EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

Telomerase Recruitment in Saccharomyces cerevisiae Is Not Dependent on Tel1-Mediated Phosphorylation of Cdc13

In Saccharomyces cerevisiae, association between the Est1 telomerase subunit and the telomere-binding protein Cdc13 is essential for telomerase to be recruited to its site of action. A current model proposes that Tel1 binding to telomeres marks them for elongation, as the result of phosphorylation of a proposed S/TQ cluster in the telomerase recruitment domain of Cdc13. However, three observations presented here argue against one key aspect of this model. First, the pattern of Cdc13 phosphatase-sensitive isoforms is not altered by loss of Tel1 function or by mutations introduced into two conserved serines (S249 and S255) in the Cdc13 recruitment domain. Second, an interaction between Cdc13 and Est1, as monitored by a two-hybrid assay, is dependent on S255 but Tel1-independent. Finally, a derivative of Cdc13, cdc13–(S/TQ)₁₁→(S/TA)₁₁, in which every potential consensus phosphorylation site for Tel1 has been eliminated, confers nearly wild-type telomere length. These results are inconsistent with a model in which the Cdc13–Est1 interaction is regulated by Tel1-mediated phosphorylation of the Cdc13 telomerase recruitment domain. We propose an alternative model for the role of Tel1 in telomere homeostasis, which is based on the assumption that Tel1 performs the same molecular task at double-strand breaks (DSBs) and chromosome termini.TELOMERE length homeostasis is a highly regulated process that must balance telomere loss (as the result of incomplete replication and/or nucleolytic degradation) with telomeric repeat addition (through the action of telomerase and/or recombination). In the budding yeast Saccharomyces cerevisiae, a key regulatory event is recruitment of telomerase to chromosome ends by the telomere end-binding protein Cdc13 (Nugent et al. 1996; Evans and Lundblad 1999; Pennock et al. 2001; Bianchi et al. 2004; Chan et al. 2008). Recruitment relies on a direct interaction between Cdc13 and the Est1 subunit of telomerase (Pennock et al. 2001), which brings the catalytic core of the enzyme to its site of action. Disruption of this interaction, due to mutations in either CDC13 (cdc13-2) or EST1 (est1-60), results in an Est (ever-shorter-telomere) phenotype, as manifested by progressive telomere shortening and an eventual senescence phenotype. The recruitment activity of Cdc13, which resides in a 15-kDa N-terminal domain (Pennock et al. 2001), is sufficient to direct telomerase even to nontelomeric sites (Bianchi et al. 2004). As predicted by the recruitment model, association of telomerase with telomeres is greatly reduced in strains expressing the recruitment-defective cdc13-2 allele (Chan et al. 2008).Telomerase action at individual telomeres is highly regulated. Using an assay that monitors telomere addition at single nucleotide resolution (single telomere extension, STEX), Lingner and colleagues showed that only ∼7% of telomeres with wild-type (i.e., 300 bp) length are elongated by telomerase during a single cell cycle (Teixeira et al. 2004). However, as telomere length declines, the extension frequency increases: ∼20% of telomeres 200 bp in length and >40% of 100-bp-long telomeres are elongated (Teixeira et al. 2004; Arneric and Lingner 2007). The mechanism by which telomerase distinguishes short from long telomeres has been the subject of intense investigation. Work from a number of laboratories has led to the proposal that Tel1-dependent phosphorylation of Cdc13 at underelongated telomeres mediates the interaction between Cdc13 and the telomerase-associated Est1 protein, thus ensuring that telomerase is directed to the shortest telomeres in a population. In support of this model, the Est1 and Est2 telomerase subunits exhibit enhanced association with telomeres that have been artificially shortened, whereas Cdc13 displays length-independent association with telomeres (Bianchi and Shore 2007; Sabourin et al. 2007). This suggests that the preferential elongation of shorter telomeres is controlled at the level of recruitment of the telomerase holoenzyme by Cdc13. Furthermore, efficient association of Est1 and Est2 with chromosome ends requires Tel1 and Mre11 (which acts in the same pathway as Tel1 for telomere length regulation; Nugent et al. 1998; Ritchie and Petes 2000) but not Mec1 (Takata et al. 2005; Goudsouzian et al. 2006). Tel1 itself is also telomere bound (Takata et al. 2004), with enhanced binding to shorter telomeres (Bianchi and Shore 2007; Hector et al. 2007; Sabourin et al. 2007; Abdallah et al. 2009), although there is considerable controversy over the degree and timing of Tel1 association with chromosome termini during the cell cycle. As expected for a key regulator of the interaction between Cdc13 and a telomerase subunit, a tel1-Δ strain has short telomeres (Lustig and Petes 1986), although telomere length is not impaired enough to confer the Est phenotype displayed by cdc13-2 and est1-60 strains.Implicit in the above proposal is that Cdc13 must be a direct substrate of Tel1. In support of this, Teng and colleagues reported several years ago that the recruitment domain of Cdc13 has a cluster of potential Tel1 (and/or Mec1) phosphorylation sites (Tseng et al. 2006). Substrates of the DNA damage kinases often contain several closely spaced phosphorylation sites, termed S/TQ cluster domains (SCDs), which are considered a structural hallmark of many DNA damage-response proteins (Traven and Heierhorst 2005). On the basis of in vitro kinase assays with GST fusions to 75- to 90-amino-acid portions of the Cdc13 recruitment domain, Tseng et al. 2006 concluded that four SQ sites in the recruitment domain of Cdc13 are overlapping substrates for both Tel1 and Mec1, leading to the proposal that telomerase recruitment in S. cerevisiae is regulated by Tel1-dependent phosphorylation of Cdc13.The above model makes a key prediction: in a tel1-Δ strain, telomerase should no longer exhibit a length-dependent pattern of elongation. However, preferential elongation of short telomeres still occurs at native chromosome ends in the absence of Tel1 (Arneric and Lingner 2007). In addition, Petes and colleagues have argued, on the basis of epistasis data, that Tel1 performs an indirect role in the telomerase pathway, rather than directly targeting a telomerase regulator (Ritchie et al. 1999; Ritchie and Petes 2000). These observations are not easily explained, if preferential recognition of short telomeres by telomerase is mediated by Tel1-dependent phosphorylation of Cdc13. In this current study, we have re-examined the evidence for phosphorylation of Cdc13 as a regulatory mechanism for telomere length homeostasis. We report on a series of observations that indicate that Tel1 contributes to telomere length control through a mechanism other than phosphorylation of the Cdc13 S/TQ cluster.     

