Identification of Streptomyces coelicolor M145 genomic region involved in biosynthesis of teichulosonic acid–cell wall glycopolymer

The cell wall of the model actinomycete Streptomyces coelicolor M145 has recently been shown to contain the novel glycopolymer teichulosonic acid. The major building block of this polymer is 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn), suggesting initial clues about the genetic control of biosynthesis of this cell wall component. Here, through genome mining and gene knockouts, we demonstrate that the sco4879–sco4882 genomic region of S. coelicolor M145 is necessary for biosynthesis of teichulosonic acid. Specifically, mutants carrying individual knockouts of sco4879, sco4880 and sco4881 genes do not produce Kdn-containing glycopolymer and instead accumulate the minor cell wall component poly(diglycosyl 1-phosphate). Our studies provide evidence that this region is at least partly responsible for biosynthesis of Kdn, whereas flanking genes might control the other steps of teichulosonic acid formation.     

Saline rivers provide arid landscapes with a considerable amount of biochemically valuable production of chironomid (Diptera) larvae

Saline rivers are supposed to be ‘hot spots’ of high biological productivity in arid landscapes. To test this, we quantified the production of chironomid larvae, because river production is known to be transferred to arid landscapes primarily by birds fed on these larvae. In addition, we studied the potential biochemical quality of the larvae for birds based on the essential highly unsaturated fatty acid (HUFA) contents in their biomass. We studied species composition and measured production of chironomid larvae in two saline rivers (Volgograd region, Russia). We also evaluated the fatty acid composition and contents of the dominant taxa and estimated the flux of HUFA from the studied saline rivers to land via chironomid potential emergence. Average monthly production of chironomids measured for only 1 month, August, was quite comparable to annual production in some freshwater rivers. All the dominant chironomid larvae had comparatively high essential eicosapentaenoic acid contents, especially Cricotopus salinophilus, which showed the highest value, reported for Chironomidae. The monthly flux of HUFA from the studied rivers to land due to the chironomid potential emergence was roughly comparable to the global average estimation of annual water–land HUFA export via emerging insects.     

Eukaryote-specific motif of ribosomal protein S15 neighbors A site codon during elongation and termination of translation

The eukaryotic ribosomal protein S15 is a key component of the decoding site in contrast to its prokaryotic counterpart, S19p, which is located away from the mRNA binding track on the ribosome. Here, we determined the oligopeptide of S15 neighboring the A site mRNA codon on the human 80S ribosome with the use of mRNA analogues bearing perfluorophenyl azide-modified nucleotides in the sense or stop codon targeted to the 80S ribosomal A site. The protein was cross-linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of eRF1 in the experiments with mRNAs bearing stop codon. Digestion of modified S15 with various specific proteolytic agents followed by identification of the resulting modified oligopeptides showed that cross-link was in C-terminal fragment in positions 131–145, most probably, in decapeptide 131-PGIGATHSSR-140. The position of cross-linking site on the S15 protein did not depend on the nature of the A site-bound codon (sense or stop codon) and on the presence of polypeptide chain release factor eRF1 in the ribosomal complexes with mRNA analogues bearing a stop codon. The results indicate an involvement of the mentioned decapeptide in the formation of the ribosomal decoding site during elongation and termination of translation. Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from eubacteria and archaea, revealed that decapeptide PGIGATHSSR in positions 131–140 is strongly conserved in eukaryotes and has minor variations in archaea but has no homology with any sequence in C-terminal part of eubacterial S19p, which suggests involvement of the decapeptide in the translation process in a eukaryote-specific manner.     

Structural motifs of the bacterial ribosomal proteins S20, S18 and S16 that contact rRNA present in the eukaryotic ribosomal proteins S25, S26 and S27A,respectively

The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively.     

