Effect of dinitroaniline herbicides on the growth of Entamoeba histolytica

The effect of the dinitroaniline herbicides oryzalin and trifluralin on the growth of Entamoeba histolytica was examined. Oryzalin inhibited the growth of E. histolytica strain HM-1:IMSS. Trifluralin was less effective than oryzalin for this parasite. Entamoeba histolytica was more resistant to these dinitroanilines than other parasitic protozoa examined so far, including Leishmania spp., Trypanosoma brucei, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum. Colchicine, a potent microtubule inhibitor of animal cells, was much less effective for E. histolytica, even at very high concentrations. A reptilian parasite, Entamoeba invadens strain IP-1, examined for comparison, was more resistant to these dinitroanilines than E. histolytica. Accumulation of E. histolytica trophozoites in mitosis was observed after culture in 100 microM oryzalin. The inhibitory effect of oryzalin on the growth of E. histolytica trophozoites was abrogated by removal of the drug after exposure to 100 microM for 2 days. In parallel to the recovery of growth after removal of the drug, the percentage of trophozoites in mitosis was reduced to a normal level. The results indicate that treatment of trophozoites with oryzalin arrests mitosis and that its effect is reversible. Therefore, oryzalin is a useful tool for studies relating to the cell cycle of this parasite.     

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA^Gln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA^Glu and Glu-tRNA^Gln. The Glu-tRNA^Gln is then converted to Gln-tRNA^Gln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA^Glu and tRNA^Gln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA^Glu/tRNA^Gln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA^Gln complex reveals the structural determinants responsible for specific tRNA^Gln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.     

Measurements of strain on single stress fibers in living endothelial cells induced by fluid shear stress

Fluid shear stress (FSS) acting on the apical surface of endothelial cells (ECs) can be sensed by mechano-sensors in adhesive protein complexes found in focal adhesions and intercellular junctions. This sensing occurs via force transmission through cytoskeletal networks. This study quantitatively evaluated the force transmitted through cytoskeletons to the mechano-sensors by measuring the FSS-induced strain on SFs using live-cell imaging for actin stress fibers (SFs). FSS-induced bending of SFs caused the SFs to align perpendicular to the direction of the flow. In addition, the displacement vectors of the SFs were detected using image correlation and the FSS-induced axial strain of the SFs was calculated. The results indicated that FSS-induced strain on SFs spanned the range 0.01-0.1% at FSSs ranging from 2 to 10 Pa. Together with the tensile property of SFs reported in a previous study, the force exerted on SFs was estimated to range from several to several tens of pN.     

Interleukin-6 as an independent predictor of future cardiovascular events in high-risk Japanese patients: comparison with C-reactive protein

Inflammation is associated with the development of atherosclerotic vascular lesions and some inflammatory parameters are used as cardiovascular (CV) risk markers. The present study was designed to assess the predictive power of interleukin (IL)-6 for future CV events. In 121 Japanese patients with multiple CV risk factors and/or disease, serum concentrations of IL-6 and high sensitive C-reactive protein (hs-CRP) were measured. During follow-up periods (mean, 2.9 years) after the baseline assessment, 50 patients newly experienced CV events such as stroke/transient ischemic attack (n=10), heart failure hospitalization (n=6), acute coronary syndrome (n=7), and revascularization for coronary artery disease (n=15) and peripheral arterial disease (n=12). The serum level of IL-6, but not hs-CRP, was significantly higher in patients who had CV events than in event-free subjects (3.9±2.6 and 3.0±2.2 pg/mL, P=0.04). When the patients were divided into three groups by tertiles of basal levels of IL-6 (<1.85, 1.85-3.77, and ≥3.77 pg/mL), cumulative event-free rates by the Kaplan-Meier method were decreased according to the increase in basal IL-6 levels (65%, 50%, and 19% in the lowest, middle, and highest tertiles of IL-6, respectively; log-rank test, P=0.002). By univariate Cox regression analysis, previous CV disease, creatinine clearance, and serum IL-6 levels were significantly associated with CV events during follow-up. Among these possible predictors, the highest tertile of IL-6 was only an independent determinant for the morbidity in the multivariate analysis (hazard ratio 2.80 vs. lowest tertile, P=0.006). These findings indicate that IL-6 is a powerful independent predictor of future CV events in high-risk Japanese patients, suggesting its predictive value is superior to that of hs-CRP.     

TRIC-A channels in vascular smooth muscle contribute to blood pressure maintenance

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.     

Identification of an allele of VAM3/SYP22 that confers a semi-dwarf phenotype in Arabidopsis thaliana

The short stem and midrib (ssm) mutants of Arabidopsis thaliana show both semi-dwarf and wavy leaf phenotypes due to defects in the elongation of the stem internodes and leaves. Moreover, these abnormalities cannot be recovered by exogenous phytohormones. ssm was originally identified as a single recessive mutant of the ecotype Columbia (Col-0), but genetic crossing experiments have revealed that this mutant phenotype is restored by another gene that is functional in the ecotype Landsberg erecta (Ler) and not in Col-0. Map-based cloning of the gene that is defective in ssm mutants has uncovered a small deletion in the sixth intron of a gene encoding a syntaxin, VAM3/SYP22, which has been implicated in vesicle transport to the vacuole. This mutation appears to cause a peptide insertion in the deduced VAM3/SYP22 polypeptide sequence due to defective splicing of the shortened sixth intron. Significantly, when compared with the wild-type Ler genome, the wild-type Col-0 genome has a single base pair deletion causing a frameshift mutation in SYP23, a gene with the highest known homology to VAM3/SYP22. These findings suggest that VAM3/SYP22 and SYP23 have overlapping functions and that the vesicle transport mediated by these syntaxins is important for shoot morphogenesis.     

The effect of protozoa on the composition of rumen bacteria in cattle using 16S rRNA gene clone libraries

The effect of the presence of protozoa on the composition of rumen bacteria was investigated in cattle. Seven castrated Holstein cattle were divided into two groups: four faunated and three unfaunated, and 16S ribosomal RNA gene (rDNA) clonal libraries were constructed. A total of 312 clones were sequenced across 1,500 bp. The 151 sequences of the faunated cattle were classified into 98 operational taxonomic units (OTUs) having at least 97% similarity. The sequences derived from the faunated cattle were classified into Firmicutes (59.7%), Bacteroidetes (34.4%), Spirochaetes (2.6%), Actinobacteria (2.0%), and Proteobacteria (1.3%). Bacteroides and Prevotella (34.4%) were the major groups in the faunated cattle. The 161 sequences in the unfaunated cattle were classified into 72 OTUs. The sequences derived from the unfaunated libraries were classified into Firmicutes (65.7%), Bacteroidetes (31.1%), Proteobacteria (1.9%), and Spirochaetes (1.2%). The Clostridium botulinum group and its relatives (36.0%) were the major groups in the unfaunated cattle.An analysis by the computer program LIBSHUFF clarified that the presence of ruminal protozoa markedly affected the composition of rumen bacteria.