Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD ₁ and nylE ₁ , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD₁) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE₁) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP⁺ as a cofactor. Phylogenic analysis revealed that NylD₁ should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE₁ should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD₁/NylE₁ coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.

     

Binding interactions of the peripheral stalk subunit isoforms from human V-ATPase

The mammalian peripheral stalk subunits of the vacuolar-type H⁺-ATPases (V-ATPases) possess several isoforms (C1, C2, E1, E2, G1, G2, G3, a1, a2, a3, and a4), which may play significant role in regulating ATPase assembly and disassembly in different tissues. To better understand the structure and function of V-ATPase, we expressed and purified several isoforms of the human V-ATPase peripheral stalk: E1G1, E1G2, E1G3, E2G1, E2G2, E2G3, C1, C2, H, a1_NT, and a2_NT. Here, we investigated and characterized the isoforms of the peripheral stalk region of human V-ATPase with respect to their affinity and kinetics in different combination. We found that different isoforms interacted in a similar manner with the isoforms of other subunits. The differences in binding affinities among isoforms were minor from our in vitro studies. However, such minor differences from the binding interaction among isoforms might provide valuable information for the future structural-functional studies of this holoenzyme.     

Day workers suffering from a wider range of sleep problems are more likely to experience suicidality

Both a higher suicide rate and widespread sleep problems are serious health concerns in Japan when compared with those of other countries. We investigated the relationship between suicidal ideation and sleep problems in Japanese day workers using the 3-dimensional sleep scale (3DSS), which measures three sleep elements (phase, quality, and quantity). Data from 635 Japanese day workers (461 mens and 174 womens) were included. The 3DSS was used to assess participants’ sleep condition. Participants were classified into eight sleep types based on scores of phase, quality, and quantity: All Good Sleep, Owl (poor phase), Inefficient (poor quality), Short (poor quantity), Owl + Inefficient (poor phase and quality), Owl + Short (poor phase and quantity), Inefficient + Short (poor quality and quantity), and All Poor Sleep. We assessed participants’ suicidal ideation using question 19 of the self-rating depression scale (SDS); 119 cases (18.7 %) had ratings of 2–4 for this question and were considered to have suicidal ideation. The higher the number of sleep problems, the higher the risk of suicidal ideation compared to sleep types not indicative of problems. All Poor Sleep had the highest risk of the eight sleep types. Individuals with Owl + Short, Inefficient + Short, or All Poor Sleep had a significant risk of suicidal ideation even after adjusting for hopelessness and nightmares. Our findings suggested that sleep problems assessed by the 3DSS were related to suicidal ideation. Analysis of various aspects of sleep could be helpful for suicide prevention.

     

Terminal settling velocity and physical properties of pollen grains in still air

Numerical simulation of wind pollination requires knowledge of pollen grain physical parameters such as size, shape factor, bulk density, and terminal settling velocity. The pollen grain parameters for Japanese cedar, Japanese cypress, short ragweed, Japanese black pine, and Japanese red pine were assessed for dry condition. Terminal settling velocities of dry pollen grains in still air were measured using image analysis of scattered light tracks in a dark settling tube. The measurement system was validated by comparing results to those obtained for standard microspheres of known size and density. Dry pollen grain shape factors indicate the resemblance of particles to spheres, except for pine pollen. Circularity factors of dry pine pollen grains were 0.90–0.86, suggesting more irregular shape than those of other pollen species. Aerodynamic diameters of dry pollen grains were calculated based on the terminal settling velocity. Aerodynamic diameters of Japanese cedar, Japanese cypress, and short ragweed closely resembled the projected area equivalent diameters, suggesting that aerodynamic behaviors of these pollen grains can be managed simply in numerical simulations. However, aerodynamic diameters of dry pine pollen grains were nearly 30 % smaller than projected area equivalent diameters. Sacci on dry pine pollen can reduce the terminal settling velocity through low density and shape effects attributed to their non-sphericity, engendering aerodynamic diameter smaller by more than 10 µm from area equivalent diameters.     

