The effects of vanilloid analogues structurally related to capsaicin on the transient receptor potential vanilloid 1 channel.

Transient receptor potential vanilloid 1 (TRPV1) is known as a receptor of capsaicin, a spicy ingredient of chili peppers. It is also sensitive to a variety of pungent compounds and is involved in nociception. Here, we focused on the structural characteristics of capsaicin, and investigated whether vanillylmanderic acid (VMA), vanillic acid (VAcid), vanillyl alcohol (VAlc), vanillyl butyl ether (VBE), and vanillin, containing a vanillyl skeleton similar to capsaicin, affected the TRPV1 activities. For detection of TRPV1 activity, intracellular Ca2+ concentration ([Ca2+]i) was measured in HEK 293 cells heterologously expressing mouse TRPV1 (mTRPV1-HEK) and in mouse sensory neurons. Except for vanillin, four vanilloid analogues dose-dependently increased [Ca2+]i in mTRPV1-HEK. The solutions that dissolved VMA, VAcid and vanillin at high concentrations were acidic, whereas those of VAlc and VBE were neutral. Neutralized VAcid evoked [Ca2+]i increases but neutralized VMA did not. Mutation of capsaicin-sensing sites diminished [Ca2+]i responses to VAcid, VAlc and VBE. VAcid, VMA, and vanillin suppressed the activation of TRPV1 induced by capsaicin. VAcid and VMA also inhibited the acid-induced TRPV1 activation. In sensory neurons, VMA diminished TRPV1 activation by capsaicin or acids. The present data indicate that these structural characteristics of chemical compounds on TRPV1 may provide strategies for the development of novel analgesic drugs targeting nociceptive TRPV1.

The difference in joint instability affects the onset of cartilage degeneration or subchondral bone changes.

OBJECTIVE: It has been debated whether the onset of knee osteoarthritis is initiated in cartilage or subchondral bone. The purpose of this study was to clarify the effects of increasing or decreasing joint instability on cartilage degeneration and subchondral bone changes in knee OA by comparing different models of joint instability. DESIGN: We used the anterior cruciate ligament transection (ACL-T) model and the destabilization of the medial meniscus (DMM) model. In addition, we created a controlled abnormal tibial translation (CATT) model and a controlled abnormal tibial rotation (CATR) model. We performed joint instability analysis, micro-computed tomography analysis, histological and immunohistological analysis in 4 and 6 weeks. RESULTS: The CATT group suppressed joint instability in the ACL-T group (6 weeks; P = 0.032), and the CATR group suppressed joint instability in the DMM group (6 weeks; P = 0.032). Chondrocyte hypertrophy in the ACL-T and DMM groups was increased compared to the Sham group (6 weeks; [ACL-T vs Sham], P = 0.002, 95%CI [5.983-33.025]; [DMM vs Sham], P = 0.022, 95%CI [1.691-28.733]). In the subchondral bone, the BV/TV in the DMM and CATR groups was increased compared to the ACL-T and CATT groups (6 weeks; [DMM vs ACL-T], P = 0.002, 95%CI [7.404-37.582]; [DMM vs CATT], P = 0.014, 95%CI [2.881-33.059]; [CATR vs ACL-T], P = 0.006, 95%CI [4.615-34.793]; [CATR vs CATT], P = 0.048, 95%CI [0.092-30.270]). CONCLUSIONS: This study showed that joint instability promotes chondrocyte hypertrophy, but subchondral bone changes were influenced by differences in ACL and meniscus function.

Controlling joint instability after anterior cruciate ligament transection inhibits transforming growth factor-beta-mediated osteophyte formation.

