Novel gallium(III) complexes transported by MDR1 P-glycoprotein: potential PET imaging agents for probing P-glycoprotein-mediated transport activity in vivo

AbstractBackground: Multidrug resistance (MDR) mediated by expression of MDR1 P-glycoprotein (Pgp) represents one of the best characterized barriers to chemotherapy in cancer patients. Positron emission tomography (PET) agents for analysis of Pgp-mediated drug transport activity in vivo would enable noninvasive assessment of chemotherapeutic regimens and MDR gene therapy.Results: Candidate Schiff-base phenolic gallium(III) complexes were synthesized from their heptadentate precursors and gallium(III)acetylacetonate. Crystal structures demonstrated a hexacoordinated central gallium with overall trans-pseudo-octahedral geometry. Radiolabeled 67Ga-complexes were obtained in high purity and screened in drug-sensitive (Pgp−) and MDR (Pgp+) tumor cells. Compared with control, lead compound 6 demonstrated antagonist-reversible 55-fold lower accumulation in Pgp-expressing MDR cells. Furthermore, compared with wild-type control, quantitative pharmacokinetic analysis showed markedly increased penetration and retention of 6 in brain and liver tissues of mdr1a/b(−/−) gene disrupted mice, correctly mapping Pgp-mediated transport activity at the capillary blood–brain barrier and hepatocellular biliary cannalicular surface in vivo.Conclusions: These results indicate that gallium(III) complex 6 is recognized by MDR1 Pgp as an avid transport substrate, thereby providing a useful scaffold to generate 68Ga radiopharmaceuticals for molecular imaging of Pgp transport activity in tumors and tissues in vivo using PET

Anticancer Gene Transfer for Cancer Gene Therapy

Gene therapy vectors are among the treatments currently used to treat malignant tumors. Gene therapy vectors use a specific therapeutic transgene that causes death in cancer cells. In early attempts at gene therapy, therapeutic transgenes were driven by non-specific vectors which induced toxicity to normal cells in addition to the cancer cells. Recently, novel cancer specific viral vectors have been developed that target cancer cells leaving normal cells unharmed. Here we review such cancer specific gene therapy systems currently used in the treatment of cancer and discuss the major challenges and future directions in this field

Jovens e associações em Moçambique: motivações e dinâmicas actuais

O presente artigo é uma reflexão sobre dinâmicas associativas de jovens no Moçambique pós colonial. O objectivo desta reflexão foi identificar e analisar as motivações de engajamento dos jovens nesses agrupamentos. Baseando-se em aproximações empíricas feitas a duas associações de jovens - Associação Aro Juvenil e Associação Positiva Juvenil - a análise demonstra que dinamica associativa de jovens mete em evidência relações complexas entre identidade, contexto, o privado, o público e o afectivo. Embora haja múltiplas motivações, a adesão dos jovens em associações associa trajectórias e expectativas individualizadas. A nível discursivo, a entrada na vida associativa representa uma forma de legitimação sóciopolítica em resposta a um discurso que considera os jovens passivos e pouco intervenientes na solução dos problemas que lhes afecta em particular e à sociedade no geral. A nível das práticas associativas quotidianas, os jovens reintrepretam e dão outro sentido às motivações do seu engajamento: para lá dos objectivos formais, pretensamente desenvolvimentistas, altruistas e humanitários, o associativismo é uma estratégia de vida e de realização de projectos individuais. Criar uma associação e/ou nela aderir pode significar maiores possibilidades de aceder e controlar recursos e capitais diversificados como emprego/profissão, dinheiro, trabalho, poder, reconhecimento e prestigio, formações entre outros que de outra forma não seria possível.This article reflects on youth associations dynamics in Postcolonial Mozambique. The aim is to identify and analyze motivations for young people's involvement in such groups. Based on empirical work with two youth associations - namely "Associação Aro Juvenil" and "Associação Positiva Juvenil" - the assessment finds that youth associations dynamics highlights intricate relationships involving identity, context, private, public and affective milieus. Although there are multiple motivations, young people's adherence to associations is combined with individual life stories and expectations. At the discourse level, entrance to the associative life represents a form of socio-political legitimation in response to other narratives that consider young people very passive and less intervening in finding solutions to their own problems, and society's in general. At the level of day-to-day practices, young people re-interpret and give a different sense to their motivations and commitment: beyond formal objectives - arguably development-oriented, altruistic and humanistic - associations are a life strategy for the accomplishment of individual achievements. To create an association and/or take part in one may imply greater possibilities of accessing and controlling diversified resources and capitals, such as a job/occupation, money, work, power, recognition, prestige, and training, among others, which would be otherwise impossible

Okazaki Fragment Processing-independent Role for Human Dna2 Enzyme during DNA Replication

