Telomere Shortening Sensitizes Cancer Cells to Selected Cytotoxic Agents: In Vitro and In Vivo Studies and Putative Mechanisms

or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner. mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening.To our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer

Metabolic networking in Brunfelsia calycina petals after flower opening

Brunfelsia calycina flowers change colour from purple to white due to anthocyanin degradation, parallel to an increase in fragrance and petal size. Here it was tested whether the production of the fragrant benzenoids is dependent on induction of the shikimate pathway, or if they are formed from the anthocyanin degradation products. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using ultraperfomance liquid chromatography–quadrupole time of flight–tandem mass specrometry (UPLC-QTOF-MS/MS). Volatiles emitted were identified by headspace solid phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS). Accumulated proteins were identified by 2D gel electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. The results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition, 146 up-regulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation were identified. A multilevel analysis suggests induction of the shikimate pathway. This pathway is the most probable source of the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. The knowledge obtained is valuable for future studies on degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species

Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

BACKGROUND: While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved. METHODOLOGY/PRINCIPAL FINDINGS: We show here the impact of PAR(1) cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR(1) activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR(1) C-tail did not prevent Shc-PAR(1) association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR(1) C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR(1)-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR(1) -C-tail, hPar1 oncogenic properties are abrogated. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that a cytoplasmic portion of the PAR(1) C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR(1)-associating region in the breast cancer signaling niche

Prediction of anisotropy from flow models: A comparison of three methods

Observations of anisotropy in Earth are used regularly as constraints for models of deformation, using various assumptions about the relationship between deformation and the resulting anisotropic fabric. We compare three methods for calculating fabric from velocity fields: tracking of finite strain ellipses, a kinematic crystallographic code, and the evolution of directors. We find that the use of the finite strain ellipse provides only limited prediction capabilities, as it cannot reproduce experimental observations that involve recrystallization. The crystallographic code we tested (a variant of the popular code D-Rex) provides a more complete fabric prediction, but at a much higher computational cost. The directors method provides an intermediate solution: while it does not include some of the more complex crystallographic processes that D-Rex does, the results of this method closely resemble those of D-Rex, at a lower computational cost. The directors are also capable of tracking anisotropy at much larger strains than D-Rex. We conclude that when computation speed is important, for example, in self-consistent geodynamic models with anisotropic rheology, the directors method provides an appropriate approximation

The development of sensitization to the psychomotor stimulant effects of amphetamine is enhanced in a novel environment

Two experiments were designed to assess the effect of a “novel” environment on the development of sensitization to the psychomotor activating effects of d -amphetamine. In the first experiment, rats with a unilateral 6-hydroxydopamine lesion of the mesostriatal dopamine system received ten daily injections of amphetamine (2 mg/kg), either in their home cages or in novel test cages. The home and novel cages were physically identical (cylindrical transparent buckets), but one group lived and were tested in these cages, whereas the other group was transported from the stainless steel hanging cages where they lived to these novel test cages, for each test session. The first injection of amphetamine produced significantly more rotational behavior in animals tested in a novel environment than in animals tested at home. In addition, animals tested in a novel environment showed greater sensitization than animals tested at home, so the difference between the two groups was even more pronounced following the last injection. In a second experiment, locomotor activity was quantified in rats that received ten injections of either saline or 1.5 mg/kg amphetamine, in their home cages or in a physically identical novel environment. Again, there was a significantly greater locomotor response to the first injection of amphetamine, and greater sensitization, in animals tested in a novel environment than in animals tested at home. These data indicate that environmental factors can exert a large effect on the susceptibility to sensitization, and mechanisms by which this may occur are discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46345/1/213_2005_Article_BF02246217.pd

Volatile Ester Formation in Roses. Identification of an Acetyl-Coenzyme A. Geraniol/Citronellol Acetyltransferase in Developing Rose Petals

The aroma of roses (Rosa hybrida) is due to more than 400 volatile compounds including terpenes, esters, and phenolic derivatives. 2-Phenylethyl acetate, cis-3-hexenyl acetate, geranyl acetate, and citronellyl acetate were identified as the main volatile esters emitted by the flowers of the scented rose var. “Fragrant Cloud.” Cell-free extracts of petals acetylated several alcohols, utilizing acetyl-coenzyme A, to produce the corresponding acetate esters. Screening for genes similar to known plant alcohol acetyltransferases in a rose expressed sequence tag database yielded a cDNA (RhAAT1) encoding a protein with high similarity to several members of the BAHD family of acyltransferases. This cDNA was functionally expressed in Escherichia coli, and its gene product displayed acetyl-coenzyme A:geraniol acetyltransferase enzymatic activity in vitro. The RhAAT1 protein accepted other alcohols such as citronellol and 1-octanol as substrates, but 2-phenylethyl alcohol and cis-3-hexen-1-ol were poor substrates, suggesting that additional acetyltransferases are present in rose petals. The RhAAT1 protein is a polypeptide of 458 amino acids, with a calculated molecular mass of 51.8 kD, pI of 5.45, and is active as a monomer. The RhAAT1 gene was expressed exclusively in floral tissue with maximum transcript levels occurring at stage 4 of flower development, where scent emission is at its peak

Immunotherapy of cerebrovascular amyloidosis in a transgenic mouse model

Cerebrovascular amyloidosis is caused by amyloid accumulation in walls of blood vessel walls leading to hemorrhagic stroke and cognitive impairment. Transforming growth factor-β1 (TGF-β1) expression levels correlate with the degree of cerebrovascular amyloid deposition in Alzheimer's disease (AD) and TGF-β1 immunoreactivity in such cases is increased along the cerebral blood vessels. Here we show that a nasally administered proteosome-based adjuvant activates macrophages and decreases vascular amyloid in TGF-β1 mice. Animals were nasally treated with a proteosome-based adjuvant on a weekly basis for 3 months beginning at age 13 months. Using magnetic resonance imaging (MRI) we found that while control animals showed a significant cerebrovascular pathology, proteosome-based adjuvant prevents further brain damage and prevents pathological changes in the blood-brain barrier. Using an object recognition test and Y-maze, we found significant improvement in cognition in the treated group. Our findings support the potential use of a macrophage immunomodulator as a novel approach to reduce cerebrovascular amyloid, prevent microhemorrhage, and improve cognition