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Volume 42 (2009)
- Issue 6 Pages 159-
- Issue 5 Pages 129-
- Issue 4 Pages 95-
- Issue 3 Pages 65-
- Issue 2 Pages 15-
- Issue 1 Pages 1-
Volume 42, Issue 2
Displaying 1-6 of 6 articles from this issue
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TECHNICAL ADVANCEMENT
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Yoshitaka Hishikawa, Shucai An, Tomomi Yamamoto-Fukuda, Yasuaki Shibat ...2009 Volume 42 Issue 2 Pages 15-21
Published: 2009
Released on J-STAGE: April 28, 2009
Advance online publication: April 23, 2009JOURNAL FREE ACCESSIn situ polymerase chain reaction (in situ PCR), which can detect a few copies of genes within a cell by amplifying the target gene, was developed to better understand the biological functions of tissues. In this study, we optimized the protocol conditions for the detection of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene in paraffin-embedded sections of mouse reproductive organs. The effects of various concentrations of proteinase K (PK) and PCR cycle numbers were examined. To label the amplified DNA, we used digoxigenin-dUTP (Dig), Cy-3-dUTP (Cy-3), or FluorX-dCTP (FluorX). The optimal concentration of PK was 50 μg/ml for the ovary and 10 μg/ml for the testis. Ten PCR cycles were optimal for Dig and 25 cycles were optimal for FluorX and Cy-3 in the ovary and testis. The signal-to-noise ratio of FluorX and Cy-3 for ovarian tissue was better than that of Dig. Using the above conditions, we detected 1–4 and 1–2 spots of pgk-1 in the nuclei of granulosa and germ cells, respectively. Our results indicate that in situ PCR is useful for detecting a specific gene in paraffin-embedded sections under optimized conditions of both PCR cycle number and PK concentration.
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REGULAR ARTICLE
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Takashi Miyakoshi, Katsuhiro Miyajima, Susumu Takekoshi, Robert Yoshiy ...2009 Volume 42 Issue 2 Pages 23-28
Published: 2009
Released on J-STAGE: April 28, 2009
Advance online publication: April 03, 2009JOURNAL FREE ACCESSBisphenol A (BPA) is a monomer use in manufacturing a wide range of chemical products which include epoxy resins and polycarbonate. It has been reported that BPA increases the cell proliferation activity of human breast cancer MCF-7 cells as well as 17-β estradiol (E2) and diethylstilbestrol (DES). However, BPA induces target genes through ER-dependent and ER-independent manners which are different from the actions induced by E2. Therefore, BPA may be unique in estrogen-dependent cell proliferation compared to other endocrine disrupting chemicals (EDCs). In the present study, to test whether ERα is essential to the BPA-induced proliferation on MCF-7 cells, we suppressed the ERα expression of MCF-7 cells by RNA interference (RNAi). Proliferation effects in the presence of E2, DES and BPA were not observed in ERα-knockdown MCF-7 cells in comparison with control MCF-7. In addition, a marker of proliferative potential, MIB-1 labeling index (LI), showed no change in BPA-treated groups compared with vehicle-treated groups on ERα-knockdown MCF-7 cells. In conclusion, we demonstrated that ERα has a role in BPA-induced cell proliferation as well as E2 and DES. Moreover, this study indicated that the direct knockdown of ERα using RNAi serves as an additional tool to evaluate, in parallel with MCF-7 cell proliferation assay, for potential EDCs.
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Jae-Sik Choi, Jea-Young Lee, Chang-Jin Jeon2009 Volume 42 Issue 2 Pages 29-38
Published: 2009
Released on J-STAGE: April 28, 2009
Advance online publication: March 31, 2009JOURNAL FREE ACCESSWe investigated the distributions of AMPA glutamate receptor subtypes GluR1 and GluR4 in the hamster superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on these distributions. We compared these labelings to those of GluR2/3 in our previous report (Park et al., 2004, Neurosci Res., 49:139–155) and calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. Anti-GluR1-immunoreactive (IR) cells were scattered throughout the SC. By contrast, anti-GluR4-IR cells formed distinct clusters within the lower lateral stratum griseum intermediale (SGI) and lateral stratum album intermediale (SAI). The GluR1- and GluR4-IR neurons varied in size and morphology. The average diameter of the GluR1-IR cells was 13.00 μm, while the GluR4-IR cells was 20.00 μm. The large majority of IR neurons were round or oval cells, but they also included stellate, vertical fusiform and horizontal cells. Monocular enucleation appeared to have no effect on the GluR1 and GluR4 immunoreactivity. Some GluR1-IR cells expressed calbindin D28K (9.50%), calretinin (6.59%), parvalbumin (2.53%), and GABA (20.54%). By contrast, no GluR4-IR cells expressed calcium-binding proteins or GABA. Although the function of the AMPA receptor subunits in SC is not yet clear, the distinct segregation of the GluR subunits, its differential colocalization with calcium-binding proteins and GABA, and differential responses to enucleation suggest the functional diversity of the receptor subunits in visuo-motor integration in the SC.
