Morphology-oriented epigenetic research

Cytosine methylation plays a major role in the regulation of sequential and tissue-specific expression of genes. De novo aberrant DNA methylation and demethylation are also crucial processes in tumorigenesis and tumor progression. The mechanisms of how and when such aberrant methylation and demethylation occur in tumor cells are still obscure, however. To evaluate subtle epigenetic alteration among minor subclonal populations, morphology-oriented epigenetic analysis is requisite, especially where heterogeneity and flexibility are as notable as in the process of cancer progression and cellular differentiation at critical stages. Therefore, establishment of reliable morphology-oriented epigenetic studies has become increasingly important in not only the experimental but also the diagnostic field. By selecting a subset of cells based on characteristic morphological features disclosed by microdissection or in situ hybridization, we discovered how methylation at certain CpG sites outside of CpG islands would play a crucial epigenetic role in the versatility and flexibility of gene expression during cancer progression. In this review, we first introduce technical aspects of two morphology-oriented epigenetic studies: (1) histoendonuclease-linked detection of methylated sites of DNA (HELMET), and (2) padlock probe and rolling circle amplification (RCA) for in situ identification of methylated cytosine in a sequence-dependent manner. We then present our observation of a novel MeCP2-mediated gene-silencing mechanism through the addition of methylation to a single-CpG-locus upstream of the TATA-box of the receptor activator of NF-κB ligand (RANKL) and of secreted frizzled-related protein 4 (SFRP4) gene promoters.     

Altered gene expression in lymphoblastoid cell lines after subculture

Lymphoblastoid cell lines (LCLs) are nearly immortalized B lymphocytes that are used as long-lasting supply of human cells for studies on gene expression analyses. However, studies on the stability of the cellular features of LCLs are scarce. To address this issue, we measured gene expression in LCLs with different passage numbers and observed that gene expression substantially changed within 10 passages. In particular, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a well-known housekeeping gene, varied considerably during subculture; thus, the use GAPDH as an internal control may be unsuitable. In conclusion, this study highlights the need for exercising caution during determination of gene expression in LCLs.     

Farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthetases during Cucurbita pepo germination

Although the geranylgeranyl pyrophosphate synthetase activity was very low compared with farnesyl pyrophosphate synthetase activity on imbibition of pumpkin seed, the former increased markedly and the latter decreased as germination proceeded.     

Role of 1-methyl-4-phenylpyridinium ion formation and accumulation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity to isolated hepatocytes

The parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is converted by isolated hepatocytes to its primary metabolite, the 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPDP+), and to its fully oxidized derivative, 1-methyl-4-phenylpyridinium ion (MPP+). Only the latter, however, accumulates in the cells. Incubation of hepatocytes in the presence of MPDP+ also results in the selective intracellular accumulation of MPP+. Conversion to MPP+ is more rapid and extensive after exposure to MPDP+, than with MPTP and the former is also more toxic. Addition of MPP+ itself is toxic to hepatocytes but only after a long lag period, which presumably reflects its limited access to the cell and its relatively slow intracellular accumulation. As previously shown with MPTP and MPP+, the cytotoxicity of MPDP+ is dose-dependent and is consistently preceeded by complete depletion of intracellular ATP. Similar to MPP+ but not MPTP, MPDP+ causes a comparable rate and extent of cytotoxicity and ATP loss in hepatocytes pretreated with the monoamine oxidase inhibitor pargyline. Pargyline blocks hepatocyte biotransformation of MPTP to MPP+, but it has no significant effect on MPP+ accumulation after exposure to either MPDP+ or MPP+. It is concluded that MPTP is toxic to hepatocytes via its monoamine oxidase-dependent metabolism and that MPP+ is likely to be the ultimate toxic metabolite which accumulates in the cell, causing ATP depletion and eventual cell death.     

