Ichiro Takasaki

Valproic acid induces up- or down-regulation of gene expression responsible for the neuronal excitation and inhibition in rat cortical neurons through its epigenetic actions

Valproic acid (VPA), a drug used to treat epilepsy and bipolar mood disorder, inhibits histone deacetylase (HDAC), which is associated with the epigenetic regulation of gene expression. Using a microarray, we comprehensively examined which genes are affected by stimulating cultured rat cortical neurons with VPA, and found that the VPA-treatment markedly altered gene expression (up-regulated; 726 genes, down-regulated; 577 genes). The mRNA expression for brain-derived neurotrophic factor (BDNF) and the alpha4 subunit of the GABA(A) receptor (GABA(A)Ralpha4), known to be involved in epileptogenesis, was up-regulated, with the increase in BDNF exon I-IX mRNA expression being remarkable, whereas that for GABA(A)Rgamma2, GAD65 and 67, and the K(+)/Cl(-) co-transporter KCC2, which are responsible for the development of GABAergic inhibitory neurons, was down-regulated. The number of GAD67-positive neurons decreased upon VPA-treatment. Similar changes of up- and down-regulation were obtained by trichostatin A. VPA did not affect the intracellular Ca(2+) concentration and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting its direct action on HDAC. The acetylation of histones H3 and H4 was increased in the promoters of up-regulated but not down-regulated genes. Thus, VPA may disrupt a balance between excitatory and inhibitory neuronal activities through its epigenetic effect.

Allodynia and hyperalgesia induced by herpes simplex virus type-1 infection in mice

&NA; Human subjects infected with herpes or varicella‐zoster viruses complain of pain, such as allodynia, in or near the region with vesicles. However, the mechanisms of the pain are unclear. We show for the first time that infection with herpes simplex virus type‐1 (HSV‐1) induces allodynia and hyperalgesia in mice. When HSV‐1 was inoculated on the hind paw of the mouse, eruption appeared on the back on day 5 post‐inoculation, and zosteriform skin lesions were developed on the inoculated side. Allodynia and hyperalgesia became apparent in the hind paw on the inoculated side on day 5 and persisted until at least day 8. HSV‐1 DNA was detected in the dorsal root ganglia from days 2 to 8 post‐inoculation, with a peak effect on day 5. The application of heat‐inactivated HSV‐1 induced no allodynia, hyperalgesia and skin lesion. When started from days 0 or 2, repeated treatment with acyclovir, anti‐HSV‐1 agent, inhibited the appearance of allodynia, hyperalgesia, eruption and the viral proliferation in the dorsal root ganglia. In contrast, when started from days 5 or 6, acyclovir treatment slightly inhibited the development of skin lesions and the viral proliferation, but not allodynia and hyperalgesia. These results suggest that the propagation of HSV‐1 in the dorsal root ganglia produces allodynia and hyperalgesia as a result of functional abnormality of the sensory neurons in mice. This may be a useful model for studying the mechanisms of herpetic pain.

Telmisartan improves insulin resistance and modulates adipose tissue macrophage polarization in high-fat-fed mice.

Diet-induced obesity is reported to induce a phenotypic switch in adipose tissue macrophages from an antiinflammatory M2 state to a proinflammatory M1 state. Telmisartan, an angiotensin II type 1 receptor blocker and a peroxisome proliferator-activated receptor-γ agonist, reportedly has more beneficial effects on insulin sensitivity than other angiotensin II type 1 receptor blockers. In this study, we studied the effects of telmisartan on the adipose tissue macrophage phenotype in high-fat-fed mice. Telmisartan was administered for 5 wk to high-fat-fed C57BL/6 mice. Insulin sensitivity, macrophage infiltration, and the gene expressions of M1 and M2 markers in visceral adipose tissues were then examined. An insulin- or a glucose-tolerance test showed that telmisartan treatment improved insulin resistance, decreasing the body weight gain, visceral fat weight, and adipocyte size without affecting the amount of energy intake. Telmisartan reduced the mRNA expression of CD11c and TNF-α, M1 macrophage markers, and significantly increased the expressions of M2 markers, such as CD163, CD209, and macrophage galactose N-acetyl-galactosamine specific lectin (Mgl2), in a quantitative RT-PCR analysis. A flow cytometry analysis showed that telmisartan decreased the number of M1 macrophages in visceral adipose tissues. In conclusion, telmisartan improves insulin sensitivity and modulates adipose tissue macrophage polarization to an antiinflammatory M2 state in high-fat-fed mice.

