PGV-1 is a Potent Antimitotic Agent

Carcinogenesis may resulted from the malfunctioning of programmed cell death. Most of the anticancer drugs incurrent use induce apoptosis in susceptible cells. The fact that disparate agent interacting with different targets seemto induce cell death through some common mechanisms suggest that anticancer activity is determined by the abilityof inhibiting cell growth. Pentagamavunon-1 (PGV-1) is one of the curcumin analogues which showed to havepotency in inhibiting proliferation of T47D human breast carcinoma cells. The effects on T47D cells growth isassociated with cell cycle arrest in G2/M phase at the concentration of 2.5 ?M, followed by hyperploidy. The data onpolymerization assay, indicated that PGV-1 interact with tubulin in different manner from taxol. PGV-1 inhibittubulin polymerization on cell culture while taxol stabilized tubulin polymerization. Immunostainning data onPGV-1 treated cells showed slightly tubulin condensation, while taxol treated cells showed tubulin condensationdistinctly at 12 minutes after releasing from depolymerizing agent.In conclusion, PGV-1 represent a new microtubule inhibitor and has the potential to be developed for antimitoticdrugKey words: Pentagamavunon-1, T47D, tubuli

Analysis of Htra Gene from Zebrafish (Danio Rerio)

HtrA which is characterized by the combination of a trypsin-like catalytic domain with at least one C-terminal PDZ domain is a highly conserved family of serine proteases found in a wide range of organisms. However the identified HtrA family numbers varies among spesies, for example the number of mammalian, Eschericia coli, fruit fly-HtrA family are 4, 3 and 1 gene respectively. One gene is predicted exist in zebrafish. Since no complete information available on zebrafish HtrA, in this paper zebrafish HtrA (zHtrA) gene was analyzed. The zHtrA is belonged to HtrA1 member and predicted encodes 478 amino acids with a signal peptide, a IGF binding domain, a Kazal-type inhibitor domain in the up stream of HtrA-bacterial homolog. At the amino acid sequence the zHtrA1 showed the 69%, 69%, 68%, 54% and 54% with the rat HtrA1, mouse HtrA1, human HtrA1, human HtrA3 and mouse HtrA4 respectively. The zHtrA1 is firstly expressed at 60 hpf and mainly in the vertebral rudiments in the tail region

Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells

An international collaborative project has been proposed to inactivate all mouse genes in embryonic stem (ES) cells using a combination of random and targeted insertional mutagenesis techniques. Random gene trapping will be the first choice in the initial phase, and gene-targeting experiments will then be carried out to individually knockout the remaining ‘difficult-to-trap’ genes. One of the most favored techniques of random insertional mutagenesis is promoter trapping, which only disrupts actively transcribed genes. Polyadenylation (poly-A) trapping, on the other hand, can capture a broader spectrum of genes including those not expressed in the target cells, but we noticed that it inevitably selects for the vector integration into the last introns of the trapped genes. Here, we present evidence that this remarkable skewing is caused by the degradation of a selectable-marker mRNA used for poly-A trapping via an mRNA-surveillance mechanism, nonsense-mediated mRNA decay (NMD). We also report the development of a novel poly-A-trap strategy, UPATrap, which suppresses NMD of the selectable-marker mRNA and permits the trapping of transcriptionally silent genes without a bias in the vector-integration site. We believe the UPATrap technology enables a simple and straightforward approach to the unbiased inactivation of all mouse genes in ES cells

PGV-1 is a potent antimitotic agent

Carcinogenesis can be involved in the malfunctioning of programmed cell death Most of the anticancer drug in current use induce apoptosis in susceptible cells. The fact that disparate agent interacting with different targets seem to induce cell death through some common mechanism suggest that anticancer activity is determined by the ability of inhibiting cell growth.Pentagamavunon-1 (PGV-1) is one of the curcumin analogue which showed to have potency in inhibiting proliferation of human breast carcinoma cell T47D. The effect on T47D growth is associated with cell cycle arrest in G2/M phase at the concentration of 2.5 mM, followed by hyperploidy. Our data on polymerization assay, indicate PGV-1 interact with tubulin in different manner from taxol. PGV-1 inhibit tubulin polymerization on cell culture while taxol stabilized tubulin polymerization. Immunostainning data on cell treated with PGV-1 showed slightly tubulin condensation, while cell treated with taxol showed tubulin condensation distinctly at 12 minutes after releasing from depolymerization agent.In conclusion, PGV-1 represent a new microtubule inhibitor and has the potential to be developed for anticancer drugKey words: Pentagamavunon-1, T47D, tubulin, antimitoti

