Adenine nucleotide metabolism in Azotobacter vinelandii. Two metabolic pathways of AMP degradation

AMP-degrading pathways in Azotobacter vinelandii cells were investigated. AMP nucleosidase (EC 3.2.2.4) was rapidly synthesized and reached a maximum at 24 h, while the activity of 5-nucleotidase (EC 3.1.3.5) specific for AMP, which was negligible during the logarithmic phase of the growth, first appeared in 24 h-cultures, and reached a maximum after complete exhaustion of sucrose from the growth medium (70 h).Cell-free extracts of A. vinelandii of 48 h-cultures hydrolyzed AMP to ribose 5-phosphate and adenine in the presence of ATP, and adenine was deaminated to hypoxanthine. When ATP was excluded, AMP was dephosphorylated to adenosine, which was further metabolized to inosine, and finally to hypoxanthine. Hypoxanthine thus formed was reutilized for the salvage synthesis of IMP under the conditions where 5-phosphoribosyl 1-pyrophosphate was able to be supplied. These results suggest that the levels of ATP can determine the rate of AMP degradation by the AMP nucleosidase- and 5-nucleotidase-pathways. The role of ATP in the AMP degradation was discussed in relation to the regulatory properties of AMP nucleosidase, inosine nucleosidase (EC 3.2.2.2) and adenosine deaminase (EC 3.5.4.4).     

Externalization of transferrin receptor in established human cell lines

The externalization of transferrin receptors was found in established human tumor cell lines at the rate of 10-35 ng/hour/10(6) cells, when they were incubated with transferrin at 37 degrees C. This externalization is inhibited by lowering the incubation temperature to 4 degrees C or eliminating the ligand from the culture medium. Metabolic inhibitors such as sodium azide, colchicine, cytochalasin B and chloroquine also decreased the rate of externalization. Almost 95% of released transferrin receptors were precipitated by centrifugation at 100,000 x g for 30 min, suggesting that transferrin receptor is externalized into the medium as a vesicular form.     

Tumor necrosis factor type alpha (cachectin) stimulates mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

To elucidate the mechanism of tumor necrosis factor alpha (TNF-alpha)-induced bone resorption, the effects of recombinant human TNF-alpha on mouse osteoblast-like cells (MC3T3-E1) were studied. TNF-alpha stimulated MC3T3-E1 cells to produce prostaglandin E2 (PGE2) and macrophage colony stimulating activity (M-CSA) in a dose-dependent manner. TNF decreased alkaline phosphatase (AL-P) activity of MC3T3-E1 cells. These TNF effects were observed at 1 ng/ml (approximately 6 X 10(-11)M). The inhibitory effect on AL-P activity was reversible and the cell growth of MC3T3-E1 cells was only slightly affected by TNF. These findings suggest that both PGE2 and M-CSA stimulated by TNF-alpha are possibly involved in osteoblast-mediated osteoclastic bone resorption, whereas inhibition of AL-P activity may lead to a decrease in bone formation.     

Quantitative Study of the Anomeric Forms of Isomaltose and Glucose Produced by Isomalto-dextranase and Glucodextranase

A gas-liquid chromatographic method was applied to the determination of anomeric forms of isomaltose and glucose produced by Arthrobacter globiformis isomalto-dextranase and glucodex- tranase. The anomeric forms of products released from isomaltotriose, panose and dextran were quantitatively determined. The isomalto-dextranase that was also capable of splitting the α-1,4- glucosidic linkage of panose was found to exclusively produce α-isomaltose from these substrates, and the glucodextranase, ^-glucose.     

Biochemical Studies on Buckwheat α-Glucosidase

The transglucosylation action of buckwheat α-glucosidase on soluble starch, maltose maltotriose and maltotetraose are described and discussed. The transglucosylation products of soluble starch were isolated by carbon-Celite column chromatography and by paper chromatography. Among the products were found follows: nigerose, maltose, kojibiose and isomaltose as disaccharides and 2-α-isomaltosylglucose, 2-α-nigerosylglucose, nigerotriose. 3-α-maltosylglucose and maltotriose as trisaccharides.

Furthermore, the existence of 6-α-nigerosylglucose, 4-α-kojibiosylglucose, panose, isopanose and 3-α-isomaItosylglucose was suspected. A new trisaccharide, 2-α-nigerosylglucose which was obtained in a crystalline form (monohydrate) melted at 186~188°C and gave [α]_D+ 178.3 (c = 0.6, in water).

These experimental results on the reaction products seem to indicate that the activated glucosyl group from the substrate (starch, in this case) is transferred to any position of C–2,3,4 or 6 of glucose released from the substrate and the same type of transglucosylation occurrs upon the non-reducing terminal of disaccharides just produced, which leads to the formation of various kinds of trisaccharide, tetrasaccharide etc. The synthesis of α-oligosaccharides from free glucose could not be detected by paper chromatography.     

