Malvone A, a phytoalexin found in Malva sylvestris (family Malvaceae)

The isolation and structure of a phytoalexin, malvone A (2-methyl-3-methoxy-5,6-dihydroxy-1,4-naphthoquinone) is reported. Malvone A formation is induced in Malva sylvestris L. by the plant pathogen Verticillium dahliae. In a turbimetric assay for toxicity to V. dahliae, it had an ED50 value of 24 microg/ml. The structure of malvone A was determined by MS and NMR spectroscopy, and by X-ray crystallographic analysis. The X-ray analysis showed water molecules were located in channels that run along the a-axis.     

Escherichia coli competence gene homologs are essential for competitive fitness and the use of DNA as a nutrient

Natural genetic competence is the ability of cells to take up extracellular DNA and is an important mechanism for horizontal gene transfer. Another potential benefit of natural competence is that exogenous DNA can serve as a nutrient source for starving bacteria because the ability to "eat" DNA is necessary for competitive survival in environments containing limited nutrients. We show here that eight Escherichia coli genes, identified as homologs of com genes in Haemophilus influenzae and Neisseria gonorrhoeae, are necessary for the use of extracellular DNA as the sole source of carbon and energy. These genes also confer a competitive advantage to E. coli during long-term stationary-phase incubation. We also show that homologs of these genes are found throughout the proteobacteria, suggesting that the use of DNA as a nutrient may be a widespread phenomenon.     

NMR structures of the selenoproteins Sep15 and SelM reveal redox activity of a new thioredoxin-like family

Selenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxin-like protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that they have redox activity. In mammals, Sep15 expression is regulated by dietary selenium, and either decreased or increased expression of this selenoprotein alters redox homeostasis. A physiological role for Sep15 and SelM as thiol-disulfide oxidoreductases and their contribution to the quality control pathways of the endoplasmic reticulum are discussed.     

An integrated morphological and molecular approach to a new species description in the Trypanosomatidae: the case of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera: Rhopalidae)

Leptomonas podlipaevi n. sp., a new trypanosomatid species, is described herein based on light microscopic, ultrastructural, and molecular phylogenetic data. The organism is pleomorphic both in host and culture, with two predominant forms-a typical promastigote with a long flagellum and a shorter promastigote with a small or barely extending flagellum. Several spliced leader RNA repeat sequences obtained from the original cultures and the clonal lines representing two types of cells were all nearly identical. These sequences formed a tight cluster in the neighbor-joining tree well separated from other trypanosomatid species. Glyceraldehyde phosphate dehydrogenase gene sequences were determined for L. podlipaevi and 10 previously described trypanosomatid species. Molecular phylogenetic analysis has demonstrated that the new species is most closely related to Leptomonas seymouri and Leptomonas pyrrhocoris. The analysis has also highlighted the polyphyly of the genus Leptomonas.     

Non-stressful death of 23S rRNA mutant G2061C defective in puromycin reaction

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.     

Properties of Ca2+ sparks revealed by four-dimensional confocal imaging of cardiac muscle

Parameters (amplitude, width, kinetics) of Ca(2+) sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x-y images in three planes at 1-μm separation (x-y-z-t mode). In 3,000 x-y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ΔF/F(0) = 0.7. The Ca(2+) release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca(2+) release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca(2+) in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca(2+)](SR)) or that different channel clusters respond to [Ca(2+)](SR) with different sensitivity.     

Dynamics of γH2AX formation and elimination in mammalian cells after X-irradiation

Phosphorylation of the replacement histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called γH2AX can be used as an effective marker for DSB repair and DNA damage response. In this study, we examined a bystander effect (BE) in locally irradiated embryonic human fibroblasts. Using fluorescence microscopy, we found that BE could be observed 1 h after X-ray irradiation (IR) and was completely eliminated 24 h after IR. Using immunohistochemistry and immunoblotting, we also studied kinetics of γH2AX formation and elimination in Syrian hamster and mouse tissues after whole body IR of animals. Analysis of hamster tissues at different times after IR at the dose 5 Gy showed that γH2AX-associated fluorescence in heart was decreased slowly with about a half level remaining 24 h after IR; at the same time, in brain, the level of γH2AX was about 3 times increased over the control level, and in liver, γH2AX level decreased to control values. We also report that in mouse heart the level of γH2AX measured by immunoblotting is lower than in brain, kidney and liver at different times after IR at the dose 3 Gy. Our observations indicate that there are significant variations in dynamics of γH2AX formation and elimination between non-proliferating mammalian tissues. These variations in γH2AX dynamics in indicated organs partially correlated with the expression level of the major kinase genes involved in H2AX phosphorylation (ATM and DNA-PK).     

Comparative study of the glycan specificities of cell-bound human tandem-repeat-type galectin-4, -8 and -9

Adhesion/growth-regulatory galectins (gals) exert their functionality by the cis/trans-cross-linking of distinct glycans after initial one-point binding. In order to define the specificity of ensuing association events leading to cross-linking, we recently established a cell-based assay using fluorescent glycoconjugates as flow cytometry probes and tested it on two human gals (gal-1 and -3). Here we present a systematic study of tandem-repeat-type gal-4, -8 and -9 loaded on Raji cells resulting in the following key insights: (i) all three gals bound to oligolactosamines; (ii) binding to ligands with Galβ1-3GlcNAc or Galβ1-3GalNAc as basic motifs was commonly better than that to canonical Galβ1-4GlcNAc; (iii) all three gals bound to 3'-O-sulfated and 3'-sialylated disaccharides mentioned above better than that to parental neutral forms and (iv) histo-blood group ABH antigens were the highest affinity ligands in both the cell and the solid-phase assay. Fine specificity differences were revealed as follows: (i) gal-8 and -9, but not gal-4, bound to disaccharide Galβ1-3GlcNAc; (ii) increase in binding due to negatively charged substituents was marked only in the case of gal-4 and (iii) gal-4 and -8 bound preferably to histo-blood group A glycans, whereas gal-9 targeted B-type glycans. Experiments with single carbohydrate recognition domains (CRDs) of gal-4 showed that the C-CRD preferably bound to ABH glycans, whereas the N-CRD associated with oligolactosamines. In summary, the comparative analysis disclosed the characteristic profiles of glycan reactivity for the accessible CRD of cell-bound gals. These results indicate the distinct sets of functionality for these three members of the same subgroup of human gals.