Preventive and curative effects of radon inhalation on chronic constriction injury-induced neuropathic pain in mice.

BACKGROUND: Radon therapy is clinically useful for the treatment of pain-related diseases. However, there have been no studies regarding the effects of radon inhalation on neuropathic pain. In this study, we aimed to determine whether radon inhalation actually induced a remission of neuropathic pain and improved the quality of life. METHODS: First, we investigated the antinociceptive effects of radon inhalation in the chronic constriction injury (CCI) model of neuropathic pain. We evaluated pain behaviour in mice before and after CCI surgery, using von Frey test. Pretreated mice received CCI surgery immediately after 24-h inhalation of radon at background (BG) concentration (c. 19 Bq/m(3) ), or at a concentration of 1000 or 2000 Bq/m(3) , and post-treated mice inhaled similar levels of radon 2 days after CCI surgery. RESULTS: CCI surgery induced mechanical allodynia and hyperalgesia on a plantar surface of mice, as assessed using von Frey test, and 2000 Bq/m(3) radon inhalation alleviated hyperalgesic conditions 22-37% compared to BG level concentration. Concurrently, CCI surgery increased norepinephrine (NE), tumour necrosis factor-alpha (TNF-α) and nitric oxide (NO) concentrations in plasma, and leukocyte migration in paws. Furthermore, CCI-induced neuropathy reduced superoxide dismutase (SOD) activity. Treatment with radon inhalation, specifically at a concentration of 2000 Bq/m(3) , produced antinociceptive effects, i.e., lowered plasma TNF-α, NE and NO levels and restored SOD activity, as well as pain-related behaviour. CONCLUSIONS: This study showed that inhalation of 2000 Bq/m(3) radon prevented and alleviated CCI-induced neuropathic pain in mice.

Enhanced specificity of HPV16 E6E7 siRNA by RNA-DNA chimera modification.

Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecific cytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1-8 and its complementary part of the passenger strand plus the 3' overhang, which is designated as a double-strand RNA-DNA chimera (dsRDC). In this study, we found that the specificity of potent siRNAs targeting human papillomavirus 16 (HPV16) E6 and E7 oncogenes, which we previously reported, could be enhanced by short dsRDC modification (first six nucleotides from the 5' end of the GS and its complementary nucleotides of the passenger strand). Such dsRDC modification reduced nonspecific cytotoxicity in two of three siRNAs (497 and 752), although not in the other (573), which correlated with their off-target effects. In addition, silencing activity was marginally impaired in two dsRDCs (497 and 573) and moderately in one (752). Finally, dsRDC-497 induced E6E7-specific growth suppression of cervical cancer cells as well as E6E7-immortalized human keratinocytes. Our results show that dsRDC modification enhances the specificity of E6E7 siRNA, which is required for use in in vivo settings.

Granulomatous prostatitis due to Cryptococcus neoformans: diagnostic usefulness of special stains and molecular analysis of 18S rDNA.

A 57-year-old Japanese man complained of pain on micturition. The prostate was of normal size but hard. Transrectal needle biopsy demonstrated granulomatous prostatitis with small focal abscesses. Staining with periodic acid-Schiff, Grocott's methenamine silver and Fontana-Masson revealed yeast-form fungus in the granulomas. The mucoid capsule of the fungus stained with mucicarmine. PCR specific for cryptococcal 18S rDNA using DNA extracted from the pathological specimen was positive, and the sequence was homologous to Cryptococcus neoformans. A diagnosis of cryptococcal granulomatous prostatitis was made. The patient was then found to suffer from meningitis and lung abscess, and was treated with amphotericin B and flucytosine. Careful histological and molecular studies are beneficial to reach the correct diagnosis and to prevent an unfavorable outcome of disseminated cryptococcosis.

New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer.

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.

Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA.

Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV small-interfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPV-positive cervical cancer.

Search for cosmic-ray antideuterons.

We performed a search for cosmic-ray antideuterons using data collected during four BESS balloon flights from 1997 to 2000. No candidate was found. We derived, for the first time, an upper limit of 1.9 x 10(-4) (m2s sr GeV/nucleon)(-1) for the differential flux of cosmic-ray antideuterons, at the 95% confidence level, between 0.17 and 1.15 GeV/nucleon at the top of the atmosphere.

Influence of apolipoprotein E genotype on the response to caloric restriction in type 2 diabetic patients with hyperlipidaemia.

