Yoshinori Muto

Biochemical properties and purification of metallo-beta-lactamase from Bacteroides fragilis.

The beta-lactamase from Bacteroides fragilis GAI-30144 hydrolyzed imipenem, oxyiminocephalosporins, cephamycins, and penicillins. Enzyme activity was inhibited by EDTA. Zinc completely reversed inactivation of the enzyme by EDTA. The molecular mass of purified enzyme was estimated to be 33,000 daltons. Images

Neuronal protein NP25 interacts with F-actin

Neuronal protein NP25 is a neuron-specific protein present in highly differentiated neural cells, but its functional properties have not been well characterized. NP25 shows high amino acid sequence homology with the smooth muscle cell cytoskeleton-associated proteins, SM22, mp20, and calponin. To gain an insight into the biological functions of NP25, we first examined its subcellular localization in the human neuroblastoma cell line, SK-N-SH. NP25 diffusely distributed in the cytoplasm and fiber-like staining was also observed. It showed that NP25 co-localized with F-actin on stress fibers. A co-sedimentation assay demonstrated that NP25 bound to filamentous actin. Further investigations using fluorescence resonance energy transfer (FRET) technique revealed intracellular binding of NP25 and actin. The significance of the interaction between NP25 and F-actin is discussed.

Phosphorylated retinoid X receptor α loses its heterodimeric activity with retinoic acid receptor β

A malfunction in retinoid X receptor (RXR) α due to phosphorylation is associated with the development of hepatocellular carcinoma. However, the precise mechanisms by which phosphorylated RXRα loses its physiological function remain unclear. In the present study we examined whether phosphorylation of RXRα affects its dimeric activity. Fluorescence resonance energy transfer studies and immunoprecipitation assays showed that the physical interaction between RXRα and retinoic acid receptor β was impaired when 293T cells were transfected with phosphomimic mutant RXRα (T82D/S260D), whereas this interaction was activated at a level similar to wild‐type RXRα‐transfected cells when the cells were transfected with an unphosphorylated mutant RXRα (T82A/S260A). Treating the T82A/S260A‐transfected cells with retinoid resulted in a significant increase in the transcriptional activities of the retinoic acid receptor responsive element and RXR responsive element promoters, whereas these transcriptional activities did not increase in the T82D/S260D‐transfected cells. Transfection with T82A/S260A enhanced both the inhibition of cell growth and the induction of apoptosis caused by retinoid, although the T82D/S260D‐transfected cells lost their responsiveness to retinoid. Moreover, transfection with T82A/S260A caused an inhibition of cell growth and a reduction of colony‐forming ability in soft agar in HuH7 human hepatocellular carcinoma cells. These findings suggest that phosphorylation of RXRα abolishes its ability to form homodimers and heterodimers with RXR and retinoic acid receptor β, thus resulting in the loss of cell growth control and the acceleration of cancer development. In conclusion, the inhibition of RXRα phosphorylation and the restoration of its original function as a master regulator of nuclear receptors might therefore be an effective strategy for controlling cancer cell growth. (Cancer Sci 2007; 98: 1868–1874)

Effects of local anesthetics on calmodulin-dependent guanylate cyclase in the plasma membrane of tetrahymena pyriformis

A highly purified preparation of Tetrahymena calmodulin activated a membrane-bound guanylate cyclase by more than 40-fold. This activation of guanylate cyclase by calmodulin was inhibited completely by local anesthetics such as dibucaine, tetracaine, lidocaine and procaine at concentrations that had no appreciable effect on the activities of basal guanylate cyclase (without calmodulin) and adenylate cyclase. The inhibition by dibucaine of calmodulin-mediated activation of the enzyme activity was not reversed by calcium but was partially overcome by increasing the concentration of calmodulin. Kinetic analysis of local anesthetic-induced inhibition of activation of guanylate cyclase demonstrated a mixed type of antagonism. These results suggest the possibility that the inhibition of calmodulin-dependent guanylate cyclase resulted, in part, from interaction of the drugs with calmodulin.

Interaction of trifluoperazine with Tetrahymena calmodulin A 19F NMR study

We have used 19F NMR to study interactions of trifluoperazine (TFP), a potent calmodulin (CaM) antagonist, with Tetrahymena calmodulin (Tet. CaM). Changes in chemical shift and bandwidth of TFP caused by adding Tet. CaM in the presence of excess Ca2+ were much smaller than those by adding porcine CaM. The spectral features of the TFP—Tet. CaM solution in the presence of excess Ca2+ were quite similar to those of the TFP—porcine CaM solution in the absence of Ca2+. The exchange rate of TFP from Tet. CaM was estimated to be nearly 20 s−1. The TFP—Tet. CaM solution in the absence of Ca2+ showed a pronounced pH dependence of the 19F NMR chemical shift, whereas the solution in the presence of excess Ca2+ showed a smaller pH dependence. Thus, it was suggested that TFP is located near a hydrophilic region of the Tet. CaM molecule in the absence of Ca2+, while TFP is located near a hydrophobic region of the Tet. CaM in the presence of excess Ca2+.

