Differential expression of HIF-1 alpha in CD44(+)CD24(-/) (low) breast ductal carcinomas

Background: Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1 alpha expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44(+)CD24(-/low) immunophenotype. Methods: Immunohistochemical analysis of CD44(+)CD24(-/low) expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). Results: In univariate analysis, CD44(+)CD24(-/low) expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44(+)CD24(-/low) immunophenotype showed relationship with HIF-1 alpha (p = 0.039) and negativity for HER-2 (p = 0.013). Conclusion: Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44(+)CD24(-/low) positive breast tumors.

In Vivo Effects on the Expression of Vascular Endothelial Growth Factor-A(165) Messenger Ribonucleic Acid of an Infrared Diode Laser Associated or Not with a Visible Red Diode Laser

Objective: This study investigated and correlated the kinetic expression of vascular endothelial growth factor (VEGF)-A(165) messenger ribonucleic acid (mRNA) with the associated use or not of an infrared laser and a visible red laser during the wound healing in rats. Background Data: There is a lack of scientific evidence demonstrating the influence of low-level laser therapy (LLLT) on the expression of VEGF mRNA in vivo. Materials and Methods: Forty-five Wistar rats were randomly allocated to one of three groups: I (n = 5, nonoperated animals), II (n = 25, operated animals), and III (n = 25, animals operated and subjected to laser irradiation). A surgical wound was performed using a scalpel in the right side of the tongue of operated animals. In group III, two sessions of laser irradiation were performed, one right after the surgical procedure (infrared laser, 780 nm, 70mW, 35 J/cm(2)) and the other 48 h later (visible red laser, 660 nm, 40mW, 5J/cm(2)). Five animals each were sacrificed 1, 3, 5, and 7 days postoperatively in groups II and III, and samples of tongue tissue were obtained. The animals of group I were sacrificed on day 7. Total RNA was extracted using guanidine-isothiocyanate-phenol-chloroform method. The results of horizontal electrophoresis after reverse transcription polymerase chain reaction permitted the ratio of VEGF-A(165) mRNA and glyceraldehyde 3-phosphate dehydrogenase mRNA expression for groups I, II, and III to be assessed (two-way analysis of variance and Tukey test, p<0.05). Results: The expression of VEGF-A(165) mRNA in group II (0.770 +/- 0.098) was statistically greater than that observed in groups I (0.523 +/- 0.164) and III (0.504 +/- 0.069) in the first day after surgery (p<0.05). Significant differences between the groups were not observed in other time periods. Conclusion: LLLT influenced the expression of VEGF-A(165) mRNA during wound healing after a surgical procedure on the tongue of Wistar rats.

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background: Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. Methods: Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. Results: In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. Conclusion: These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Genes up- and down-regulated by dermcidin in breast cancer: a microarray analysis

Dermcidin (DCD) is a human gene mapped to chromosome 12q13 region, which is co-amplified with multiple oncogenes with a well-established role in the growth, survival and progression of breast cancers. Here, we present a summary of a DNA microarray-based study that identified the genes that are up- and down-regulated in a human MDA-361 pLKO control clone and three clones expressing short hairpin RNA against three different regions of DCD mRNA. A list of 235 genes was differentially expressed among independent clones (> 3-fold change and P < 0.005). The gene expression of 208 was reduced and of 27 was increased in the three DCD-RNAi clones compared to pLKO control clone. The expression of 77 genes (37%) encoding for enzymes involved in amino acid metabolism, glucose metabolism and oxidoreductase activity and several genes required for cell survival and DNA repair were decreased. The expression of EGFR/ErbB-1 gene, an important predictor of outcome in breast cancer, was reduced together with the genes for betacellulin and amphiregulin, two known ligands of EGFR/ErbB receptors. Many of the 27 genes up-regulated by DCD-RNAi expression have not yet been fully characterized; among those with known function, we identified the calcium-calmodulin-dependent protein kinase-II delta and calcineurin A alpha. We compared 132 up-regulated and 12 down-regulated genes in our dataset with those genes up- and down-regulated by inhibitors targeting various signaling pathway components. The analysis showed that the genes in the DCD pathway are aligned with those functionally influenced by the drugs sirolimus, LY-294002 and wortmannin. Therefore, DCD may exert its function by activating the PI3K/AKT/mTOR signaling pathway. Together, these bioinformatic approaches suggest the involvement of DCD in the regulation of genes for breast cancer cell metabolism, proliferation and survival.

