Hisamitsu Ogawa

Constitutive NADPH-Dependent Electron Transferase Activity of the Nox4 Dehydrogenase Domain

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47phox and p67phox and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a Km for NADPH of 55 ± 10 μM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of ∼200 mol of H2O2 min−1 (mol of Nox4)−1. A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H2O2 production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min−1, largely superoxide dismutase-independent), ferricyanide (470 min−1), and other electron acceptors (artificial dyes and cytochrome b5). Rates were similar to those observed for H2O2 production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

Functional Properties of the unc-64 Gene Encoding aCaenorhabditis elegans Syntaxin

Phenotypes of Caenorhabditis elegans unc-18 and unc-64 gene mutations are similar. Whileunc-18 is known to be essential for normal synaptic transmission (Hosono, R., Hekimi, S., Kamiya, Y., Sassa, T., Murakami, S., Nishiwaki, S., Miwa, J., Taketo, A., and Kodaira, K.-I. (1992)J. Neurochem. 58, 1517–1525), the function ofunc-64 remains unclear. Here we describe the cloning, and the molecular and genetic characterization of the unc-64gene, especially in relation to unc-18. unc-64 encodes a protein (C. elegans syntaxin) showing sequence and structural similarities to mammalian syntaxin 1A. Fromunc-64, at least three types of poly(A)+ RNA are transcribed, which encode two types of syntaxin that differ in the deduced transmembrane domain. In gene expression, unc-64closely resembles unc-18, that is, both are expressed in neural cells, especially in motor neurons and neurons constituting head ganglions. C. elegans syntaxin binds to UNC-18 with high affinity. The unc-64 (e246) mutation producing a mild phenotype causes an Ala → Val conversion in the conserved COOH-terminal region in mammalian syntaxin 1A or Drosophilasyntaxin-1A whose site is included in three types of transcripts. The binding of the mutant C. elegans syntaxin to UNC-18 is greatly reduced, indicating the mutation site contributes to the binding.

A GCM Motif Protein Is Involved in Placenta-specific Expression of Human Aromatase Gene

A new cis-element, trophoblast-specific element 2 (TSE2) is located in the placenta-specific enhancer of the human aromatase gene that dictates its tissue-specific expression. In the minimum enhancer region, an element similar to the trophoblast-specific element (TSE), originally described for the human chorionic gonadotropin α-subunit gene, also exists (Yamada, K., Harada, N., Honda, S., and Takagi, Y. (1995)J. Biol. Chem. 270, 25064–25069). The co-presence of TSE and TSE2 is required to direct trophoblast-specific expression driven by a heterologous thymidine kinase promoter. A 2562-base pair cDNA clone encoding a 436-amino acid protein that binds to TSE2 was isolated from a human placental cDNA library using a yeast one-hybrid system with the TSE2 as a reporter sequence. The protein was revealed to be identical to hGCMa, a mammalian homologue of theDrosophila GCM (glia cells missing) protein. Expression of hGCMa is restricted to the placenta. The protein also binds to PLE1 in the leptin promoter among other cis-elements reported to confer placenta-specific expression, suggesting that hGCMa is a placenta-specific transcription regulator, possibly involved in the expression of multiple placenta-specific genes.

Activation of NADPH oxidase 1 in tumour colon epithelial cells

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.

The synaptic protein UNC-18 is phosphorylated by protein kinase C.

The C. elegans unc-18 encoded protein UNC-18 is implicated in the interactions between synaptic vesicles and presynaptic plasma membrane. To further characterize the neural protein, we investigated the phosphorylation in vitro of the protein expressed in Spodoptera frugiperda Sf21 cells. The UNC-18 protein is selectively phosphorylated by protein kinase C (PKC) but not by casein kinase II and cyclic AMP-dependent protein kinase. The presumed phosphorylation sites determined by manual Edman degradation were serine-2, serine-322, threonine-462 and serine-515, of which the last is highly conserved as a consensus phosphorylation site for PKC in Drosophila and the mammalian homologue. Phosphorylated UNC-18 extracted from C. elegans was also detected, indicating that it has a physiological role in intact nerve terminals. Therefore, the phosphorylation by PKC may play a physiological role in the regulation.

MICROHETEROGENEITY AND INTRAHEPATIC LOCALIZATION OF HUMAN AND RAT LIVER CYTOSOLIC ALANINE AMINOTRANSFERASE

Native human cytosolic alanine aminotransferase (EC 2.6.1.2) was found to be a homodimer consisting of two 55 kDa subunits. The human enzyme was more cationic and more susceptible to heat inactivation and heavy metal-inhibition than its rat homologue. Isoelectric focussing separated three human isoforms and four rat isoforms of cALT that differed in their isoelectric points. Two subtypes of the enzyme which differed in apparent molecular weight on sodium dodecylsulfate polyacrylamide gel electrophoresis were detected in rat, the liver type and the muscular type. This microheterogeneity was not found for human cytosolic alanine aminotransferase. Immunohistochemical studies revealed that the rat liver enzyme was exclusively localized in periportal hepatocytes, consistent with its role in gluconeogenesis. In human hepatocytes the plasma membrane was intensely stained, implicating an intracellular localization near the plasma membrane.

