Reprimo as a modulator of cell migration and invasion in the MDA-MB-231 breast cancer cell line

BACKGROUND: Reprimo (RPRM), a highly glycosylated protein, is a new downstream effector of p53-induced cell cycle arrest at the G2/M checkpoint, and a putative tumor suppressor gene frequently silenced via methylation of its promoter region in several malignances. The aim of this study was to characterize the epigenetic inactivation and its biological function in BC cell lines. METHODS: The correlation between RPRM methylation and loss of mRNA expression was assessed in six breast cancer cell lines by methylation specific PCR (MSP), 5'-Aza-2'-deoxycytidine treatment and RT-PCR assays. MDA-MB-231 cells were chosen to investigate the phenotypic effect of RPRM in cell proliferation, cell cycle, cell death, cell migration and invasion. RESULTS: In the cancer methylome system (CMS) (web-based system for visualizing and analyzing genome-wide methylation data of human cancers), the CpG island region of RPRM (1.1 kb) was hypermethylated in breast cancer compared to normal breast tissue; more interesting still was that ERa(+) tumors showed higher methylation intensity than ERa(-). Downregulation of RPRM mRNA by methylation was confirmed in MDA-MB-231 and BT-20 cell lines. In addition, overexpression of RPRM in MDA-MB-231 cells resulted in decreased rates of cell migration, wound healing and invasion in vitro. However, RPRM overexpression did not alter cell viability, phosphatidylserine (PS) translocation or G2/M cell cycle transition. CONCLUSION: Taken together, these data suggest that RPRM is involved in decreased cell migration and invasion in vitro, acting as a potential tumor suppressor gene in the MDA-MB-231 cell line.

Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

Cysteine-Rich Secretory Proteins (CRISP) and their role in mammalian fertilization

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.

Glicoproteína del virus rábico: Estructura, inmunogenicidad y rol en la patogenia / Rabies vims glycoprotein: Structure, immunogenicity and pathogenic role

Rabies glycoprotein is the only exposed protein which is inserted in the viral lipidie envelope. This 65-67 kda protein is a N-glycosilated transmembrane protein forming trimers on the viral surface. It has been identified as the major pathogenicity determinant, playing a role in the budding, viral axonal transport during infection, apoptosis and immune evasion. It is also the major antigen responsible for the protective immune response and it is been used in commercial recombinant vaccines. Its structure, antigenicity and pathogenic role have been well studied, identifying main antigenic sites that have the responsibility for virulence, cellular receptors attachment and epitope acquisition.

La glicoproteína del virus rábico es la única proteína viral expuesta, encontrándose inserta en la envoltura lipídica. Esta molécula de 65-67 kda corresponde a una proteína trans-membrana N-glicosilada que se dispone en forma de trímeros en la superficie viral. Ha sido identificada como el mayor determinante de pato-genicidad, participando además en procesos de yemación, flujo axonal del virion durante la infección, apoptosis y evasión de la respuesta inmune. Es también el principal antígeno inductor de la respuesta inmune protectora siendo utilizado en vacunas recom-binantes comerciales. Su estructura, antigenicidad e implicancias en la patogenia han sido bien estudiadas identificándose los principales sitios antigénicos responsables de la patogenicidad, unión a receptores celulares y formación de epitopos.

The Effect of Interleukin-4 and Amphiregulin on the Proliferation of Human Airway Smooth Muscle Cells and Cytokine Release