Membrane topology of the chromate transporter ChrA of Pseudomonas aeruginosa

The membrane topology of the plasmid-encoded Pseudomonas aeruginosa ChrA protein, which effluxes chromate ions, was determined by the analysis of translational fusions with reporter enzymes alkaline phosphatase and beta-galactosidase. A novel 13-TMS (transmembrane segments) topology, with the N-terminus located in the cytoplasm and the C-terminus in the periplasmic space, was consistent with the enzyme activities determined in both Escherichia coli and P. aeruginosa. Alignment of the two halves of ChrA showed significant sequence homology, with TMS I, II, III, IV, V and VI displaying similarity to TMS VIII, IX, X, XI, XII and XIII, respectively, although with opposite membrane orientations. This suggests that ChrA arose from the duplication of a gene encoding a 6-TMS ancestral protein, followed by the insertion of extra TMS VII. These data also suggest that the two halves of ChrA may carry out distinct functions for the transport of chromate.     

Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.     

Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica

In vitro exposure of Entamoeba histolytica trophozoites to the sex steroids 17beta-estradiol, progesterone, and dehydrotestosterone had little effect on parasite viability or proliferation. However, treatment with the adrenal steroid dehydroepiandrosterone (DHEA) markedly inhibited parasite proliferation, adherence and motility, and at a certain dose it induced trophozoite lysis. The opposite effect on proliferation was found when the trophozoites were exposed to cortisol. Moreover, DHEA decreased while cortisol increased the parasite's DNA synthesis determined by 3H-thymidine incorporation. Trophozoite lysis by DHEA appeared to be caused by a necrotic rather than an apoptotic process, as observed in propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assays. A possible mechanisms of action was derived from experiments demonstrating that the activity of a putative 3-hydroxy-3-methyl glutaryl CoA reductase detected in trophozoite extracts was inhibited in the presence of DHEA. Contrary to its in vitro inhibitory effect, in vivo administration of DHEA to infected hamsters resulted in exacerbation of the amebic liver abscesses. These results demonstrated that androgen steroids act directly upon E. histolytica growth and viability, and may shed new light on some age and gender differences in disease progression, as well as finding application in the drug treatment of human amebiasis.     