OBJECTIVE: Abnormal joint instability contributes to cartilage damage and osteophyte formation. We investigated whether controlling joint instability inhibited chronic synovial membrane inflammation and delayed osteophyte formation and examined the role of transforming growth factor-beta (TGF-ß) signaling in the associated mechanism. DESIGN: Rats (n = 94) underwent anterior cruciate ligament (ACL) transection. Anterior tibial instability was either controlled (CAM group) or allowed to continue (SHAM group). At 2, 4, and 8 weeks after surgery, radiologic, histopathologic, immunohistochemical, immunofluorescent, and enzyme-linked immunosorbent assay examinations were performed to evaluate osteophyte formation and TGF-ß signaling. RESULTS: Joint instability increased cartilage degeneration score and osteophyte formation, and cell hyperplasia and proliferation and synovial thickening were observed in the synovial membrane. Major findings were increased TGF-ß expression and Smad2/3 following TGF-ß phosphorylation in synovial membarene, articular cartilage, and the posterior tibial growth plate (TGF-ß expression using ELISA: 4 weeks; P = 0.009, 95% CI [260.1-1340.0]) (p-Smad2/3 expression density: 4 weeks; P = 0.024, 95% CI [1.67-18.27], 8 weeks; P = 0.034, 95% CI [1.25-25.34]). However, bone morphogenetic protein (BMP)-2 and Smad1/5/8 levels were not difference between the SHAM model and the CAM model. CONCLUSIONS: This study showed that the difference between anterior tibial instability caused a change in the expression level of TGF in the posterior tibia and synovial membrane, and the reaction might be consequently involved in osteophyte formation.

Myeloperoxidase in blood neutrophils during normal and abnormal menstrual cycles in women of reproductive age.

INTRODUCTION: We previously reported that granulocyte colony-stimulating factor (G-CSF) plays a critical role in ovulation, suggesting that neutrophils may maintain ovulation. We assessed myeloperoxidase (MPO), a major and specific enzyme of neutrophils, in women with abnormal and normal menstrual cycles to clarify the relationship between MPO and ovulation. METHODS: We analyzed MPO activity in blood neutrophils of women with abnormal menstrual cycles (indicative of anovulation, n = 12) and age- and body mass index-matched normal menstrual cycles (indicative of ovulation, n = 24) using two parameters as a marker of MPO, Neut X and mean peroxidase index (MPXI). RESULTS: MPO of women with abnormal menstrual cycles was significantly lower than that of women with normal menstrual cycles [Neut X: 62.6 ± 1.1 (mean ± standard error of the mean) vs. 66.2 ± 0.3, P = 0.009; MPXI: -0.54 ± 1.66 vs. 4.91 ± 0.53, P = 0.008]. Among women with normal menstrual cycles, MPO was highest in the follicular phase (Neut X: 67.0 ± 0.3; P = 0.033). CONCLUSION: The difference in MPO between women with abnormal and normal menstrual cycles and the upregulation of MPO before ovulation suggest that neutrophils and MPO are closely related to ovulation.

Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

Extracted ion current density in close-coupling multi-antenna type radio frequency driven ion source: CC-MATIS.

Positive ions are extracted by using a small extractor from the Close-Coupling Multi-Antenna Type radio frequency driven Ion Source. Two types of RF antenna are used. The maximum extracted ion current density reaches 0.106 A/cm(2). The RF net power efficiency of the extracted ion current density under standard condition is 11.6 mA/cm(2)/kW. The efficiency corresponds to the level of previous beam experiments on elementary designs of multi-antenna sources, and also to the efficiency level of a plasma driven by a filament in the same chamber. The multi-antenna type RF plasma source is promising for all metal high density ion sources in a large volume chamber.

In vivo eradication of MLL/ENL leukemia cells by NK cells in the absence of adaptive immunity.

It remains unclear how the immune system affects leukemia development. To clarify the significance of the presence of immune systems in leukemia development, we transferred MLL/ENL leukemia cells into immune-competent or immune-deficient mice without any preconditioning including irradiation. The wild-type mice did not develop leukemia, whereas all the Rag2(-/-)γc(-/-) mice lacking both adaptive immune cells and natural killer (NK) cells developed leukemia, indicating that leukemia cells were immunologically rejected. Interestingly, leukemia cells were also rejected in 60% of the Rag2(-/-) mice that lacked adaptive immune cells but possessed NK cells, suggesting that NK cells play a substantial role in the rejection of leukemia. Moreover, engraftment of leukemia cells was enhanced by NK cell depletion in Rag2(-/-) recipients and inhibited by transfer of NK cells into Rag2(-/-)γc(-/-) recipients. Upregulation of NKG2D (NK group 2, member D) ligands in MLL/ENL leukemia cells caused elimination of leukemia cells by NK cells. Finally, we found that leukemia cells resistant to elimination by NK cells had been selected during leukemia development in Rag2(-/-) recipients. These results demonstrate that NK cells can eradicate MLL/ENL leukemia cells in vivo in the absence of adaptive immunity, thus suggesting that NK cells can play a potent role in immunosurveillance against leukemia.