Equilibrium Frequency of Endosymbionts in Multiple Infections Based on the Balance between Vertical Transmission and Cytoplasmic Incompatibility

Cytoplasmic incompatibility (CI)-inducing endosymbiotic bacteria, such as Wolbachia and Cardinium, have been well studied through field data and validations on the basis of numerical simulations. However, the analytically derived equilibrium frequency of multiple infections has not yet been determined, although the equilibrium for cases of single infection has been reported. In this study, we considered the difference equation for endosymbionts using three parameters: the probability of the failure of vertical transmission (), CI strength (), and the level of host inbreeding (). To analyze this model, we particularly focused on , i.e., the frequency of host individuals completely infected with all -bacterial strains in the population. , at the equilibrium state, was analytically calculated in the cases where and is any arbitrary value. We found that can be described using two parameters: and , which is identical to . has a larger value in a system with a smaller . In addition, determines the maximum number of strains that infect a single host. Our results revealed the following: i) three parameters can be reduced to a single parameter, i.e., and ii) the threshold of the maximum number of infections is defined by , which prevents additional invasions by endosymbionts.     

Atypical gaze patterns in children and adults with autism spectrum disorders dissociated from developmental changes in gaze behaviour

Eye tracking has been used to investigate gaze behaviours in individuals with autism spectrum disorder (ASD). However, traditional analysis has yet to find behavioural characteristics shared by both children and adults with ASD. To distinguish core ASD gaze behaviours from those that change with development, we examined temporo-spatial gaze patterns in children and adults with and without ASD while they viewed video clips. We summarized the gaze patterns of 104 participants using multidimensional scaling so that participants with similar gaze patterns would cluster together in a two-dimensional plane. Control participants clustered in the centre, reflecting a standard gaze behaviour, whereas participants with ASD were distributed around the periphery. Moreover, children and adults were separated on the plane, thereby showing a clear effect of development on gaze behaviours. Post hoc frame-by-frame analyses revealed the following findings: (i) both ASD groups shifted their gaze away from a speaker earlier than the control groups; (ii) both ASD groups showed a particular preference for letters; and (iii) typical infants preferred to watch the mouth rather than the eyes during speech, a preference that reversed with development. These results highlight the importance of taking the effect of development into account when addressing gaze behaviours characteristic of ASD.     

Longitudinal epidemiological study of subjective sleep quality in Japanese adolescents to investigate predictors of poor sleep quality

Sleep and Biological Rhythms - To clarify the predictors of poor sleep quality in Japanese adolescents. In 2010, baseline surveys were administered to students at 24 randomly selected...     

Mutational analysis of a catalytically important loop containing active site and substrate-binding site in Escherichia coli phytase AppA

A phytase from Escherichia coli, AppA, has been the target of protein engineering to reduce the amount of undigested phosphates from livestock manure by making phosphorous from phytic acid available as a nutrient. To understand the contribution of each amino acid in the active site loop to the AppA activity, alanine and glycine scanning mutagenesis was undertaken. The results of phytase activity assay demonstrated loss of activity by mutations at charged residues within the conserved motif, supporting their importance in catalytic activity. In contrast, both conserved, non-polar residues and non-conserved residues tended to be tolerant to Ala and/or Gly mutations. Correlation analyses of chemical/structural characteristics of each mutation site against mutant activity revealed that the loop residues located closer to the substrate have greater contribution to the activity of AppA. These results may be useful in efficiently engineering AppA to improve its catalytic activity.

Abbreviations: AppA: pH 2.5 acid phosphatase; CSU: contacts of structural units; HAPs: histidine acid phosphatases; SASA: solvent accessible surface area; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SSM: site-saturation mutagenesis; WT: wild type     

A small molecule inhibitor of p53-inducible protein phosphatase PPM1D

PPM1D is a p53-inducible Ser/Thr protein phosphatase. PPM1D gene amplification and overexpression have been reported in a variety of human tumors, including breast cancer and neuroblastoma. Because the phosphatase activity of PPM1D is essential for its oncogenic role, PPM1D inhibitors should be viable anti-cancer agents. In our current study, we showed that SPI-001 was a potent and specific PPM1D inhibitor. SPI-001 inhibited PPM1D phosphatase activity in PPM1D-overexpressing human breast cancer cells and increased phosphorylation of p53. Furthermore, SPI-001 suppressed cell proliferation by inducing apoptosis. Our present study suggested that SPI-001 was a potential lead compound in developing anti-cancer drugs.