Comparative Analysis of Activation Phenotype, Proliferation, and IFN-gamma Production by Spleen NK1.1(+) and NK1.1(-) T Cells During Plasmodium chabaudi AS Malaria

The NK1.1 molecule participates in NK, NKT, and T-cell activation, contributing to IFN-gamma production and cytotoxicity. To characterize the early immune response to Plasmodium chabaudi AS, spleen NK1.1(+) and NK1.1(-) T cells were compared in acutely infected C57BL/6 mice. The first parasitemia peak in C57BL/6 mice correlated with increase in CD4(+)NK1.1(+)TCR-alpha beta(+), CD8(+)NK1.1(+)TCR-alpha beta(+), and CD4(+)NK1.1(-)TCR-alpha beta(+) cell numbers per spleen, where a higher increment was observed for NK1.1(+) T cells compared to NK1.1(-) T cells. According to the ability to recognize the CD1d-alpha-GalCer tetramer, CD4(+)NK1.1(+) cells in 7-day infected mice were not predominantly invariant NKT cells. At that time, nearly all NK1.1(+) T cells and around 30% of NK1.1(-) T cells showed an experienced/activated (CD44(HI)CD69(HI)CD122(HI)) cell phenotype, with high expression of Fas and PD-L1 correlating with their low proliferative capacity. Moreover, whereas IFN-gamma production by CD4(+)NK1.1(+) cells peaked at day 4 p.i., the IFN-gamma response of CD4(+)NK1.1(-) cells continued to increase at day 5 of infection. We also observed, at day 7 p.i., 2-fold higher percentages of perforin(+) cells in CD8(+)NK1.1(+) cells compared to CD8(+)NK1.1(-) cells. These results indicate that spleen NK1.1(+) and NK1.1(-) T cells respond to acute P. chabaudi malaria with different kinetics in terms of activation, proliferation, and IFN-gamma production.

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 angstrom, alpha = 63.45, beta = 80.66, gamma = 94.55 degrees. xfGST crystals diffracted to 2.23 angstrom resolution on a rotating-anode X-ray source.

Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Background: Schistosoma mansoni is the major causative agent of schistosomiasis. The parasite takes advantage of host signals to complete its development in the human body. Tumor necrosis factor-alpha (TNF-alpha) is a human cytokine involved in skin inflammatory responses, and although its effect on the adult parasite's metabolism and egg-laying process has been previously described, a comprehensive assessment of the TNF-alpha pathway and its downstream molecular effects is lacking. Methodology/Principal Findings: In the present work we describe a possible TNF-alpha receptor (TNFR) homolog gene in S. mansoni (SmTNFR). SmTNFR encodes a complete receptor sequence composed of 599 amino acids, and contains four cysteine-rich domains as described for TNFR members. Real-time RT-PCR experiments revealed that SmTNFR highest expression level is in cercariae, 3.5 (+/- 0.7) times higher than in adult worms. Downstream members of the known human TNF-alpha pathway were identified by an in silico analysis, revealing a possible TNF-alpha signaling pathway in the parasite. In order to simulate parasite's exposure to human cytokine during penetration of the skin, schistosomula were exposed to human TNF-alpha just 3 h after cercariae-to-schistosomula in vitro transformation, and large-scale gene expression measurements were performed with microarrays. A total of 548 genes with significantly altered expression were detected, when compared to control parasites. In addition, treatment of adult worms with TNF-alpha caused a significantly altered expression of 1857 genes. Interestingly, the set of genes altered in adults is different from that of schistosomula, with 58 genes in common, representing 3% of altered genes in adults and 11% in 3 h-old early schistosomula. Conclusions/Significance: We describe the possible molecular elements and targets involved in human TNF-alpha effect on S. mansoni, highlighting the mechanism by which recently transformed schistosomula may sense and respond to this host mediator at the site of cercarial penetration into the skin.

Crystallization and preliminary X-ray diffraction analysis of human IL-22 bound to its soluble decoy receptor IL-22BP

Interleukin-22 (IL-22) is a pleiotropic cytokine that is involved in inflammatory responses. Human IL-22 was incubated with its soluble decoy receptor IL-22BP (IL-22 binding protein) and the IL-22 -IL-22BP complex was crystallized in hanging drops using the vapour-diffusion method. Suitable crystals were obtained from polyethylene glycol solutions and diffraction data were collected to 2.75 angstrom resolution. The crystal belonged to the tetragonal space group P41, with unit-cell parameters a = b = 67.9, c = 172.5 angstrom, and contained two IL-22-IL- 22BP complexes per asymmetric unit.