Expression and putative function of kisspeptins and their receptors during early development in medaka.

Kisspeptins (Kiss1 and Kiss2) and their receptors (putatively Gpr54-1 and Gpr54-2) have emerged as key players in vertebrate reproduction owing to their stimulatory effect on the brain-pituitary-gonadal axis. Virtually nothing is known, however, about their role during embryogenesis. Using medaka (Teleostei) as a model system, we report, for the first time in vertebrates, an early developmental expression and putative function of kisspeptins. Expression analyses and knockdown experiments suggest that early actions of kisspeptins are probably mediated by binding to maternally supplied Gpr54-1 and late action by both Gpr54-1 and Gpr54-2. Knockdown of maternally provided kiss1 and gpr54-1 arrested development during gastrulation, before establishment of any germ layers, whereas knockdown of zygotically provided kiss1 and gpr54-1 disrupted brain development. A similar phenotype was observed for gpr54-2 knockdown embryos, suggesting a critical role for kiss1, gpr54-1, and gpr54-2 in neurulation. These data demonstrate that kisspeptin signaling is active both maternally and zygotically and is involved in embryonic survival and morphogenesis.

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients.

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

A close-coupling multi-antenna type radio frequency driven ion source.

A newly close coupling multi-antenna type radio frequency driven ion source is tested for the purpose of essentially improving plasma coupling on the basis of our old type ion source, which reuses a NNBI (negative ion source for neutral beam injection) ion source used in 1∕5th scale of the Large Helical Device NNBI. The ion source and the antenna structure are described, and the efficient plasma production in terms of the positive ion saturation current (the current density) is studied. The source is made of a metal-walled plasma chamber which is desirable from the point of view of the structural toughness for fusion and industrial application, etc. At around 160 kW of rf input power, the ion saturation current density successfully reaches the 5 A∕cm(2) level with a gas pressure of 0.6-2 Pa in hydrogen for 10 ms pulse duration. The rf power efficiency of the plasma production with a close coupling configuration of the antenna is improved substantially compared to that with the previous antenna unit in the old type ion source. The power efficiency is assessed as competing with that of other types of sources.

Steroid sensitive kiss2 neurones in the goldfish: evolutionary insights into the duplicate kisspeptin gene-expressing neurones.

The KISS1/Kiss1/kiss1 gene product kisspeptin is suggested to be involved in the steroid feedback system in vertebrates. In addition to kiss1, kiss2 has been identified in many vertebrates, including some mammals, suggesting that the both genes were originally expressed in the common ancestor of teleosts and tetrapods. Moreover, peptides from both genes have been shown to activate the kisspeptin receptors. To investigate the involvement of kiss1 or kiss2 neurones in steroid feedback, we used a seasonal breeder, the goldfish (Carassius auratus). We found that kiss2 is expressed in the preoptic area (POA), nucleus lateralis tuberis and nucleus recessus lateralis, and that kiss1 is expressed in the habenula. Greater mRNA expression in breeding than in nonbreeding condition animals and conspicuous up-regulation of gene expression by gonadal steroids was seen only in the kiss2 neurones of the POA. Furthermore, double in situ hybridisation suggested that these neurones express oestrogen receptors. Given that amphibians express kiss2 in POA and mammalian anteroventral periventricular nucleus/POA Kiss1 neurones show similar expression dynamics as goldfish POA Kiss2 neurones, we hypothesise that kiss1 and kiss2 share the same evolutionary origin; and, after the loss of kiss2, kiss1 became active for steroid feedback in mammals.

Effects of losartan/hydrochlorothiazide on serum uric acid levels and blood pressure in hypertensive patients.

