Angiotensin-(1-7) can promote cell migration and tumor growth of clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common kidney malignancy, accounting for 3% of all cancers. Despite significant advances in targeted therapies and immunotherapy, many patients with RCC develop resistance to available drugs. Angiotensin-(1-7) (Ang-(1-7)) is a heptapeptide and a member of the renin-angiotensin system which regulates the cardiovascular and the renal system. It has been proposed as a potential anticancer agent for the treatment of various types of cancers, but data regarding its efficiency against RCC are conflicting. The aim of our study was to evaluate the effects of Ang-(1-7) in RCC models in vitro and in vivo. We performed a series of in vitro experiments investigating the effects of Ang-(1-7) on cell viability and migration in Caki-1 and Caki-2 cell lines. In addition, we carried out an in vivo study in xenografts of Caki-1 cells in nude mice. In results: Ang-(1-7) or A779, an antagonist of its receptor MasR (Mas receptor), showed no effect on cell viability. Ang-(1-7) promoted cell migration in a dose-dependent manner by inducing the activation of MasR. It also promoted tumor growth in vivo, and this effect was not inhibited by the blockade of MasR. No effects on cell proliferation or tumor vessel density were observed. The results suggest that Ang-(1-7) can exert protumorigenic activity in RCC, however, further research on other RCC models is needed to better recapitulate the heterogeneity of the disease.

Detection of circulating tumor cells in blood by shell-isolated nanoparticle - enhanced Raman spectroscopy (SHINERS) in microfluidic device.

Isolation and detection of circulating tumor cells (CTCs) from human blood plays an important role in non- invasive screening of cancer evolution and in predictive therapeutic treatment. Here, we present the novel tool utilizing: (i) the microfluidic device with (ii) incorporated photovoltaic (PV) based SERS-active platform, and (iii) shell-isolated nanoparticles (SHINs) for simultaneous separation and label-free analysis of circulating tumour cells CTCs in the blood specimens with high specificity and sensitivity. The proposed microfluidic chip enables the efficient size - based inertial separation of circulating cancer cells from the whole blood samples. The SERS-active platform incorporated into the microfluidic device permits the label-free detection and identification of isolated cells through the insight into their molecular and biochemical structure. Additionally, the silver nanoparticles coated with an ultrathin shell of silica (Ag@SiO2) was used to improve the detection accuracy and sensitivity of analysed tumor cells via taking advantages of shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS). The empirical analysis of SHINERS spectra revealed that there are some differences among studied (HeLa), renal cell carcinoma (Caki-1), and blood cells. Unique SHINERS features and differences in bands intensities between healthy and cancer cells might be associated with the variations in the quantity and quality of molecules such as lipid, protein, and DNA or their structure during the metastasis cancer formation. To demonstrate the statistical efficiency of the developed method and improve the differentiation for circulating tumors cells detection the principal component analysis (PCA) has been performed for all SHINERS data. PCA method has been applied to recognize the most significant differences in SHINERS data among the three analyzed cells: Caki-1, HeLa, and blood cells. The proposed approach challenges the current multi-steps CTCs detection methods in the terms of simplicity, sensitivity, invasiveness, destructivity, time and cost of analysis, and also prevents the defragmentation/damage of tumor cells and thus leads to improving the accuracy of analysis. The results of this research work show the potential of developed SERS based tool for the separation of tumor cells from whole blood samples in a simple and minimally invasive manner, their detection and molecular characterization using one single technology.

Photovoltaic cells as a highly efficient system for biomedical and electrochemical surface-enhanced Raman spectroscopy analysis.

Surface-enhanced Raman scattering (SERS) has been intensively used recently as a highly sensitive, non-destructive, chemical specific, and label-free technique for a variety of studies. Here, we present a novel SERS substrate for: (i) the standard ultra-trace analysis, (ii) detection of whole microorganisms, and (iii) spectroelectrochemical measurements. The integration of electrochemistry and SERS spectroscopy is a powerful approach for in situ investigation of the structural changes of adsorbed molecules, their redox properties, and for studying the intermediates of the reactions. We have developed a conductive SERS platform based on photovoltaic materials (PV) covered with a thin layer of silver, especially useful in electrochemical SERS analysis. These substrates named Ag/PV presented in this study combine crucial spectroscopic features such as high sensitivity, reproducibility, specificity, and chemical/physical stability. The designed substrates permit the label-free identification and differentiation of cancer cells (renal carcinoma) and pathogens (Escherichia coli and Bacillus subtilis). In addition, the developed SERS platform was adopted as the working electrode in an electrochemical SERS approach for p-aminothiophenol (p-ATP) studies. The capability to monitor in real-time the electrochemical changes spectro-electro-chemically has great potential for broadening the application of SERS.

Global analysis of gene expression in maize leaves treated with low temperature. II. Combined effect of severe cold (8 °C) and circadian rhythm.

