Mechanism of NO-mediated oxidative and nitrative DNA damage in hamsters infected with Opisthorchis viverrini: a model of inflammation-mediated carcinogenesis.

Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.     

Bionic epidural stimulation restores arterial pressure regulation during orthostasis.

A bionic baroreflex system (BBS) is a computer-assisted intelligent feedback system to control arterial pressure (AP) for the treatment of baroreflex failure. To apply this system clinically, an appropriate efferent neural (sympathetic vasomotor) interface has to be explored. We examined whether the spinal cord is a candidate site for such interface. In six anesthetized and baroreflex-deafferentiated cats, a multielectrode catheter was inserted into the epidural space to deliver epidural spinal cord stimulation (ESCS). Stepwise changes in ESCS rate revealed a linear correlation between ESCS rate and AP for ESCS rates of 2 pulses/s and above (r2, 0.876-0.979; slope, 14.3 +/- 5.8 mmHg.pulses(-1).s; pressure axis intercept, 35.7 +/- 25.9 mmHg). Random changes in ESCS rate with a white noise sequence revealed dynamic transfer function of peripheral effectors. The transfer function resembled a second-order, low-pass filter with a lag time (gain, 16.7 +/- 8.3 mmHg.pulses(-1).s; natural frequency, 0.022 +/- 0.007 Hz; damping coefficient, 2.40 +/- 1.07; lag time, 1.06 +/- 0.41 s). On the basis of the transfer function, we designed an artificial vasomotor center to attenuate hypotension. We evaluated the performance of the BBS against hypotension induced by 60 degrees head-up tilt. In the cats with baroreflex failure, head-up tilt dropped AP by 37 +/- 5 mmHg in 5 s and 59 +/- 11 mmHg in 30 s. BBS with optimized feedback parameters attenuated hypotension to 21 +/- 2 mmHg in 5 s (P < 0.05) and 8 +/- 4 mmHg in 30 s (P < 0.05). These results indicate that ESCS-mediated BBS prevents orthostatic hypotension. Because epidural stimulation is a clinically feasible procedure, this BBS can be applied clinically to combat hypotension associated with various pathophysiologies.     

A new species of the genus Acropoma (Perciformes: Acropomatidae) from the Philippines

Acropoma boholensis sp. nov. is described on the basis of two specimens collected from Dumaguete city market, Negros Island, Philippines. It is distinguished from other species of Acropoma by the following combination of characters: anus situated closer to the origin of pelvic fin than to that of anal fin; luminous gland extending from isthmus to anal fin base; scales cycloid except weakly ctenoid scales around anterior part of lateral line and on ventral side of body; proximal radial of first anal fin pterygiophore lacking trough or hollow; body depth 31%–33% SL; head length 40%–42% SL; length of orbit 13%–14% SL. Received: November 14, 2000 / Revised: September 12, 2001 / Accepted: October 10, 2001     

Demography and selection analysis of the incipient adaptive radiation of a Hawaiian woody species

Ecological divergence in a species provides a valuable opportunity to study the early stages of speciation. We focused on Metrosideros polymorpha, a unique example of the incipient radiation of woody species, to examine how an ecological divergence continues in the face of gene flow. We analyzed the whole genomes of 70 plants collected throughout the island of Hawaii, which is the youngest island with the highest altitude in the archipelago and encompasses a wide range of environments. The continuous M. polymorpha forest stands on the island of Hawaii were differentiated into three genetic clusters, each of which grows in a distinctive environment and includes substantial genetic and phenotypic diversity. The three genetic clusters showed signatures of selection in genomic regions encompassing genes relevant to environmental adaptations, including genes associated with light utilization, oxidative stress, and leaf senescence, which are likely associated with the ecological differentiation of the species. Our demographic modeling suggested that the glaberrima cluster in wet environments maintained a relatively large population size and two clusters split: polymorpha in the subalpine zone and incana in dry and hot conditions. This ecological divergence possibly began before the species colonized the island of Hawaii. Interestingly, the three clusters recovered genetic connectivity coincidentally with a recent population bottleneck, in line with the weak reproductive isolation observed in the species. This study highlights that the degree of genetic differentiation between ecologically-diverged populations can vary depending on the strength of natural selection in the very early phases of speciation.     

