Simultaneous determination of ascorbic acid and dehydroascorbic acid in fish tissues by high-performance liquid chromatography

An high-performance liquid chromatographic method with post-column derivatization has been developed for the simultaneous determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fish tissues. Extracted AA and DHAA were separated by a Shim-pack SCR-101H column within 20 min, reacted with sodium hydroxide containing sodium borohydride and monitored at 300 nm. The detection limits for both AA and DHAA were 0.1 μg/ml.     

Extensive proliferation of oligodendrocyte precursors in the parenchyma of the embryonic chick central nervous system

The proliferation of oligodendrocyte lineage cells in the chick embryo central nervous system (CNS) was examined by double-immunolabeling with a lineage marker monoclonal antibody (mAb) O4 or mAb O1 and 5-bromo-3'-deoxyuridine (BrdU). In all regions examined, the first O4-positive (O4+) cells appeared in restricted regions of the ventricular zone (VZ), regarded as a site of oligodendrocyte origin. Within the O4+ focus, less than 20% of the O4+ cells incorporated BrdU. In contrast, O4+ cells in the parenchyma were mitotically active; for example, 40-50% of early O4+ cells were labeled with BrdU. Some of these were unipolar in shape, indicative of migratory precursor cells. The frequency of O4+/BrdU+ cell appearance decreased to less than 20% with further development. O1+ oligodendrocytes were largely mitotically inactive, with only approximately 5% of O1+ cells incorporating BrdU. These results clearly demonstrated that the VZ generates relatively few precursor cells and that these oligodendrocyte precursors actively generate their cohort in the parenchyma of the CNS.     

Onset of amnesia is delayed with a decrease in inhibition of protein synthesis during odor-taste associative learning in the terrestrial slug Limax valentianus

Slugs can retain odor-taste associative memory for several weeks, and this requires protein synthesis. We examined the dose-dependency of the onset time of amnesia caused by the protein synthesis inhibitor anisomycin, and showed that with reduced dose, the onset is shifted from 2 days to 3 days after conditioning; we could not shift the onset delay later than 3 days. Our results suggest that the mechanism underling memory retention is different in the period up to 3 days, versus the period later than 3 days. Our results also suggest that sustained inhibition of protein synthesis in the period from zero to 3 hr after conditioning is necessary to cause amnesia.     

Histochemical analysis of glycoconjugates in the eccrine glands of the raccoon digital pads

The distribution and selectivity of complex carbohydrates in the eccrine glands of the digital pads in the North American raccoon (Procyon lotor) were studied using light and electron microscopic histochemical methods, particularly lectin histochemistry. In the eccrine glands, the dark cells exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine and N-acetyl-neuraminic acid); the clear cells contained numerous glycogen particles and showed a distinct reaction of alpha-L-fucose. The presence of complex carbohydrates with various terminal sugars was evident in the excretory duct cells. In addition, beta-D-galactose and N-acetyl-neuraminic acid residues were mainly observed in the luminal secretion. The glycoconjugates produced by the eccrine glands of the raccoon digital pads may protect the epidermis against physical damage or microbial contamination. In this way, the normal functioning of the sensory apparatus of the foot pads is ensured.     

Spinnokinetic analyses of blood disposition and biliary excretion of nitric oxide (NO)-Fe(II)-N-(dithiocarboxy)sarcosine complex in rats: BCM-ESR and BEM-ESR studies

Nitric oxide (NO) is well known to have a wide variety of biological and physiological functions in animals. On the basis of the fact that Fe(II)-dithiocarbamates react with NO, a Fe(II)-N-(dithiocarboxy)sarcosine complex (Fe(II)-DTCS) was proposed as a trapping agent for endogenous NO. However, quantitative pharmacokinetic investigation for NO-Fe(II)-dithiocarbamate complexes in experimental animals has been quite limited. This paper describes the results on the quantitative pharmacokinetic features of a NO-Fe(II)-N-DTCS in both the blood and bile of rats following intravenous (i.v.) administration of the complex. For this purpose, we applied two in vivo methods, i.e. (1) in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) which previously developed, and (2) in vivo biliary excretion monitoring-electron spin resonance (BEM-ESR). We monitored real-time ESR signals due to nitrosyl-iron species in the circulating blood and bile flow. The ESR signal due to NO-Fe(II)-DTCS was stable in biological systems such as the fresh blood and bile. In in vivo BCM- and BEM-ESR, the pharmacokinetic parameters were calculated on the basis of the two-compartment and hepatobiliary transport models. The studies also revealed that the compound is widely distributed in the peripheral organs and partially excreted into the bile. We named a kinetic method to follow spin concentrations as spinnokinetics and this method will be useful for detecting and quantifying the endogenously generated NO in Fe(II)-DTCS administered animals.     