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G2 phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation

Contributions Made by CDC25 Phosphatases to Proliferation of Intestinal Epithelial Stem and Progenitor Cells

The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells

Heart and systemic effects of statin pretreatment in a rat model of abdominal sepsis. Assessment by Tc99m-sestamibi biodistribition

PURPOSE: To evaluate the heart and the Tc-99m-sestamibi biodistribution after statin pretreatment in a rat model of abdominal sepsis. METHODS: Twenty-four Wistar rats were randomly distributed into four groups (n=6 per group): 1) sepsis with simvastatin treatment, 2) sepsis with vehicle, 3) sham control with simvastatin and 4) sham control with vehicle. 24 hours after cecal ligation and puncture rats received 1.0MBq of Tc-99m-sestamibi i.v. 30min after, animals were euthanized for ex-vivo tissue counting and myocardium histological analysis. RESULTS: Myocardial histologic alterations were not detected 24 hours post-sepsis. There was significantly increased cardiac Tc-99m-sestamibi activity in the sepsis group with simvastatin treatment (1.9$\pm$0.3%ID/g, p<0.001) in comparison to the sepsis group+vehicle (1.0$\pm$0.2% ID/g), control sham group+ simvastatin (1.2$\pm$0.3% ID/g) and control sham group (1.3$\pm$0.2% ID/g). Significant Tc-99m-sestamibi activity in liver, kidney and lungs was also detected in the sepsis group treated with simvastatinin comparison to the other groups. CONCLUSIONS: Statin treatment altered the biodistribution of Tc-99m-sestamibi with increased cardiac and solid organ activity in rats with abdominal sepsis, while no impact on controls. Increased myocardial tracer activity may be a result of a possible protection effect due to increased tissue perfusion mediated by statins

Biology-driven cancer drug development: back to the future

Most of the significant recent advances in cancer treatment have been based on the great strides that have been made in our understanding of the underlying biology of the disease. Nevertheless, the exploitation of biological insight in the oncology clinic has been haphazard and we believe that this needs to be enhanced and optimized if patients are to receive maximum benefit. Here, we discuss how research has driven cancer drug development in the past and describe how recent advances in biology, technology, our conceptual understanding of cell networks and removal of some roadblocks may facilitate therapeutic advances in the (hopefully) near future

A Freeze Frame View of Vesicular Stomatitis Virus Transcription Defines a Minimal Length of RNA for 5′ Processing

The RNA synthesis machinery of vesicular stomatitis virus (VSV) comprises the genomic RNA encapsidated by the viral nucleocapsid protein (N) and associated with the RNA dependent RNA polymerase, the viral components of which are a large protein (L) and an accessory phosphoprotein (P). The 241 kDa L protein contains all the enzymatic activities necessary for synthesis of the viral mRNAs, including capping, cap methylation and polyadenylation. Those RNA processing reactions are intimately coordinated with nucleotide polymerization such that failure to cap results in termination of transcription and failure to methylate can result in hyper polyadenylation. The mRNA processing reactions thus serve as a critical check point in viral RNA synthesis which may control the synthesis of incorrectly modified RNAs. Here, we report the length at which viral transcripts first gain access to the capping machinery during synthesis. By reconstitution of transcription in vitro with highly purified recombinant polymerase and engineered templates in which we omitted sites for incorporation of UTP, we found that transcripts that were 30-nucleotides in length were uncapped, whereas those that were 31-nucleotides in length contained a cap structure. The minimal RNA length required for mRNA cap addition was also sufficient for methylation since the 31-nucleotide long transcripts were methylated at both ribose-2′-O and guanine-N-7 positions. This work provides insights into the spatial relationship between the active sites for the RNA dependent RNA polymerase and polyribonucleotidyltransferase responsible for capping of the viral RNA. We combine the present findings with our recently described electron microscopic structure of the VSV polymerase and propose a model of how the spatial arrangement of the capping activities of L may influence nucleotide polymerization

The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas

<p>Abstract</p> <p>Background</p> <p>14-3-3 sigma (σ) promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown.</p> <p>Methods</p> <p>We investigated the 14-3-3σ expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome.</p> <p>Results</p> <p>In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3σ protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3σ in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (<it>p </it>= 0.001, <it>p </it>= 0.002 and <it>p </it>= 0.001, respectively) and deep invasion (<it>p </it>= 0.01, <it>p </it>= 0.001 and <it>p </it>= 0.007, respectively). Variations of 14-3-3σ protein expression were not associated to disease-specific survival.</p> <p>Conclusion</p> <p>Our results indicate that 14-3-3σ may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3σ protein. Neither cytoplasmic nor nuclear level of 14-3-3σ expression was associated with prognosis.</p