AIMS: Hyperlipidaemia is a major predisposing factor to atherosclerosis in patients with type 2 diabetes. Apolipoprotein (apo) E polymorphism influences lipoprotein metabolism, and the present study was undertaken to explore the relation, in type 2 diabetics, between apo E genotype and the plasma lipid response to dietary therapy. METHODS: The subjects were 104 patients with type 2 diabetes and hyperlipidaemia, and the difference, due to apo E genotype, in dietary response was followed for 4-6 weeks. The caloric intake was maintained in the range 20-25 kcal/kg, and the medications for diabetes were not changed during the follow-up period. RESULTS: Plasma total cholesterol (TC) and triglyceride (TG) levels were significantly lowered by the dietary treatment in patients with apo E genotypes of epsilon3/3, epsilon2/3 and epsilon3/4; however, the lipid levels in patients with epsilon2/4 did not respond to the diet. CONCLUSIONS: apo E genotype should be taken into consideration in the treatment of hyperlipidaemia in diabetic patients.

Measurements of cosmic-ray low-energy antiproton and proton spectra in a transient period of solar field reversal.

The energy spectra of cosmic-ray low-energy antiprotons ( *p's) and protons ( p's) have been measured by BESS in 1999 and 2000, during a period covering reversal at the solar magnetic field. Based on these measurements, a sudden increase of the *p/p flux ratio following the solar magnetic field reversal was observed, and it generally agrees with a drift model of the solar modulation.

Isolation of X and Y chromosome-specific DNA markers from a liverwort, Marchantia polymorpha, by representational difference analysis.

The liverwort Marchantia polymorpha has X and Y chromosomes in the respective female and male haploids. Here we report the successful exploitation of representational difference analyses to isolate DNA markers for the sex chromosomes. Two female-specific and six male-specific DNA fragments were genetically confirmed to originate from the X and Y chromosomes, respectively.

Plasma levels of soluble vascular adhesion molecule-1 and cholesterol oxidation product in type 2 diabetic patients with nephropathy.

Functional impairment of the vascular endothelium is an early event in the development of atherosclerosis, and soluble adhesion molecules in plasma are regarded as an indicator of the endothelial damage in diabetes mellitus. We compared the soluble vascular adhesion molecule levels in the patients with diabetic nephropathy in concerning with plasma 7-ketocholesterol levels, which is major cholesterol auto-oxidation products. Average value of plasma VCAM-1 in 31 patients with type 2 diabetes mellitus was 297.6+/-10.2 ng/ml (mean+/-SE), and the value was significantly higher than that in 8 age-matched healthy controls (231.9+/-15.0 ng/ml). Among the 31 diabetic patients, the group with macroalbuminuria (n = 8) had the higher levels of plasma VCAM-1 (349.5+/-26.0 ng/ml) than the levels in the group with normoalbuminuria (n=15; 280.6+/-12.3 ng/ml). The levels of plasma 7-ketocholesterol in diabetes (26.9+/-1.5 ng/ml) or the patients with macroalbuminuria (31.4+/-3.3 ng/ml) were significantly higher than the control (22.5+/-1.8 ng/ml). The level of soluble VCAM-1 showed significant correlation between the values of 7-ketocholesterol (r=0.42, p=0.024), TC (r=0.42, p=0.014) and LDL-C (r=0.38, p=0.044). However no correlation was demonstrated with HbA1c nor creatinine level. We conclude that soluble VCAM-1 in plasma may be an indicator of oxidative stress and vascular injury in diabetic nephropathy.

The Y chromosome in the liverwort Marchantia polymorpha has accumulated unique repeat sequences harboring a male-specific gene.

The haploid liverwort Marchantia polymorpha has heteromorphic sex chromosomes, an X chromosome in the female and a Y chromosome in the male. We here report on the repetitive structure of the liverwort Y chromosome through the analysis of male-specific P1-derived artificial chromosome (PAC) clones, pMM4G7 and pMM23-130F12. Several chromosome-specific sequence elements of approximately 70 to 400 nt are combined into larger arrangements, which in turn are assembled into extensive Y chromosome-specific stretches. These repeat sequences contribute 2-3 Mb to the Y chromosome based on the observations of three different approaches: fluorescence in situ hybridization, dot blot hybridization, and the frequency of clones containing the repeat sequences in the genomic library. A novel Y chromosome-specific gene family was found embedded among these repeat sequences. This gene family encodes a putative protein with a RING finger motif and is expressed specifically in male sexual organs. To our knowledge, there have been no other reports for an active Y chromosome-specific gene in plants. The chromosome-specific repeat sequences possibly contribute to determining the identity of the Y chromosome in M. polymorpha as well as to maintaining genes required for male functions, as in mammals such as human.

A novel differentiation-inducing therapy for acute promyelocytic leukemia with a combination of arsenic trioxide and GM-CSF.

