High expression of focal adhesion kinase (p125(FAK)) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome

INTRODUCTION: Focal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy. METHODS: We analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis. RESULTS: Elevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01). CONCLUSIONS: Immunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src

Anti-prion drug mPPIg5 inhibits PrP(C) conversion to PrP(Sc).

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future

Transition of plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein pumilio

Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism

Agronomic Management of Indigenous Mycorrhizas

Many of the advantages conferred to plants by arbuscular mycorrhiza (AM) are associated to the ability of AM plants to explore a greater volume of soil through the extraradical mycelium. Sieverding (1991) estimates that for each centimetre of colonized root there is an increase of 15 cm3 on the volume of soil explored, this value can increase to 200 cm3 depending on the circumstances. Due to the enhancement of the volume of soil explored and the ability of the extraradical mycelium to absorb and translocate nutrients to the plant, one of the most obvious and important advantages resulting from mycorrhization is the uptake of nutrients. Among of which the ones that have immobilized forms in soil, such as P, assume particular significance. Besides this, many other benefits are recognized for AM plants (Gupta et al, 2000): water stress alleviation (Augé, 2004; Cho et al, 2006), protection from root pathogens (Graham, 2001), tolerance to toxic heavy metals and phytoremediation (Audet and Charest, 2006; Göhre and Paszkowski, 2006), tolerance to adverse conditions such as very high or low temperature, high salinity (Sannazzaro et al, 2006), high or low pH (Yano and Takaki, 2005) or better performance during transplantation shock (Subhan et al, 1998). The extraradical hyphae also stabilize soil aggregates by both enmeshing soil particles (Miller e Jastrow, 1992) and producing a glycoprotein, golmalin, which may act as a glue-like substance to adhere soil particles together (Wright and Upadhyaya, 1998). Despite the ubiquous distribution of mycorrhizal fungi (Smith and Read, 2000) and only a relative specificity between host plants and fungal isolates (McGonigle and Fitter, 1990), the obligate nature of the symbiosis implies the establishment of a plant propagation system, either under greenhouse conditions or in vitro laboratory propagation. These techniques result in high inoculum production costs, which still remains a serious problem since they are not competitive with production costs of phosphorus fertilizer. Even if farmers understand the significance of sustainable agricultural systems, the reduction of phosphorus inputs by using AM fungal inocula alone cannot be justified except, perhaps, in the case of high value crops (Saioto and Marumoto, 2002). Nurseries, high income horticulture farmers and no-agricultural application such as rehabilitation of degraded or devegetated landscapes are examples of areas where the use of commercial inoculum is current. Another serious problem is quality of commercial available products concerning guarantee of phatogene free content, storage conditions, most effective application methods and what types to use. Besides the information provided by suppliers about its inoculum can be deceiving, as from the usually referred total counts, only a fraction may be effective for a particular plant or in specific soil conditions. Gianinazzi and Vosátka (2004) assume that progress should be made towards registration procedures that stimulate the development of the mycorrhizal industry. Some on-farm inoculum production and application methods have been studied, allowing farmers to produce locally adapted isolates and generate a taxonomically diverse inoculum (Mohandas et al, 2004; Douds et al, 2005). However the inocula produced this way are not readily processed for mechanical application to the fields, being an obstacle to the utilization in large scale agriculture, especially row crops, moreover it would represent an additional mechanical operation with the corresponding economic and soil compaction costs. It is well recognized that inoculation of AM fungi has a potential significance in not only sustainable crop production, but also environmental conservation. However, the status quo of inoculation is far from practical technology that can be widely used in the field. Together a further basic understanding of the biology and diversity of AM fungi is needed (Abbott at al, 1995; Saito and Marumoto, 2002). Advances in ecology during the past decade have led to a much more detailed understanding of the potential negative consequences of species introductions and the potential for negative ecological consequences of invasions by mycorrhizal fungi is poorly understood. Schwartz et al, (2006) recommend that a careful assessment documenting the need for inoculation, and the likelihood of success, should be conducted prior to inoculation because inoculations are not universally beneficial. Agricultural practices such as crop rotation, tillage, weed control and fertilizer apllication all produce changes in the chemical, physical and biological soil variables and affect the ecological niches available for occupancy by the soil biota, influencing in different ways the symbiosis performance and consequently the inoculum development, shaping changes and upset balance of native populations. The molecular biology tools developed in the latest years have been very important for our perception of these changes, ensuing awareness of management choice implications in AM development. In this context, for extensive farming systems and regarding environmental and economic costs, the identification of agronomic management practices that allow controlled manipulation of the fungal community and capitalization of AM mutualistic effect making use of local inoculum, seem to be a wise option for mycorrhiza promotion and development of sustainable crop production

