Establishment of a New Quality Control and Vaccine Safety Test for Influenza Vaccines and Adjuvants Using Gene Expression Profiling

We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.     

Mest but Not MiR-335 Affects Skeletal Muscle Growth and Regeneration

When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs. In this study, we examined whether Mest, one of paternally expressed imprinted genes that regulates body size during development, and miR-335 located in the second intron of the Mest gene play roles in muscle regeneration. We generated miR-335-deficient mice, and found that miR-335 is a paternally expressed imprinted microRNA. Although both Mest and miR-335 are highly expressed during muscle development and regeneration, only Mest^+/- (maternal/paternal) mice show retardation of body growth. In addition to reduced body weight in Mest^+/-; DMD-null mice, decreased muscle growth was observed in Mest^+/- mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration. Moreover, expressions of H19 and Igf2r, maternally expressed imprinted genes were affected in tibialis anterior muscle of Mest^+/-; DMD-null mice compared to DMD-null mice. Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.     

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.     

Immunohistochemical Localization of Manserin,a Novel Neuropeptide Derived from Secretogranin II,in Rat Adrenal Gland,and its Upregulation by Physical Stress

We recently identified a novel 40-amino acid neuropeptide designated manserin from the rat brain (Yajima in NeuroReport 15: 1755–1759, 2004). Manserin is highly expressed in pituitary and hypothalamic nuclei, which suggests that it plays a role in the endocrine system. In this study, we employed immunohistochemical methods to investigate the presence of manserin in rat adrenal glands, as well as its regulation by physical stress. Immunohistochemical analysis using anti-manserin antibody showed that manserin is present in the rat adrenal medulla but not in the cortex. When the colocalization of manserin and phenylethanolamine N-methyltransferase (PNMT), an epinephrine-synthesizing enzyme, was examined, virtually all PNMT-positive cells expressed manserin. Interestingly, the immunoreactivity of manserin was significantly increased when the rats were exposed to water-immersion restraint stress. These results demonstrate for the first time that adrenal manserin, a novel neuropeptide, may have a potential physiological role under stress-inducing conditions.     

Differential epigenetic regulation of BDNF and NT-3 genes by trichostatin A and 5-aza-2′-deoxycytidine in Neuro-2a cells

To understand epigenetic regulation of neurotrophins in Neuro-2a mouse neuroblastoma cells, we investigated the alteration of CpG methylation of brain-derived neurotrophic factor (BDNF) promoter I and neurotrophin-3 (NT-3) promoter IB and that of histone modification in Neuro-2a cells. Bisulfite genomic sequencing showed that the CpG sites of BDNF promoter I were methylated in non-treated Neuro-2a cells and demethylated following 5-aza-2′-deoxycytidine (5-aza-dC) treatment. In contrast, methylation status of the NT-3 promoter IB did not change by 5-aza-dC treatment in Neuro-2a cells. Furthermore, we demonstrated that BDNF exon I-IX mRNA was induced by trichostatin A (TSA) treatment. However, NT-3 exon IB-II mRNA was not induced by TSA treatment. Chromatin immunoprecipitation assays showed that the levels of acetylated histones H3 and H4 on BDNF promoter I were increased by TSA. These results demonstrate that DNA methylation and/or histone modification regulate BDNF gene expression, but do not regulate NT-3 gene expression in Neuro-2a cells.     

Occurrence of unusual heterogeneous lipid-containing granule-storing cells in the main excretory duct epithelium of the male mouse submandibular gland

Summary The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supranuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity.

The recent discovery that point mutations in the beta/A4 amyloid precursor protein may be the cause of certain forms of familial Alzheimer's disease provides strong support for the view that a thorough understanding of the metabolism of this protein may elucidate the pathogenesis of most forms of the disease and thus serve as a basis for rational prevention and therapy. Here we show that overexpression of a portion of the amyloid precursor protein molecule produces at least four distinct fragments of the COOH-terminus of amyloid precursor protein, suggesting altered proteolysis of amyloid precursor protein, and that such overexpression is associated with cytotoxicity. The degree of toxicity in the P19 cell culture model (differentiating mouse embryonal carcinoma cells) is shown to be related to the two larger novel COOH-terminal protein fragments (16 and 14 kilodalton), as well as to levels of expression of these two fragments. The toxicity is manifested in several differentiated cell lineages, including neuronal cells.     

Increases of Calcium,Phosphorus, and Magnesium in Both the Right and Left Fibrous Trigones of Human Heart with Aging

We previously reported that reduced platelet endogenous antioxidant enzymes activities are related to the low plasma zinc level in patients with end-stage renal failure (ESRF). In this study, we attempt to evaluate whether dietary zinc deprivation reduces the activities of endogenous antioxidant and then enhances oxidative stress in the unstimulated platelet of normal and 5/6 nephrectomized (Nx) rats because increased platelet oxidative stress is suggested to involve in the incidence of thrombotic and atherosclerotic diseases. Male Sprague–Dawley rats (n = 48) were fed a zinc-deficient diet and deionized distilled water for 1 week to induce reduction of plasma zinc level. Half of the rats continued on this diet for 4 weeks as zinc-deplete group, and the other half were maintained on the same diet but with zinc-supplemented water (120 mg/L zinc sulfate solution) to correct the reduction of plasma zinc level as zinc-replete group. Half of each group underwent 5/6 Nx, while the other half underwent sham operation. Another 12 normal rats were fed standard rat chow (containing 23.4% protein and 50 ppm zinc) and drank deionized distilled water as normal control rats. In zinc-deplete rats including sham-operated and 5/6 Nx rats exhibited lower endogenous antioxidant enzymes activities such as reduced glutathione (GSH), superoxide dismutase (SOD), and glutathione peroxidase (GPX) and higher malondialdehyde (MDA) levels than normal control rats in the unstimulated platelets. However, in zinc-replete rats including sham-operated and 5/6 Nx rats have a normal endogenous antioxidant enzymes activity and normal MDA levels in the unstimulated platelets. We suggest that in uremia, the low plasma zinc level may be a risk factor for thrombotic and atherosclerotic diseases because it reduces the activities of endogenous antioxidant enzymes and increases oxidative stress in the unstimulated platelet. Supported by grant 92-117 from Taipei Veterans General Hospital