Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Natural Products from Cyanobacteria with Antimicrobial and Antitumor Activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

INSEL: an in silico method for optimizing and exploring biorecognition assays

A practical in silico method for optimizing and exploring biointeraction-based events is developed.

Binding of antioxidant flavone isovitexin to human serum albumin investigated by experimental and computational assays

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Redox-tagged peptide for capacitive diagnostic assays

Early detection assays play a key role in the successful treatment of most diseases. Redox capacitive biosensors were recently introduced as a potential electroanalytical assay platform for point-of-care applications but alternative surfaces (besides a mixed layer containing ferrocene and antibody receptive component) for recruiting important clinical biomarkers are still needed. Aiming to develop alternative receptive surfaces for this novel electrochemical biosensing platform, we synthesized a ferrocene redoxtagged peptide capable of self-assembly into metallic interfaces, a potentially useful biological surface functionalization for bedside diagnostic assays. As a proof of concept we used C-reactive protein (CRP), as a model biomarker, and compared the obtained results to those of previously reported capacitive assays. The redox-tagged peptide approach shows a limit of detection of 0.8 nmol L 1 (same as 94 ng mL 1 ) and a linear range (R2 ∼98%) with the logarithm of the concentration of the analyte comprising 0.5–10.0 nmol L 1 , within a clinical relevant range for CRP.

Using the comet and micronucleus assays for genotoxicity studies: a review

Physical, chemical and biological agents can act in the DNA, resulting in mutation involved in cancer. Thus, genotoxic tests are required by regulatory agencies in order to evaluate potential risk of cancer. Among these tests, the comet assay (CA) and micronucleus assay (MNA) are the most commonly used. However, there are different protocols and recommendations already published. This is the first review, after the inclusion of CA in S2R1 guidance and OECD 489, which summarizes the main technical recommendations of both CA and MNA.

Brazilian green propolis induced apoptosis in human lung cancer A549 cells through mitochondrial-mediated pathway

Propolis effect on the growth and apoptosis of human lung adenocarcinoma (A549 cells) was investigated as well as its mechanisms. Cells were incubated with propolis for 72 h, and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were employed to assess cell viability and the inhibitory concentration (IC). Apoptosis was detected by Acridine Orange/Ethidium Bromide and 4',6-diamidino-2-phenylindole staining after 24 and 48 h of incubation with ¼ IC50 of propolis by testing the mitochondrial membrane potential (ΔΨm) and the expression of apoptosis-related genes (p53, Caspase-3, Bax, Bcl-2, Bcl-XL , Noxa, Puma and p21) by reverse transcription polymerase chain reaction. Propolis displayed antiproliferative and cytotoxic effects on A549 cells in a dose- and time-dependent manner, but it did not suppress the growth of normal Vero cells. An enhanced apoptosis was seen in A549 propolis-treated cells after 48 h compared with the control cells. Propolis decreased mitochondrial membrane potential by overexpression of pro-apoptotic genes (Bax and Noxa) and reduction of the antiapoptotic gene Bcl-XL . The expression level of other genes remained unchanged (p53, Caspse-3 and Bax), whereas p21 expression was increased. Propolis induced caspase-independent apoptosis through a p53-independent mitochondrial pathway, and cell cycle arrest by upregulation of p21. Although propolis induces apoptosis mainly by p53-independent manner, it may be induced by another pathway, and new insights may arise for preventing or treating lung cancer.