Asymmetric temporal properties in the receptive field of retinal transient amacrine cells

The speed of signal conduction is a factor determining the temporal properties of individual neurons and neuronal networks. We observed very different conduction velocities within the receptive field of fast-type On-Off transient amacrine cells in carp retina cells, which are tightly coupled to each other via gap junctions. The fastest speeds were found in the dorsal area of the receptive fields, on average five times faster than those detected within the ventral area. The asymmetry was similar in the On- and Off-part of the responses, thus being independent of the pathway, pointing to the existence of a functional mechanism within the recorded cells themselves. Nonetheless, the spatial decay of the graded-voltage photoresponse within the receptive field was found to be symmetrical, with the amplitude center of the receptive field being displaced to the faster side from the minimum-latency location. A sample of the orientation of varicosity-laden polyaxons in neurobiotin-injected cells supported the model, revealing that approximately 75% of these processes were directed dorsally from the origin cells. Based on these results, we modeled the velocity asymmetry and the displacement of amplitude center by adding a contribution of an asymmetric polyaxonal inhibition to the network. Due to the asymmetry in the conduction velocity, the time delay of a light response is proposed to depend on the origin of the photostimulus movement, a potentially important mechanism underlying direction selectivity within the inner retina.     

Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine

We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 micro g per 10(4) cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 x 10(6) PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.     

Serine proteases increase oxidative stress in lung cells

Several serine proteases are directly cytotoxic. We investigated whether the cytotoxic effects of proteases are associated with increased levels of reactive oxygen species (ROS) in cells. We found that treatment of lung fibroblasts or bronchial epithelial cells with relatively high concentrations (0.1--100 U/ml) of neutrophil elastase, trypsin, and Pronase increased ROS levels in the mitochondria and cytoplasm. The protease-induced increase in ROS was associated with oxidative cellular injury as determined by generation of 8-hydroxy-2'-deoxyguanosine and malonaldehyde plus 4-hydroxyalkenal. The protease-induced increase in ROS was not merely due to cell detachment because the proteases also caused an increase in ROS in suspended cells, which precluded attachment to the extracellular matrix. The protease-induced increase in ROS appears to contribute to cytotoxicity because cell death induced by proteases was attenuated by treatment with catalase, a decomposer of H(2)O(2), and accelerated by treatment with aminotriazole, a catalase inhibitor. These results suggest that several proteases increase oxidative stress, indicating a direct interaction between proteases and ROS in mediating cytotoxicity.     

Female multiple mating as a genetic bet-hedging strategy when mate choice criteria are unreliable

Female multiple mating (or polyandry) is considered to act as a genetic bet-hedging mechanism, by which females can reduce the assessment error in regard to mates genetic quality when only uncertain information is available. In spite of frequent verbal arguments, no theoretical examination has been carried out to determine the effectiveness of bet-hedging by multiple mating. In the present paper, I show that three factors, female population size, remating costs and environmental fluctuation, all affect the effectiveness of bet-hedging. A mathematical model predicts that bet-hedging effectively works only in small populations, and computer simulations were used to confirm this prediction. The results of simulations differed according to the degree of environmental fluctuation. In relatively stable environments, if there is no remating cost, the fixation probability of a multiple mating strategy is slightly higher than that of a single mating strategy, independent of female population size. However, with very slight fitness costs, multiple mating drastically loses its advantage as population size increases, and almost always becomes extinct within large populations. This means that the evolution of polyandry solely by the mechanism of bet-hedging is unlikely in stable environments. However, in unpredictable environments, or when negative frequency-dependent selection on fitness-related loci is introduced, a multiple mating strategy is sometimes successful against a single mating strategy, even if it entails a small fitness cost. Therefore, female multiple mating may possibly evolve only in these limited conditions. In most cases, some deterministic mechanisms such as postcopulatory sperm selection by multiply mated females (or direct material benefits) are more reasonable as the evolutionary causes of polyandry.     

A back-up glycosylase in Nth1 knock-out mice is a functional Nei (endonuclease VIII) homologue

Thymine glycol, a potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth (endonuclease III), or its mammalian homologue NTH1. We have found previously that mice deleted in the Nth homologue still retain at least two residual glycosylase activities for thymine glycol. We report herein that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E. coli Fpg (MutM) and Nei (endonuclease VIII). Tissue expression of this mouse Nei-like (designated as Neil1) gene is ubiquitous but much lower than that of mNth1 except in heart, spleen, and skeletal muscle. Recombinant NEIL1 can remove thymine glycol and 5-hydroxyuracil in double- and single-stranded DNA much more efficiently than 8-oxoguanine and can nick the strand by an associated (beta-delta) apurinic/apyrimidinic lyase activity. In addition, the mouse NEIL1 has a unique DNA glycosylase/lyase activity toward mismatched uracil and thymine, especially in U:C and T:C mismatches. These results suggest that NEIL1 is a back-up glycosylase for NTH1 with unique substrate specificity and tissue-specific expression.