Airway smooth muscle (ASM) hyperplasia and angiogenesis are important features associated with airway remodeling. We investigated the effect of IL-4 and amphiregulin, an epidermal growth factor family member, on the proliferation of human ASM cells and on the release of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1 from human ASM cells. Human ASM cells were growth-arrested for 48 hr and incubated with platelet-derived growth factor (PDGF)- BB, interleukin (IL)-4, amphiregulin, and VEGF to evaluate cell proliferation. The cells were treated with PDGF, IL-4 and amphiregulin to evaluate the release of VEGF, MCP-1. IL-4 suppressed unstimulated and PDGF-stimulated ASM cell proliferation. Amphiregulin stimulated ASM cell proliferation in a dose-dependent manner. VEGF did not have any influence on ASM cell proliferation. IL-4 stimulated VEGF secretion by the ASM cells in a dose-dependent manner and showed added stimulatory effects when co-incubated with PDGF. Amphiregulin did not promote VEGF secretion. IL-4 and amphiregulin showed no stimulatory effects on MCP-1 secretion. The results of this study showed that IL-4 had bifunctional effects on airway remodeling, one was the suppression of the proliferation of the ASM cells and the other was the promotion of VEGF release by the ASM cells, and amphiregulin can promote human ASM cell proliferation.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Anti-Müllerian hormone in disorders of sex determination and differentiation

A masculinização dos genitais internos e externos durante o desenvolvimento fetal depende da existência de dois hormônios testiculares distintos: a testosterona secretada pelas células de Leydig conduz à diferenciação dos dutos de Wolff, do seio urogenital e dos genitais externos, enquanto que o hormônio anti-Mülleriano (HAM) produzido pelas células de Sertoli provoca a regressão dos dutos de Müller. A falta de ação do HAM no início da vida fetal resulta na formação de tubas uterinas, útero e terço superior da vagina. Em fetos 46,XY, a falta do HAM pode resultar de disgenesia testicular, afetando tanto as células de Leydig quanto as de Sertoli: nesse caso, a presença de remanescentes Müllerianos está associada a genitais externos femininos ou ambíguos. Por outro lado, distúrbios na ação do HAM podem resultar de mutações em genes que codificam o HAM ou seu receptor: nessa afecção, conhecida como síndrome da Persistência dos Dutos de Müller, a produção de testosterona é normal e os genitais externos são virilizados normalmente. Finalmente, o HAM costuma ser secretado normalmente em pacientes com intersexo decorrente de defeitos restritos à síntese ou à ação de andrógenos, resultando em indivíduos com genitais externos femininos ou ambíguos com ausência de derivados de Müller.

Fertilization, embryonic development and oviductal environment: role of estrogen induced oviductal glycoprotein.

Mammalian oviduct is the physiological site for sperm capacitation, gamete fertilization and early embryonic development. The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP) in response to estrogen. The protein has been shown to interact with gametes and early embryo. Several key functions have been postulated particularly its role in pre-implantation events which would have far reaching implications in assisted reproductive technology and in the development of non-hormonal contraceptive vaccine. The intention of this article is to discuss the current status of the protein and analyze how far the postulated function of OGP has been borne out by the available data.

Moléculas de adhesión y su importancia en odontología: revisión de la literatura / Adhesion molecules and its importance in dentistry

En la actualidad se reconoce que las interacciones que se suceden entre las células entre sí y de éstas con los componentes de la matriz extracelular se producen por la intervención de las Moléculas de Adhesión. Las Moléculas de adhesión son glucoproteínas distribuidas en gran cantidad de células que le permiten al organismo realizar funciones tanto fisiológicas, como la adhesión entre las células epiteliales, como fisiopatológicas, por ejemplo la inflamación. Es por ello que se hace imperativo conocer estas familias, para así entender los procesos que se desarrollan en las diversas actividades, normales o patológicas, de la cavidad bucal

Expression of a cell death marker (clusterin) in muscle target fibers

Descrevemos, pela primeira vez, a expressäo de imunorreatividade para clusterina em músculo esquelético. Clusterina, proteína relacionada ao processo de morte celular programada (apoptosis), encontrou-se especificamente muito aumentada em fibras musculares com "target" (FT). Todas as FT encontradas em 50 biópsias musculares de pacientes com diferentes doenças neuromusculares apresentaram alta concentraçäo de clusterina no centro das FT. Clusterina näo esteve semelhantemente aumentada em fibras com "targetóide" ou com "core". A alta expressäo de clusterina em "target" indica que FT podem ser o início de apoptosis e que há intima relaçäo entre FT e desnervaçäo aguda