The effect of a mixed formulation of 50 mg losartan (LOS) and 12.5 mg hydrochlorothiazide (HCTZ) on blood pressure and the uric acid metabolism was analyzed in 73 patients who switched to this formulation from other antihypertensive drugs. Eight patients who switched to the formulation from the regular dose of renin-angiotensin (RA) inhibitor (angiotensin receptor blocker [ARB] or angiotensin-converting enzyme [ACE] inhibitor) only showed a significant decrease in blood pressure, from 156.9 ± 14.1/88.6 ± 9.7 mmHg to 128.3 ± 16.0/76.1 ±10.7 mmHg (p = 0.007), and a significant increase in serum uric acid levels, from 5.2 ± 1.1 mg/dL to 6.8 ± 0.7 mg/dL (p = 0.02). In the other 50 patients who switched from a combination of the regular dose of RA inhibitor and calcium channel blocker (CCB), their blood pressure significantly increased, from 126.0 ± 13.8/72.0 ± 10.0 mmHg to 132.5 ± 16.4/76.5 ± 11.3 mmHg (p = 0.02), and their serum uric acid levels also significantly increased, from 5.6 ± 1.1 mg/dL to 6.1 ± 1.3 mg/dL (p = 0.0002). Considering that guidelines recommend using antihypertensive therapies that do not lead to an increase in serum uric acid levels, we conclude that using the ARB/HCTZ combination is less suitable than the regular dose of the ARB/CCB combination due to its effect on hypertension and serum uric acid levels.

A single-stranded DNA-cross-reactive immunogenic epitope of human homocysteine-inducible endoplasmic reticulum protein.

The mechanism involved in generating anti-DNA antibodies (Abs) remains unclear, as DNA is poorly immunogenic. Molecular mimicry between DNA and non-DNA substances has been implicated as a possible mechanism. We previously reported that homocysteine-inducible endoplasmic reticulum protein (Herp), which is induced by endoplasmic reticulum stress, is recognized by anti-double-stranded DNA (dsDNA) IgG from patients with systemic lupus erythematosus and that immunization with Herp elicits anti-dsDNA Abs in BALB/c mice. In this study, we observed that anti-single-stranded DNA (ssDNA) Abs were also generated in Herp-immunized BALB/c mice and established an anti-Herp monoclonal antibody (mAb), HT4, which specifically cross-reacted with ssDNA. The epitope of the HT4 mAb on Herp, 'EPAGSNR', was identified by screening a synthetic peptide library. The binding of the HT4 mAb to the peptide was competitively inhibited by ssDNA. Immunization of the epitope peptide elicited anti-ssDNA Abs in BALB/c mice. These results indicate that the epitope exists in a human self-protein, mimics ssDNA and shows antigenicity for anti-ssDNA Abs in normal mice. Anti-ssDNA Abs are often found in patients with drug-induced lupus erythematosus. Treatment with representative drugs that cause drug-induced lupus (chlorpromazine, procainamide and hydralazine) induced Herp expression and apoptosis in HeLa cells. These findings suggest that molecular mimicry between Herp and ssDNA is involved in anti-ssDNA Ab production in drug-induced lupus.

Expression of vesicular glutamate transporter-2.1 in medaka terminal nerve gonadotrophin-releasing hormone neurones.

There are three paralogous genes for gonadotrophin-releasing hormone (GnRH) peptides of vertebrates in general. GnRH1, the protein product of gnrh1 gene, is the hypophysiotrophic neuropeptide, and is a critical regulator of gonadotrophin secretion, whereas GnRH2 and GnRH3 are regarded to have neuromodulatory functions. In some teleost species, the terminal nerve (TN) GnRH3 neuronal system, which expresses GnRH3, has been shown to project extensively throughout the brain and regulate the motivational state for some behavioural repertoires. In recent years, it has been considered that most, if not all, peptidergic and aminergic neurones synthesise and release more than one neurotransmitter, and the cotransmission of conventional small-molecule neurotransmitters, such as GABA, glutamate or acetylcholine together with neuropeptides, is regarded as a common feature of such neurones. For a functional characterisation of the GnRH3 neuronal system, we examined the possible co-expression of conventional neurotransmitters, GABA, acetylcholine and glutamate, in addition to GnRH in the TN-GnRH3 neurone by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridisation of recently identified marker genes for neurotransmitters using a teleost fish medaka (Oryzias latipes). By RT-PCR and dual-label in situ hybridisation, we demonstrated the co-expression of GnRH3 and vesicular transporter for glutamate (VGluT) 2.1. in a single TN-GnRH3 neurone. We therefore suggest that the TN-GnRH3 neurones use glutamate as a cotransmitter of GnRH.

