Rapid and simple detection of PCR product DNA: a comparison between Southern hybridization and fluorescence polarization analysis

The essential aim of this study was to compare two different methods, Southern hybridization and fluorescence polarization (FP) assay. They both detect specific hybridization and were examined using common asymmetric PCR products and probes. FP assay clearly showed the hybridization of probe DNAs with the asymmetric PCR products of their target genes. Southern blot patterns presented excellent consistency with the results of FP assay. In both methods, two types of Shiga toxin (vero toxin) genes held in enterohaemorrhagic Escherichia coli (EHEC) were used as target genes. For detection of the two genes, stx1 and stx2, two respective DNA probes were synthesized. Both in FP assay and in Southern hybridization, the probe for stx1 hybridized only with the product of stx1 and vice versa. The results of the DNA detection using different methods were completely in agreement. Moreover, FP assay makes it possible to detect the hybridization rapidly. In our high NaCl concentration condition, hybridization between the probes and the asymmetric PCR products could be monitored within about 15min.     

Contribution of diet to the dosing time-dependent change of vitamin D3-induced hypercalcemia in rats

We have recently reported that the degree of hypercalcemia as an adverse effect induced by a single large-dose of active vitamin D3 varied with its dosing time without alteration in therapeutic effect for secondary hyperparathyroidism in patients with chronic renal failure. The present study was conducted to elucidate an effect of intestinal calcium (Ca) absorption on the chronopharmacological profiles of vitamin D3. 1, 25-dihydroxy-cholecalciferol (D3, 2 microg/kg) or vehicle alone was orally administered at two different times (2 and 14 hours after lights on; HALO) to male Wistar rats (n= 10) kept in rooms with a 12 h light-dark cycle. Blood samples for serum Ca concentration were taken before and 3, 6, 9, and 12 hours after the administration. Urine was collected for 6 hours after dosing. An identical protocol was repeated using the same animals after 16 hours fasting by a cross-over fashion. Under free-fed condition, basal concentration of serum Ca was higher at a resting period (lights on) than during an active period (lights off). Serum Ca reached its peak at 6 hours after dosing in both timings, while the value was significantly higher in the 2 HALO trial than in the 14 HALO trial. Area under the serum Ca concentration-time curve from 0 to 12 hours (AUC0-12h) and urinary excretion of Ca for 6 hours were also significantly higher in the 2 HALO trial than in the 14 HALO trial. When fasted, basal Ca concentration was reduced compared with the free-fed condition, while the daily variation was maintained. Serum Ca concentration profiles from 3 to 12 hours after dosing were not significantly different between the 2 HALO and 14 HALO trials. The AUC0-12h of serum Ca or its urinary excretion was not different between both trials. Serum concentrations of parathyroid hormone and total protein, measured before and 6 hours after the dosing were not affected by the dosing schedule. We have concluded that intestinal Ca absorption is a major factor for the chronopharmacological phenomenon of D3-induced hypercalcemia in intact rats, while intestinal and renal involvement may be relatively small in the mechanism of the intrinsic diurnal variation of serum Ca.     

“Nojirimycin” as a Potent Inhibitor of Glucosidase

Glucose-6-phosphate dehydrogenase in a yeast, Hansenula mrakii IFO 0895 is induced when the cells are cultured in a medium containing lipid hydroperoxide. The enzyme was purified from H. mrakii to the homogeneous state on polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be approximately 52kDa by SDS-PAGE and 130 kDa by Sephadex G-150column chromatography, respectively. The enzyme was specific to glucose-6-phosphate and NADP⁺, and K_mvalues for glucose-6-phosphate and NADP⁺ were 293µM and 24.1 µM, respectively. The enzyme activity was inhibited by diethylpyrocarbonate and 2, 4, 6-trinitrobenzene sulfonate, and by metal ions such as Zn^{2 +}, Cd^{2 +}, Cu^{2 +}, and Al^{3 +} . tert-Butyl hydroperoxide, a kind of lipid hydroperoxide, slightly(approximately 20%) increased the enzyme activity.     

In vitro culture of a root parasite,Aeginetia indica L.

