Yo Tabuse

Tetraspanin protein (TSP-15) is required for epidermal integrity in Caenorhabditis elegans

Epidermal integrity is essential for animal development and survival. Here, we demonstrate that TSP-15, a member of the tetraspanin protein family, is required for epithelial membrane integrity in Caenorhabditis elegans. Reduction of tsp-15 function by mutation or by RNA interference elicits abnormalities of the hypodermis, including dissociation of the cuticle and degeneration of the hypodermis. Lethality during molting often results. Examination of GFP transgenic animals, genetic mosaic analysis and rescue assays revealed that TSP-15 functions in hyp7, a large syncytium that composes most of the hypodermis. Assays with a membrane-impermeable dye or leakage analysis of a hypodermal-specific marker indicate that the barrier function of the hypodermal membrane is impaired owing to the loss or reduction of TSP-15. These results indicate that TSP-15 functions in the maintenance of epithelial cell integrity.

Long-Term Nicotine Adaptation in Caenorhabditis elegans Involves PKC-Dependent Changes in Nicotinic Receptor Abundance

Chronic exposure to nicotine leads to long-term changes in both the abundance and activity of nicotinic acetylcholine receptors, processes thought to contribute to nicotine addiction. We have found that in Caenorhabditis elegans, prolonged nicotine treatment results in a long-lasting decrease in the abundance of nicotinic receptors that control egg-laying. In naive animals, acute exposure to cholinergic agonists led to the efficient stimulation of egg-laying, a response mediated by a nicotinic receptor functionally expressed in the vulval muscle cells. Overnight exposure to nicotine led to a specific and long-lasting change in egg-laying behavior, which rendered the nicotine-adapted animals insensitive to simulation of egg-laying by the nicotinic agonist and was accompanied by a promoter-independent reduction in receptor protein levels. Mutants defective in the gene tpa-1, which encodes a homolog of protein kinase C (PKC), failed to undergo adaptation to nicotine; after chronic nicotine exposure they remained sensitive to cholinergic agonists and retained high levels of receptor protein in the vulval muscles. These results suggest that PKC-dependent signaling pathways may promote nicotine adaptation via regulation of nicotinic receptor synthesis or degradation.

Protein expression profile characteristic to hepatocellular carcinoma revealed by 2D-DIGE with supervised learning

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Although several major risks related to HCC, e.g., hepatitis B and/or hepatitis C virus infection, aflatoxin B1 exposure, alcohol drinking and genetic defects have been revealed, the molecular mechanisms leading to the initiation and progression of HCC have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to detect the proteins which are associated with tumor progression and may be useful as potential therapeutic or diagnosis targets. However, previous studies have not yet revealed the associations among HCC cells, histological grade and AFP. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) combined with MS for 18 HCC patients. To focus not on individual proteins but on multiple proteins associated with pathogenesis, we introduce the supervised feature selection based on stochastic gradient boosting (SGB) for identifying protein spots that discriminate HCC/non HCC, histological grade of moderate/well and high alpha-fetoprotein (AFP)/low AFP level without arbitrariness. We detected 18, 25 and 27 protein spots associated with HCC, histological grade and AFP level, respectively. We confirmed that SGB is able to identify the known HCC-related proteins, e.g., heat shock proteins, carbonic anhydrase 2. Moreover, we identified the differentially expressed proteins associated with histological grade of HCC and AFP level and found that aldo-keto reductase 1B10 (AKR1B10) is related to well differentiated HCC, keratin 8 (KRT8) is related to both histological grade and AFP level and protein disulfide isomerase-associated 3 (PDIA3) is associated with both HCC and AFP level. Our pilot study provides new insights on understanding the pathogenesis of HCC, histological grade and AFP level.

Caenorhabditis elegans DAF-21 (HSP90) is characteristically and predominantly expressed in germline cells: Spatial and temporal analysis

Three monoclonal antibodies against antigens that exist in the Caenorhabditis elegans germ line have previously been described. In the present study, a full‐length mRNA for one of these antigens was isolated, and by sequencing its corresponding cDNA, it was predicted that the protein would show a high homology with the 90 kDa heat shock protein (HSP90) in other species, and with the protein of daf‐21, a previously identified hsp90 homologue. The spatial and temporal distribution of the antigen (DAF‐21) was analyzed in C. elegans, and the localization of daf‐21 mRNA, as detected by in situ hybridization, agreed with that detected by the monoclonal antibody. Under normal conditions, daf‐21 mRNA is characteristically distributed in postembryonic germ cells derived from Z2 and Z3 cells in both hermaphrodites and males. Under heat stress conditions, however, daf‐21 mRNA was not only detected in germ cells, but also apparently expressed all over the body. In addition, the DAF‐21 protein seemed to be localized in the perinuclear region of somatic cells.

Tumor promoters specifically and reversibly disturb development and behavior of Caenorhabditis elegans.

SummaryThe effect of phorbol ester tumor promoters on the development and behavior of a free-living soil nematode, Caenorhabditis elegans, was studied. When young developing C. elegans were grown on E. coli-seeded agar with low concentrations (0.1 μg/ml) of 12-0-tetradecanoyl-phorbol-13-acetate or phorbol-12,13-didecanoate, their growth was arrested. These tumor promoters reduced the brood size when gravid adults were treated and caused uncoordinated movement in animals treated at any stage of development. The effects of these tumor promoters on nematode development and behavior were partially reversible. The nonpromoting derivatives phorbol and 4α-phorbol-12,13-didecanoate showed no effect on the animals.

Comparative Analysis of Proteome and Transcriptome in Human Hepatocellular Carcinoma using 2D-DIGE and SAGE

Proteome analysis of human hepatocellular carcinoma was conducted using two-dimensional difference gel electrophoresis, and the protein expression profiles were compared to the mRNA expression profiles made from serial analysis of gene expression (SAGE) in identical samples from a single patient. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the protein expression profiles. A total of 188 proteins were identified, and the expression profiles of 164 proteins which had the corresponding SAGE data were compared to the mRNA expression profiles. Among them, 40 proteins showed significant differences in the mRNA expression levels between non HCC and HCC. We compared expression changes of proteins with those of mRNAs. We found that the expression tendency of 24 proteins were similar to that of mRNA, whereas 16 proteins showed different or opposite tendency to the mRNA expression.

CePKC: Protein kinase C (C. elegans) (TPA-1)

The chapter discusses the protein kinase C ( and the subunit structure and isoforms, domain structure, and amino acid sequence. The tpa-1 gene mediates the action of tumor-promoting phorbol esters in the nematode Caenorhabditis elegans. Analysis of cDNA clones for tpa-1 predicts a protein (TPA-1) of 557 amino acids, which is homologous to protein kinase C in other animals. Like mammalian PKCs, TPA-1 has a C-terminal kinase domain and tandem Cys-rich repeats in the N-terminal region, which may represent the phorbol ester-binding region. Just N-terminal to the first Cys-rich repeat is a putative pseudosubstrate site. TPA-1 is homologous to PKC isoforms in other species (PKC, DmPKC, ScPKC). TPA-1 may be assayed using histone HI as for mammalian PKC.