Effect of tryptophan metabolites on fluorescent granules in the Malpighian tubules of eye color mutants of Drosophila melanogaster.

Abstract Fluorescent granules that are stained with Nile blue sulfate are present in larval Malpighian tubules in the wild type strain of Drosophila melanogaster, Oregon-R. These granules emit a weak blue fluorescence and most of them are about 2 &mgr;m or more in diameter. The ommochrome precursor 3-hydroxy-kynurenine (3-HK) is actively transferred into the tubules of Oregon-R. Changes in the fluorescent granules in the Malpighian tubules on administration of ommochrome precursors were investigated in eye color mutants of Drosophila. The fluorescent granules in the tubules of the nonautonomous mutants v;bw and cn bw emit a strong blue fluorescence and most of them are about 1&mgr;m or less in diameter. When v;bw and cn bw larvae were cultured on medium supplemented with kynurenine or 3-HK, respectively, the fluorescence intensity of their granules decreased, and their size increased. These additions resulted in almost equal accumulation of 3-HK to that in Oregon-R. On the other hand, no 3-HK accumulated in the tubules of larvae of the autonomous mutants bw;st, ltd bw and w, which lack the fluorescent granules. These findings indicate that the fluorescent granules are an important intracellular site for uptake or storage of ommochrome precursors in larval Malpighian tubules of Drosophila.

Neurotrophic action of lipocortin 1 derived from astrocytes on cultured rat cortical neurons

The lipocortins are a family of structurally related proteins, namely an annexin family, that exerts a variety of cellular functions through Ca2+-dependent binding to phospholipase A2 [EC 3.1. 1.4], including a crucial role in the central nervous system (CNS) such as antipyrogenic, thermoregulatory and neuroprotective agents in vivo. To elucidate the paradigm of lipocortin 1 functions in the CNS, we have first demonstrated (1) the induction and subsequent extracellular secretion of LC1 by glucocorticoid in cultured rat astrocytes, and (2) neurotrophic activities (survival-promoting, neuritogenic and synaptogenic actions on rat cortical neurons) of recombinant LC1. Time-and dose-dependent experiments of a synthetic glucocorticoid, dexamethasone (DEX), on rat cortical astrocytes in culture revealed that the expression of the intracellular LC1 mRNA and protein were significantly augmented by DEX (1 microM). In addition, DEX evoked an extracellular secretion of LC1 without its cytotoxic effects. Furthermore, the recombinant LC1 appeared to promote not only the survival and neurite outgrowth but also the synaptogenesis of embryonal rat cortical neurons. These results suggest that LC1 induced and selectively released from astrocytes by either endogenously or exogenously introduced glucocorticoids may play a specific and essential role on development and regeneration of the central nervous system.

Expression, purification and characterization of recombinant C. Elegans UNC-18.

The Caenorhabditis elegans unc-18-encoded protein (UNC-18) is implicated in the processes of vesicle targeting, docking, and/or fusion. To further characterize the properties of this important neural protein, we expressed it at a high level in Spodoptera frugiperda Sf21 cells using a baculovirus expressing system. A cDNA containing the coding sequence for UNC-18 was inserted into the transfer vector pBlueBac to yield the recombinant virus pAcNPV/unc-18. At maximal expression, the recombinant virus produces a protein of 67 kDa, which constitutes about one-third of total cell protein. The UNC-18 protein was highly purified and its biochemical and functional properties were assessed. The protein is globular with an isoelectric point of 6.95. Circular dichroism spectroscopy indicated that the alpha-helix and beta-sheet account for 10.0 and 59.0%, respectively. Immunolabeling the Sf21 cells expressing UNC-18 showed that the expressed UNC-18 is predominantly localized in the cytoplasm as a soluble monomer. The protein is phosphorylated by protein kinase C and binds to the recombinant C. elegans syntaxin in vitro. These findings suggest that in vesicle traffic UNC-18 is a regulator factor associated with the plasma membrane through syntaxin, although intrinsically cytoplasmic.

Expression and transport into mitochondria of bovine cytochrome P-450(SCC) in insect cells using the baculovirus expression system

Bovine cytochrome P-450(SCC) introduced with the baculovirus host vector system was found to be expressed in Spodoptera frugiperda cells. Cell fractionation analysis indicated that the P-450(SCC) expressed as the precursor form was transported into mitochondria and converted to a mature form. However, this form did not exhibit definite activity for cholesterol side chain cleavage. These findings suggest that most of the P-450(SCC) expressed by this system is an inactive protein within mitochondria that is not folded to the conformation of the active enzyme and/or does not incorporate heme appropriately.