Effect of innoculated spores of Clostridium estertheticum and Clostridium gasigenes on vaccum packaged meat and ability of Clostridium estertheticum to form biofilm

Orientador: Arnaldo Yoshiteru KuayeDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Casos de estufamento de carne bovina embalada à vácuo, causado por Clostridium estertheticum e Clostridium gasigenes, mantida sob temperatura de refrigeração vêm sendo observados em diversas regiões do Brasil, principalmente no Centro-Oeste, onde foi detectada a presença destes microrganismos em ambientes de abatedouros-frigoríficos. A complexidade analítica da técnica utilizada em pesquisas com esses microrganismos faz com que sejam escassos os trabalhos que visam o controle desse tipo de deterioração. Diante do exposto e considerando-se a importância da exportação de carne para a economia brasileira, o presente estudo teve como objetivo avaliar o efeito da inoculação de esporos de Clostridium estertheticum e Clostridium gasigenes em carne bovina embalada a vácuo e a eficiência esporicida in vitro do uso de ácidos orgânicos sobre estes microrganismos. Além disso, avaliou-se a capacidade de formação de biofilmes por C. estertheticum em superfície de aço inoxidável, e a eficiência de agentes químicos para sua remoção. Observou-se em carnes embaladas a vácuo, que C. gasigenes produzem maiores quantidades de gases, promovendo o estufamento da embalagem mais rápido em relação ao C. estertheticum. O primeiro indício da formação de bolhas por C. gasigenes foi observado aos 21 dias de incubação à temperatura de 7°C de armazenamento, enquanto que para C. estertheticum observou-se somente aos 35 dias na mesma temperatura. Notou-se uma produção de gás mais intensa à temperatura de armazenamento mais alta. Na carne inoculada com C. gasigenes houve um aumento progressivo no valor do pH, atingindo valores maiores em temperaturas mais elevadas, por volta de 49-77 dias, seguido por uma queda. Por sua vez, a inoculação com C. estertheticum não promoveu a mesma variação de pH, notando-se uma acentuada queda até 21-35 dias, seguido por um progressivo aumento. A utilização, de ácidos orgânicos mostrou reduzido ou nenhum efeito sobre esporos, de C. gasigenes e C. estertheticum, não sendo viável sua aplicação em cortes de carnes bovinas com a finalidade de inibir ou minimizar o blown pack. Observou-se que tanto a cepa de C. estertheticum padrão DSM 8809T quanto a cepa LHCE-13 isolada de equipamento (rolete de retirada do couro) foram capazes de formar biofilmes após 07 dias de contato com a superfície de aço inoxidável e os sanitizantes, ácido peracético (500mg/L) e peróxido de hidrogênio (200 mg/L) em contato por 15 minutos, mostraram-se eficientes no controle destes. São necessários programas de higienização mais rigorosos e efetivos para o controle de C. estertheticum e C. gasigenes nos abatedouros-frigoríficos, pois eles podem estar presentes no ambiente em forma de esporos e também como biofilmes, em locais com grande acúmulo de material orgânico ou em associações com outros microrganismosAbstract: Occurrence of blown pack in vacuum packaged refrigerated meat caused by Clostridium estertheticum and Clostridium gasigenes have been reported by studies from several Brazilian states where these microorganisms were detected in abattoir environment such as the Midwestern region. Analytical complexity of the researches with C. estertheticum e C. gasigenes makes the studies to prevent or to control this spoilage scarce. For these reasons and it is considering the importance of Brazil¿s beef export to economy, the current study aim to assess the effect of inoculated spores of C. estertheticum and C. gasigenes on vaccum packaged meat and the efficiency in vitro of organic acid on reported microorganisms spores. Furthermore this study tested, for the first time, the ability of C. estertheticum to form biofilm in stainless steel surface and the use of some sanitizers to remove it. Results obtained from behavior analysis of C. estertheticum and C. gasigenes spores inoculated in vacuum packaged meat, showed us differences in growth between these microorganisms. C. gasigenes produced more amount of gas, leading the blown pack faster than C. estertheticum. The first evidence of gas bubble production by C. gasigenes was observed after 21 storage days at 7°C, whereas C. estertheticum started to produce bubbles only after 35 days at the same storage conditions. Gas production was intense in higher storage temperatures. Through the pH measurement it was possible verify that C. gasigenes increases progressively the pH, reaching maximum values after 49-77 days at higher storage temperatures, followed by decreasing. Moreover C. estertheticum do not cause variation in the pH, but it may be seen an accentuated decrease until 21-35 days, followed by an increase of the values, probably because of C. estertheticum ability to revert a falling pH through fast lactate fermentation, started when availability of glucose ends. Regarding to use of organic acids, there is reduced or none effect on C. gasigenes and C. estertheticum spores, it becomes this technique no viable to reduce or control blown pack in vacuum packaged meat. For the first time is demonstrate that C. estertheticum forms biofilm. Strains of C. estertheticum DSM 8809T and C. estertheticum LHCE-13 isolated from abattoir equipment (leather removal drums) are able to colonize surfaces of stainless steel in 07 days. The sanitizers peracetic acid (500mg/L) and hydrogen peroxide (200 mg/L) exposure in 5-25 day-old biofilms at least 15 minutes were effective to control them. Severe and effective hygienization program is needed to control C. estertheticum and C. gasigenes in abattoir environment, mainly because they may be present on equipment surfaces in spore forms and also through biofilm formation in places with high organic material presenceMestradoEngenharia de AlimentosMestre em Tecnologia de Alimento