Sex differences in aromatase gene expression in the medaka brain.

The brain of teleost fish exhibits a significant degree of sexual plasticity, even in adulthood. This unique feature is almost certainly attributable to a teleost-specific sexual differentiation process of the brain, which remains largely unknown. To dissect the molecular basis of sexual differentiation of the teleost brain, we searched for genes differentially expressed between both sexes in the medaka brain. One gene identified in the screen, cyp19a1b, which encodes the steroidogenic enzyme aromatase, was selected for further analysis. As opposed to the situation in most vertebrates, medaka cyp19a1b is expressed at higher levels in the adult female brain than the male brain. The female-biased expression in the brain is consistent regardless of reproductive or diurnal cycle. Medaka cyp19a1b is expressed throughout the ventricular zones in wide areas of the brain, where, in most regions, females have a greater degree of expression compared to males, with the optic tectum exhibiting the most conspicuous predominance in females. Contrary to what is known in mammals, cyp19a1b expression exhibits neither a transient elevation nor a sex difference in medaka embryos. It is not until just before the onset of puberty that cyp19a1b expression in the medaka brain is sexually differentiated. Finally, cyp19a1b expression in the medaka brain is not under the direct control of sex chromosome genes but relies mostly, if not solely, on oestrogen derived from the gonad. These unique properties of aromatase expression in the brain probably contribute substantially to the less rigid sexual differentiation process, thus ensuring remarkable sexual plasticity in the teleost brain.

Functional and evolutionary insights into vertebrate kisspeptin systems from studies of fish brain.

The kiss1 gene product kisspeptin is now considered to be an essential regulator of the hypothalamic-pituitary-gonadal (HPG) axis in most vertebrate species. Recent findings in fishes are beginning to set a new stage for the kisspeptin study; the existence of paralogous kisspeptin genes as well as kisspeptin receptor (formerly called GPR54) genes has quite recently been reported in several fish and amphibian species. The fishes may provide excellent animal models for the study of general principles underlying the kisspeptin and kisspeptin receptor systems of vertebrates from the evolutionary viewpoint. Unlike placental and marsupial mammalian species mainly studied so far, many teleost species have two paralogous genes of kisspeptin, kiss1 and kiss2. Medaka, Oryzias latipes, in which kiss1 and kiss2 are expressed in distinctive hypothalamic neuron populations, is a good model system for the study of central regulation of reproduction. Here, the kiss1 system but not the kiss2 system shows expression dynamics strongly indicative of its direct involvement in the HPG axis regulation via its actions on GnRH1 neurons. On the other hand, the kiss1 gene is missing, and only kiss2 is expressed in some fish species. Also, there are some recent reports that Kiss2 peptide may be a potent regulator of reproduction in some fish species. The ancestral vertebrate probably already had two paralogous kiss genes, and their main function was the HPG axis regulation. In the species that retained both paralogues during evolution, either Kiss1 or Kiss2 predominantly retains its ability for the HPG axis regulation, while the other may assume new non-reproductive functions (neofunctionalization). Alternatively, both the paralogues may assume complementary functions in the HPG axis regulation (subfunctionalization). After the divergence of teleost and tetrapod lineages, either one of the two paralogues, or even both in birds, have been lost (degradation) or became a pseudogene (non-functionalization), but the remaining paralogue retained its original function of HPG axis regulation. The identification of multiple forms of kisspeptin receptors and the rather promiscuous ligand-receptor relationships has led to the further proposal that such promiscuousness may be the basis for the functional robustness of kisspeptin and kisspeptin receptor systems in the HPG axis regulation, when one or both paralogous genes are lost or functionally partitioned during evolution.