Regional scale analysis of landform configuration with base-level (isobase) maps

Base-level maps (or ""isobase maps"", as originally defined by Filosofov, 1960), express a relationship between valley order and topography. The base-level map can be seen as a ""simplified"" version of the original topographic surface, from which the ""noise"" of the low-order stream erosion was removed. This method is able to identify areas with possible tectonic influence even within lithologically uniform domains. Base-level maps have been recently applied in semi-detail scale (e.g., 1:50 000 or larger) morphotectonic analysis. In this paper, we present an evaluation of the method's applicability in regional-scale analysis (e.g., 1:250 000 or smaller). A test area was selected in northern Brazil, at the lower course of the Araguaia and Tocantins rivers. The drainage network extracted from SRTM30_PLUS DEMs with spatial resolution of approximately 900 m was visually compared with available topographic maps and considered to be compatible with a 1:1,000 000 scale. Regarding the interpretation of regional-scale morphostructures, the map constructed with 2nd and 3rd-order valleys was considered to present the best results. Some of the interpreted base-level anomalies correspond to important shear zones and geological contacts present in the 1:5 000 000 Geological Map of South America. Others have no correspondence with mapped Precambrian structures and are considered to represent younger, probably neotectonic, features. A strong E-W orientation of the base-level lines over the inflexion of the Araguaia and Tocantins rivers, suggest a major drainage capture. A N-S topographic swath profile over the Tocantins and Araguaia rivers reveals a topographic pattern which, allied with seismic data showing a roughly N-S direction of extension in the area, lead us to interpret this lineament as an E-W, southward-dipping normal fault. There is also a good visual correspondence between the base-level lineaments and geophysical anomalies. A NW-SE lineament in the southeast of the study area partially corresponds to the northern border of the Mosquito lava field, of Jurassic age, and a NW-SE lineament traced in the northeastern sector of the study area can be interpreted as the Picos-Santa Ines lineament, identifiable in geophysical maps but with little expression in hypsometric or topographic maps.

Functional microarray analysis suggests repressed cell-cell signaling and cell survival-related modules inhibit progression of head and neck squamous cell carcinoma

Background: Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease. Methods: Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results: The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions: The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the Delta hrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the Delta hrcU mutant complemented with HrcU(AAAH), suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the Delta hrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.

Real-time PCR investigation on the expression of sboA and ituD genes in Bacillus spp

Aims: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. Methods and Results: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT-PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42 degrees C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. Conclusions: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. Significance and Impact of the Study: Monitoring the expression of essential biosynthetic genes by qRT-PCR is a valuable tool for optimization of the production of antimicrobial peptides.

Analysis of Single-Nucleotide Polymorphisms in the crt-o and mdr1 Genes of Plasmodium vivax among Chloroquine-Resistant Isolates from the Brazilian Amazon Region

Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the Brazilian Amazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.

LOW-INTENSITY PULSED ULTRASOUND PRODUCED AN INCREASE OF OSTEOGENIC GENES EXPRESSION DURING THE PROCESS OF BONE HEALING IN RATS

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery. (E-mail: a.renno@unifesp.br) (C) 2010 Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology.

Low-Intensity Electrical Stimulation Counteracts the Effects of Ovariectomy on Bone Tissue of Rats: Effects on Bone Microarchitecture, Viability of Osteocytes, and Nitric Oxide Expression

Low Intensity Electrical Stimulation (LIES) has been used for bone repair, but little is known about its effects on bone after menopause. Osteocytes probably play a role in mediating this physical stimulus and they could act as transducers through the release of biochemical signals, such as nitric oxide (NO). The aim of the present study was to investigate the effects of LIES on bone structure and remodeling, NOS expression and osteocyte viability in ovariectomized (OVX) rats. Thirty rats (200-220 g) were divided into 3 groups: SHAM, OVX, and OVX subjected to LIES (OVX + LIES) for 12 weeks. Following the protocol, rats were sacrificed and tibias were collected for histomorphometric analysis and immunohistochemical detection of endothelial NO synthase (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). OVX rats showed significant (p < 0.05 vs. SHAM) decreased bone volume (10% vs. 25%) and trabecular number (1.7 vs. 3.9), and increased eroded surfaces (4.7% vs. 3.2%) and mineralization surfaces (15.9% vs. 7.7%). In contrast, after LIES, all these parameters were significantly different from OVX but not different from SHAM. eNOS and iNOS were similarly expressed in subperiosteal regions of tibiae cortices of SHAM, not expressed in OVX, and similarly expressed in OVX + LIES when compared to SHAM. In OVX, the percentage of apoptotic osteocytes (24%) was significantly increased when compared to SHAM (11%) and OVX + LIES (8%). Our results suggest that LIES counteracts some effects of OVX on bone tissue preserving bone structure and microarchitecture, iNOS and eNOS expression, and osteocyte viability.