Expression of recombinant alpha and beta tubulins from the yew Taxus cuspidata and analysis of the microtubule assembly in the presence of taxol

Taxol was originally isolated from the yew Taxus brevifolia. Because taxol inhibits the depolymerization of microtubules, the presence of a self-resistance mechanism in Taxus spp. was hypothesized. The cloning of the cDNA for alpha and beta tubulins from Taxus cuspidata and those from the human embryonic kidney cell line HEK293T revealed that the ²⁶Asp, ³⁵⁹Arg, and ³⁶¹Leu residues in the human beta tubulin, which are important for taxol binding, were replaced with Glu, Trp, and Met in the beta tubulin of T. cuspidata, respectively. The microtubule assembly of the recombinant alpha and beta tubulins was monitored turbidimetrically, and the results clearly demonstrated that the microtubule from T. cuspidata is less sensitive to taxol than that from HEK293T cells. The Taxus microtubule composed of the wild-type alpha tubulin and the beta tubulin with the E26D mutation restored the sensitivity to taxol. We thus postulated that the mutation identified in the beta tubulin of T. cuspidata plays a role in the self-resistance of this species against taxol.     

Diarylheptanoids with inhibitory effects on melanogenesis from the rhizomes of Curcuma comosa in B16 melanoma cells

The methanolic extract from the dried rhizomes of Curcuma comosa cultivated in Thailand was found to inhibit melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the methanolic extract, three new diarylheptanoids, diarylcomosols I–III, were isolated together with 12 known diarylheptanoids. Their chemical structures were elucidated on the basis of chemical and physicochemical evidence. The diarylheptanoids inhibited melanogenesis, and several structural requirements of the active constituents for the inhibition were clarified. In particular, (3R)-1,7-bis(4-hydroxyphenyl)-(6E)-6-hepten-3-ol exhibited stronger inhibitory effect [IC₅₀ = 0.36 μM] without inducing cytotoxicity. The biological effect was much stronger than that of a reference compound, arbutin [IC₅₀ = 174 μM]. We conclude that diarylheptanoid analogs are promising therapeutic agents for the treatment of skin disorders.     

Delayed production of arachidonic acid contributes to the delay of proteinase-activated receptor-1 (PAR1)-triggered prostaglandin E2 release in rat gastric epithelial RGM1 cells

Proteinase-activated receptor-1 (PAR1), upon activation, exerts prostanoid-dependent gastroprotection, and increases prostaglandin E(2) (PGE(2)) release through cyclooxygenase-2 (COX-2) upregulation in rat gastric mucosal epithelial RGM1 cells. However, there is a big time lag between the PAR1-triggered PGE(2) release and COX-2 upregulation in RGM1 cells; that is, the former event takes 18 h to occur, while the latter rapidly develops and reaches a plateau in 6 h. The present study thus aimed at clarifying mechanisms for the delay of PGE(2) release after PAR1 activation in RGM1 cells. Although a PAR1-activating peptide, TFLLR-NH(2), alone caused PGE(2) release at 18 h, but not 6 h, TFLLR-NH(2) in combination with arachidonic acid dramatically enhanced PGE(2) release even for 1-6 h. TFLLR-NH(2) plus linoleic acid caused a similar rapid response. CP-24879, a Δ(5)/Δ(6)-desaturase inhibitor, abolished the PGE(2) release induced by TFLLR-NH(2) plus linoleic acid, but not by TFLLR-NH(2) alone. The TFLLR-NH(2)-induced PGE(2) release was not affected by inhibitors of cytosolic phospholipase A(2) (cPLA(2)), Ca(2+)-independent PLA(2) (cPLA(2)) or secretory PLA(2) (sPLA(2)), but was abolished by their mixture or a pan-PLA(2) inhibitor. Among PLA(2) isozymes, mRNA of group IIA sPLA(2) (sPLA(2)-IIA) was upregulated following PAR1 stimulation for 6-18 h, whereas protein levels of PGE synthases were unchanged. These data suggest that the delay of PGE(2) release after COX-2 upregulation triggered by PAR1 is due to the poor supply of free arachidonic acid at the early stage in RGM1 cells, and that plural isozymes of PLA(2) including sPLA(2)-IIA may complementarily contribute to the liberation of free arachidonic acid.     