Seed germination and further growth of seedlings of an obligate root parasiteAeginetia indica L. are described. (1) Germination: dormancy of fresh seeds can be broken by sodium hypochlorite treatment, but seeds stored for 1–5 years at 5 C did not necessarily require halogen treatment for good germination. (2) Formation of the tendril, which facilitated the organic connection with the host, was stimulated greatly byMiscanthus root extract. (3) Septation (cell division) of tendril was promoted by addition of sucrose to the basal medium. (4) Rhizogenesis of the seedlingin vitro was stimulated by deproteinized coconut milk.     

Intragranular co-storage of neuropeptide Y and arginine vasopressin in the paraventricular magnocellular neurons of the rat hypothalamus

Summary Certain populations of arginine vasopressin (AVP) neurons in the magnocellular paraventricular nucleus became immunoreactive for neuropeptide Y (NPY) when rats were treated with colchicine or monosodium glutamate (MSG). The co-storage of these peptides was examined by empooying a post-embedding electron-microscopic immunohistochemistry technique using goldlabeled antibodies to the two peptides. In colchicinetreated rats, the neuronal perikarya contained numerous secretory granules showing co-storage of the two peptides. The cells of the MSG-treated rats were characterized by having well-developed Golgi bodies with the granular structures also co-storing the two peptides, although the secretory granules in the perikarya were rather fewer than in the colchicine-treated rats. It is concluded that the destruction of the arcuate nucleus by MSG-treatment may potentiate the synthesis of NPY in AVP neurons, the synthesis of which is latent in intact animals.     

Single nucleotide polymorphisms of thrifty genes for energy metabolism: evolutionary origins and prospects for intervention to prevent obesity-related diseases

The "thrifty" genotype and phenotype that save energy are detrimental to the health of people living in affluent societies. Individual differences in energy metabolism are caused primarily by single nucleotide polymorphisms (SNPs), some of which promote the development of obesity/type 2 diabetes mellitus. In this review, four major questions are addressed: (1) Why did regional differences in energy metabolism develop during evolution? (2) How do genes respond to starvation and affluence? (3) Which SNPs correspond to the hypothetical "thrifty genes"? (4) How can we cope with disease susceptibility caused by the "thrifty" SNPs? We examined mtDNA and genes for energy metabolism in people who live in several parts of Asia and the Pacific islands. We included 14 genes, and the SNP frequencies of PPAR gamma 2, LEPR, and UCP3-p and some other genes differ significantly between Mongoloids and Caucasoids. These differences in SNPs may have been caused by natural selection depending on the types of agriculture practiced in different regions. Interventions to counteract the adverse effects of "thrifty" SNPs have been partially effective.     

Daily Rhythms of P‐glycoprotein Expression in Mice

Recent studies have shown the gene expression of several transporters to be circadian rhythmic. However, it remains to be elucidated whether the expression of P‐glycoprotein, which is involved in the transport of many medications, undergoes 24 h rhythmicity. To address this issue, we investigated daily profiles of P‐glycoprotein mRNA and protein levels in peripheral mouse tissues. In the liver and intestine, but not in the kidney, Abcb1a mRNA expression showed clear 24 h rhythmicity. On the other hand, Abcb1b and Abcb4, the other P‐glycoprotein genes, did not exhibit significant rhythmic expression in the studied tissues. In the intestine, levels of whole P‐glycoprotein also exhibited a daily rhythm, with a peak occurring in the latter half of the light phase and a trough at the onset of the light phase. Consistent with the day‐night change of P‐glycoprotein level, the ex vivo accumulation of digoxin, an Abcb1a P‐glycoprotein substrate, into the intestinal segments at the onset of dark phase was significantly lower than it was at the onset of the light phase. Thus, Abcb1a P‐glycoprotein expression, and apparently its function, are 24 h rhythmic at least in mouse intestine tissue. This circadian variation might be involved in various chronopharmacological phenomena.     

LET and ion species dependence for cell killing in normal human skin fibroblasts

We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/microm. The RBE of carbon ions at 98 keV/microm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/microm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*2/beta2 (where Z* is the effective charge and beta is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*2/beta2 curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*2/beta2, it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.     

Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass

* Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. * Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. * One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. * Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.