Subpopulation Primers Essential for Exhaustive Detection of Diverse Hemagglutinin Genes of H5 Subtype Avian Influenza Viruses by Loop-Mediated Isothermal Amplification Method.

Loop-mediated isothermal amplification (LAMP) is a potential screening test for avian influenza (AI), but its narrow detection spectrum limits its applications. To improve this narrow detection spectrum, 3 types of primers were compared for detection of diverse H5 subtype hemagglutinin (HA) genes. Four and 6 genes, of 10 genetically different H5 HA genes tested, were detected with S primers specific for A/duck/Tsukuba/9/2005 (H5N2) and with M primers (which contained mixed bases), respectively. In contrast, all 10 HA genes became positive with population primers (P primers) (a mixture of primers designed for each subpopulation of 2,202 HA genes). Our study indicated that the P primers for the forward inner primer (FIP) and backward inner primer (BIP) sites were essential for exhaustive detection, whereas those for the F3, forward loop (FL), backward loop (BL), and B3 sites were exchangeable with M primers. A base mismatch experiment demonstrated that HA genes with ≤2 base mismatches per primer site and ≤10 base mismatches per HA gene were amplifiable. Reverse transcription-LAMP was broadly reactive, specific for H5 subtype HA genes, and applicable to field samples, with the sensitivity of real-time PCR. The in silico analysis suggested that most H5 HA genes (2,586 positive genes/2,588 genes tested) registered in the GenBank database might be amplifiable. These results indicate that the use of subpopulation primers in LAMP allows exhaustive detection of diverse HA genes and H5 LAMP can be used as a reliable AI screening test in general diagnostic laboratories.

Distribution of phosphoneuroprotein 14 (PNP 14) in vertebrates: its levels as determined by enzyme immunoassay.

We have established an enzyme immunoassay for phosphoneuroprotein 14 (PNP 14) which is mainly localized in the cytoplasmic matrix in presynaptic axon terminals and which is phosphorylated in vivo, as well as in vitro. Fab' prepared from rabbit IgG antibodies against bovine PNP 14 was conjugated with maleimide-horseradish peroxidase. The enzyme-conjugated Fab' was used as a second antibody in a sandwich enzyme immunoassay. This assay was able accurately to quantify 0.5-100 ng of rat PNP 14, as well as bovine PNP 14, and it was used for the determination of concentrations of PNP 14 in various rat tissues, neuroblastoma cells, and brains of other vertebrates. The concentrations of PNP 14 in the rat cerebrum, cerebellum, and testis were 1.1, 1.0, and 0.28 micrograms/mg of protein, respectively, and those in other tissues examined were less than 0.1 microgram/mg of protein. PNP 14 was also found in cultured cells, such as rat pheochromocytoma PC12 cells, NG108-15 cells, which are a hybrid between a mouse neuroblastoma and a rat glioma, mouse neuroblastoma Neuro-2a cells, and human neuroblastoma IMR32 cells. Furthermore, PNP 14-specific immunoreactivity was evaluated in the brains of various vertebrates, such as fish, frog, snake and chicken by immunoblot and enzyme immunoassay. The results revealed the immunoreactivity in the brains of all vertebrates examined and the levels were determined to be 0.6-2.1 micrograms bovine PNP 14 equivalents per mg of protein, suggesting that PNP 14 might be an essential component of the central nervous systems of vertebrates.

A new visual method for evaluating multiple data (Diagnostic Radar Chart) in general toxicological study.

A Diagnostic Radar Chart method was devised for use in the general assessment of the toxicity of drugs on the basis of visual patterns in toxicity studies. Its usefulness was evaluated on the findings of 32 laboratory items in 3 groups of animal models with experimental pathological conditions. The chart was found to facilitate intuitive discrimination among three types of pathological animal models, evaluation of sexual differences and evaluation of individual differences among the animals for each item. Therefore, if patterns are constructed on the Diagnostic Radar Chart for each of the drugs usable for preparing animal models with typical experimental pathological conditions, the chart is thought to be useful for evaluation or identification of the toxicity of an unknown drug.

[Strong opioids].

Oral route morphine should be first choice for moderate or strong cancer pain. Morphine must be administered essentially at fixed interval. It is important to keep effective plasma morphine concentration. When a patient can not take morphine via oral route, morphine must be administered by intravenous or subcutaneous infusion. Respiratory rate per minute of patients always must be measured during administration of morphine. Patients taking morphine have to take laxatives and antiemetics simultaneously. It is crucial to establish the cause of pain and choose other proper treatment when morphine is not effective.

Lower ß-endorphin content of peripheral blood mononuclear cells in patients with complex regional pain syndrome.