Temperature-controlled atmospheric-pressure plasma treatment induces protein uptake via clathrin-mediated endocytosis in tobacco cells

Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescent-tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N₂ plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N₂ plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.     

Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase

A three-dimensional structural model of Escherichia coli fructosamine 6-kinase (FN6K), an enzyme that phosphorylates fructosamines at C6 and catalyzes the production of the fructosamine 6-phosphate stable intermediate, was generated using the crystal structure of 2-keto-3-deoxygluconate kinase isolated from Thermus thermophilus as template. The putative active site region was then investigated by site-directed mutagenesis to reveal several amino acid residues that likely play important roles in the enzyme reaction. Met220 was identified as a residue that plays a role in substrate recognition when compared to Bacillus subtilis derived FN6K, which shows different substrate specificity from the E. coli FN6K. Among the various Met220-substituted mutant enzymes, Met220Leu, which corresponded to the B. subtilis residue, resulted in an increased activity of fructosyl-valine and decreased activity of fructosyl-lysine, thus increasing the specificity for fructosyl-valine by 40-fold.     

Lamellarin-inspired potent topoisomerase I inhibitors with the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one scaffold

A new class of topoisomerase I inhibitors containing the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (abbreviated as BBPI) ring system have been developed based on structure-activity relationship studies of the cytotoxic marine alkaloid lamellarin D. The pentacyclic BBPI scaffold was constructed from N-tert-butoxycarbonylpyrrole by sequential and regioselective functionalization of the pyrrole core using directed lithiation, conventional electrophilic substitution, and palladium-catalyzed cross-coupling reactions. Further N-alkylation of the scaffold followed by selective deprotection of the O-isopropyl group produced a range of N-substituted BBPI derivatives. The BBPIs thus prepared exhibited potent topoisomerase I inhibitory activity in DNA relaxation assays. The activities of BBPIs were higher than those of lamellarin D and camptothecin; they showed potent and selective antiproliferative activity in the panel of 39 human cancer cell lines established by Japanese Foundation for Cancer Research. COMPARE analyses indicated that the inhibition patterns of the BBPIs correlated well with those of the known topoisomerase I inhibitors such as SN-38 and TAS-103. The water-soluble valine ester derivative exhibited antitumor activity in vivo against murine colon carcinoma colon 26. The activity was comparable to that of the approved anticancer agent irinotecan.     

Metabolic and Biosynthetic Alterations in Cultured Astrocytes Exposed to Hypoxia/Reoxygenation

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to ± 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.     

Bioelectronic sniffers for ethanol and acetaldehyde in breath air after drinking

Two kinds of bioelectronic gas sensors (bio-sniffer) incorporating alcohol oxidase (AOD) and aldehyde dehydrogenase (ALDH) were developed for the convenient analysis of ethanol and acetaldehyde in expired gas, respectively. The sniffer devices for gaseous ethanol and acetaldehyde were constructed by immobilizing enzyme on electrodes covered with filter paper and hydrophilic PTFE membrane, respectively. The AOD and ALDH sniffers were used in the gas phase to measure ethanol vapor from 1.0 to 500 ppm, and acetaldehyde from 0.11 to 10 ppm covering the concentration range encountered in breath after alcohol consumption. Both bio-sniffers displayed good gas selectivity which was attributed to the substrate specificity of the relevant enzymes (AOD and ALDH) as gas recognition material. From the results of physiological application, the bio-sniffers could monitor the concentration changes in breath ethanol and acetaldehyde after drinking. The ethanol and acetaldehyde concentrations in expired air from ALDH2 [-] (aldehyde dehydrogenase type 2 negative) subjects were higher than that of the ALDH2 [+] (positive) subjects. The results indicated that the lower activity of ALDH2 induced an adverse effect on ethanol metabolism, leading to ethanol and acetaldehyde remaining in the human body, even human expired air.