Inhibition by ATP and activation by ADP in the regulation of flagellar movement in sea urchin sperm

ATP and ADP are known to play inhibitory and activating roles, respectively, in the regulation of dynein motile activity of flagella. To elucidate how these nucleotide functions are related to the regulation of normal flagellar beating, we examined their effects on the motility of reactivated sea urchin sperm flagella at low pH. At pH 7.0-7.2 which is lower than the physiological pH of 8, about 90% of reactivated flagella were motionless at 1 mM ATP, while about 60% were motile at 0.02 mM ATP. The motionless flagella at 1 mM ATP maintained a single large bend or an S-shaped bend, indicating formation of dynein crossbridges in the axoneme. The ATP-dependent inhibition of flagellar movement was released by ADP, and was absent in outer arm-depleted flagella. Similar inhibition was also observed at 0.02 mM ATP when demembranated flagella were reactivated in the presence of Li+ or pretreated with protein phosphatase 1 (PP1). ADP also released this type of ATP-inhibition. In PP1-pretreated axonemes the binding of a fluorescent analogue of ADP to dynein decreased. Under elastase-treatment at pH 8.0, the beating of demembranated flagella at 1 mM ATP and 0.02 mM ATP lasted for approximately 100 and 45 s, respectively. The duration of beating at 0.02 mM ATP was prolonged by Li+, and that at 1 mM ATP was shortened by removal of outer arms. These results indicate that the regulation of on/off switching of dynein motile activity of flagella involves ATP-induced inhibition and ADP-induced activation, probably through phosphorylation/dephosphorylation of outer arm-linked protein(s).     

Gene expression profile during the life cycle of the urochordate Ciona intestinalis

Recent whole-genome studies and in-depth expressed sequence tag (EST) analyses have identified most of the developmentally relevant genes in the urochordate, Ciona intestinalis. In this study, we made use of a large-scale oligo-DNA microarray to further investigate and identify genes with specific or correlated expression profiles, and we report global gene expression profiles for about 66% of all the C. intestinalis genes that are expressed during its life cycle. We succeeded in categorizing the data set into 5 large clusters and 49 sub-clusters based on the expression profile of each gene. This revealed the higher order of gene expression profiles during the developmental and aging stages. Furthermore, a combined analysis of microarray data with the EST database revealed the gene groups that were expressed at a specific stage or in a specific organ of the adult. This study provides insights into the complex structure of ascidian gene expression, identifies co-expressed gene groups and marker genes and makes predictions for the biological roles of many uncharacterized genes. This large-scale oligo-DNA microarray for C. intestinalis should facilitate the understanding of global gene expression and gene networks during the development and aging of a basal chordate.     

Recombinant human thioredoxin-1 becomes oxidized in circulation and suppresses bleomycin-induced neutrophil recruitment in the rat airway

Thioredoxin-1 (TRX) is a redox-active protein with anti-inflammatory effects. This study investigated the optimal delivery method and the mechanisms of recombinant human TRX (rhTRX) to suppress neutrophil recruitment in a rat bleomycin (BLM)-induced sustained acute lung injury model. In male Wister rats intratracheally administered with 0.125 mg/kg BLM, 8 mg/kg/day rhTRX was intravenously administered on days 3-6 using one of three protocols: daily bolus injection, 3 h daily infusion or continuous infusion for 96 h. Only the continuous-infusion of rhTRX significantly reduced the neutrophil infiltration compared with the other two methods. The BLM-induced down-regulation of L-selectin expression on circulating neutrophils was inhibited by rhTRX. Oxidized rhTRX showed a comparable effect with reduced rhTRX and rhTRX incubated with plasma or circulating in plasma was more than 99% oxidized. These results suggest that rhTRX becomes oxidized in circulation and continuous infusion of rhTRX suppresses neutrophil recruitment in the airway.     