Genetic networks responsive to sodium butyrate in colonic epithelial cells

We performed microarray and computational gene network analyses to identify the detailed mechanisms by which sodium butyrate (SB) induces cell growth arrest and the differentiation of mouse colonic epithelial MCE301 cells. Two thousand six hundred four differentially expressed probe sets were identified in the cells treated with 2 mM SB and were classified into four groups. Of these, the gradually increased group and the gradually and remarkably decreased group contained the genetic networks for cellular development and cell cycles or canonical pathways for fatty acid biosynthesis and pyrimidine metabolism, respectively. The present results provide a basis for understanding the detailed molecular mechanisms of action of SB in colonic epithelial cells.

DNA Double-Strand Breaks Induced by Cavitational Mechanical Effects of Ultrasound in Cancer Cell Lines

Ultrasonic technologies pervade the medical field: as a long established imaging modality in clinical diagnostics; and, with the emergence of targeted high intensity focused ultrasound, as a means of thermally ablating tumours. In parallel, the potential of [non-thermal] intermediate intensity ultrasound as a minimally invasive therapy is also being rigorously assessed. Here, induction of apoptosis in cancer cells has been observed, although definitive identification of the underlying mechanism has thus far remained elusive. A likely candidate process has been suggested to involve sonochemical activity, where reactive oxygen species (ROS) mediate the generation of DNA single-strand breaks. Here however, we provide compelling new evidence that strongly supports a purely mechanical mechanism. Moreover, by a combination of specific assays (neutral comet tail and staining for γH2AX foci formation) we demonstrate for the first time that US exposure at even moderate intensities exhibits genotoxic potential, through its facility to generate DNA damage across multiple cancer lines. Notably, colocalization assays highlight that ionizing radiation and ultrasound have distinctly different signatures to their respective γH2AX foci formation patterns, likely reflecting the different stress distributions that initiated damage formation. Furthermore, parallel immuno-blotting suggests that DNA-PKcs have a preferential role in the repair of ultrasound-induced damage.

Parathyroid hormone 1 (1–34) acts on the scales and involves calcium metabolism in goldfish

The effect of fugu parathyroid hormone 1 (fugu PTH1) on osteoblasts and osteoclasts in teleosts was examined with an assay system using teleost scale and the following markers: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Synthetic fugu PTH1 (1-34) (100pg/ml-10ng/ml) significantly increased ALP activity at 6h of incubation. High-dose (10ng/ml) fugu PTH1 significantly increased ALP activity even after 18h of incubation. In the case of TRAP activity, fugu PTH1 did not change at 6h of incubation, but fugu PTH1 (100pg/ml-10ng/ml) significantly increased TRAP activity at 18h. Similar results were obtained for human PTH (1-34), but there was an even greater response with fugu PTH1 than with human PTH. In vitro, we demonstrated that both the receptor activator of the NF-κB ligand in osteoblasts and the receptor activator NF-κB mRNA expression in osteoclasts increased significantly by fugu PTH1 treatment. In an in vivo experiment, fugu PTH1 induced hypercalcemia resulted from the increase of both osteoblastic and osteoclastic activities in the scale as well as the decrease of scale calcium contents after fugu PTH1 injection. In addition, an in vitro experiment with intramuscular autotransplanted scale indicated that the ratio of multinucleated osteoclasts/mononucleated osteoclasts in PTH-treated scales was significantly higher than that in the control scales. Thus, we concluded that PTH acts on osteoblasts and osteoclasts in the scales and regulates calcium metabolism in goldfish.

Ultrasound-induced apoptosis in the presence of Sonazoid and associated alterations in gene expression levels: a possible therapeutic application.

Ultrasound (US) has been shown to induce apoptosis and cell lysis in cancer cells. In this study, we report on the potential of using Sonazoid, a new echo-contrast agent, that is chemically more stable and US-resistant in combination with US in cancer therapy. The biological effects and their mechanisms in the presence or absence of ultrasonic exposure in vitro were investigated. In addition, the impact of the agent on the expression levels of genes responsive to US was studied using global-scale microarrays and computational gene expression analysis tools. Our results show that the combination led to enhanced cell killing in the presence of 1 MHz acoustic field. The apoptosis induction was shown to be mediated by the mitochondrial pathway. The occurrence of US-induced DNA damage was also observed. Despite these findings, the agent at concentrations similar to those clinically used can be considered as well tolerated. Furthermore, Sonazoid enhanced expression of genes that related to apoptosis and are responsive to US, although it alone had almost no effect. These results indicate the potential of Sonazoid for US contrast enhancement as well as the possibility of its use in US-aided therapies.

