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Belo Horizonte, Brazil

Urbanavicius J.,Laboratory of Cell Biology | Scorza C.,Institute Investigaciones Biologicas Clemente Estable
Behavioural Pharmacology

Melanin-concentrating hormone (MCH) administered within the rat dorsal raphe nucleus (DRN) has been shown to elicit prodepressive behaviors in the forced-swim test. The present study was designed to evaluate the time course (30 and 60min) and dose dependence (25-100ng) of this effect, and whether it would be antagonized by an intra-DRN microinjection of the MCH-1 receptor antagonist ATC0175 (ATC, 1mmol/l) or intraperitoneal pretreatment with the noradrenergic antidepressant nortriptyline (20mg/kg). The results showed that the behavioral effect of MCH was time and dose dependent as immobility was increased, and climbing decreased, only by the 50ng MCH dose at T30. The effect was mediated by MCH-1 receptors as a significant blockade of this behavioral response was observed in ATC-pretreated animals. ATC did not by itself modify animal behavior. Nortriptyline also prevented the prodepressive-like effect of MCH. Concomitantly, the effect of MCH (50ng) at T30 on anxiety-related behaviors was assessed using the elevated plus-maze. Interestingly, these behaviors were unchanged. In conclusion, MCH administration within the DRN elicits, through the MCH-1 receptor, a depression-related behavior that is not accompanied by changes in anxiety and that is prevented by a noradrenergic antidepressant. © 2014 Wolters kluwer Health. Source

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Researchers at the University of Massachusetts Medical School have designed a nanoparticle that mimics the bacterium Salmonella and may help to counteract a major mechanism of chemotherapy resistance. Working with mouse models of colon and breast cancer, Beth McCormick, Ph.D., and her colleagues demonstrated that when combined with chemotherapy, the nanoparticle reduced tumor growth substantially more than chemotherapy alone. The results of their research were published in Nature Communications. A membrane protein called P-glycoprotein (P-gp) acts like a garbage chute that pumps waste, foreign particles, and toxins out of cells. P-gp is a member of a large family of transporters, called ATP-binding cassette (ABC) transporters, that are active in normal cells but also have roles in cancer and other diseases. For instance, cancer cells can co-opt P-gp to rid themselves of chemotherapeutic agents, severely limiting the efficacy of these drugs. In previous work, McCormick and her colleagues serendipitously discovered that Salmonella enterica, a bacterium that causes food poisoning, decreases the amount of P-gp on the surface of intestinal cells. Because Salmonella has the capacity to grow selectively in cancer cells, the researchers wondered whether there was a way to use the bacterium to counteract chemotherapy resistance caused by P-gp. “While trying to understand how Salmonella invades the human host, we made this other observation that may be relevant to cancer therapeutics and multidrug resistance,” explains McCormick. To determine the specific bacterial component responsible for reducing P-gp levels, the researchers engineered multiple Salmonella mutant strains and tested their effect on P-gp levels in colon cells. They found that a Salmonella strain lacking the bacterial protein SipA was unable to reduce P-gp levels in the colon of mice or in a human colon cancer cell line. Salmonella secretes SipA, along with other proteins, to help the bacterium invade human cells. The researchers then showed that treatment with SipA protein alone decreased P-gp levels in cell lines of human colon cancer, breast cancer, bladder cancer, and lymphoma. Because P-gp can pump drugs out of cells, the researchers next sought to determine whether SipA treatment would prevent cancer cells from expelling chemotherapy drugs. When they treated human colon cancer cells with the chemotherapy agents doxorubicin or vinblastine, with or without SipA, they found that the addition of SipA increased drug retention inside the cells. SipA also increased the cancer cells’ sensitivity to both drugs, suggesting that it could possibly be used to enhance chemotherapy. “Through millions of years of co-evolution, Salmonella has figured out a way to remove this transporter from the surface of intestinal cells to facilitate host infection,” says McCormick. “We capitalized on the organism’s ability to perform that function.” It would not be feasible to infect people with the bacterium, and SipA on its own will likely deteriorate quickly in the bloodstream, coauthor Gang Han, Ph.D., of the University of Massachusetts Medical School, explained in a press release. The researchers therefore fused SipA to gold nanoparticles, generating what they refer to as a nanoparticle mimic of Salmonella. They designed the nanoparticle to enhance the stability of SipA, while retaining its ability to interact with other proteins. In an effort to target tumors without harming healthy tissues, the researchers used a nanoparticle of specific size that should only be able to access the tumor tissue due to its “leaky” architecture. “Because of this property, we are hoping to be able to avoid negative effects to healthy tissues,” says McCormick. Another benefit of this technology is that the nanoparticle can be modified to enhance tumor targeting and minimize the potential for side effects, she added. The researchers showed that this nanoparticle was 100 times more effective than SipA protein alone at reducing P-gp levels in a human colon cancer cell line. The enhanced function of the nanoparticle is likely due to stabilization of SipA, explained the researchers. The team then tested the nanoparticle in a mouse model of colon cancer, because this cancer type is known to express high levels of P-gp. When they treated tumor-bearing mice with the nanoparticle plus doxorubicin, P-gp levels dropped and the tumors grew substantially less than in mice treated with the nanoparticle or doxorubicin alone. The researchers observed similar results in a mouse model of human breast cancer. There are concerns about the potential effect of nanoparticles on normal tissues. “P-gp has evolved as a defense mechanism” to rid healthy cells of toxic molecules, says Suresh Ambudkar, Ph.D., deputy chief of the Laboratory of Cell Biology in NCI’s Center for Cancer Research. It plays an important role in protecting cells of the blood-brain barrier, liver, testes, and kidney. “So when you try to interfere with that, you may create problems,” he said. The researchers, however, found no evidence of nanoparticle accumulation in the brain, heart, kidney, or lungs of mice, nor did it appear to cause toxicity. They did observe that the nanoparticles accumulated in the liver and spleen, though this was expected because these organs filter the blood, says McCormick. The research team is moving forward with preclinical studies of the SipA nanoparticle to test its safety and toxicity, and to establish appropriate dosage levels. However, Ambudkar notes, “the development of drug resistance in cancer cells is a multifactorial process. In addition to the ABC transporters, other phenomena are involved, such as drug metabolism.” And because there is a large family of ABC transporters, one transporter can compensate if another is blocked, he explained. For the last 25 years, clinical trials with drugs that inhibit P-gp have failed to overcome chemotherapy resistance, Ambudkar says. Tackling the issue of multidrug resistance in cancer, he continues, “is not something that can be solved easily.” McCormick and her team are also pursuing research to better characterize and understand the biology of SipA. “We are not naïve about the complexity of the problem," she says. "However, if we know more about the biology, we believe we can ultimately make a better drug.”