KEY MESSAGE: In maize seedlings, severe cold results in dysregulation of circadian pattern of gene expression causing profound modulation of transcription of genes related to photosynthesis and other key biological processes. Plants live highly cyclic life and their response to environmental stresses must allow for underlying biological rhythms. To study the interplay of a stress and a rhythmic cue we investigated transcriptomic response of maize seedlings to low temperature in the context of diurnal gene expression. Severe cold stress had pronounced effect on the circadian rhythm of a substantial proportion of genes. Their response was strikingly dual, comprising either flattening (partial or complete) of the diel amplitude or delay of expression maximum/minimum by several hours. Genes encoding central oscillator components behaved in the same dual manner, unlike their Arabidopsis counterparts reported earlier to cease cycling altogether upon cold treatment. Also numerous genes lacking circadian rhythm responded to the cold by undergoing up- or down-regulation. Notably, the transcriptome changes preceded major physiological manifestations of cold stress. In silico analysis of metabolic processes likely affected by observed gene expression changes indicated major down-regulation of photosynthesis, profound and multifarious modulation of plant hormone levels, and of chromatin structure, transcription, and translation. A role of trehalose and stachyose in cold stress signaling was also suggested. Meta-analysis of published transcriptomic data allowed discrimination between general stress response of maize and that unique to severe cold. Several cis- and trans-factors likely involved in the latter were predicted, albeit none of them seemed to have a major role. These results underscore a key role of modulation of diel gene expression in maize response to severe cold and the unique character of the cold-response of the maize circadian clock.

SURVEY AND SUMMARY: exon-intron organization of genes in the slime mold Physarum polycephalum.

The slime mold Physarum polycephalum is a morphologically simple organism with a large and complex genome. The exon-intron organization of its genes exhibits features typical for protists and fungi as well as those characteristic for the evolutionarily more advanced species. This indicates that both the taxonomic position as well as the size of the genome shape the exon-intron organization of an organism. The average gene has 3.7 introns which are on average 138 bp, with a rather narrow size distribution. Introns are enriched in AT base pairs by 13% relative to exons. The consensus sequences at exon-intron boundaries resemble those found for other species, with minor differences between short and long introns. A unique feature of P.polycephalum introns is the strong preference for pyrimidines in the coding strand throughout their length, without a particular enrichment at the 3'-ends.

Ras protein of the slime mold Physarum polycephalum is farnesylated in vitro.

Physarum Ppras1 protein was efficiently prenylated by prenyltransferases of spinach. Surprisingly in spite of the C-terminal sequence (CLLL) specific for geranylgeranylation the protein was preferentially farnesylated. Consequences of this observation are discussed.

PPRAS1PPRAS2 AND PPRAP1 GENES, MEMBERS OF A RAS GENE FAMILY FROM THE TRUE SLIME MOLD PHYSARUM POLYCEPHALUM ARE DEVELOPMENTALLY REGULATED

The expression patterns of two true ras genes, Ppras1 and Ppras2and one rap gene, Pprap1were examined in four Physarum polycephalum developmental stages: uninucleate amoebae, plasmodia (multinucleate syncytia), spherules (a vegetative, dormant stage) and fruiting bodies. Ppras1 and Pprap1 are expressed in all stages examined with the maximum levels of their transcripts in amoebae and fruiting bodies, respectively, and the minimum levels in plasmodia, whereas the Ppras2 transcript is only detectable in amoebae and fruiting bodies. The results obtained indicate that P. polycephalum is an organism possessing a developmentally regulated ras gene family and presents a convenient system to study the role of ras/rap genes in control of growth and differentiation of lower eukaryotic organisms.Copyright 1997 Academic Press Limited Copyright 1997Academic Press Limited

Identification and sequence analysis of a rap gene from the true slime mold Physarum polycephalum.

A member of the ras gene superfamily, belonging to the rap family and designated Pprap1, was isolated from a cDNA library from the true slime mold Physarum polycephalum by plaque hybridization in combination with 5'-RACE. The assembled nucleotide sequence of Pprap1 (1062 bp) has an open reading frame coding for a protein of 188 amino acids of a calculated M(r) of 21035. This protein exhibits: (i) a highly conserved GTP binding domain containing a putative effector domain, with the threonine-for-glutamine substitution characteristic of rap proteins, (ii) a hypervariable domain, and (iii) the CAAX motif. Analysis of the C-terminal amino acid sequence of Pprap1 shows that it presumably undergoes geranylgeranylation but is not palmitoylated; however, it contains a lysine-rich domain which might serve as the second membrane localization signal. Pprap1 exhibits significantly high amino acid homology within the GTP binding domain with its homologues: Ddrap1 from Dictyostelium discoideum (92%) and human Rap1A (83%), and relatively low homology (59%) with the Saccharomyces cerevisiae homologue, RSR1. It has also 59% and 61% homology with the P. polycephalum Ppras1 and Ppras2 proteins, respectively. This gene is the third member of the ras gene superfamily identified in P. polycephalum so far.

Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene, a homologue of the ras protooncogene.

We have cloned the genomic copy of the Ppras1 gene, a homologue of the ras proto-oncogene, from the true slime mold Physarum polycephalum. Ppras1 contains five small introns, four of which have a high content of pyrimidines. The (dC)-homopolymers present in introns 4 and 5 may be responsible for the observed recA-independent deletion in Ppras1 upon amplification of the Ppras1-bearing plasmid by choramphenicol. Although Ppras1 exhibits amino acid and nucleotide homologies with the DdrasG gene, a homologue of ras from another slime mold, Distyostelium discoideum, locations and sequences of their introns are quite different. This discordance suggests that introns of the ras genes in these species were acquired independently.