Rapid and onsite BOD sensing system using luminous bacterial cells-immobilized chip

A BOD monitoring system based on a bio-chip which immobilized luminous bacterium in micrometer-order holes were arrayed and fabricated by micro-machine techniques, was developed. The acrylic chip (3 cmx3 cm) comprises nine micro-holes (diameter: 700 microm or 1 mm, depth: 100 microm) arranged in a three by three array. Cells of the marine luminous bacterium, Photobacterium phosphoreum IFO 13896, which was grown at 15 degrees C for 15 h, were immobilized with 3% or 15% sodium alginate gel. BOD standard solutions or actual sample solution (approximately 10 microl) was fallen onto the cell-arrayed chip, and then the chip was incubated at 25 degrees C for 25 min. After incubation, bioluminescence from the each hole was gray-scaled and measured by a chemi-imager or newly developed onsite-type-monitoring system using a digital camera and a mobile-type personal computer. BOD values less than 16 ppm could detect by the chip, in particular, linear relationship at the concentrations between 0 and 16 ppm could be observed when luminous cells were immobilized with 3% sodium alginate gel. Steady bioluminescence was observed on the chip in the presence of BOD standard solution (GGA solution) which contained mineral elements. Furthermore, simultaneous detection of BOD values in various samples could be employed in the single chip. These results showed that the monitoring system with bio-chip could achieve high-through-put and onsite BOD detection. Our newly developed onsite-type BOD detection system which was used a digital camera and a (mobile) laptop computer was applied to measure and detect organic pollution due to biodegradable substances in wastewater treatment system. The same performance as the chemi-imager system was obtained for data of bioluminescence. The obtained BOD values showed a similar correlation with that of the conventional method for BOD determination (BOD5). These results suggested for successful achievement of high-though-put and onsite detection of BOD in practical.     

Modulation of the venation pattern of cotyledons of transgenic tobacco for the tumorigenic <Emphasis Type="Italic">6b</Emphasis> gene of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> AKE10

Neoplastic plant-tissue formation, termed crown gall disease, is induced on infection with Agrobacterium tumefaciens. The tumorous tissues develop an extensive vascular system, with a venation pattern distinct from that of native host plants. We report here that the plant-tumorigenic 6b gene of the A. tumefaciens strain AKE10 is capable of inducing extensive vein formation in transgenic tobacco seedlings with distinct pattern formation. Unlike the wild-type cotyledons, transgenic cotyledons had wavy and striate veins depending on the extent of severity of leaf morphology. Graph analysis of the transgenic cotyledonous vein patterns revealed an increase in the number of branch points of veins, end-points of veins, and areas surrounded by the veins. Histological analysis showed abnormal tissue growth on the abaxial side of the cotyledon blades and continual formation of adventitious veins. These adventitiously formed veins included inverted dorso-ventrality and formation of a radial axis.     

L-NAME augments PAF-induced venoconstriction in isolated perfused livers of rat and guinea pig, but not mouse

Platelet-activating factor (PAF), one of vasoconstrictive lipid mediators, is involved in systemic anaphylaxis. On the other hand, nitric oxide (NO) is known to attenuate anaphylactic venoconstriction of the pre-sinusoids in isolated guinea pig and rat livers. However, it is not known whether NO attenuates PAF-induced hepatic venoconstriction. We therefore determined the effects of L-NAME, a NO synthase inhibitor, on PAF-induced venoconstriction in blood- and constant flow-perfused isolated livers of mice, rats and guinea pigs. The sinusoidal pressure was measured by the double occlusion pressure (Pdo), and was used to determine the pre- (Rpre) and post-sinusoidal (Rpost) resistances. PAF (0.01-1 microM) concentration-dependently caused predominant pre-sinusoidal constriction in all livers of three species studied. The guinea pig livers were the most sensitive to PAF, while the mouse livers were the weakest in responsiveness. L-NAME pretreatment selectively increased the basal Rpre in all of three species. L-NAME also significantly augmented the PAF-induced increases in Rpre, but not in Rpost, in rat and guinea pig livers. This augmentation was stronger in rat livers than in guinea pig livers at the high concentration of 0.1 microM PAF. However, L-NAME did not augment PAF-induced venoconstriction in mouse livers. In conclusion, in rat and guinea pig livers, NO may be released selectively from the pre-sinusoids in response to PAF, and then attenuate the PAF-induced pre-sinusoidal constriction. In mouse liver, PAF-induced venoconstriction is weak and not modulated by NO.     

Expression of engrailed-family genes in the jumping bristletail and discussion on the primitive pattern of insect segmentation

It has been shown that segmentation in the short-germ insects proceeds by a two-step mechanism. The anterior region is simultaneously segmented in a manner similar to that in Drosophila, which is apparently unique to insects, and the rest of the posterior region is segmented sequentially by a mechanism involving a segmentation clock, which is derived from the common ancestor of arthropods. In order to propose the evolutionary scenario of insect segmentation, we examined segmentation in the jumping bristletail, the basalmost extant insect. Using probes for engrailed-family genes for in situ hybridization, we found no sign of simultaneous segmentation in the anterior region of the jumping bristletail embryos. All segments except the anteriormost segment are formed sequentially. This condition shown in the jumping bristletail embryos may represent the primitive pattern of insect segmentation. The intercalating formation of the intercalary segment is assumed to be a synapomorphic trait shared among all insects after the branching of the jumping bristletail.     

Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation

Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.     

CpG site degeneration triggered by the loss of functional constraint created a highly polymorphic macaque drug-metabolizing gene, CYP1A2

Background

Duplicated genes frequently experience asymmetric rates of sequence evolution. Relaxed selective constraints and positive selection have both been invoked to explain the observation that one paralog within a gene-duplicate pair exhibits an accelerated rate of sequence evolution. In the majority of studies where asymmetric divergence has been established, there is no indication as to which gene copy, ancestral or derived, is evolving more rapidly. In this study we investigated the effect of local synteny (gene-neighborhood conservation) and codon usage on the sequence evolution of gene duplicates in the S. cerevisiae genome. We further distinguish the gene duplicates into those that originated from a whole-genome duplication (WGD) event (ohnologs) versus small-scale duplications (SSD) to determine if there exist any differences in their patterns of sequence evolution.

Results

For SSD pairs, the derived copy evolves faster than the ancestral copy. However, there is no relationship between rate asymmetry and synteny conservation (ancestral-like versus derived-like) in ohnologs. mRNA abundance and optimal codon usage as measured by the CAI is lower in the derived SSD copies relative to ancestral paralogs. Moreover, in the case of ohnologs, the faster-evolving copy has lower CAI and lowered expression.

Conclusions

Together, these results suggest that relaxation of selection for codon usage and gene expression contribute to rate asymmetry in the evolution of duplicated genes and that in SSD pairs, the relaxation of selection stems from the loss of ancestral regulatory information in the derived copy.     

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.