Alu pair exclusions in the human genome

<p>Abstract</p> <p>Background</p> <p>The human genome contains approximately one million <it>Alu </it>elements which comprise more than 10% of human DNA by mass. <it>Alu </it>elements possess direction, and are distributed almost equally in positive and negative strand orientations throughout the genome. Previously, it has been shown that closely spaced <it>Alu </it>pairs in opposing orientation (inverted pairs) are found less frequently than <it>Alu </it>pairs having the same orientation (direct pairs). However, this imbalance has only been investigated for <it>Alu </it>pairs separated by 650 or fewer base pairs (bp) in a study conducted prior to the completion of the draft human genome sequence.</p> <p>Results</p> <p>We performed a comprehensive analysis of all (> 800,000) full-length <it>Alu </it>elements in the human genome. This large sample size permits detection of small differences in the ratio between inverted and direct <it>Alu </it>pairs (I:D). We have discovered a significant depression in the full-length <it>Alu </it>pair I:D ratio that extends to repeat pairs separated by ≤ 350,000 bp. Within this imbalance bubble (those <it>Alu </it>pairs separated by ≤ 350,000 bp), direct pairs outnumber inverted pairs. Using PCR, we experimentally verified several examples of inverted <it>Alu </it>pair exclusions that were caused by deletions.</p> <p>Conclusions</p> <p>Over 50 million full-length <it>Alu </it>pairs reside within the I:D imbalance bubble. Their collective impact may represent one source of <it>Alu </it>element-related human genomic instability that has not been previously characterized.</p

Modeling Activity and Target-Dependent Developmental Cell Death of Mouse Retinal Ganglion Cells Ex Vivo

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death

RGD-avidin–biotin pretargeting to αvβ3 integrin enhances the proapoptotic activity of TNFα related apoptosis inducing ligand (TRAIL)

Recombinant TNF-related apoptosis-inducing ligand (TRAIL) is considered a powerful and selective inducer of tumor cell death. We hypothesize that TRAIL’s potential as anticancer agent can be enhanced further by promoting its accumulation in tumor tissue. For this purpose, we developed TRAIL complexes that bind to angiogenic endothelial cells. We employed an avidin–biotin pretargeting approach, in which biotinylated TRAIL interacted with RGD-equipped avidin. The assembled complexes killed tumor cells (Jurkat T cells) via apoptosis induction. Furthermore, we demonstrated that the association of the RGD-avidin-TRAIL complex onto endothelial cells enhanced the tumor cell killing activity. Endothelial cells were not killed by TRAIL nor its derived complexes. Our approach can facilitate the enrichment of TRAIL onto angiogenic blood vessels, which may enhance intratumoral accumulation. Furthermore, it offers a versatile technology for the complexation of targeting ligands with therapeutic recombinant proteins and by this a novel way to enhance their specificity and activity

The Roles of Transmembrane Domain Helix-III during Rhodopsin Photoactivation

Background: Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11- cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear. Principal Findings: We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 49-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. Significance: Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.National Institutes of Health (U.S.) (grant GM28289)National Eye Institute (Grant Grant EY11716)National Science Foundation (U.S.) (grant EIA-0225609