New functions of histamine found in histidine decarboxylase gene knockout mice

Gene targeting techniques have revolutionized the investigation of the effects of bioactive substances in pathological and physiological conditions. Histamine synthesis is uniquely catalyzed by L-histidine decarboxylase. The knockout mice of this gene express no histamine-producing activity and lack histamine. These mice have been used to examine the mechanisms of histamine in several known phenotypes, e.g., gastric acid secretion, contraction of smooth muscles, vascular permeability, and awakening, and have also been used to explore unreported effects of histamine in the whole body. First, we will review the former mechanisms and then move to the latter, new effects. Especially, in the latter mechanisms, we focus on several important roles of histamine in angiogenesis, neutrophil and eosinophil recruitment, bacterial infection, and systemic anaphylaxis in this review. Moreover, to our surprise, the morphology of mast cells in the knockout mice was severely affected by the absence of histamine in terms of their granules.     

Resonance Raman applications in investigations of cytochrome c oxidase

Recent applications of resonance Raman (RR) spectroscopy in investigations of cytochrome c oxidase (CcO) are reviewed. Red-excited RR spectra for the fully oxidized "as-isolated" CcO tuned to the ligand-to-metal charge transfer absorption band at 655nm exhibit a Raman band at 755cm(-1) assignable to the ν(OO) stretching mode of a peroxide. Binding of CN(-) diminishes the RR band concomitant with the loss of the charge transfer absorption band. This suggests that a peroxide forms a bridge between heme a(3) and Cu(B). Time-resolved RR spectroscopy of whole mitochondria identified a band at 571cm(-1) arising from the oxygenated intermediate at Δt=0.4, 0.6 and 1.4ms. Bands at 804 and 780cm(-1) of the P and F intermediates were observed at Δt=0.6 and 1.4ms, respectively. The coordination geometries of the three intermediates are essentially the same as the respective species observed for solubilized CcO. However, the lifetime of the oxygenated intermediate in mitochondria was significantly longer than the lifetime of this intermediate determined for solubilized CcO. This phenomenon is due either to the pH effect of mitochondrial matrix, the effect of ΔpH and/or ΔΨ across the membrane, or the effect of interactions with other membrane components and/or phospholipids.     

Polygalacturonase mRNA of Tomato: Size and Content in Ripe Fruits

In ripe tomato fruits, polygalacturonase (PG) mRNA comprisedabout 1% of the translatable RNAs in the poly(A)(+)RNA fraction.Sucrose density gradient centrifugation showed that this PGmRNA is similar in size to 18S rRNA, which suggests the presenceof a non-coding region. (Received June 19, 1984; Accepted October 23, 1984)     

Combined genotoxic effects of radiation and a tobacco-specific nitrosamine in the lung of gpt delta transgenic mice

It is important to evaluate the health effects of low-dose-rate or low-dose radiation in combination with chemicals as humans are exposed to a variety of chemical agents. Here, we examined combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. In this mouse model, base substitutions and deletions can be separately analyzed by gpt and Spi- selections, respectively. Female gpt delta mice were either treated with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or combined with NNK treatments at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. In the gpt selection, the NNK treatments enhanced the mutation frequencies (MFs) significantly, but no obvious combined effects of gamma-irradiation were observable at any given radiation dose. In contrast, NNK treatments appeared to suppress the Spi- large deletions. In the Spi- selection, the MFs of deletions more than 1 kb in size increased in a dose-dependent manner. When NNK treatments were combined, the dose-response curve became bell-shaped where the MF at the highest radiation dose decreased substantially. These results suggest that NNK treatments may elicit an adaptive response that eliminates cells bearing radiation-induced double-strand breaks in DNA. Possible mechanisms underlying the combined genotoxicity of radiation and NNK are discussed, and the importance of evaluation of combined genotoxicity of more than one agent is emphasized.     

Differential type I IFN-inducing abilities of wild-type versus vaccine strains of measles virus

Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-beta production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By gene-silencing analysis, DI RNA activated the RIG-I/MDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-beta mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-beta mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells.     

Upregulation of the glutamine transporter through transactivation mediated by cAMP/protein kinase A signals toward exacerbation of vulnerability to oxidative stress in rat neocortical astrocytes

In the present study, we have evaluated the possible functionality in astrocytes of the glutamine (Gln) transporter (GlnT) known to predominate in neurons for the neurotransmitter pool of glutamate. Sustained exposure to the adenylyl cyclase activator forskolin for 24 h led to a significant increase in mRNA expression of GlnT among different membrane transporters capable of transporting Gln, with an increase in [(3)H]Gln accumulation sensitive to a system A transporter inhibitor, in cultured rat neocortical astrocytes, but not neurons. Forskolin drastically stimulated GlnT promoter activity in a manner sensitive to a protein kinase A (PKA) inhibitor in rat astrocytic C6 glioma cells, while deletion mutation analysis revealed that the stimulation was mediated by a cAMP responsive element (CRE)/activator protein-1 (AP-1) like site located on GlnT gene promoter. Forskolin drastically stimulated the promoter activity in a fashion sensitive to a PKA inhibitor in C6 glioma cells transfected with a CRE or AP-1 reporter plasmid, in association with the phosphorylation of CRE binding protein on serine133. Transient overexpression of GlnT significantly exacerbated the cytotoxicity of hydrogen peroxide in cultured astrocytes. These results suggest that GlnT expression is upregulated by cAMP/PKA signals for subsequent exacerbation of the vulnerability to oxidative stress in astrocytes.     

Meiotic association between Spo11 regulated by Rec102, Rec104 and Rec114

Meiotic recombination is initiated by DNA double-stranded break (DSB) formation catalyzed by Spo11, a type-II topoisomerase-like transesterificase, presumably via a dimerization-mediated mechanism. We demonstrate the existence of in vivo interactions between Spo11 proteins carrying distinct tags, and the chromatin-binding and DSB activity of tagged Spo11at innate and targeted DSB sites upon fusion to the Gal4 DNA-binding domain. First we identified the interaction between Spo11-3FLAG and Gal4BD-Spo11 proteins, and established that this interaction specifically occurs at the time of DSB formation. We then observed that presence of the Gal4BD-spo11Y135F (nuclease-deficient) protein allows Spo11-3FLAG recruitment at the GAL2 locus, indicative of the formation of a hetero-complex near the GAL2 UAS sites, but no formation of double- or single-strand breaks. Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114. Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated. The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.     

Design, synthesis, and evaluation of isoindolinone-hydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors

We designed and synthesized hydroxamic acid derivatives bearing a 4-(3-pyridyl)phenyl group as a cap structure, and found that they exhibit potent histone deacetylase (HDAC) inhibitory activity. A representative compound, 17a, showed more potent growth-inhibitory activity against pancreatic cancer cells and greater upregulation of p21(WAF1/CIP1) expression than the clinically used HDAC inhibitor suberoylanilide hydroxamic acid (Zolinza).     

Detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) using a fully automated system with a nano-scale engineered biomagnetite

A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples.