Kinetic analysis of the shift of aerobic to anaerobic metabolism in rats during acute cyanide poisoning

Blood concentrations of cyanide, lactate, glucose, oxypurines and allantoin were determined in rats sampled at 10 min after the intraperitoneal administration of various concentrations of potassium cyanide. Lactate and oxypurines in plasma increased biquadratically with increase in the cyanide concentration in blood. The concentrations of cyanide for half maximal effect were 1.63 μg/ml for lactate and 2.09 μg/ml for oxypurines. Plasma glucose increased quadratically with increase in the cyanide concentration, and the marked increase was observed where plasma lactate concentration became near maximal. Plasma allantoin concentrations were not significantly changed throughout the experiments. The present results indicate that determination of plasma oxypurines as well as lactate is an excellent parameter for tissue hypoxia.     

Aluminum-induced apoptosis in PC12D Cells

The addition of aluminum-maltol complex to PC12D cells induced a time-dependent and concentration-dependent growth inhibition as well as cell death, whereas aluminum chloride or maltol alone did not affect the viability of PC12D cells. Apoptosis of differentiated PC12D cells was assessed by using terminal deoxynucleotidyltransferase-mediated 2-deoxyuridine-5-triphosphate nick end labeling (TUNEL) technique to detect DNA strand breaks in situ. The number of TUNEL-positive cells treated with aluminum-maltol increased with time in the treatment cultures. The ability of aluminum ion to elevate intracellular reactive oxygen species was determined by fluorescence in PC12D cells loaded with the oxidant-sensitive dye 2,7-dichlorofluorescin diacetate. Aluminum ion incorporated to PC12D cells causes apoptotic cell death by enhancing the generation of reactive oxygen species.     

Stress-induced factor involved in flower formation of Lemna is an alpha-ketol derivative of linolenic acid

A stress-induced substance(s) (factor C) incubated with norepinephrine (NE) has strong flower-inducing activity in Lemna paucicostata. We isolated an essential component (FIF) of factor C, and clarified its chemical structure as 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid, an alpha-ketol derivative of linolenic acid, which is formed via 9-hydroperoxy linolenic acid. Synthesized FIF showed flower-inducing activity after incubation with NE (factor C activity) equivalent to that formed in the stressed Lemna. Jasmonic acid and 13-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoic acid (12,13-alpha-ketol linolenic acid), both of which are formed via 13-hydroperoxide of linolenic acid and all other derivatives of FIF synthesized by chemical and enzymatic processes failed to show the factor C activity. These results suggest that the molecular structure of FIF is very specific for the factor C activity.     

Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function

Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one μg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice.     

Hemin reduces cellular sensitivity to imatinib and anthracyclins via Nrf2

Heme plays an important biomodulating role in various cell functions. In this study, we examined the effects of hemin on cellular sensitivity to imatinib and other anti-leukemia reagents. Hemin treatment of human BCR/ABL-positive KCL22 leukemia cells increased IC(50) values of imatinib, that is, the drug resistance, in a dose-dependent manner without any change in the BCR/ABL kinase activity. Imatinib-induced apoptosis was also suppressed by hemin treatment in KCL22 cells. Hemin treatment increased the activity of gamma-glutamylcysteine synthetase (gamma-GCS) light subunit gene promoter, which contains a Maf recognition element (MARE). Protein levels of gamma-GCS and heme oxygenase-1 (HO-1), two MARE-containing genes, were also increased after hemin treatment. Knockdown of Nrf2 expression by RNA interference largely abolished the effect of hemin on imatinib-treated cells, suggesting that Nrf2 recognition of MARE is essential for the hemin-mediated protective effect. Similar to hemin, treatment of cells with delta-aminolevulinic acid (delta-ALA), the obligatory heme precursor, also increased IC(50) values of imatinib. In contrast, inhibition of cellular heme synthesis by succinylacetone increased the sensitivity of cells to imatinib in two imatinib-resistant cell lines, KCL22/SR and KU812/SR. Hemin treatment also decreased the sensitivity of cells to four anthracyclins, daunorubicin, idarubicin, doxorubicin, and mitoxantrone, in BCR/ABL-negative leukemia U937 and THP-1 cells, as well as in KCL22 cells. These findings thus indicate that cellular heme level plays an important role in determining the sensitivity of cells to imatinib and certain other anti-leukemia drugs and that the effect of heme may be mediated via its ability to upregulate Nrf2 activity.     

Description ofGerres chrysops sp. nov. From Thailand and redescription ofGerres setifer (Hamilton, 1822) andG. Decacanthus (Bleeker, 1865) (perciformes: Gerreidae)

Gerres chrysops, a new gerreid species from the Gulf of Thailand, is described on the basis of 29 specimens, 58–83 mm in standard length (SL). A small-sized species (less than 100 mm SL), it is characterized by a silvery-gold sheen on the head and trunk, vivid yellow or yellowish-hyaline fins in life, two supraneural bones (formula 0/0/2/) and dorsal fin rays usually IX, 10. The new species is similar toG. decacanthus (Bleeker, 1865) andG. setifer (Hamilton, 1822), which are redescribed. being similarly small valid gerreid species characterized by two supraneural bones. Together, the three species comprise “theGerres setifer complex.”Gerres chrysops differs from bothG. decacanthus andG. setifer in life and fresh colors, the body being silvery-gold with vivid yellow or yellowish dorsal, caudal, anal and pelvic fins, and yellowish-hyaline pectoral fins (vs. silver body with hyaline fins in the latter two species).Gerres setifer differs fromG. chrysops andG. decacanthus in having the last dorsal fin spine longer than the penultimate spine (vs. almost same length or shorter), usually ten dorsal fin spines and nine soft dorsal rays (vs. usually IX, 10), and 8 or 9 lower series gill rakers (vs. usually 7).Gerres decacanthus differs fromG. chrysops andG. setifer in having a shorter head, lesser body depth at the first anal fin spine base, lesser body width at the pectoral fin base, and shorter second dorsal and third anal fin spines. The new species is currently known only from Angsilla, near Bangsaen, and around Si Chang Island, northeastern Gulf of Thailand.Gerres decacanthus inhabits southern Chinese waters andG. setifer is currently known from the Bay of Bengal to the Andaman Sea.     

Investigation of the recognition of an important uridine in an internal loop of a hairpin ribozyme prepared using post-synthetically modified oligonucleotides.

We introduced 4-thio- ((4S)U), 2-thio- ((2S)U), 4- O -methyluridine ((4Me)U) and cytidine substitutions for U+2, which is an important base for cleavage in a substrate RNA. Oligonucleotides containing 4-thio- and 4- O -methyluridine were prepared by a new convenient post-synthetic modification method using a 4- O - p -nitrophenyl-uridine derivative. A hairpin ribozyme cleaved the substrate RNA with either C+2, (4S)U+2 or (4Me)U+2 at approximately 14-, 6- and 4-fold lower rates, respectively, than that of the natural substrate. In contrast, the substrate with a (2S)U+2 was cleaved with the same activity as the natural substrate. These results suggest that the O4 of U+2 is involved in hydrogen bonding at loop A, but the O2 of U+2 is not recognized by the active residues. Circular dichroism data of the ribozymes containing (4S)U+2 and (2S)U+2, as well as the susceptibility of the thiocarbonyl group to hydrogen peroxide, suggest that a conformational change of U+2 occurs during the domain docking in the cleavage reaction. We propose here the conformational change of U+2 from the ground state to the active molecule during the reaction.     

Association of habitual dietary intake with morningness-eveningness and rotating shift work in Japanese female nurses

Rotating shift workers are associated with imbalanced dietary intakes. Rotating shift workers and dietary intakes in adults who do not engage in night work have also been shown to be associated with chronotype. However, no studies have examined associations between morningness-eveningness (i.e., the degree to which people prefer to be active in the morning or the evening), rotating shift work and dietary intakes. Therefore, our first purpose was to elucidate the association between morningness-eveningness and habitual food group intakes in rotating shift workers. The second purpose was to elucidate the association of morningness-eveningness and rotating shift work with food group intakes, considering habitual sleep durations. Japanese nurses (1095 day workers and 1464 rotating shift workers) were studied using a self-administered questionnaire. The questionnaire covered habitual dietary intakes, morningness-eveningness and demographic characteristics of the participants. A Japanese version of the Morningness-Eveningness Questionnaire (MEQ) was used to measure self-rated morningness-eveningness. Dietary intakes over the previous 1 month were evaluated using a semi-quantitative food frequency questionnaire. Intakes of pulses, green/yellow vegetables, white vegetables, fruits, algae, eggs, confectioneries/savory snacks and sugar-sweetened beverages were significantly (p < 0.05) associated with the MEQ score in rotating shift workers. Among these food groups, intakes of green/yellow vegetables, white vegetables, fruits and algae were significantly (p < 0.05) lower in rotating shift workers than in day workers, and intakes of confectioneries/savory snacks and sugar-sweetened beverages were significantly (p < 0.05) higher in rotating shift workers than in day workers. Intakes of these food groups were also significantly (p < 0.05) associated with the MEQ score in day workers. In addition, the MEQ score was significantly (p < 0.05) lower in rotating shift workers than in day workers, indicating greater eveningness among rotating shift workers. Multivariate linear regression revealed that the MEQ scores were significantly (p < 0.05) associated with intakes of these food groups, while rotating shift work was associated only with confectioneries/savory snacks. These results suggest that morningness-eveningness is associated with unbalanced dietary intakes in rotating shift workers as well as day workers, which may partially explain associations between rotating shift work and unfavorable dietary intakes. These findings have important implications for the development of novel strategies for preventing poor health caused by imbalanced dietary intakes in rotating shift workers.     

Highly efficient catalytic RNA cleavage by the cooperative action of two Cu(II) complexes embodied within an antisense oligonucleotide

Based on our recent studies of RNA cleavage by oligonucleotide–terpyridine·Cu(II) complex 5′- and/or 3′-conjugates, we designed 2′-O-methyloligonucleotides with two terpyridine-attached nucleosides at contiguous internal sites. To connect the 2′-terpyridine-modified uridine residue at the 5′-side to the 5′-O-terpyridyl nucleoside residue at the 3′-side, a dimethoxytrityl derivative of 5-hydroxypropyl-5′-O-terpyridyl-2′-deoxyuridine-3′-phosphoramidite was newly synthesized. Using this unit, we constructed two terpyridine conjugates, with either an unusual phophodiester bond or the bond extended by a propanediol(s)-containing linker. Cleavage reactions of the target RNA oligomer, under the conditions of conjugate excess in the presence of Cu(II), indicated that the conjugates precisely cleaved the RNA at the predetermined site and that one propanediol-containing linker was the most appropriate for inducing high cleavage activity. Furthermore, a comparison of the activity of the propanediol agent with those of the control conjugates with one complex confirmed that the two complexes are required for efficient RNA cleavage. The reaction of the novel cleaver revealed a bell-shaped pH–rate profile with a maximum at pH ~7.5, which is a result of the cooperative action of the complexes. In addition, we demonstrated that the agent catalytically cleaves an excess of the RNA, with the kinetic parameter k_cat/K_m = 0.118 nM^–1 h^–1.