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Yasuko Okumura, Noriyuki Sugiyama, Susumu Tanimura, Masashi Nishida, K ...2009 Volume 42 Issue 2 Pages 39-45
Published: 2009
Released on J-STAGE: April 28, 2009
Advance online publication: April 03, 2009JOURNAL FREE ACCESSNephronophthisis (NPHP) is the most frequent genetic cause of end-stage kidney disease in children and young adults. Inv mice are a model for human nephronophthisis type 2 (NPHP2) and characterized by multiple renal cysts and situs inversus. Renal epithelial cells in inv cystic kidneys show increased cell proliferation. We studied the ERK pathway to understand the mechanisms that induce cell proliferation and renal cyst progression in inv kidneys. We studied the effects of ERK suppression by administering PD184352, an oral mitogen-activated protein kinase kinase (MEK) inhibitor on renal cyst expansion, extracellular signal-regulated protein kinase (ERK) activity, bromo-deoxyuridine (BrdU) incorporation and expression of cell-cycle regulators in invΔC kidneys. Phosphorylated ERK (p-ERK) level increased along with renal cyst enlargement. Cell-cycle regulators showed a high level of expression in invΔC kidneys. PD184352 successfully decreased p-ERK level and inhibited renal cyst enlargement. The inhibitor also decreased expression of cell-cycle regulators and BrdU incorporation in renal epithelial cells. The present results showed that ERK regulated renal cell proliferation and cyst expansion in inv mutants.
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Takahiro Naka, Satoshi Yokose2009 Volume 42 Issue 2 Pages 47-53
Published: 2009
Released on J-STAGE: April 28, 2009
Advance online publication: March 31, 2009JOURNAL FREE ACCESSThe development of mouse submandibular gland (SMG) begins at embryonic day 11.5–12 (E11.5–12), during which successive rounds of epithelial clefting and branching create complex epithelial tree-like structures. Homeobox genes regulate place-dependent morphogenesis, including epithelial-mesenchymal interactions, and control the expression patterns of signaling molecules. The Barx2 containing Homeobox exerts several key roles in development. Some studies have shown that the Barx2 plays important roles in the epithelial-mesenchymal interactions of organogenesis. However, the mechanisms of Barx2 associated with the development of SMG are obscure. In this study, we demonstrated for the first time the exact spatial and temporal Barx2 expression pattern in SMG epithelial tissue during development using immunohistochemical staining and Real-Time quantitative PCR. Barx2 was expressed in the nucleus of the epithelial cells located in the proliferative and differentiative regions of the developing SMG during the early development stages (E11.5–E13.5). After the E14.5-time period, the expression gradually decreased, and at E16.5, expression mostly disappeared despite the fact that evidence of cytodifferentiation, such as the appearance of proacinar cells, distinct lumen formation, and secretory products, was beginning to be observed. Results of Real-Time PCR demonstrated that the amount of Barx2 mRNA expression in SMG was maximal on E14.5, and gradually decreased by E18.5. These results indicate that Barx2 is associated with early stage epithelial tissue development, and can be a useful epithelial marker of the SMG during early developmental stages.
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Keiko Miyoshi, Yuki Akazawa, Taigo Horiguchi, Takafumi Noma2009 Volume 42 Issue 2 Pages 55-64
Published: 2009
Released on J-STAGE: April 28, 2009
Advance online publication: April 07, 2009JOURNAL FREE ACCESSAdenylate kinase (AK) is a key enzyme in the high-energy phosphoryl transfer reaction in living cells. Of its isoforms, AK4 has a similar sequence and subcellular localization to that of AK3 in the mitochondrial matrix. However, unlike AK3, AK4 lacks the guanosine triphosphate: adenosine monophosphate phosphotransferase activity. To elucidate the physiological role of AK4, we explored the protein localization of AK4 in various mouse tissues by immunohistochemical analysis. AK4 protein was detected in the kidney, liver, brain, heart, stomach, intestine, and gonads but not in the lung and spleen. Interestingly, cell-type specific expression was evident in the brain, gastrointestinal tract, and gonads. In the cerebellum, AK4 was detected in granular cells but not in Purkinje cell bodies. In the gastrointestinal tract, AK4 was highly expressed in epithelia. In the ovary, AK4 was detected in oocytes and corpora lutea. In the testis, AK4 was detected in spermatocytes but not in spermatogonia. Our findings demonstrate that AK4 localizes uniquely in a cell-type and tissue-specific manner in mouse tissues.
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