Constructing and testing the thermodynamic limits of synthetic NAD(P)H:H2 pathways

NAD(P)H:H₂ pathways are theoretically predicted to reach equilibrium at very low partial headspace H₂ pressure. An evaluation of the directionality of such near‐equilibrium pathways in vivo, using a defined experimental system, is therefore important in order to determine its potential for application. Many anaerobic microorganisms have evolved NAD(P)H:H₂ pathways; however, they are either not genetically tractable, and/or contain multiple H₂ synthesis/consumption pathways linked with other more thermodynamically favourable substrates, such as pyruvate. We therefore constructed a synthetic ferredoxin‐dependent NAD(P)H:H₂ pathway model system in Escherichia coli BL21(DE3) and experimentally evaluated the thermodynamic limitations of nucleotide pyridine‐dependent H₂ synthesis under closed batch conditions. NADPH‐dependent H₂ accumulation was observed with a maximum partial H₂ pressure equivalent to a biochemically effective intracellular NADPH/NADP⁺ ratio of 13:1. The molar yield of the NADPH:H₂ pathway was restricted by thermodynamic limitations as it was strongly dependent on the headspace : liquid ratio of the culture vessels. When the substrate specificity was extended to NADH, only the reverse pathway directionality, H₂ consumption, was observed above a partial H₂ pressure of 40 Pa. Substitution of NADH with NADPH or other intermediates, as the main electron acceptor/donor of glucose catabolism and precursor of H₂, is more likely to be applicable for H₂ production.     

Roles of plasma membrane-associated sialidase NEU3 in human cancers

Altered sialylation of glycosphingolipids is observed in cancer as a ubiquitous phenotype, leading to the appearance of tumor-associated antigens, aberrant adhesion and disturbance of transmembrane signaling. To understand the pathological significance of aberrant alterations of gangliosides in cancer, our studies have been focused on sialidase, which is responsible for the removal of sialic acids from glycoproteins and glycolipids. Among human sialidases so far identified, sialidase NEU3 is a key enzyme for ganglioside degradation because of its uniqueness both in its localization in the plasma membrane and in specifically hydrolyzing gangliosides. NEU3 is markedly up-regulated in many types of cancers including colon and renal carcinomas and suppresses apoptosis of cancer cells. The present paper briefly summarizes our recent results on the sialidase alterations and their significance in cancer. NEU3 is indeed closely related to malignancy and thus may be a potential target for cancer diagnosis and therapy.     

Immunohistochemical evidence for the existence of rat cytosolic sialidase in rat skeletal muscles

 Histochemical evidence is required to demonstrate the presence of biochemically defined cytosolic sialidase. To meet this requirement, we examined the immunohistochemical localization of the enzyme in rat skeletal muscles. Sections of chemically fixed tissues were incubated with a polyclonal antibody raised against a synthetic peptide which corresponded to a part of the enzyme protein. After incubation with the primary antibody, cryosections for fluorescence microscopy and resin sections for electron microscopy were incubated with a fluorochrome- and colloidal gold-labeled secondary antibody, respectively. Immunofluorescence was diffusely distributed in the muscle fibers and was also found in the perimysium and blood vessels. Many immunogold particles were scattered over the sarcoplasm, myofibrils, nucleoplasm, and matrix of mitochondria. The immunogold particles were also found in the equivalent compartments of axons, Schwann cells, and cells of endomysium and blood vessels. The specificity of the primary antibody was elucidated by immunoblotting and an immunoprecipitation test. These findings clearly indicate that this type of sialidase is essentially located in the cytosolic compartment. Consequently, the name, cytosolic sialidase, will be appropriate for this enzyme. Additionally it is indicated that this enzyme is also present in cells other than skeletal muscle fibers. Accepted: 29 January 1997     

Histone H3 peptides incorporating modified lysine residues as lysine-specific demethylase 1 inhibitors

Lysine-specific demethylase 1 (LSD1) is a flavin-dependent enzyme that removes methyl groups from mono- or dimethylated lysine residues at the fourth position of histone H3. We have previously reported several histone H3 peptides containing an LSD1 inactivator motif at Lys-4. In this study, histone H3 peptides having a trans-2-phenylcyclopropylamine (PCPA), a 2,5-dihydro-1H-pyrrole, and a 1,2,3,6-tetrahydropyridine moiety at Lys-4 were prepared along with related compounds possessing a shorter side chain at the fourth position. Enzymatic assays showed that PCPA peptides containing a longer side chain, which can react with FAD in the active site, are potent LSD1-selective inhibitors.     

HZE radiation effects for hereditary renal carcinomas.

Eker rat known as a model of hereditary renal carcinoma (RC) is an example of Mendelian dominantly inherited predisposition to a specific cancer in experimental animals. We investigate the effects of simulated space radiation on carcinogenesis using HIMAC. We estimated RBE from the Eker rats exposed to the heavy-ions, C (290 MeV/u) and Fe (500 MeV/u) ions, comparing to the effects of X-ray irradiation. Pregnant rats were exposed to C and Fe ions and X-rays with a single dose of 1 Gy, 2 Gy, 3 Gy on day 19 of gestation. The offspring were sacrificed at 8 weeks of age. We evaluated organ weights and tumor genesis. The weights of thymus, lung, liver, spleen were found to be no difference from the control at 1 Gy irradiation but 50% decrease at 3 Gy irradiation. We found in the irradiated animal that kidney, brain and testis were very sensitive organs of which the weight decreased to approximately 80% at 1 Gy and to 40% at 3 Gy irradiations. Based on the dose-response relationship of the radiation-induced carcinoma, averaged RBE ware calculated to be 1.1 for C-ion, 1.6 for Fe-ion.     

Effects of heavy ion to the primary [correction of rimary] culture of mouse brain cells.

To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.