Novel Technique for Quantifying Adhesion of Metarhizium anisopliae Conidia to the Tick Cuticle

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of Metarhizium anisopliae var. anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged Rhipicephalus annulatus (Say) (Arachnida: Ixodidae) females and was also verified for M. anisopliae var. acridum Driver et Milner (Hypocreales: Clavicipitaceae) and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female R. annulatus and tick mortality. However, no correlation was observed between a tick''s susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.The entomopathogenic fungus Metarhizium anisopliae var. anisopliae (Metschn.) Sorokîn (1883) infects a broad range of arthropod hosts and can be used as a biopesticide against different insect and tick species (8, 22, 35, 36). The adhesion of the conidia of entomopathogenic fungi to the host cuticle is the initial stage of the pathogenic process and includes both passive and active events (5, 10). The hydrophobic epicuticular lipid layer plays an important role during both the attachment process and the germination of the conidia on the surface of the host (15, 19). According to Boucias et al. (7), the attachment of conidia to the host cuticle is based on nonspecific hydrophobic and electrostatic forces. The conidia of most entomopathogenic fungi, including M. anisopliae, have an outer cell layer made up of rodlets (6). The hydrophobins, specific proteins present in the rodlet layer, mediate the passive adhesion of conidia to hydrophobic surfaces, such as the cuticles of arthropods (16, 45, 46). However, as germination commences, the hydrophobins are replaced by an adhesion-like protein, Mad1, which promotes tighter and more-specific adhesion between the conidia and the host (44). Many factors may affect the adhesion and persistence of conidia on the host cuticle (i.e., characteristics of the pathogen, including its virulence [2, 18, 48], conditions under which the pathogen is cultured [17], type of spores [7, 16], topographical and chemical properties of the host cuticle [9, 38, 42], host surface hydrophobicity [15, 23], host behavior [31, 33], and environmental conditions [33]). Conidia of M. anisopliae have shown an affinity to cuticular regions containing setae or spines (7, 38) and to highly hydrophobic cuticle regions, such as mosquitoes'' siphon tubes (23) and intersegmental folds (43). Sites with higher numbers of adhered conidia varied among host species. However, in general, the membranous intersegmental regions were often particularly attractive sites for conidial attachment (26). Variation in the distribution of conidia across different anatomical regions has also been noted in studies of several tick species inoculated with entomopathogenic fungi (3, 21, 22). An evaluation of the attachment of Beauveria bassiana conidia to three tick species, Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis, demonstrated that the distribution patterns of the different conidia on the ticks'' bodies were not uniform (22). The density of the conidia and their germination varied dramatically across different anatomical regions of Amblyomma maculatum and A. americanum that had been inoculated with B. bassiana (21). Arruda et al. (3) demonstrated that mass adhesion of M. anisopliae conidia to engorged Boophilus microplus females occurs predominantly on ticks'' legs, suggesting its association with the presence of setae.There are a few approaches for assessing the adhesion of conidia to the host cuticle that are based on direct observation of the conidia on the arthropod cuticle. They involve examining a few areas on the surface of an arthropod by means of scanning electron microscopy (SEM) (11, 15, 30), transmission electron microscopy (TEM) (4), or fluorescence microscopy following vital staining of the conidia (2, 28, 29, 37). These methods are expensive, time-consuming, and relatively inaccurate due to the uneven distribution of conidia on the host surface.In this work, we describe a quantitative method for determining the total amount of conidia that have adhered to a whole host cuticle. This method is based on removing adhered conidia from the tick cuticle using an organic solvent, separating the conidia from the extract by centrifugation, resuspending the conidia in a detergent solution, and then counting the conidia in a hemocytometer. The efficacy of the method was evaluated by comparing the results of this procedure with those of a supplementary examination of conidial removal using SEM.The term “adhered” is often used to define conidia in different states: washed or unwashed after inoculation, present on the host cuticle immediately after inoculation, or kept for several hours (1, 2, 38). In this paper, the term “adhered conidia” refers to conidia that remained on the cuticle after washing by vortexing the inoculated and dried host in an aqueous solution of Triton X-100 and rinsing of the material under tap water.     

