electron microscopy showed that the alpha glucan that is usually localized to the outer portion of the cell wall was absent, the outer region of the wall was highly disorganized, and the inner region was hypertrophic. Analysis of cell wall composition demonstrated complete loss of alpha glucan accompanied by a compensatory increase in chitin/chitosan and a redistribution of beta glucan between cell wall fractions. The mutants were unable to grow ina mouse model of infection, but caused death in nematodes. These studies integrate morphological and biochemical investigations of the role of alpha glucan in the cryptococcal cell wall.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Double knockdown of α1,6-fucosyltransferase (<Emphasis Type="Italic">FUT8</Emphasis>) and GDP-mannose 4,6-dehydratase (<Emphasis Type="Italic">GMD</Emphasis>) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC

Background  

Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies.