Different phospholipase-C-coupled receptors differentially regulate capacitative and non-capacitative Ca2+ entry in A7r5 cells.

Several receptors, including those for AVP (Arg8-vasopressin) and 5-HT (5-hydroxytryptamine), share an ability to stimulate PLC (phospholipase C) and so production of IP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol) in A7r5 vascular smooth muscle cells. Our previous analysis of the effects of AVP on Ca2+ entry [Moneer, Dyer and Taylor (2003) Biochem. J. 370, 439-448] showed that arachidonic acid released from DAG stimulated NO synthase. NO then stimulated an NCCE (non-capacitative Ca2+ entry) pathway, and, via cGMP and protein kinase G, it inhibited CCE (capacitative Ca2+ entry). This reciprocal regulation ensured that, in the presence of AVP, all Ca2+ entry occurred via NCCE to be followed by a transient activation of CCE only when AVP was removed [Moneer and Taylor (2002) Biochem. J. 362, 13-21]. We confirm that, in the presence of AVP, all Ca2+ entry occurs via NCCE, but 5-HT, despite activating PLC and evoking release of Ca2+ from intracellular stores, stimulates Ca2+ entry only via CCE. We conclude that two PLC-coupled receptors differentially regulate CCE and NCCE. We also address evidence that, in some A7r5 cells lines, AVP fails either to stimulate NCCE or inhibit CCE [Brueggemann, Markun, Barakat, Chen and Byron (2005) Biochem. J. 388, 237-244]. Quantitative PCR analysis suggests that these cells predominantly express TRPC1 (transient receptor potential canonical 1), whereas cells in which AVP reciprocally regulates CCE and NCCE express a greater variety of TRPC subtypes (TRPC1=6>2>3).

Alpha CP-4, encoded by a putative tumor suppressor gene at 3p21, but not its alternative splice variant alpha CP-4a, is underexpressed in lung cancer.

alpha CP-4 is an RNA-binding protein coded by PCBP4, a gene mapped to 3p21, a common deleted region in lung cancer. In this study we characterized the expression of alpha CP-4 and alpha CP-4a, an alternatively spliced variant of alpha CP-4, in lung cancer cell lines and non-small cell lung cancer (NSCLC) samples from early stage lung cancer patients. In NSCLC biopsies, an immunocytochemical analysis showed cytoplasmic expression of alpha CP-4 and alpha CP-4a in normal lung bronchiolar epithelium. In contrast, alpha CP-4 immunoreactivity was not found in 47% adenocarcinomas and 83% squamous cell carcinomas, whereas all of the tumors expressed alpha CP-4a. Besides, lack of alpha CP-4 expression was associated with high proliferation of the tumor (determined by Ki67 expression). By fluorescence in situ hybridization, >30% of NSCLC cell lines and tumors showed allelic losses at PCBP4, correlating with the absence of the protein. On the other hand, no mutations in the coding region of the gene were found in any of the 24 cell lines analyzed. By Northern blotting and real-time reverse transcription-PCR, we detected the expression of alpha CP-4 and alpha CP-4a messages in NSCLC and small cell lung cancer cell lines. Our data demonstrate an abnormal expression of alpha CP-4 in lung cancer, possibly associated with an altered processing of the alpha CP-4 mRNA leading to a predominant expression of alpha CP-4a. This may be considered as an example of alternative splicing involved in tumor suppressor gene inactivation. Finally, induction of alpha CP-4 expression reduced cell growth, in agreement with its proposed role as a tumor suppressor, and suggesting an association of this RNA-binding protein with lung carcinogenesis.

Altered patterns of expression of members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family in lung cancer.

hnRNP A2/B1 has been suggested as a useful early detection marker for lung carcinoma. hnRNP A2/B1 is a member of a large family of heterogeneous nuclear ribonucleoproteins (hnRNP proteins) involved in a variety of functions, including regulation of transcription, mRNA metabolism, and translation. In lung cancer, we have evaluated the expression and cellular localization of several members of the hnRNP family, hnRNP A1, A2, B1, C1, C2 and K. 16 cell lines (SCLC and NSCLC) and biopsies from 32 lung cancer patients were analyzed. Our results suggest that, besides hnRNP A2/B1, the expression of other members of the hnRNP family is altered both in SCLC and NSCLC. In the biopsies, negative or low expression of the hnRNP proteins analyzed was observed in normal epithelial cells whereas lung cancer cells showed highly intense nuclear or cytoplasmic immunolocalization. In all the lung cancer cell lines, the mRNA for all the hnRNP proteins was detected. In general, higher levels of hnRNP mRNAs were found in SCLC as compared with NSCLC. Our results also suggest that the expression and processing of each hnRNP protein in lung cancer is independently regulated and is not exclusively related to proliferation status. In SCLC cell lines, hnRNP A1 protein expression correlated with that of Bcl-x(L). In the lung cancer cell lines, hnRNP K protein localization varied with the cellular confluence.