In vitro maturation and early developmental capacity of bovine oocytes cultured in pure follicular fluid and supplementation with follicular wall

Mammalian oocytes mature in follicular fluid (FF), surrounded by follicular cells. In the present study, in vitro maturation of bovine oocytes cultured in FF from dominant follicles 15-17mm in diameter (with various forms of heat pretreatment) and supplementation with follicular wall from follicles 3-5mm in diameter (FW1) were examined. Heat pretreatment of FF was as follows: (1) no treatment (FF1); (2) 56 degrees C for 30min (FF2); and (3) 100 degrees C for 20s (FF3). After IVM in FF1, oocytes underwent IVF and IVC and embryo development was assessed (up to the morula stage). The rate of oocyte maturation was decreased in pure FF1 versus control (44.5% versus 62.8%, P<0.001). In the control medium, FW1 did not significantly affect nuclear maturation. By contrast, the addition of FW1 to FF1 increased the rate of matured oocytes approximately two-fold (85.9% versus 45.6%, P<0.001). Furthermore, the maturation rate in the FF+FW1 system declined (from 85.9 to 71.0%, P<0.001), whereas that in the FF system increased (from 45.6 to 71.6%, P<0.001) with increased temperature of the FF treatment. Supplementation of the control medium with FW1 increased the yield of morulae (42.6% versus 13.7%, P<0.001). However, the stimulatory effect of FW1 on the morula rate was much higher in pure FF1 (72.5% versus 31.7%, P<0.001). These findings indicated, for the first time, the stimulatory impact of FW1 on in vitro maturation and early developmental capacity of bovine oocytes cultured in pure FF from dominant follicles. We also inferred that bovine FF constituents affecting bovine oocyte maturation and the meiosis-promoting ability of the FW were heat-labile.     

Radiation-induced lung adenocarcinoma is associated with increased frequency of genes inactivated by promoter hypermethylation

Epigenetic inactivation of genes by promoter hypermethylation, a major mechanism in the initiation and progression of tobacco-induced cancer, has also been associated with lung cancer induced through environmental and occupational exposures. Our previous study of gene methylation in workers from the MAYAK nuclear enterprise identified a significantly higher prevalence for methylation of the p16 gene (CDKN2A) in adenocarcinomas from workers compared to tumors from non-worker controls. The purpose of this investigation was to determine whether genes in addition to p16 are "targeted" for silencing and whether overall gene methylation was more common in radiation-induced adenocarcinoma. A significant increase in the prevalence of methylation of GATA5 was seen in tumors from workers compared to tumors from controls. The prevalence for methylation of PAX5 beta and H-cadherin did not differ in tumors from workers and controls. Evaluating the frequency for methylation of a five-gene panel revealed that 93% of adenocarcinomas from workers compared to 66% of tumors from controls were methylated for at least one gene. Moreover, a twofold increase was seen in the number of tumors methylated for three or more genes for tumors from workers compared to controls. Increased frequency for inactivation of genes by promoter hypermethylation and targeting of tumor suppressor genes such as GATA5 may be factors that contribute to the increased risk for lung cancer associated with radiation exposure.     

Suppression of TRPC3 leads to disappearance of store-operated channels and formation of a new type of store-independent channels in A431 cells

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.     

Protoporphyrin IX interacts with wild-type p53 protein in vitro and induces cell death of human colon cancer cells in a p53-dependent and -independent manner

Photodynamic therapy (PDT) of cancer is an alternative treatment for tumors resistant to chemo- and radiotherapy. It induces cancer cell death mainly through generation of reactive oxygen species by a laser light-activated photosensitizer. It has been suggested that the p53 tumor suppressor protein sensitizes some human cancer cells to PDT. However, there is still no direct evidence for this. We have demonstrated here for the first time that the photosensitizer protoporphyrin IX (PpIX) binds to p53 and disrupts the interaction between p53 tumor suppressor protein and its negative regulator HDM2 in vitro and in cells. Moreover, HCT116 colon cancer cells exhibited a p53-dependent sensitivity to PpIX in a dose-dependent manner, as was demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and fluorescence-activated cell sorter (FACS) analysis of cell cycle profiles. We have also observed induction of p53 target pro-apoptotic genes, e.g. puma (p53-up-regulated modulator of apoptosis), and bak in PpIX-treated cells. In addition, p53-independent growth suppression by PpIX was detected in p53-negative cells. PDT treatment (2 J/cm2) of HCT116 cells induced p53-dependent activation of pro-apoptotic gene expression followed by growth suppression and induction of apoptosis.