APOPTOSIS INDUCTION EFFECT OF CURCUMIN AND ITS ANALOGS PENTAGAMAVUNON-0 AND PENTAGAMAVUNON-1 ON CANCER CELL LINES

ABSTRACTObjectives: This experiment aims to investigate the apoptosis effect of curcumin and its analogs pentagamavunon-0 (PGV-0) and PGV-1 on normaland other cancer cell lines.Methods: Growth inhibition effect was investigated using the MTT method. Double staining used acridine orange, 2-(4-aminodiphenyl)-6-indolcarbamidine dihydrochloride and ethidium bromide was performed to determine morphological changes of cells. Detection of PARP, caspase-3,PUMA and BAX using a western blot method was conducted to elucidate the apoptosis effect of the compounds.Results: PGV-1 (2.5 μM) and PGV-0 (5.0 μM) could inhibit T47D-cell growth on 72 h observation, but not for curcumin. DNA staining showed PGV-1has the strongest apoptosis induction effect on T47D-cells compared to PGV-0 and curcumin as well. Western blot analysis resulted in cleavage PARP(83 kD) on HeLa, T47D, and MCF-7 cells treated with PGV-1 (2.5 μM), PGV-0 (5.0 μM). Curcumin (10.0 μM) just induced apoptosis on T47D-cell andMCF-7 cell, but not HeLa cell. Cleavage PARP resulted by apoptosis process in the cell. PGV-1 (2.5 μM) had a stronger apoptosis effect compared toPGV-0 (5.0 μM) and curcumin (10.0 μM) based on cleaved PARP result qualitatively. On the normal cell (NH3T3), cells that were treated with thecompounds resulted in a negative cleavage PARP. This result indicated that the compounds were part of a selectively induced cancer cell line apoptosisprocess.Conclusion: Curcumin, PGV-0 and PGV-1 could inhibit cell growth by induce apoptosis on cancer cells but not on normal cells, which PGV-1 hasstrongest apoptosis induction effect on cancer cell lines.Keywords: Curcumin and analogs, Apoptosis, Cancer cell lines