Experience of nursing students upon their first care encounter with terminally ill patients.

OBJECTIVE: This work seeks to describe the experiences endured by third- and fourth-year nursing students upon their first care encounter with a terminally ill patient. METHODOLOGY: This was a descriptive qualitative study with methodology of analysis of contents of written testimonies at the end of the care experience. The study had the participation of 65 students from a private university in Santiago, Chile. RESULTS: Emerging themes were classified into seven categories: life learning is accomplished, feelings regarding the encounter, loving care, interdisciplinary work with comprehensive care, sense to nursing, incorporation of the family in caring, and development of communication skills. CONCLUSION: The experiences of the nursing students show they have difficulties in facing the care of an individual in process of death. Educational strategies should be posed to improve undergraduate formation on end-of-life care.

Experience of Nursing Students upon Their First Care Encounter with Terminally ill Patients / Experiencia de estudiantes de enfermería ante su primer encuentro de cuidado con enfermos terminales / Experiência de estudantes de enfermagem ante seu primeiro encontro de cuidado com doentes terminais

Objective. This work seeks to describe the experiences endured by third- and fourth-year nursing students upon their first care encounter with a terminally ill patient. Methodology. This was a descriptive qualitative study with methodology of analysis of contents of written testimonies at the end of the care experience. The study had the participation of 65 students from a private university in Santiago, Chile. Results. Emerging themes were classified into seven categories: life learning is accomplished, feelings regarding the encounter, loving care, interdisciplinary work with comprehensive care, sense to nursing, incorporation of the family in caring, and development of communication skills. Conclusion. The experiences of the nursing students show they have difficulties in facing the care of an individual in process of death. Educational strategies should be posed to improve undergraduate formation on end-of-life care.

Objetivo. Describir la experiencia vivida por estudiantes de enfermería de 3° y 4° año, ante su primer encuentro de cuidado con un enfermo terminal. Metodología. Estudio cualitativo descriptivo con metodología de análisis de contenido de testimonios escritos al final de la experiencia de cuidado. Participaron 65 alumnos de una universidad privada de Santiago, Chile. Resultados. Los temas emergentes se clasificaron en siete categorías: se logra un aprendizaje de vida, sentimientos antes del encuentro, cuidado amoroso, trabajo interdisciplinario con atención integral, sentido a la enfermería, incorporación de la familia en el cuidado, y desarrollo de habilidades comunicativas. Conclusión. Las experiencias de los estudiantes de enfermería muestran que estos tienen dificultades para enfrentar el cuidado de una persona en proceso de muerte. Se deben plantear estrategias educativas que mejoren la formación en pregrado del futuro profesional sobre el cuidado del enfermo terminal.

Objetivo. Descrever a experiência vivida por estudantes de enfermagem de 3° e 4° ano, ante seu primeiro encontro de cuidado com um doente terminal. Metodologia. Estudo qualitativo descritivo com metodologia de análise de conteúdo de depoimentos escritos ao final da experiência de cuidado. Participaram 65 alunos de uma universidade privada em Santiago, Chile. Resultados. Os temas emergentes se classificaram em sete categorias: consegue-se uma aprendizagem de vida, sentimentos antes do encontro, cuidado amoroso, trabalho interdisciplinares com atendimento integral, sentido à enfermagem, incorporação da família no cuidado, e desenvolvimento de habilidades comunicativas. Conclusão. As experiências dos estudantes de enfermagem mostram que têm dificuldades para enfrentar o cuidado de uma pessoa em processo de morte. Devem-se propor estratégias educativas que melhorem a formação da graduação sobre o cuidado do fim da vida.

The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer.

The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses.

Histone H3 and H4 modification profiles in a Rett syndrome mouse model.

