Cytoskeleton of human mononuclear cells as a possible peripheral marker for phenylalanine neurotoxicity in PKU

In this work we tested human mononuclear cells as a peripheral marker to study neurotoxicity of phenylalanine (Phe). Slices of cerebral cortex of rats or human mononuclear cells were incubated with different concentrations of Phe and/or Ala in the presence of ³²P-orthophosphate, the cytoskeletal fraction was extracted, and the radioactivity incorporated into intermediate filament proteins was measured. Our results show that 2 mM Phe as well as 1 mM Ala are effective in increasing the ³²P in vitro incorporation into IFs in both tissues. When cerebral cortex slices or mononuclear cells were incubated with different concentrations of Phe and/or Ala, the effects on the ³²P in vitro incorporation into IF proteins was compatible with an antagonistic mechanism of action of the two amino acids on the enzymes of the phosphorylating system. In addition, these blood cells may be a possible peripheral marker to study neurotoxicity of Phe in patients with PKU.     

Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes

Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear "viral compartment." We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.     

Gene expression databases and data mining

The DNA microarray technology has arguably caught the attention of the worldwide life science community and is now systematically supporting major discoveries in many fields of study. The majority of the initial technical challenges of conducting experiments are being resolved, only to be replaced with new informatics hurdles, including statistical analysis, data visualization, interpretation, and storage. Two systems of databases, one containing expression data and one containing annotation data are quickly becoming essential knowledge repositories of the research community. This present paper surveys several databases, which are considered "pillars" of research and important nodes in the network. This paper focuses on a generalized workflow scheme typical for microarray experiments using two examples related to cancer research. The workflow is used to reference appropriate databases and tools for each step in the process of array experimentation. Additionally, benefits and drawbacks of current array databases are addressed, and suggestions are made for their improvement.     

Replication error repair,microsatellites, and cancer

Some common human tumors are characterized by inactivating alterations of mismatch repair (MMR) genes that lead to an inability to recognize and repair errors that occur during DNA replication. These alterations are either inherited in the so-called hereditary non polyposis colorectal cancer (HNPCC) syndrome or can occur sporadically in 10-15% of colorectal, gastric, or endometrial tumors. Because of their repetitive nature, microsatellite sequences are particularly prone to mutation in tumors with MMR deficiency. Thousands of microsatellite alterations accumulate in MMR deficient cancers and these are referred to as MSI-H tumors (high level of microsatellite instability). MSI-H tumors have different clinicopathological features compared to cancers without this phenotype, and the repertoire of genetic events involved in their tumoral progression is also thought to be different. Many of the genetic alterations observed in MSI-H tumors affect nucleotide repeat tracks contained within genes thought to have a putative oncogenic function. These alterations are believed to play an important role during MSI-H carcinogenesis, since they can be either inactivating or activating events that are selected for in a recessive or dominant manner. We provide here an overview of the genetic changes that occur in MSI-H tumors and that appear to constitute a new genetic mutator pathway leading a normal cell to become malignant.     

Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.     

Histone phosphorylation: how to proceed

Among all posttranslational modifications that occur on histone tails, phosphorylation is the one that establishes a direct link between chromatin remodeling and intracellular signaling pathways. Specific, conserved serine residues are present on the N-terminal tails of each histone. These are phosphoacceptor sites for a number of kinases, whose identification is essential to decipher the transduction routes leading to various physiological responses. In the case of histone H3, phosphorylation at the Ser10 residue may lead to either activated gene expression or chromatin condensation during mitosis. In addition, phosphorylation at specific sites may be coupled to other distinct modifications, such as acetylation and methylation, generating the so-called "histone code" which postulates that well defined combinatorial modifications at histone tails correspond to specific physiological responses. Here we describe a number of experimental methodologies that are essential for the study of histone phosphorylation. While chromatin immunoprecipitation is useful in recognizing gene targets, the in-gel kinase assay is a first, essential step in establishing the identity of the kinase(s) that operates in response to a specific signaling pathway. The subsequent use of in vitro kinase assays is helpful in validating the implication of a candidate kinase. These powerful approaches are important as identification of the signaling transduction routes leading to chromatin remodeling is critical to an understanding of all cellular processes.