Engineering the substrate specificity of porcine kidney D-amino acid oxidase by mutagenesis of the "active-site lid"

Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-N225 is substituted by R216-G220 of DDO, showed D-aspartate-oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that I215-N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220-Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220-Y224. All of the mutants exhibited decreased apparent K(m) values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, k(cat app)/K(m app), for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil.     

Foot odor due to microbial metabolism and its control

To characterize foot odor, we analyzed its components by sensory tests, isolated microorganisms that produce it, and evaluated the mechanism of the occurrence of foot odor. As a result, foot odor was found to be derived from isovaleric acid, which is produced when Staphylococcus epidermidis, a resident species of the normal cutaneous microbial flora, degrades leucine present in sweat. In addition, Bacillus subtilis was detected in the plantar skin of subjects with strong foot odor, and this species was shown to be closely associated with increased foot odor. Therefore, we screened various naturally occurring substances and fragrant agents that inhibit microbial production of foot odor without disturbing the normal microbial flora of the human skin. As a result, we identified citral, citronellal, and geraniol as fragrant agents that inhibit the generation of isovaleric acid at low concentrations.     

En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2�CE3 pairs

Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6–RAD18 (an E2–E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2–UBC13 (a UEV–E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ub_n) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ub_n+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.     

Polarity and asymmetry in the arrangement of dynein and related structures in the Chlamydomonas axoneme

Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer-inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella.     

Contribution of p38 MAPK, NF-κB and glucocorticoid signaling pathways to ER stress-induced increase in retinal endothelial permeability

Diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. Endoplasmic reticulum (ER) stress is known to play a pathogenic role in vascular impairment in DR. The present study demonstrated that the treatment of human retinal endothelial cells with ER stress inducers such as thapsigargin (Tg) and tunicamycin (Tm) significantly increased the permeability of exogenously added FITC-dextran, accompanied by a decrease of transendothelial electrical resistance (TEER). The expression of claudin-5 among tight junction proteins was significantly decreased by the treatment with Tg or Tm. A p38 MAPK inhibitor, SB203580, and an NF-κB inhibitor, dexamethasone, significantly suppressed the Tg-induced down-regulation of claudin-5, decrease of TEER and leakage of added FITC-dextran. The translocation of NF-κB p65 subunit to the nucleus was also inhibited by the addition of SB203580 or dexamethasone. The effects of dexamethasone are thought to be due to the transrepression of the above signaling and direct regulation of claudin-5 gene.     

Demographic History of Shorea curtisii (Dipterocarpaceae) Inferred from Chloroplast DNA Sequence Variations

We assessed the variability of chloroplast DNA sequences in populations of the dipterocarp forest tree, Shorea curtisii. This species is widely distributed in hill and coastal hill dipterocarp forests of the Malay Peninsula, whereas isolated populations are found in the coastal hills of north Borneo. Two chloroplast DNA regions (1555 bp of trnH‐psbA‐trnK and 925 bp of trnL‐trnF) were sequenced from 123 individuals collected from six Malay Peninsula and two Bornean populations. There were 15 chloroplast haplotypes derived from 16 polymorphic sites. A haplotype network revealed two distinct haplogroups that correlate with two geographic regions, the Malay Peninsula and Borneo. These two haplogroups differed by a number of mutations, and no haplotypes were shared between populations from the different geographic regions. This suggests an ancient diversification of these haplogroups, and that long‐distance seed dispersal was unlikely to have occurred during the Pleistocene when the Sunda Shelf was a contiguous landmass. Phylogenetic analysis of the haplotypes together with those found in other Shorea species showed that two haplogroups in S. curtisii appear in different positions of the phylogenetic tree. This could be explained by the persistence of ancestral polymorphisms or by ancient chloroplast capture. Low levels of genetic differentiation were found between populations within each geographic region. Signature of a bottleneck followed by demographic expansion was detected in the Malay Peninsula haplogroup. The presence of two distinct evolutionary lineages in the different regions suggests that they should be managed independently to conserve the major sources of genetic diversity in S. curtisii.     

Ciliates expel environmental legionella-laden pellets to stockpile food

When Tetrahymena ciliates are cultured with Legionella pneumophila, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmental L. pneumophila, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human L. pneumophila isolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental L. pneumophila for stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance.