Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

Photobiocatalyzed asymmetric reduction of ketones using Chlorella sp. MK201

Aromatic ketones were reduced using suspension culture of Chlorella sp. MK201 under fluorescent light illumination producing the corresponding chiral alcohols in high yields with excellent enantiomeric excess (ee). For example, 2′,3′,4′,5′,6′-pentafluoroacetophenone at 0.25?mg/ml was converted to the corresponding (S)-alcohol in 80?% yield with?>99?% ee by 1?mg dry wt of Chlorella/ml in 12?h illumination (2,000 lux).     

A theoretical study of the conformational behavior of analogues of alpha-L-rhamnose-1-phosphate

The conformational behavior of methyl(2-O-methyl-alpha-L-rhamnopyranosyl)phosphate, together with a group of potentially more stable analogues, was investigated through a DFT approach at the B3LYP/6-31G(d) level; the energy of all the optimized structures was recalculated using a continuum solvent model, C-PCM, choosing water as the solvent. The compounds exhibited several, sometimes tenths of populated conformations so that the overall properties of flexibility and mobility were evaluated. The analogue in which the pyranose oxygen atom is replaced by a methylene group emerges as the best candidate as a mimic of the reference 1-phosphate, in spite of the fact that it lacks the anomeric and exo-anomeric effects. The other analogues result poorer mimics because of a conformational equilibrium at the pyranose ring or of an excessive rigidity of the aglycone moiety.     

Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations

The medaka, Oryzias latipes, has an XX/XY sex determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a prime candidate for the sex-determining gene. Furthermore, the crucial role of DMY during male development was established by studying two wild-derived XY female mutants. In this study, to find new DMY and sex-determination related gene mutations, we conducted a broad survey of the genotypic sex (DMY-negative or DMY-positive) of wild fish. We examined 2274 wild-caught fish from 40 localities throughout Japan, and 730 fish from 69 wild stocks from Japan, Korea, China, and Taiwan. The phenotypic sex type agreed with the genotypic sex of most fish, while 26 DMY-positive (XY) females and 15 DMY-negative (XX) males were found from 13 and 8 localities, respectively. Sixteen XY sex-reversals from 11 localities were mated with XY males of inbred strains, and the genotypic and phenotypic sexes of the F(1) progeny were analyzed. All these XY sex-reversals produced XY females in the F(1) generation, and all F(1) XY females had the maternal Y chromosome. These results show that DMY is a common sex-determining gene in wild populations of O. latipes and that all XY sex-reversals investigated had a DMY or DMY-linked gene mutation.     

Inhibition of sphingomyelinase activity helps to prevent neuron death caused by ischemic stress

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.     

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α_s1-casein-specific proliferation and IFN-γ productivity of CD⁴⁺ T cells, did not affect the proliferative response of CD8⁺ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8⁺ T cells, we established α_s1-casein-specific CD8⁺ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8⁺ T cells in oral tolerance is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Apoptosis-inducing neurotoxicity of dopamine and its metabolites via reactive quinone generation in neuroblastoma cells

Neurotoxic properties of L-dopa and dopamine (DA)-related compounds were assessed in human neuroblastoma SH-SY5Y cells with reference to their structural relationship. L-Dopa and its metabolites containing two free hydroxyl residues on their benzene ring showed toxicity in the cell, which was prevented by superoxide dismutase (SOD) and reduced glutathione (GSH), but not by catalase. Furthermore, a synthetic derivative of DA, 3-hydroxy-4-methoxyphenethylamine (HMPE) containing methoxy residue at position 4 in the benzene ring, exerted partial cytotoxicity, which was not prevented by SOD, GSH or catalase. However, the metabolites containing methoxy residue at position 3 failed to show a toxic effect in the SH-SY5Y cells. Moreover, DA induced apoptotic cell death, which was observed by nuclear and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and measurement of caspase-3 activity; this compound up-regulated apoptotic factor p53 while down-regulating anti-apoptotic factor Bcl-2. In the cell-free in vitro electron spin resonance (ESR) spectrometry, DA possessing two hydroxyl groups showed generation of DA-semiquinone radicals, which were markedly prevented by addition of SOD or GSH but not by catalase. On the other hand, methylation of one of the hydroxyl residues on the benzene ring of DA converted DA to an unoxidizable compound (3-MT or HMPE), and caused it to lose the property to produce semiquinone radicals. It has been previously reported that SOD acting as a superoxide:semiquinone oxidoreductase prevents quinone formation, and that reduced GSH through forming a complex with DA-quinone prevents quinone binding to the thiol group of the intact protein. Therefore, the present results suggest that DA and its metabolites containing two hydroxyl residues exert cytotoxicity mainly due to generation of highly reactive quinones.     

Brief exposure to carbon monoxide preconditions cardiomyogenic cells against apoptosis in ischemia-reperfusion

We examined whether and how pretreatment with carbon monoxide (CO) prevents apoptosis of cardioblastic H9c2 cells in ischemia-reperfusion. Reperfusion (6 h) following brief ischemia (10 min) induced cytochrome c release, activation of caspase-9 and caspase-3, and apoptotic nuclear condensation. Brief CO pretreatment (10 min) or a caspase-9 inhibitor (Z-LEHD-FMK) attenuated these apoptotic changes. Ischemia-reperfusion increased phosphorylation of Akt at Ser472/473/474, and this was enhanced by CO pretreatment. A specific Akt inhibitor (API-2) blunted the anti-apoptotic effects of CO in reperfusion. In normoxic cells, CO enhanced generation, which was inhibited by a mitochondrial complex III inhibitor (antimycin A) but not by a NADH oxidase inhibitor (apocynin). The CO-enhanced Akt phosphorylation was suppressed by an scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of , which is then converted by SOD to H₂O₂, and subsequent Akt activation by H₂O₂ attenuates apoptosis in ischemia-reperfusion.     

Identification of 57 conventional RFLP and 6 VNTR systems with 32 DNA clones on chromosome 11p15

Fifty-four clones containing human inserts were selected from a cosmid library constructed from a somatic cell hybrid containing chromosome 11p15.3-p15.5 as its only human complement. In 32 of these clones, 63 polymorphic systems were identified with a panel of restriction enzymes: 57 conventional RFLP systems and 6 highly polymorphic VNTR systems. Although we examined the cosmid with only seven enzymes, 18 clones (including 6 VNTRs) were polymorphic with three or more enzymes. The results suggested that DNA sequences on the peritelomeric region of chromosome 11p tend to be highly variable. Because these markers are highly informative, they will be excellent resources for investigations of hereditary diseases and tumor suppressor genes in this region of chromosome 11.