Rett syndrome (RTT) is a complex neurodevelopmental disorder that has been associated with mutations of methyl-CpG binding protein 2 (MeCP2). MeCP2 acts as a transcriptional repressor and binds to histone modifier proteins, which prompted us to wonder whether MeCP2 disruption affects global histone modification patterns. Taking a two-fold approach of using high-performance capillary electrophoresis (HPCE) and western blot, we analyzed the acetylation and methylation status of histones H3 and H4 in a mouse model of RTT where the MeCP2 locus is genetically disrupted. The comparison of cortex, midbrain and cerebellum in wild-type and MeCP2-knock out mice did not reveal any significant difference in the global H3 and H4 histone modification patterns. Our results suggest that MeCP2 deficiency involves local and gene-specific chromatin changes rather than massive histone modification changes.

Altered expression of adhesion molecules and epithelial-mesenchymal transition in silica-induced rat lung carcinogenesis.

Loss of the epithelial phenotype and disruption of adhesion molecules is a hallmark in the epithelial-mesenchymal transition (EMT) reported in several types of cancer. Most of the studies about the relevance of adhesion and junction molecules in lung cancer have been performed using established tumors or in vitro models. The sequential molecular events leading to EMT during lung cancer progression are still not well understood. We have used a rat model for multistep lung carcinogenesis to study the status of adherens and tight junction proteins and mesenchymal markers during EMT. After silica-induced chronic inflammation, rats sequentially develop epithelial hyperplasia, preneoplastic lesions, and tumors such as adenocarcinomas and squamous cell carcinomas. In comparison with normal and hyperplastic bronchiolar epithelium and with hyperplastic alveolar type II cells, the expression levels of E-cadherin, alpha-catenin and beta-catenin were significantly reduced in adenomatoid preneoplastic lesions and in late tumors. The loss of E-cadherin in tumors was associated with its promoter hypermethylation. alpha- and beta-catenin dysregulation lead to cytoplasmic accumulation in some carcinomas. No nuclear beta-catenin localization was found at any stage of any preneoplastic or neoplastic lesion. Zonula occludens protein-1 was markedly decreased in 66% of adenocarcinomas and in 100% squamous cell carcinomas. The mesenchymal-associated proteins N-cadherin and vimentin were analyzed as markers for EMT. N-cadherin was de novo expressed in 32% of adenocarcinomas and 33% of squamous cell carcinomas. Vimentin-positive tumor cells were found in 35% of adenocarcinomas and 88% of squamous cell carcinomas. Mesenchymal markers were absent in precursor lesions, both hyperplastic and adenomatoid. The present results show that silica-induced rat lung carcinogenesis is a good model to study EMT in vivo, and also provide in vivo evidence suggesting that the changes in cell-cell adhesion molecules are an early event in lung carcinogenesis, while EMT occurs at a later stage.

Relative amounts of antagonistic splicing factors, hnRNP A1 and ASF/SF2, change during neoplastic lung growth: implications for pre-mRNA processing.

Pre-mRNA processing is an important mechanism for globally modifying cellular protein composition during tumorigenesis. To understand this process during lung cancer, expression of two key pre-mRNA alternative splicing factors was compared in a mouse model of early lung carcinogenesis and during regenerative growth following reversible lung injury. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and alternative splicing factor/splicing factor 2 (ASF/SF2) act antagonistically to modulate splice site selection. Both hnRNP A1 and ASF/SF2 contents rose in adenomas and during injury-induced hyperplasia compared to control lungs, as measured by immunoblotting. While both proteins increased similarly during compensatory hyperplasia, hnRNP A1 increased to a much greater extent than ASF/SF2 in tumors, resulting in a 6-fold increase of the hnRNP A1 to ASF/SF2 ratio. Immunohistochemical analysis showed that hnRNP A1 localized exclusively within tumor nuclei, while ASF/SF2 appeared in cytoplasm and/or nuclei, depending on the growth pattern of the tumor cells. We also demonstrated cancer-associated changes in the pre-mRNA alternative splicing of CD44, a membrane glycoprotein involved in cell-cell and cell-extracellular matrix interactions. hnRNP A1 and ASF/SF2 expression is thus differentially altered in neoplastic lung cells by mechanisms that do not strictly arise from increased cell division. These changes are influenced by tumor histology and may be associated with production of variant CD44 mRNA isoforms.