Antimicrobial potential of four Lactobacillus strains isolated from breast milk

AIMS: The antimicrobial potential of four lactobacilli (Lactobacillus salivarius CECT5713, Lactobacillus gasseri CECT5714, L. gasseri CECT5715 and Lactobacillus fermentum CECT5716), isolated from fresh human breast milk, was evaluated in this study and compared with Lactobacillus coryniformis CECT5711, a reuterin-producing strain isolated from an artisan goat's cheese. METHODS AND RESULTS: Agar diffusion tests, competitive adhesion assays and mucin expression assays were carried out in order to value the antibacterial properties of the lactobacilli strains. The antibacterial capability of the strains was tested in vivo by using a murine infection model with Salmonella choleraesuis. The results revealed that all the strains studied, displayed antibacterial properties against pathogenic bacteria. However, the antibacterial potential varied among the lactobacilli tested and, in fact, L. salivarius CECT5713 showed not only the best in vitro antibacterial activity, but also the highest protective effect against a Salmonella strain in the murine infection model. CONCLUSION: The four breast-milk lactobacilli, and particularly L. salivarius CECT5713, possess potent antibacterial activities that result in a higher protection against S. choleraesuis CECT4155 in a mouse infection model. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that lactobacilli from breast milk could contribute to an anti-infective protection in neonates and would be excellent candidates for the development of infant probiotic products.     

Effects of endogenous salicylic acid on nodulation in the model legumes Lotus japonicus and Medicago truncatula

The exogenous addition of salicylic acid (SA) was previously shown to inhibit indeterminate but not determinate-type nodulation. We sought to extend these results by modulating endogenous levels of SA through the transgenic expression of salicylate hydroxylase (NahG) in both stably transformed Lotus japonicus and composite Medicago truncatula plants. NahG expression in L. japonicus resulted in a marked reduction of SA levels. This reduction correlated with an increase in the number of infections and mean nodule number when compared to controls. However, a complicating factor was that NahG-expressing plants had greater root growth. Spot inoculations of NahG-expressing L. japonicus plants confirmed increased nodulation in these plants. Consistent with the reported inhibitory effects of exogenous SA on indeterminate-type nodulation, NahG expression in M. truncatula plants led to enhanced nodulation and infection. These data point to an important role for SA-mediated plant defense pathways in controlling nodule formation on both determinate and indeterminate nodule-forming hosts.     

Prevalence of Helicobacter pylori in Georgian patients with dyspepsia

BACKGROUND: Georgia has showed a high prevalence of peptic ulcer disease (PUD), but the prevalence of Helicobacter pylori in this country is practically unknown. The purpose of this study was to determine the prevalence of H. pylori and specific genotypes in different populations in Georgia. MATERIALS AND METHODS: We studied 62 patients from several hospitals in Tbilisi, Georgia. More than 55% of patients had PUD. We determined H. pylori presence as well as specific genotypes cagA and vacA by polymerase chain reaction. In addition, we studied serum samples from 94 healthy persons to determine H. pylori and CagA prevalence by ELISA. RESULTS: We found a high prevalence of H. pylori and CagA in the healthy population (70.2 and 57.4%, respectively) and a high prevalence of CagA among the H. pylori-positive persons (71.2%). Prevalence increased with age as reported in other countries (p = .05). Among symptomatic persons, we found nearly the same high prevalence of H. pylori (64.5%) as in the asymptomatic population. Furthermore, in symptomatic H. pylori patients, we found 65.0 and 67.5% prevalence of cagA and vacA, respectively. For 33 patients with PUD, 24 patients (72.7%) were H. pylori positive and 66.7% of them were cagA positive. In contrast, among the patients with non-ulcer dyspepsia (NUD), 16 (55.2%) were H. pylori positive and 62.5% of them were colonized with cagA-positive strains. H. pylori and cagA prevalence were not significantly different between PUD and patients with NUD. CONCLUSIONS: We confirmed that among individuals in Georgia, the prevalence of H. pylori is high and cagA-positive strains were equally present among H. pylori-positive patients with PUD and NUD and asymptomatic persons.     

Anatomy of corpus callosum in prenatally malnourished rats

The effect of prenatal malnutrition on the anatomy of the corpus callosum was assessed in adult rats (45-52 days old). In the prenatally malnourished animals we observed a significant reduction of the corpus callosum total area, partial areas, and perimeter, as compared with normal animals. In addition, the splenium of corpus callosum (posterior fifth) showed a significant decrease of fiber diameters in the myelinated fibers without changing density. There was also a significant decrease in diameter and a significant increase in density of unmyelinated fibers. Measurements of perimeter's fractal dimensions from sagittal sections of the brain and corpus callosum did not show significant differences between malnourished and control animals. These findings indicate that cortico-cortical connections are vulnerable to the prenatal malnutrition, and suggest this may affect interhemispheric conduction velocity, particularly in visual connections (splenium).