Effects of Analgesics on Delayed Postherpetic Pain in Mice

Background Postherpetic neuralgia is pain that persists long after the disappearance of the cutaneous lesions of herpes zoster. However, the mechanisms of this delayed pain are unclear. Herpes simplex virus infection induces cutaneous lesions and pain-related responses in mice. The authors examined whether such responses would persist after the disappearance of the cutaneous lesions and whether some analgesics would be effective against them. Methods Female BALB/c mice were inoculated with herpes simplex virus type 1 on the unilateral hind paw. Pain-related responses of hind paw were determined using von Frey filaments. Beginning 5 days after inoculation, mice were given perorally the antiherpes agent acyclovir five times a day for 7 days. Effects of morphine (3–5 mg/kg subcutaneously), gabapentin (30–100 mg/kg perorally), mexiletine (10–30 mg/kg intraperitoneally), and diclofenac (30 mg/kg intraperitoneally) on pain-related responses were examined on days 25–35 after inoculation. Results Viral inoculation induced cutaneous lesions and pain-related responses beginning on day 5 after inoculation. Acyclovir treatment healed all skin lesions by day 15 after inoculation. Approximately half of the mice given acyclovir showed pain-related responses at least until day 40 after inoculation. Morphine, gabapentin, and mexiletine dose-dependently inhibited pain-related responses, but diclofenac had no effects. Conclusions The authors show a mouse model of delayed postherpetic pain. This may be useful for manifesting the mechanisms of postherpetic neuralgia and the factors contributing to the transition from acute herpetic pain to delayed postherpetic pain. This may also be useful for the development of new analgesics against postherpetic neuralgia.

Genes and genetic networks responsive to mild hyperthermia in human lymphoma U937 cells

In this study, to better understand the molecular mechanism underlying cellular responses to mild hyperthermia, we investigated gene expression patterns and genetic networks in human myelomonocytic lymphoma U937 cells using high-density oligonucleotide microarrays and computational gene expression analysis tools. The cells were incubated at 41°C for 30 min (mild hyperthermia treatment) and then at 37°C for 0–6 h. Although the mild hyperthermia treatment of the cells did not induce apoptosis, significant increases in the protein expression levels of heat shock proteins (HSPs), namely, Hsp27, Hsp40 and Hsp70, were observed following the activation of heat shock factor-1. Of the 22,283 probe sets analyzed, 423 probe sets were up-regulated and 515 probe sets were down-regulated by >1.5-fold in the cells 3 h post-treatment. Computational gene network analysis demonstrated that the significant genetic network A that contained many HSPs such as DNAJB1, HSPA1A, and HSPA1B was associated with cellular function and maintenance, post-transcriptional modification, or protein folding. Moreover, the significant genetic network B whose core contained v-myc myelocytomatosis viral oncogene homolog (MYC) was associated with cell morphology, cell cycle, and cellular development. The expression levels of nine selected genes were comparable to those determined by microarray analysis with real-time quantitative PCR assay. The present results indicate that mild hyperthermia affects the expression of a large number of genes and provides additional novel insights into the molecular basis of mild hyperthermia in cells.

Genetic networks responsive to low-intensity pulsed ultrasound in human lymphoma U937 cells.

To clarify the detailed molecular mechanism underlying cellular responses to nonthermal low-intensity pulsed ultrasound (LIPUS), gene expression patterns and genetic networks in human lymphoma U937 cells were examined using global-scale microarrays and computational gene expression analysis tools. Six hours after LIPUS treatment (0.3W/cm(2) for 1min), apoptosis (14+/-3.8%, mean+/-SD) without no cell lysis was observed. Of 22,283 probe sets analyzed, LIPUS down-regulated 193 genes and up-regulated 201 genes by >1.5-fold. For down-regulated genes, the significant genetic network D was associated with cellular growth and proliferation, gene expression, or cellular development. For up-regulated genes, the significant genetic network U was associated with cellular movement, cell morphology, and cell death. The present results indicate that LIPUS affects the expression of many genes and will provide novel insight into the biomolecular mechanisms of LIPUS in therapeutic application for cancer therapy.