Antiproliferative activity of cardenolide glycosides from Asclepias subulata

Asclepias subulata Decne. is a shrub occurring in Sonora-Arizona desert (Mexico-USA). The ethnic groups, Seris and Pimas, use this plant for the treatment of sore eyes, gastrointestinal disorders and cancer. To isolate the compounds responsible for antiproliferative activity of the methanol extract of A. subulata. A bioguided fractionation of methanol extract of A. subulata was performed using MTT assay to measure the antiproliferative activity of different compounds on three human cancer cell lines (A549, LS 180 and PC-3), one murine cancer cell line (RAW 264.7) and one human normal cell line (ARPE-19). The methanol extract was partitioned with hexane, ethyl acetate and ethanol. The active fractions, ethanol and residual, were fractioned by silica-column chromatography and active sub-fractions were separated using HPLC. The chemical structures of isolated compounds were elucidated with different chemical and spectroscopic methods. A new cardenolide glycoside, 12, 16-dihydroxycalotropin, and three known, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, were isolated of active sub-fractions. All isolated compounds showed a strong antiproliferative activity in human cancer cells. Calotropin was the more active with IC50 values of 0.0013, 0.06 and 0.41 µM on A549, LS 180 and PC-3 cell lines, respectively; while 12, 16-dihydroxycalotropin reached values of 2.48, 5.62 and 11.70 µM, on the same cells; corotoxigenin 3-O-glucopyranoside had IC50 of 2.64, 3.15 and 6.62 µM and desglucouzarin showed values of 0.90, 6.57 and 6.62, µM. Doxorubicin, positive control, showed IC50 values of 1.78, 6.99 and 3.18 µM, respectively. The isolated compounds had a weak effect on murine cancer cells and human normal cells, exhibiting selectivity to human cancer cells. In this study, we found that 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin are responsible of antiproliferative properties of A. subulata, and that these compounds are highly selective to human cancer cells. Further studies are needed in order to establish the action mechanisms of the isolated compounds.

Bisphenol-A promotes antiproliferative effects during neonatal prostate development in male and female gerbils

Conselho Nacional de Desenvolvimento Científico e Tecnol[ogico (CNPq)

Screening da atividade antiproliferativa e investigação toxicológica in vitro e in vivo de compostos de coordenação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of Blood Assays for Detection of Mycobacterium bovis in White-Tailed Deer (Odocoileus virginianus) in Michigan

Surveillance and control activities related to bovine tuberculosis (TB) in free-ranging, Michigan white-tailed deer (Odocoileus virginianus) have been underway for over a decade, with significant progress. However, foci of higher TB prevalence on private lands and limited agency ability to eliminate them using broad control strategies have led to development and trial of new control strategies, such as live trapping, testing, and culling or release. Such strategies require a prompt, accurate live animal test, which has thus far been lacking. We report here the ability of seven candidate blood assays to determine the TB infection status of Michigan deer. Our aims were twofold: to characterize the accuracy of the tests using field-collected samples and to evaluate the feasibility of the tests for use in a test-and-cull strategy. Samples were collected from 760 deer obtained via five different surveys conducted between 2004 and 2007. Blood samples were subjected to one or more of the candidate blood assays and evaluated against the results of mycobacterial culture of the cranial lymph nodes. Sensitivities of the tests ranged from 46% to 68%, whereas specificities and negative predictive values were all .92%. Positive predictive values were highly variable. An exploratory analysis of associations among several host and sampling-related factors and the agreement between blood assay and culture results suggested these assays were minimally affected. This study demonstrated the capabilities and limitations of several available blood tests for Mycobacterium bovis on specimens obtained through a variety of field surveillance methods. Although these blood assays cannot replace mass culling, information on their performance may prove useful as wildlife disease managers develop innovative methods of detecting infected animals where mass culling is publicly unacceptable and cannot be used as a control strategy.

Antiproliferative Effects of Fluoxetine on Colon Cancer Cells and in a Colonic Carcinogen Mouse Model

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

Antimicrobial Activity of Essential Oils against Streptococcus mutans and their Antiproliferative Effects

This study aimed to evaluate the activity of essential oils (EOs) against Streptococcus mutans biofilm by chemically characterizing their fractions responsible for biological and antiproliferative activity. Twenty EO were obtained by hydrodistillation and submitted to the antimicrobial assay (minimum inhibitory (MIC) and bactericidal (MBC) concentrations) against S. mutans UA159. Thin-layer chromatography and gas chromatography/mass spectrometry were used for phytochemical analyses. EOs were selected according to predetermined criteria and fractionated using dry column; the resulting fractions were assessed by MIC and MBC, selected as active fractions, and evaluated against S. mutans biofilm. Biofilms formed were examined using scanning electron microscopy. Selected EOs and their selected active fractions were evaluated for their antiproliferative activity against keratinocytes and seven human tumor cell lines. MIC and MBC values obtained for EO and their active fractions showed strong antimicrobial activity. Chemical analyses mainly showed the presence of terpenes. The selected active fractions inhibited S. mutans biofilm formation (P < 0.05) did not affect glycolytic pH drop and were inactive against keratinocytes, normal cell line. In conclusion, EO showed activity at low concentrations, and their selected active fractions were also effective against biofilm formed by S. mutans and human tumor cell lines.