Abnormal development of placenta in HtrA1-deficient mice

AbstractAbnormal levels of High temperature requirement A1 (HtrA1) protein have been repeatedly observed in sera and placentas of preeclampsia patients. To understand the functions of HtrA1 in placentation and in the etiology of preeclampsia, we established HtrA1−/− mice. HtrA1−/− mice show intrauterine growth retardation, and their placentas are small due to a reduced size of the junctional zone and aberrant vascularization in the labyrinth at the mid-gestation stage. HtrA1 is expressed by Tpbpa-positive trophoblast precursors in the outer ectoplacental cone and junctional zone from embryonic day 7.5 to 10.5. In the HtrA1−/− placenta, Tpbpa-positive cell precursors are decreased in the early stage. Spongiotrophoblasts and glycogen trophoblast cells, both of which differentiate from Tpbpa-positive precursors, are consequently decreased in the junctional zone. Fewer spiral artery-associated trophoblast giant cells, another cell type derived from Tpbpa-positive precursors, invade the decidua and associate with maternal arteries in the HtrA1−/− placenta than in the wild type placenta. Maternal arteries in the HtrA1−/− decidua have narrower lumens, thicker arterial walls, and more vascular smooth muscle cells remaining in the walls than those in the wild type decidua, indicating impaired remodeling of maternal arteries. These results indicate that HtrA1 plays important roles in the differentiation of trophoblasts from Tpbpa-positive precursors in the ectoplacental cone. Insufficient levels of HtrA1 cause poor placental development and intrauterine growth retardation, due to aberrant trophoblast differentiation and consequent defects in maternal artery remodeling, and may contribute to the onset of preeclampsia

T47D cells arrested at G2M and Hyperploidy Formation Induced by a Curcumin’s Analogue PGV-1

its chemical structure than curcumin. As a curcumin analogue, PGV-1 was considered to have anticanceractivities. This research was conducted to study the effect of PGV-1 on the cycle progression of T47D cells. Cytotoxiceffects of PGV-1 on T47D cells were determined using MTT assay, and the the effect on cell cycle progressionwas carried out using flowcytometry. Western blot analysis was used to analyze protein expression correspondingto cell cycle progression. The result showed that at the concentration of 2.5 μM PGV-1 inhibited cell cycleprogression through G2/M arrest and induced of cells hyperploidy formation. The hyperploidy formation inducedby PGV-1 was related to the increase of cdc-2 expression. PGV-1 2.5 μM elevated the level of p21 CIP/KIPthrough p53- independent manner. Apoptosis was also induced by PGV-1 at early phase of treatment indicated byPARP cleavage due to activation of caspase-3/7 after 12 h treatment. The results above suggest that PGV-1 inhibitsthe growth of T47D cells targeted on microtubules.Keywords: PGV-1, G2/M arrest, apoptosis, p2

THE ETHYL ACETATE FRACTION OF Gynura procumbens SENSITIZES WiDr COLON CANCER CELL LINE AGAINST 5-FLUOROURACIL BUT SHOWS ANTAGONISM WITH CISPLATIN

Our recent study has evaluated fraction of ethyl acetate of Gynura procumbens (FEG) as co-chemotherapeutic agent in combination with 5-fluorouracil (5-FU) and cisplatin (CISP) against colon cancer cell line, WiDr cells. This study aims to assess whether FEG show synergism with 5-FU and CISP and to evaluate its regulation on proliferation, cell cycle, and apoptosis on WiDr colon cancer cells. (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay was performed to determine the growth inhibitory effect of both single (FEG, 5-FU, or CISP) and combination treatments. FEG (25-500 μg/mL),  5-FU (25-1000 μM)  and CISP (5-100 μM) inhibited cells growth in a dose dependent manner and exhibited IC50 value of 125  μg/mL, 848 mM and 43 mM, respectively. FEG sensitizes WiDr cells that was treated by 5-FU, boosting its therapeutic potential. Conversely when FEG was combined with CISP, it caused antagonism.  The antiproliferative effect of single and combination treatment was determined by studying the cell proliferation kinetics under MTT assay. Flowcytometry and (4’,6-diamidino-2-phenylindole) DAPI staining was used to disclose the mechanism of cell cycle arrest and apoptosis. FEG inhibited cell proliferation, induced G1 and S phase arrest and apoptosis. The inhibitory effect was enhanced when FEG was combined with 5-FU, differing from CISP. According to the datas obtained, FEG appeared to possess sensitizing properties, and caused cell cycle arrest and apoptosis on WiDr cells. FEG demonstrated a possibility of additive to synergism properties when combined with 5-FU but not with CISP.   Keywords: Gynura procumbens; WiDr; G1 and S phase arrest; apoptosis