Substrate and metabolic promiscuities of d‐altronate dehydratase family proteins involved in non‐phosphorylative d‐arabinose,sugar acid,l‐galactose and l‐fucose pathways from bacteria

The gene context in microorganism genomes is of considerable help for identifying potential substrates. The C785_RS13685 gene in Herbaspirillum huttiense IAM 15032 is a member of the d‐ altronate dehydratase protein family, and which functions as a d‐ arabinonate dehydratase in vitro, is clustered with genes related to putative pentose metabolism. In the present study, further biochemical characterization and gene expression analyses revealed that l‐ xylonate is a physiological substrate that is ultimately converted to α‐ketoglutarate via so‐called Route II of a non‐phosphorylative pathway. Several hexonates, including d‐ altronate, d‐ idonate and l‐ gluconate, which are also substrates of C785_RS13685, also significantly up‐regulated the gene cluster containing C785_RS13685, suggesting a possibility that pyruvate and d‐ or l‐ glycerate were ultimately produced (novel Route III). On the contrary, ACAV_RS08155 of Acidovorax avenae ATCC 19860, a homologous gene to C785_RS13685, functioned as a d‐ altronate dehydratase in a novel l‐ galactose pathway, through which l‐ galactonate was epimerized at the C5 position by the sequential activity of two dehydrogenases, resulting in d‐ altronate. Furthermore, this pathway completely overlapped with Route III of the non‐phosphorylative l‐ fucose pathway. The ‘substrate promiscuity’ of d‐ altronate dehydratase protein(s) is significantly expanded to ‘metabolic promiscuity’ in the d‐ arabinose, sugar acid, l‐ fucose and l‐ galactose pathways.     

Dynamic range compression in the honey bee auditory system toward waggle dance sounds

Honey bee foragers use a "waggle dance" to inform nestmates about direction and distance to locations of attractive food. The sound and air flows generated by dancer's wing and abdominal vibrations have been implicated as important cues, but the decoding mechanisms for these dance messages are poorly understood. To understand the neural mechanisms of honey bee dance communication, we analyzed the anatomy of antenna and Johnston's organ (JO) in the pedicel of the antenna, as well as the mechanical and neural response characteristics of antenna and JO to acoustic stimuli, respectively. The honey bee JO consists of about 300-320 scolopidia connected with about 48 cuticular "knobs" around the circumference of the pedicel. Each scolopidium contains bipolar sensory neurons with both type I and II cilia. The mechanical sensitivities of the antennal flagellum are specifically high in response to low but not high intensity stimuli of 265-350 Hz frequencies. The structural characteristics of antenna but not JO neurons seem to be responsible for the non-linear responses of the flagellum in contrast to mosquito and fruit fly. The honey bee flagellum is a sensitive movement detector responding to 20 nm tip displacement, which is comparable to female mosquito. Furthermore, the JO neurons have the ability to preserve both frequency and temporal information of acoustic stimuli including the "waggle dance" sound. Intriguingly, the response of JO neurons was found to be age-dependent, demonstrating that the dance communication is only possible between aged foragers. These results suggest that the matured honey bee antennae and JO neurons are best tuned to detect 250-300 Hz sound generated during "waggle dance" from the distance in a dark hive, and that sufficient responses of the JO neurons are obtained by reducing the mechanical sensitivity of the flagellum in a near-field of dancer. This nonlinear effect brings about dynamic range compression in the honey bee auditory system.     

Mating rates and the prevalence of male‐killing Spiroplasma in Harmonia axyridis (Coleoptera: Coccinellidae)

Maternally inherited bacteria that kill male but not female hosts during embryogenesis have been widely reported in invertebrates. Harmonia axyridis is one of the species infected by male‐killing Spiroplasma. The presence of male‐killers in host populations can lead to the occurrence of extremely female‐biased sex ratios. Furthermore, infected females may have fewer chances to mate if males can discriminate between infected and uninfected females and prefer the latter. Although there have been many investigations of male‐killer infection rates in H. axyridis, little is known about the influence of host mating on male‐killer infection dynamics. We investigated copulation rates and changes in infection frequency in a wild population of H. axyridis in western Japan. Almost all infected females collected each year laid fertilized eggs and had therefore mated. Mean infection rates of females collected each year were 13% in 2003, 15% in 2012 and 23% in 2013. Statistical analysis showed that neither the copulation rate nor the infection rate differed significantly among years. These results suggest that the infection rate of H. axyridis with male‐killing Spiroplasma is kept approximately constant and that there is no difference in the chance of mating with infected and uninfected females.     

Eudesmane-type sesquiterpene lactones inhibit multiple steps in the NF-κB signaling pathway induced by inflammatory cytokines

Inflammatory cytokines, such as interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), induce the intracellular signaling pathway leading to the activation of nuclear factor κB (NF-κB). A series of eudesmane-type sesquiterpene lactones possessing an α-methylene γ-lactone group and/or an α-bromo ketone group were synthesized and evaluated for their inhibitory effects on the NF-κB-dependent gene expression and signaling pathway. Our present study reveals that eudesmane-type α-methylene γ-lactones and α-bromo ketones inhibit multiple steps in the NF-κB signaling pathway induced by IL-1α and TNF-α.     

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.