m6A‐mediated alternative splicing coupled with nonsense‐mediated mRNA decay regulates SAM synthetase homeostasis

Alternative splicing of pre‐mRNAs can regulate gene expression levels by coupling with nonsense‐mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS‐NMD) in an organism, we performed long‐read RNA sequencing of poly(A)⁺ RNAs from an NMD‐deficient mutant strain of Caenorhabditis elegans, and obtained full‐length sequences for mRNA isoforms from 259 high‐confidence AS‐NMD genes. Among them are the S‐adenosyl‐L‐methionine (SAM) synthetase (sams) genes sams‐3 and sams‐4. SAM synthetase activity autoregulates sams gene expression through AS‐NMD in a negative feedback loop. We furthermore find that METT‐10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3′ splice site (3′SS) in␣vitro. Direct RNA sequencing coupled with machine learning confirms m⁶A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m⁶A modification at the 3′SS of the sams genes.     

Longitudinal Study of Spatially Heterogeneous Emphysema Progression in Current Smokers with Chronic Obstructive Pulmonary Disease

Background

Cigarette smoke is the main risk factor for emphysema, which is a key pathology in chronic obstructive pulmonary disease (COPD). Low attenuation areas (LAA) in computed tomography (CT) images reflect emphysema, and the cumulative size distribution of LAA clusters follows a power law characterized by the exponent D. This property of LAA clusters can be explained by model simulation, where mechanical force breaks alveolar walls causing local heterogeneous lung tissue destruction. However, a longitudinal CT study has not investigated whether continuous smoking causes the spatially heterogeneous progression of emphysema.

Methods

We measured annual changes in ratios of LAA (LAA%), D and numbers of LAA clusters (LAN) in CT images acquired at intervals of ≥3 years from 22 current and 31 former smokers with COPD to assess emphysema progression. We constructed model simulations using CT images to morphologically interpret changes in current smokers.

Results

D was decreased in current and former smokers, whereas LAA% and LAN were increased only in current smokers. The annual changes in LAA%, D, and LAN were greater in current, than in former smokers (1.03 vs. 0.37%, p = 0.008; −0.045 vs. −0.01, p = 0.004; 13.9 vs. 1.1, p = 0.007, respectively). When LAA% increased in model simulations, the coalescence of neighboring LAA clusters decreased D, but the combination of changes in D and LAN in current smokers could not be explained by the homogeneous emphysema progression model despite cluster coalescence. Conversely, a model in which LAAs heterogeneously increased and LAA clusters merged somewhat in relatively advanced emphysematous regions could reflect actual changes.

Conclusions

Susceptibility to parenchymal destruction induced by continuous smoking is not uniform over the lung, but might be higher in local regions of relatively advanced emphysema. These could result in the spatially heterogeneous progression of emphysema in current smokers.     

Visual shape representation with geometrically characterized contour partitions

This paper proposes a biologically plausible matching method to recognize general shapes based on contour curvature information. The human visual system recognizes general shapes flexibly in real-world scenes through the ventral pathway. The pathway is typically modeled using artificial neural networks. These network models, however, do not construct a shape representation that satisfies the following required constraints: (1) The original shape should be represented by a group of partitioned contours in order to retrieve the whole shape (global information) from the partial contours (local information). (2) Coarse and fine structures of the original shapes should be individually represented in order for the visual system to respond to shapes as quickly as possible based on the least number of their features, and to discriminate between shapes based on detailed information. (3) The shape recognition realized with an artificial visual system should be invariant to geometric transformation such as expansion, rotation, or shear. In this paper, we propose a visual shape representation with geometrically characterized contour partitions described on multiple spatial scales.