Arsenic trioxide (As2O3) effectively induces clinical remission via apoptosis in relapsed acute promyelocytic leukemia (APL). However, because this new anti-leukemic drug is also considered to be a poison, its possible adverse effects are a highly important issue related to its clinical use. We here investigated, both in vitro and in vivo, the effects of a combination of As2O3 and GM-CSF as a novel therapeutic approach for the treatment of APL. Treatment of both retinoic acid (RA)-sensitive and -resistant APL cell lines (NB4 and UF-1 cells, respectively), as well as primary APL cells with a combination of As2O3 and GM-CSF for 4 days resulted in inducing differentiation, but not apoptosis, to mature granulocytes. In addition, a combination of both agents induced degradation of the PML/RARalpha protein. GM-CSF was found to be associated with increased tyrosine phosphorylation of Jak2 kinase in both NB4 and UF-1 cells, and a specific inhibitor of Jak2, AG490, completely blocked the ability of GM-CSF to prevent apoptosis and induce differentiation of As2O3-treated UF-1 cells. In in vivo analysis, As2O3 induced differentiation of APL cells in a RA-resistant APL model of human GM-CSF-producing transgenic SCID mice that had a high level of human GM-CSF in their sera. In contrast, As2O3 alone diminished tumors in UF-1 cells transplanted into NOD/SCID mice via induction of apoptosis. In conclusion, a combination of As2O3 and GM-CSF appears to be a novel differentiation-inducing therapy in patients with APL, including relapsed or RA-resistant cases.

Activation of the p21(CIP1/WAF1) promoter by bone morphogenetic protein-2 in mouse B lineage cells.

BMPs exert a negative growth effect on various types of cells. We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21(CIP1/WAF1) expression. In the present study, we demonstrated that BMP-2 activated the mouse p21(CIP1/WAF1) promoter in HS-72 cells, and that a 29-base pair (b) region of the promoter (-1928/-1900 relative to the TATA box), conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors. Furthermore, an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2-stimulated HS-72 cells. These results suggested that BMP-2 might activate p21(CIP1/WAF1) transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells.

The "botryoid sign": a characteristic feature of rhabdomyosarcomas in the head and neck.

We investigated nine patients with rhabdomyosarcoma in the head and neck (6-53 years of age), using CT and MRI. The tumours originated in the paranasal sinuses (3), cheek (2), soft palate (1), orbit (1), sternocostoclavicular muscle (1) and parapharyngeal space (1). The histological subtype was embryonal in five, alveolar in three and pleomorphic in one case. The tumours enhanced markedly and heterogeneous on CT and MRI. The masses were isointense or gave slightly higher signal than surrounding muscles on T1- and heterogeneously high signal on T2-weighted images. In four tumours, multiple ring enhancement resembling bunches of grapes. This appears to be characteristic of rhabdomyosarcoma and probably reflects a component of botryoid-type rhabdomyosarcoma in which mucoid-rich stroma is covered with a thin layer of tumour cells. We have named this imaging feature the "botryoid sign".

Mcl-1, an early-induction molecule, modulates activin A-induced apoptosis and differentiation of CML cells.

Activin A, one member of the transforming growth factor (TGF)-beta superfamily, is known to be a commitment factor for cell death and differentiation. In the present study, we demonstrate that human chronic myeloid leukemia (CML) cell lines, KU812 and K562 cells, either induced apoptosis or differentiation, respectively, by treatment with activin A. During these cell fate decisive events caused by activin A, rapid and transient up-regulation of Mcl-1 was observed in both cell lines. In activin A-induced apoptosis of KU812 cells, continuous up-regulation of Bax was observed. After the decrease in Mcl-1 expression had occurred, activation of caspase-9 and caspase-3 and cleavage of DFF45 were shown to take place in KU812 cells, resulting in the fragmentation of the genomic DNA of the cells. In contrast, the down-regulation of Mcl-1 without up-regulation of Bax caused accumulation of hemoglobin (Hb) contents in activin A-treated K562 cells. Interestingly, erythropoietin (EPO) prevented activin A-induced apoptosis with continuous expression of Mcl-1 and caused KU812 cells to undergo erythroid differentiation. To address the role of Mcl-1 in activin A-treated CML cells, KU812 and K562 cells were stably transfected with cDNA encoding Mcl-1 (designated as KU812/mcl and K562/mcl cells). As in combined effect of activin A and EPO on the parental KU812 cells, activin A induced differentiation, but not apoptosis, of KU812/mcl cells without modulating Bax levels. Activin A-treated K562/mcl cells, as well as parental cells, were only differentiated to erythroid cells. These results suggest that Mcl-1 is an early inducible gene activated by the activin A signaling pathway for both cellular differentiation and apoptosis, and continuous expression of Mcl-1 may be contributed to differentiation signals to the erythroid lineage in CML cells.

An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endothelial cells in culture.

Treatment of human umbilical vein endothelial cells (HUVECs) with 7-ketocholesterol resulted in an increased release of soluble vascular cell adhesion molecule-1 (VCAM-1) into culture medium. 7-Ketocholesterol did not enhance the expression of mRNA for VCAM-1. 7 beta-Hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels. Western blot analysis revealed that soluble VCAM-1, in the conditioned medium of both 7-ketocholesterol-stimulated and control cells, had a molecular size of 100 kDa. Stimulation of the TNF-alpha-treated HUVECs with 7-ketocholesterol further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA level, which was enhanced by TNF-alpha. Pretreatment of the cells with tissue inhibitor of membrane metalloproteinase-2 (TIMP-2) completely inhibited the release of VCAM-1 in response to 7-ketocholesterol but TIMP-1 had no effect. Adherence of mononuclear cells to TNF-stimulated HUVEC monolayers was slightly inhibited by 7-ketocholesterol, but this oxysterol did not affect the basal adherence to non-stimulated HUVECs. Immunofluorescent staining of the cells confirmed diffuse perinuclear distribution of VCAM-1 in HUVECs treated with TNF-alpha, but 7-ketocholesterol did not affect the intensity or distribution of immunofluorescence. We conclude that 7-ketocholesterol releases VCAM-1 from the endothelium probably by a proteolytic process.

Osteoclast differentiation is associated with transient upregulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1).

Osteoclasts, bone-resorbing multinucleated cells, develop from monocyte-macrophage lineage cells in the presence of osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) and macrophage colony-stimulating factor (M-CSF). M-CSF-dependent bone marrow macrophages (M-BMMPhis) from mouse bone marrow cells have been shown to differentiate into osteoclast-like multinucleated cells (OCLs) in the presence of soluble ODF/RANKL (sODF/RANKL) and M-CSF within 3 days. In this study, we found that stimulation of M-BMMPhis with sODF/RANKL induced a transient expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21(WAF1/CIP1) and p27(KIP1) by 24 h. The CDK inhibitor proteins disappeared by 48 h. Tumor necrosis factor alpha (TNF-alpha), which is reported to stimulate OCL differentiation, stimulated p21(WAF1/CIP1) and p27(KIP1) expression in M-BMMPhis as well. However, M-CSF alone did not stimulate the expression of the two CDK inhibitors. To clarify the role of p21(WAF1/CIP1) and p27(KIP1) in osteoclastogenesis, accumulation of these CDK inhibitors was aborted by antisense oligonucleotides. Treatment with p21(WAF1/CIP1) antisense oligonucleotide alone, or p27(KIP1) antisense oligonucleotide alone, showed a limited inhibitory effect on OCL formation. However, treatment with a mixture of these two antisense oligonucleotides strongly inhibited OCL formation. These results suggest that a combined modulation of the CDK inhibitors p21(WAF1/CIP1) and p27(KIP1) may be involved in osteoclast differentiation induced by ODF/RANKL.

Intracellular apoptosis-inducing factor is induced by a vacuolar type H+-ATPase inhibitor in B lineage cells.

We previously reported that concanamycin A, a specific inhibitor of vacuolar type H+-ATPases, induces DNA fragmentation in B cell hybridoma HS-72 cells. In the present study, we found that the cytosol from concanamycin A-treated HS-72 cells had a cytotoxic effect on intact cells in a cell viability assay. While activin A also induced apoptosis in HS-72 cells, the cytosol from activin A-treated HS-72 cells had no effect on cell viability. We purified the cytosol from concanamycin A-treated HS-72 cells by a four-step procedure: ultracentrifugation; HiTrap heparin column chromatography; HiTrap Q column chromatography; and reverse-phase high performance liquid chromatography on a C18 hydrophobic support. The biologically active fraction, which was used as partially purified cytosol, gave a specific band of protein with a molecular mass of 33 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis in cells cultured with the partially purified cytosol. The overexpression of human Bcl-2 suppressed apoptosis, indicating that the cytosol from concanamycin A-treated HS-72 cells induces apoptosis by a Bcl-2-inhibiting mechanism. These findings suggest that concanamycin A, a vacuolar type H+-ATPase inhibitor, produces intracellular apoptosis-inducing factor in B cell hybridoma.

Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha.

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.

Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L.

Thalli of the haploid liverwort Marchantial polymorpha were successfully used for direct particle bombardment with plasmid pMT, which carries a hygromycin phosphotransferase gene (hpt) controlled by the CaMV 35S promoter and the NOS polyadenylation region. Hygromycin-resistant cell masses arose from the thallus surface and developed directly into hygromycin-resistant thalli. Southern blot analyses indicated that these thalli carried at least 1-4 copies of the hpt gene, which were stably transmitted to their asexual thallus progenies via gemma propagation for three generations. This transformation and direct plant regeneration protocol is expected to be a valuable tool for the molecular analysis of this lower land plant.