In vitro activation of immune cells by a mycolic acid-containing glycolipid, trehalose-2,3,6'-trimycolate, derived from Gordona aurantiaca

A mycolic acid-containing glycolipid, trehalose-2,3,6'-trimycolate (GaGM), derived from Gordona aurantiaca, an acid-fast bacteria closely related taxonomically to Mycobacterium, was investigated for its immune adjuvant activity in vitro. The liposomes containing GaGM showed strong mitogenic effects on murine spleen cells at the doses used (25-100 micrograms/ml), but not on T-cell-depleted spleen cells or macrophage-depleted spleen cells. These results suggest that the mitogenic property of liposomes containing GaGM differs from that of such as lipopolysaccharide, a B-cell mitogen and that its mitogenic effects depend on the presence of macrophages. In addition, liposomes containing GaGM augmented the mixed lymphocyte reaction (MLR) and in vitro induction of cytotoxic T-lymphocytes (CTLs) against allogeneic tumor cells. These results suggest that liposomes containing GaGM have immune adjuvant properties in vitro and the adjuvant activity may be related to such cytokines as interleukin-1 and -2.     

Infrared laser‐mediated local gene induction in medaka,zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.     

Colonization of leaf patches at topographically different locations by insect shredders in a small mountain stream

We examined physical constraints on the colonization of leaf patches by shredder individuals by comparing the colonizations of artificially standardized leaf patches placed at different locations within a stream reach (i.e., riffles, middles and edges of pools). Stonefly taxa (Nemoura, Protonemura) colonized riffle patches 2–10 times more often than pool (middle, edge) patches, whereas caddisfly taxa (two species of Lepidostoma, Nothopsyche) almost exclusively colonized pool patches. Colonization also differed between the middle and edge patches in pools for most taxa; it was 2–5 times greater in edge patches for Nemoura and in middle patches for Lepidostoma. The abilities of species to cope with low oxygen circulation and high shear stress appear to determine differences in colonization between riffle and pool patches, whereas species-specific dispersion behavior (e.g., return time from drift) may differentiate colonization between middle and edge patches in pools. Our results suggest that changes in leaf distribution within a reach can affect the suitability of stream reaches in terms of food acquisition for shredder individuals.     

c-kitpos GATA-4 high rat cardiac stem cells foster adult cardiomyocyte survival through IGF-1 paracrine signalling

Background

Resident c-kit positive (c-kit^pos) cardiac stem cells (CSCs) could be considered the most appropriate cell type for myocardial regeneration therapies. However, much is still unknown regarding their biological properties and potential.

Methodology/Principal Findings

We produced clones of high and low expressing GATA-4 CSCs from long-term bulk-cultured c-kit^pos CSCs isolated from adult rat hearts. When c-kit^pos GATA-4 high expressing clonal CSCs (cCSCs) were co-cultured with adult rat ventricular cardiomyocytes, we observed increased survival and contractility of the cardiomyocytes, compared to cardiomyocytes cultured alone, co-cultured with fibroblasts or c-kit^pos GATA-4 low expressing cCSCs. When analysed by ELISA, the concentration of IGF-1 was significantly increased in the c-kit^pos GATA-4 high cCSC/cardiomyocyte co-cultures and there was a significant correlation between IGF-1 concentration and cardiomyocyte survival. We showed the activation of the IGF-1 receptor and its downstream molecular targets in cardiomyocytes co-cultured with c-kit^pos GATA-4 high cCSCs but not in cardiomyocytes that were cultured alone, co-cultured with fibroblasts or c-kit^pos GATA-4 low cCSCs. Addition of a blocking antibody specific to the IGF-1 receptor inhibited the survival of cardiomyocytes and prevented the activation of its signalling in cardiomyocytes in the c-kit^pos GATA-4 high cCSC/cardiomyocyte co-culture system. IGF-1 supplementation or IGF-1 high conditioned medium taken from the co-culture of c-kit^pos GATA-4 high cCSCs plus cardiomyocytes did extend the survival and contractility of cardiomyocytes cultured alone and cardiomyocytes co-cultured with c-kit^pos GATA-4 low cCSCs.

Conclusion/Significance

c-kit^pos GATA-4 high cCSCs exert a paracrine survival effect on cardiomyocytes through induction of the IGF-1R and signalling pathway.