Importance of renal mitochondria in the reduction of TEMPOL,a nitroxide radical

Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol.     

Electrochemical characterization of lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium

Electrochemical analysis of lignin peroxidase (LiP) was performed using a pyrolytic graphite electrode coated with peroxidase-embedded tributylmethyl phosphonium chloride membrane. The formal redox potential of ferric/ferrous couples of LiP was −126 mV (versus SHE), which was comparable with that of manganese peroxidase (MnP) and horseradish peroxidase (HRP). Yet, only LiP is capable of oxidizing non-phenolic substrates with a high redox potential. Since with decreasing pH, the redox potential increased, an incredibly low pH optimum of LiP as peroxidase at 3.0 or lower was proposed as the clue to explain LiP mechanisms. A low pH might be the key for LiP to possess a high redox potential. The pK_a values for the distal His in peroxidases were calculated using redox data and the Nernst equation, to be 5.8 for LiP, 4.7 for MnP, and 3.8 for HRP. A high pK_a value of the distal His might be crucial for LiP compound II to uptake a proton from the solvent. As a result, LiP is able to complete its catalytic cycle during the oxidation of non-proton-donating substrates. In compensation, LiP has diminished its reactivity toward hydrogen peroxide.     

Isolation and characterization of novel tachykinins from the posterior salivary gland of the common octopus Octopus vulgaris

Two novel tachykinins (OctTK-I: Lys-Pro-Pro-Ser-Ser-Ser-Glu-Phe-Ile-Gly-Leu-Met-NH(2) and OctTK-II: Lys-Pro-Pro-Ser-Ser-Ser-Glu-Phe-Val-Gly-Leu-Met-NH(2)) were isolated from the posterior salivary gland of the octopus (Octopus vulgaris) using a contraction assay of the carp rectum. These peptides had in common the pentapeptide sequence -Phe-X-Gly-Leu-Met-NH(2) at the C-terminal and induced immediate contractions on the carp rectum and the guinea-pig ileum. cDNAs encoding their precursor proteins were cloned. The OctTK gene was expressed in the posterior salivary gland and the expression was localized in mucus-secreting cells of the gland. The results suggested that OctTKs might be secreted as a venomous substance acting on vertebrates such as fishes, which are the prey or natural enemies of the octopus.     

Synthesis and characterization of the sweet protein brazzein

The sweet protein brazzein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon is 2000-500 times sweeter than sucrose, and consists of 54 amino acid residues with four intramolecular disulfide bonds. Brazzein was prepared by the fluoren-9-yl-methoxycarbonyl solid-phase method, and was identical to natural brazzein by high performance liquid chromatography, mass spectroscopy, peptide mapping, and taste evaluation. The D enantiomer of brazzein was also synthesized, and was shown to be the mirror image of brazzein. The D enantiomer (ent-brazzein) was devoid of any sweetness and was essentially tasteless. © 1996 John Wiley & Sons, Inc.     

Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase

Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.     

Double-stranded RNA Mediates Selective Gene Silencing of Protein Phosphatase Type 1 Delta Isoform in HEK-293 Cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1δ) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP18. This RNA interference did not affect the expression of α and γ1 isoforms of PP1. Transfection of antisense RNA specific for PP1δ also suppressed the expression of PP1δ. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Characteristic Cerebrospinal Fluid Cytokine/Chemokine Profiles in Neuromyelitis Optica,Relapsing Remitting or Primary Progressive Multiple Sclerosis

Background

Differences in cytokine/chemokine profiles among patients with neuromyelitis optica (NMO), relapsing remitting multiple sclerosis (RRMS), and primary progressive MS (PPMS), and the relationships of these profiles with clinical and neuroimaging features are unclear. A greater understanding of these profiles may help in differential diagnosis.

Methods/Principal Findings

We measured 27 cytokines/chemokines and growth factors in CSF collected from 20 patients with NMO, 26 with RRMS, nine with PPMS, and 18 with other non-inflammatory neurological diseases (OND) by multiplexed fluorescent bead-based immunoassay. Interleukin (IL)-17A, IL-6, CXCL8 and CXCL10 levels were significantly higher in NMO patients than in OND and RRMS patients at relapse, while granulocyte-colony stimulating factor (G-CSF) and CCL4 levels were significantly higher in NMO patients than in OND patients. In NMO patients, IL-6 and CXCL8 levels were positively correlated with disability and CSF protein concentration while IL-6, CXCL8, G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ were positively correlated with CSF neutrophil counts at the time of sample collection. In RRMS patients, IL-6 levels were significantly higher than in OND patients at the relapse phase while CSF cell counts were negatively correlated with the levels of CCL2. Correlation coefficients of cytokines/chemokines in the relapse phase were significantly different in three combinations, IL-6 and GM-CSF, G-CSF and GM-CSF, and GM-CSF and IFN-γ, between RRMS and NMO/NMOSD patients. In PPMS patients, CCL4 and CXCL10 levels were significantly higher than in OND patients.

Conclusions

Our findings suggest distinct cytokine/chemokine alterations in CSF exist among NMO, RRMS and PPMS. In NMO, over-expression of a cluster of Th17- and Th1-related proinflammatory cytokines/chemokines is characteristic, while in PPMS, increased CCL4 and CXCL10 levels may reflect on-going low grade T cell and macrophage/microglia inflammation in the central nervous system. In RRMS, only a mild elevation of proinflammatory cytokines/chemokines was detectable at relapse.     

Microsatellite polymorphism in the human heme oxygenase-1 gene promoter and its application in association studies with Alzheimer and Parkinson disease

Oxidative stress has been suggested to be involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer disease (AD) and Parkinson disease (PD). Heme oxygenase-1 (HO-1), a key enzyme in heme catabolism, also functions as an antioxidant enzyme. Here, we show that a (GT)_n repeat in the human HO-1 gene promoter region is highly polymorphic, although no particular alleles are associated with AD or PD. This newly identified genetic marker should allow us to study the possible involvement of HO-1 in certain human diseases. Received: 5 November 1996 / Accepted: 18 February 1997     

Development of mouse models for analysis of human virus infections

Viruses usually exhibit strict species‐specificity as a result of co‐evolution with the host. Thus, in mouse models, a great barrier exists for analysis of infections with human‐tropic viruses. Mouse models are unlikely to faithfully reproduce the human immune response to viruses or viral compounds and it is difficult to evaluate human therapeutic efficacy with antiviral reagents in mouse models. Humans and mice essentially have different immune systems, which makes it difficult to extrapolate mouse results to humans. In addition, apart from immunological reasons, viruses causing human diseases do not always infect mice because of species tropism. One way to determine tropism would be a virus receptor that is expressed on affected cells. The development of gene‐disrupted mice and Tg mice, which express human receptor genes, enables us to analyze several viral infections in mice. Mice are, indeed, susceptible to human viruses when artificially infected in receptor‐supplemented mice. Although the mouse cells less efficiently permit viral replication than do human cells, the models for analysis of human viruses have been established in vivo as well as in vitro, and explain viral pathogenesis in the mouse systems. In most systems, however, nucleic acid sensors and type I interferon suppress viral propagation to block the appearance of infectious manifestation. We herein review recent insight into in vivo antiviral responses induced in mouse infection models for typical human viruses.