Biochemical characterization of (Ca2+ + Mg2+)-ATPase in Tetrahymena microsomes

Abstract The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase ((Ca2+ + Mg2+)-ATPase, EC 3.6.1.3) was found to be high in Tetrahymena microsomes. The (Ca2+ + Mg2+)-ATPase has an apparent Km of 0.17 μM for free calcium and a maximum reaction velocity of 74 nmol ATP hydrolysed/mg protein per min. The enzyme was dependent on 1–3 mM magnesium and the pH optimum was pH 7.2. Among the nucleoside triphosphates tested, ATP was the best substrate, with an apparent Km of 63 μM. The (Ca2+ + Mg2+)-ATPase activity was unaffected by K+, Na+, dicyclohexylcarbodiimide, oligomycin, NaN3 and ouabain, but was potently inhibited by orthovanadate. Although the calmodulin antagonist, trifluoperazine, inhibited the enzyme activity, addition of calmodulin isolated from Tetrahymena did not elicit any stimulatory effect on the activity. Tetrahymena microsomes also displayed ATP-dependent uptake of calcium which lead to the accumulation to a maximal level of 9.2 nmol/mg microsomal protein. And this activity was inhibited by vanadate at a similar concentration range to that required to inhibit (Ca2+ + Mg2+)-ATPase. These observations suggest that the (Ca2+ + Mg2+)-ATPase in microsomes may act as a calcium-pumping ATPase in this cell.

Blepharismins, produced by the protozoan, Blepharisma japonicum, form ion‐permeable channels in planar lipid bilayer membranes

Blepharismins are polycyclic quinones found in the pigment granules of the ciliated protozoan, Blepharisma. Exposure to purified blepharismins results in lethal damage to several other ciliates. We here report that, at cytotoxic concentrations, blepharismins formed cation‐selective channels in planar phospholipid bilayer membranes. The channels formed in a diphytanoylphosphatidylcholine bilayer had a K+/Cl− permeability ratio of 6.6:1. Single channel recordings revealed the conductance to be quite heterogeneous, ranging from 0.2 to 2.8 nS in solutions containing 0.1 M KCl, possibly reflecting different states of aggregation of blepharismin. Our observations suggest that channel formation is a cytotoxic mechanism of blepharismins action against predatory protozoa.

Emergence of fluoroquinolone resistance in Bacteroides fragilis accompanied by resistance to beta-lactam antibiotics.

The in vitro susceptibilities of a group of 93 isolates of Bacteroides fragilis collected from 1983 to 1984 to six fluoroquinolones were compared with those of a group of 93 isolates collected from 1986 to 1987. The recently collected strains were less susceptible than the older strains to all of the agents tested. Norfloxacin-resistant (MIC, greater than 100 micrograms/ml) strains were less susceptible to cefoxitin and piperacillin than norfloxacin-susceptible strains.

Climacostol inhibits Tetrahymena motility and mitochondrial respiration

Climacostol is a resorcinol derivative that is produced by the ciliate Climacostomum virens. Exposure to purified climacostol results in lethal damage to the predatory ciliate Dileptus margaritifer and several other ciliates. To elucidate the mechanism of climacostol toxic action, we have investigated the effects of this compound on the swimming behavior of Tetrahymena thermophila and the respiratory system of isolated rat liver mitochondria. When added to living T. thermophila cells, climacostol markedly increased the turning frequency that was accompanied by a decrease in swimming velocity and subsequently by cell death. Observations by DIC and fluorescence microscopy showed morphological alterations in climacostol treated T. thermophila, indicating that climacostol might exert cytotoxic action on this organism. In the experiment with isolated rat liver mitochondria, climacostol was found to inhibit the NAD-linked respiration, but had no apparent effect on succinate-linked respiration. This finding indicates that climacostol specifically inhibits respiratory chain complex I in mitochondria. The combination of results suggest that the inhibition of mitochondrial respiration may be the cytotoxic mechanism of climacostol’s defenses against predatory protozoa.

An evolutionarily conserved leucine-rich repeat protein CLERC is a centrosomal protein required for spindle pole integrity

The centrosome functions as the microtubule-organizing center (MTOC) and plays a vital role in organizing spindle poles during mitosis. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle, and this feature is essential for the establishment of spindle bipolarity. Here we describe the molecular characterization of a novel protein called CLERC (Centrosomal leucine-rich repeat and coiled-coil containing protein) which is a human ortholog of Chlamydomonas Vfl1 protein. CLERC is a protein of 1032 amino acids with a calculated molecular mass of 120 kDa and possesses leucine-rich repeat and coiled-coil domains. Database searches revealed that CLERC has homologs in a wide variety of eukaryotes and is evolutionarily conserved. Endogenous CLERC protein associated with the centrosomes throughout the cell cycle and accumulated during mitosis. RNAi-mediated depletion of CLERC blocked formation of normal mitotic spindles and led to multipolar spindles. Moreover, many of the spindle poles in CLERC depleted cells contained only one centriole, indicating that centrosomes split into fractions containing a single centriole. These data indicate that the major function of CLERC during mitosis is to maintain the structural integrity of centrosomes, thereby contributing to spindle bipolarity.