Vasilyev S.A.,Cancer Research Institute | Vasilyev S.A.,Institute of Medical Genetics | Kubes M.,Laboratory of Cell Biology | Markova E.,Cancer Research Institute | And 2 more authors.
International Journal of Radiation Biology

Purpose: Human hematopoietic stem cells (HSC) are thought to be a major target of radiation-induced leukemogenesis and also provide a relevant cellular model for assessing cancer risk. Cluster of designation 133+ (CD133+) is a marker found in human progenitor and hematopoietic stem cells. Our study examined the repair of radiation-induced DNA double-strand breaks (DSB) in CD133 + umbilical cord blood cells (UCBC). Materials and methods: After γ-irradiation, endogenous and induced DSB were evaluated in CD133 + UCBC, CD133 - UCBC and peripheral blood lymphocytes (PBL) in terms of phosphorylated histone 2A family member X (γH2AX) and tumor suppressor p53 binding protein 1 (53BP1) foci. Results: We found that repair signaling in CD133 + UCBC is different from CD133 - UCBC and PBL. These differences include lower endogenous DSB levels and higher 53BP1 recruitment. Conclusions: Our data, together with a recent report on radiation-induced γH2AX and 53BP1 foci in CD34 + cells, indicate enhanced DNA repair capacity in HSC as compared to mature lymphocytes. © 2013 Informa UK, Ltd. Source

Martina J.A.,U.S. National Institutes of Health | Martina J.A.,Laboratory of Cell Biology | Chen Y.,U.S. National Institutes of Health | Gucek M.,U.S. National Institutes of Health | And 2 more authors.

The mammalian target of rapamycin (MTOR) protein kinase complex is a key component of a pathway that regulates cell growth and proliferation in response to energy levels, hypoxia, nutrients and insulin. Inhibition of MTORC1 strongly induces autophagy by regulating the activity of the ULK protein kinase complex that is required for the formation of autophagosomes. However, the participation of MTORC1 in the expression of autophagy genes has not been characterized. Here we show that MTORC1 regulates nuclear localization and activity of the transcription factor EB (TFEB), a member of the bHLH leucine-zipper family of transcription factors that drives expression of autophagy and lysosomal genes. Under normal nutrient conditions, TFEB is phosphorylated in Ser211 in an MTORC1-dependent manner. This phosphorylation promotes association of TFEB with members of the YWHA (14-3-3) family of proteins and retention of the transcription factor in the cytosol. Pharmacological or genetic inhibition of MTORC1 causes dissociation of the TFEB/ YWHA complex and rapid transport of TFEB to the nucleus where it increases transcription of multiple genes implicated in autophagy and lysosomal function. Active TFEB also associates with late endosomal/lysosomal membranes through interaction with the LAMTOR/RRAG/MTORC1 complex. Our results unveil a novel role for MTORC1 in the maintenance of cellular homeostasis by regulating autophagy at the transcriptional level. © 2012 Landes Bioscience. Source

Hoelz A.,California Institute of Technology | Debler E.W.,Laboratory of Cell Biology | Blobel G.,Laboratory of Cell Biology | Blobel G.,Howard Hughes Medical Institute
Annual Review of Biochemistry

In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies. © 2011 by Annual Reviews. All rights reserved. Source

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