Recent studies have demonstrated that immune cell-derived ß-endorphin inhibits peripheral nociception. Changes in the ß-endorphin content of peripheral blood mononuclear cells (PBMC) were also reported in various human disorders. These findings suggest the modulation of pain by immuno-neural interaction through opioid-dependent mechanisms. The aim of this study, therefore, was to determine whether the levels of ß-endorphin in PBMC of patients with complex regional pain syndrome (CRPS) differ from those of healthy subjects. Heparinized venous blood was collected from ten CRPS patients (7 women and 3 men; mean age 39.4 ± 13.0 years) and 13 age-matched healthy volunteers (6 women and 7 men; mean age 38.4 ± 10.8 years). PBMC were separated by density gradient centrifugation. ß- endorphin was extracted from the cells in a commercial cell lysis buffer and its concentration was measured by enzyme immunoassay technique. Immunoreactive ß-endorphin levels in PBMC from the CRPS patients were significantly lower than those from the healthy volunteers (101.5 ± 57.5 versus 222.1 ± 77.6, P < 0.001), and were not correlated to the present pain intensity or pain duration. The results indicate an altered condition of the immune-linked opioid system underlying CRPS. Further immunological approaches may provide new insight into the pathophysiology of CRPS.

Development of a Simple Cell Invasion Assay System.

Cell invasion assay is important for studying various biological events, such as inflammation, cancer metastasis, and angiogenesis. In this study, we developed a simple method for the quantification of cell invasion by using a culture insert with fluorescence blocking micropore membrane (FBM). Fluorescence labeled cells were simply added to a culture insert with a 8 micrompore FBM precoated with Matrigel and incubated for an appropriate duration. Then, the FBM was examined under a fluorescence microscope to count the invaded cell number. By this method, accurate invasion assay is easily performed without the steps of fixation and staining of cells and removal of cells which do not invade.

Digestibility of the hydrogenated derivative of an isomaltooligosaccharide mixture by rats.

The digestibility of the hydrogenated derivative of an isomaltooligosaccharide mixture (IMO-H) was investigated. In an in vitro experiment, the digestibility of IMO-H was examined by models of the digestive system. IMO-H was resistant to two types of alpha-amylase and to artificial gastric juice. Enzymes in the rat small intestinal mucosa hydrolyzed tri-, tetra- and higher saccharide alcohols to disaccharide alcohol, removing successive glucose units from the non-reducing ends of the chains. The hydrolysis ratio for IMO-H was intermediate between the values for maltose and maltitol. In an in vivo study, growing rats were fed on an experimental diet containing IMO-H, maltitol, or hydrogenated palatinose in the range from 5% to 20%. The growth parameters of the rats fed on the test sugar show that the availability of IMO-H was about 1.2 to 1.25 times that of maltitol or hydrogenated palatinose.

[Comparison of urine volume, glomerular filtration rate, excretion and fraction of reabsorption of major substances in urine in three different rat strains].

Urine volume (UV), Glomerular filtration rate (GFR) and Fraction of reabsorption (FR) of water, Ca, IP, Na+, K+, Cl-, Glu and UN, which are significant in kidney function analysis, were investigated and compared in three rat strains (WI, SD and F344). UV/body weight ratios were higher in SD rats as compared to other strains. The GFR difference was not significant, but the FRH2O difference was significant in three rat strains. FR of Ca, K+ and Glu showed no significant differences in three different rat strains. SD and F344 were significantly lower than WI in FR of IP. FR of Na+, Cl- and UN were significantly different in three rat strains without Cl- between SD and F344, and UN between WI and SD.

The vasocontractive action of norepinephrine and serotonin in deep arterioles of rat cerebral gray matter.

To examine the direct effects of norepinephrine (NE) and serotonin (5-HT) on the contractility of arterioles in the gray matter of the rat cerebrum, we micro-perfused arterioles in vitro and observed the changes in luminal diameter under the stop-flow condition with constant intraluminal pressure. While the average diameter of the lumen of arterioles was 39.9 +/- 9.7 microm (n=7) in Hepes-buffered saline, the average in 10(-7) M NE in the extraluminal solution changed into smaller in saline by 21.1 +/- 5.4% (n=7). The contractile effect of NE shows a dose-dependent curve between the 10(-7) and 10(-5) M. The contractile response to 10(-6) M NE was significantly reduced by yohinbin, an alpha2 blocker. 10(-6) M NE applied to the lumen also caused contraction of arterioles by 12.4 +/- 5.3% in diameter (n=5). 5-HT at 10(-7) M in the extraluminal solution caused contraction of arterioles by 10.9 +/- 4.4% in diameter (n=7). 5-HT in the extraluminal solution caused contraction of arterioles in a dose dependent manner between 10(-10) and 10(-6) M. The contractile effect of 5-HT at 10(-6) M was strongly reduced by 10(-6) M ketanserin, a 5-HT2 receptor antagonist. 5-HT applied to the lumen had no effect at all (n=6), however NE applied to the lumen caused contraction. These results strongly suggest that 5-HT plays a significant role in arteriolar contractility only from the cerebrospinal fluid (CSF) side, while NE is an important regulator of arteriolar contractility from both the CSF and blood circulation sides.