LPA5 signaling is involved in multiple sclerosis-mediated neuropathic pain in the cuprizone mouse model

Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain.     

Push-Pull Controlled Drug Release Systems: Effect of Molecular Weight of Polyethylene Oxide on Drug Release

First, an elementary osmotic pump (EOP) with a simple structure was prepared using polyethylene oxide (PEO) and NaCl as an excipient, and the influence of the molecular weight (Mw) of PEO on drug release was investigated. In the dissolution test of EOP, it was observed that the gelated core tablet was pushed out through the orifice. The dissolution profile of EOP was sigmoidal, and despite the short time, a zero-order release region was observed. The gel swelling rate in the zero-order region was independent of the Mw of PEO. It was also found that higher the Mw of PEO, the larger the saturated swelling amount. Next, a push-pull pump (PPP) with almost identical formulation to that of EOP was prepared, and its drug release characteristics were investigated. PPPs were prepared by varying the combination of Mws of PEO in both layers, and their dissolution profiles were compared. It was found that PPP using a low-Mw PEO for the drug layer and PEO with a high-Mw in the push layer showed the longest dissolution profile of the linear region. The obtained findings suggested that the properties of PEO and its hydrogel play a crucial role in the drug release of PPP.     

In vitro evaluation of the activity of β-lactams,new quinolones,and other antimicrobial agents againstStreptococcus pneumoniae

The in vitro activities of the β-lactam and quinolone antibiotics panipenem, cefpodoxime, cefdinir, cefditoren, faropenem, tosufloxacin, levofloxacin and grepafloxacin were compared with similar conventional antibiotics against penicillin-resistantStreptococcus pneumoniae (PRSP). Pneumococcal isolates collected from October 1994 to March 1995 (n=1283) consisted of penicillin-susceptibleS. pneumoniae (PSSP; 59.2%), penicillin-intermediately-resistantS. pneumoniae (PISP;11.2%), and PRSP (29.6%). The isolates were highly susceptible to panipenem, faropenem and cefditoren with MIC₉₀ values of 0.125 μg/mL, 0.5 μg/mL and 0.5 μg/mL, respectively. Correlation coefficients for the relationships between the MICs of these β-lactam agents and that of penicillin G ranged from γ=0.7652 to γ=0.8022. These new β-lactam agents produced excellent bactericidal responses at concentrations greater than their MICs for PSSP concomitant with appropriate cellular morphologic changes. However, the bactericidal action of these antibiotics against PRSP was less pronounced and fewer instances of cell lysis were observed. The MIC₉₀ of cefpodoxime was similar to that of cefaclor, whereas that of cefdinir was between those of faropenem and cefpodoxime. The MIC distribution of the new quinolone agents showed 1 peak, but the MIC₉₀ values of tosufloxacin and grepafloxacin were both 0.5 μg/mL and that of levofloxacin was 2.0 μg/mL. Only 1% of all isolates demonstrated cross-resistance to all quinolone agents.     

Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes

Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu²⁺ inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 μM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1 h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.     

Epidemiological,clinical, and genetic landscapes of hypomyelinating leukodystrophies

To determine the epidemiological, clinical, and genetic characteristics of congenital hypomyelinating leukodystrophies, including Pelizaeus–Merzbacher disease (PMD), we conducted a nationwide epidemiological survey in Japan. A two-step survey targeting all medical institutions specializing in pediatric neurology and childhood disability (919 institutes) in Japan was performed. Detailed information was collected for 101 patients (86 males and 15 females) with congenital hypomyelinating leukodystrophies. The prevalence of congenital hypomyelinating disorders was 0.78 per 100,000 people (0–19 years old), and the incidence was 1.40 per 100,000 live births. Molecular testing was performed in 75 % of patients, and PLP1 gene abnormalities were observed in 62 %. The incidence of PMD with PLP1 mutations was estimated to be 1.45 per 100,000 male live births and that for congenital hypomyelinating disorders with unknown cause to be 0.41 per 100,000 live births. Patients with PLP1 mutations showed a higher proportion of nystagmus and hypotonia, both of which tend to disappear over time. Our results constitute the first nationwide survey of congenital hypomyelinating disorders, and provide the epidemiological, clinical, and genetic landscapes of these disorders.     

Steroid profile analysis and UGT2B17 genotyping of the same urine sample to determine testosterone abuse

When testing a urine sample for testosterone abuse, a ratio of testosterone glucuronide (T) to epitestosterone glucuronide (ET) of 4.0 or above is considered suspicious. A degree of variation, however, has been observed in T/ET ratio between individuals from both the same and different ethnic backgrounds. The majority of this variation might be due to UGT2B17 deletion genotype (UGT2B17 deletion-type). The aim of this study was to investigate the use of the same urine sample for the analysis of T/ET ratio and UGT2B17 deletion-type. Japanese men were deletion-typed via a UGT2B17 copy number assay using DNA from blood. Urinary T and ET levels were determined using gas chromatography–mass spectrometry before (n = 112) and after a testosterone injection (n = 25). Basal T level and the increase in T/ET ratio after injection were dependent on UGT2B17 deletion-type, being lower in subjects with deletion (del/del) than nondeletion (ins/del or ins/ins) genotype. UGT2B17 deletion-typing was first performed using DNA from urine cryopreserved for 1–1.5 years (n = 66). The concentration of DNA required for discrimination between the deletion and nondeletion genotype by copy number assay was more than 0.1 ng/ml urine. Discrimination was possible in 94.0 % of urine samples (5–7 ml each). These findings show that T/ET ratio and UGT 2B17 deletion-type can be analyzed exclusively via urine samples, removing the need for the collection of other samples, such as blood or buccal cells. The combination of T/ET ratio and UGT 2B17 deletion-type may help inform decisions regarding a genotype-specific T/ET cutoff ratio.     

Effect of prostaglandin E2 on urokinase‐type plasminogen activator production by human lung fibroblasts

Human lung fibroblasts are components of stromal tissue and produce various proteins as occasion demands, such as extracellular matrix (ECM) components and proteases. Pulmonary tumour cells produce high levels of prostaglandin E₂ (PGE₂), which regulates tumour growth and metastasis. Urokinase‐type plasminogen activator (uPA) is essential in the degradation of peritumour ECM. Furthermore, uPA is an important protease believed responsible for several tumour characteristics through its activation of certain proteases and growth factors. We hypothesized that the PGE₂ overexpression from tumour cells would have some effect on uPA expression in lung fibroblasts. In this study, the influence of PGE₂ on uPA expression in human lung fibroblasts was investigated using two lines of such fibroblasts. Although the cell surface uPA level was comparable to that of PGE₂ untreated cells, the expression of uPA mRNA and production was increased by the addition of PGE₂ in both lines of fibroblasts. These fibroblasts expressed both the EP₂ and EP₄ PGE₂ receptor mRNAs. Pretreatment with EP₂ and/or EP₄ receptor antagonists reduced the intercellular and cell surface uPA expression of the human lung fibroblasts. These results indicated that there is a relationship between the PGE₂ system and uPA production in human lung fibroblasts operating through EP₂ and/or EP₄ receptor signalling. uPA induced by PGE₂ from stromal fibroblasts surrounding lung tumour thus appears to play an important role through these EP receptors. Inhibition of EPs in tumour tissue might be a useful strategy for anti‐metastasis therapy.     

Pyoderma gangrenosum associated with paroxysmal nocturnal hemoglobulinuria and monoclonal gammopathy

Pyoderma gangrenosum developed in a man with a five-year history of paroxysmal nocturnal hemoglobinuria and monoclonal gammopathy. He had multiple walnut sized ulcers on his back and extremities, plasma IgM-k type M-protein and low erythrocytic CD55 expression. This is an extremely rare association. However, clonal expansion of plasma cells and chimeric expression of hematopoietic cell glycosylphosphatidylinositol (GPI)-anchored proteins may represent somatic mutations of hematopoietic stem cells in PG as well as PNH. PNH is based on abnormalities in the GPI-anchor formation on various hematopoietic and non-hematopoietic cells. Since the GPI-anchored proteins have pleiotropic functions in complement mediated cell lysis, leukocyte motility, and coagulation systems, the present case may indicate the possible involvement of a GPI-anchored protein abnormality in the pathogenesis of PG.