Histone acetyltransferase MOZ acts as a co-activator of Nrf2-MafK and induces tumour marker gene expression during hepatocarcinogenesis

HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway.     

Direct comparison between ICSI-mediated DNA transfer and pronuclear DNA microinjection for producing transgenic rats.

Production efficiency of transgenic rats was compared directly between the routine pronuclear microinjection of exogenous DNA solution (PNMI-Tg method) and the ooplasmic injection of sperm cells exposed to exogenous DNA solution (ICSI-Tg method) using six DNA constructs. The overall production efficiency per treated oocyte in the ICSI-Tg method (mean 1.1%, range 0.2 to 3.1%) was similar to that in the PNMI-Tg method (mean 1.1%, range 0 to 2.4%). An advantage of the ICSI-Tg method in the production of transgenic rats is noted in cases in which a low yield of pronuclear zygotes is an inevitable fate of the rat strain.     

Immunoelectron Microscopic Localization of Sperm-specific Nuclear Basic Proteins during Spermatogenesis in Anuran Amphibians

Antisera were raised in rabbits against sperm-specific nuclear basic proteins (SPs) of Bufo japonicus and Xenopus laevis . The localizations of these proteins in spermatogenic cells were then studied by electron microscopy with colloidal gold labeled antibodies as probes. The numbers of gold particles counted on ultra-thin sections of cells at various spermatogenic stages were corrected for the density per unit area, on the basis of areas determined with a digitizer. No grains were deposited during early nuclear elongation stages. Grains appeared on nuclei at the beginning of chromatin granulation, and their density increased first gradually and then sharply at the last step of spermiogenesis. Recalculation of grain counts according to the estimated nuclear volumes of Bufo spermatogenic cells also indicated a sharp increase in the amount of SPs per nucleus in the last step of spermiogenesis. No significant localization of grains in the cytoplasm was observed at any stage of spermatogenesis.     

Visualization of acidic compartments in cultured osteoclasts by use of an acidotrophic amine as a marker for low pH

Using the acidotrophic amine 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP) as a marker for low pH and immunofluorescence cytochemistry, we examined acidic compartments of osteoclasts cultured on cover glasses or bone slices, where they could resorb the bone surface, forming resorptive lacunae. DAMP-positive structures were seen as vesicular and tubular forms in the cytoplasm, indicating lysosomes and endosomes. Not only the osteoclastic cytoplasm but also the extracellular area around the ruffled border and resorptive lacunae were stained with DAMP, suggesting acidic regions. Immunofluorescence was localized predominantly on the substratum side of actively resorbing osteoclasts, whereas an evenly distributed staining pattern was seen in the nonactive cell. The most intensive reaction was seen at the advancing front of resorptive lacunae within the actively resorbing osteoclasts. The distribution pattern of DAMP seemed to be correlated with the osteoclastic activity, since osteoclasts exhibit alternating resorption and migration phases during the bone-remodeling cycle. In this culture system, the resorptive lacunae were left behind after the osteoclasts had completed resorption and migrated along the bone surface. These exposed resorptive lacunae were also stained with DAMP, which were presumably kept at an acidic pH. The effect of treatment with monensin, chloroquine, ammonium chloride, or nigericin was varied in terms of the immunoreactivity for DAMP, but not complete abolition of the staining was obtained. Weak bases such as chloroquine or ammonium chloride inhibited both intra- and extracellular immunoreactivity. Immunoreactivity for the vacuolar type of proton ATPase (V-ATPase) was demonstrable in the cytoplasm of the osteoclasts but was weakened by the addition of bafilomycin. Immunofluorescence of the resorptive lacunae was still retained even after the treatment with bafilomycin and acetazolamide. Besides, both bafilomycin and acetazolamide reversibly inhibited cellular acidity as judged by DAMP immunocytochemistry, which agrees with the fact that ostoeclastic acidification results from the action of vacuolar proton-pump ATPase coupled with carbonic anhydrase.     

Morphological and molecular characterization of Tulasnella spp. fungi isolated from the roots of Epidendrum secundum,a widespread Brazilian orchid

Tulasnella spp. are the main fungal symbionts of Brazilian Epidendrum orchids. The taxonomy of these fungi is largely based on ITS rDNA similarity, but culture dependent techniques are still essential to establish the true biological entity of the mycobiont. The aim of this study was to characterize morphologically and molecularly 16 Tulasnella spp. fungi isolated from three different populations of E. secundum and to test the coincidences between morphological and molecular characterization. Two uninucleate rhizoctonia fungi, obtained from Oncidium barbaceniae, and two phytopathogenic isolates were included as outgroups. Qualitative and quantitative morphological characteristics were analyzed using multivariate statistics and were able to distinguish Ceratobasidium, Tulasnella and Thanatephorus genera and separate the isolates of Tulasnella spp. into two groups. Analysis of RAPD (Random Amplified Polymorphic DNA) and ITS rDNA sequences validated the morphological data. Symbionts of O. barbaceniae presented identity to ITS sequences of Ceratobasidium genus, while E. secundum isolates presented identity to two species of Tulasnella. We observed homogeneity among Tulasnella spp. obtained from a single population and from neighboring populations, but there was higher variability among isolates obtained from populations of regions that were farther apart. Morphological data associated with multivariate statistics proved to be a useful tool in the multi-level taxonomy of these orchid-associated fungi and in estimating the diversity of orchid mycorrhizal fungi.     

Chelation of Extracellular Calcium-Induced Cell Death was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

Calcium ion is a secondary messenger that mediates a variety of physiological responses of neurons, including cell survival responses. To determine the role of calcium in regulating neuronal survival and death, we examined whether chelation of extracellular calcium with EGTA induces caspase-dependent apoptotic cell death and whether glycogen synthase kinase-3 is involved in EGTA-induced cell death in PC12 cells. EGTA increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation and fragmentation accompanied by caspase activation. EGTA increased GRP78 protein expression, suggesting that EGTA induces ER stress. Glycogen synthase kinase-3 inhibitors prevented EGTA-induced apoptosis. In addition, nerve growth factor and insulin growth factor-I completely blocked EGTA-induced cell death. Moreover, caspase-3 activation was inhibited by glycogen synthase kinase-3 inhibitors. These results suggest that chelation of extracellular calcium with EGTA induces caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.