Identification and functional analysis of a Masculinizer orthologue in Trilocha varians (Lepidoptera: Bombycidae).

We recently showed that the Masculinizer gene (Masc) plays a primary role in sex determination in the lepidopteran model insect Bombyx mori. However, it remains unknown whether this Masc protein-dependent sex determination system is conserved amongst lepidopteran insects or within the family Bombycidae. Here we cloned and characterized a Masc homologue (TvMasc) in Trilocha varians (Lepidoptera: Bombycidae), a species closely related to B. mori. To elucidate the role of TvMasc in the sex determination cascade of T. varians, TvMasc expression was knocked down in early embryos by the injection of small interfering RNAs (siRNAs) that targeted TvMasc mRNAs. Both female- and male-type splice variants of Tvdsx, a doublesex (dsx) homologue in T. varians were observed in control siRNA-injected embryos. By contrast, only female-type splice variants were observed in TvMasc siRNA-injected embryos. These results indicate that the TvMasc protein directly or indirectly regulates the splicing patterns of Tvdsx. Furthermore, we found that male-type splice variants of B. mori dsx (Bmdsx) were produced in TvMasc-overexpressing BmN4 cells. The mRNA level of B. mori Imp, a gene whose product induces male-specific Bmdsx splicing also increased. These results suggest that Masc genes play similar roles in the sex-determination cascade in Bombycidae.

Positional cloning of the sex-linked giant egg (Ge) locus in the silkworm, Bombyx mori.

The giant egg (Ge) locus is a Z-linked mutation that leads to the production of large eggs. Cytological observations suggest that an unusual translocation of a large fragment of the W chromosome bearing a putative egg size-determining gene, Esd, gave rise to giant egg mutants. However, there is currently no molecular evidence confirming either a W-Z translocation or the presence of Esd on the W chromosome. To elucidate the origin of giant egg mutants, we performed positional cloning. We observed that the Bombyx mori. orthologue of the human Phytanoyl-CoA dioxygenase domain containing 1 gene (PHYHD1) is disrupted in giant egg mutants. PHYHD1 is highly conserved in eukaryotes and is predicted to be a Fe(II) and 2-oxoglutarate-dependent oxygenase. Exon skipping in one of the two available Ge mutants is probably caused by the insertion of a non-long terminal repeat transposon into intron 4 in the vicinity of the 5' splice site. Segmental duplication in Ge(2) , an independent allele, was caused by unequal recombination between short interspersed elements inserted into introns 3 and 5. Our results indicate that (1) Bombyx PHYHD1 is responsible for the Ge mutants and that (2) the Ge locus is unrelated to the W-linked putative Esd. To our knowledge, this is the first report describing the phenotypic defects caused by mutations in PHYHD1 orthologues.

Risk factors for infantile atopic dermatitis and recurrent wheezing.

BACKGROUND: The pathogenic mechanisms of atopic dermatitis (AD) and recurrent wheezing (RW) during infancy are not fully understood. OBJECTIVE: We evaluated immunological markers associated with AD and RW during infancy. METHODS: We followed a cohort (n = 314) from birth to 14 months of age. Some of the participants underwent a physical examination and blood test at 6 and 14 months of age. Univariate and multivariate logistic regression analysis and receiver operating characteristic curve analysis were performed to find which immunological markers could be risk factors for AD and RW. RESULTS: Of 16 immunological markers found in cord blood, only immunoglobulin (Ig) E was associated with AD at 6 months of age (adjusted OR [aOR], 1.607). None of the markers was associated with AD or RW at 14 months of age. Of 23 immunological markers at 6 months of age, total IgE (aOR, 1.018) and sensitization to egg white (aOR, 23.246) were associated with AD at 14 months of age. Phytohemagglutinin (PHA)-induced production of interleukin (IL) 4 from peripheral blood mononuclear cells (PBMCs) (aOR, 1.043) was associated with RW at 14 months of age. CONCLUSION: Cord blood IgE was a risk factor for AD at 6 months of age. Total IgE and sensitization to egg white at 6 months of age were risk factors for AD at 14 months of age. PHA-induced IL-4 production in PBMCs at 6 months of age was a risk factor for RW at 14 months of age.

The severity of sevoflurane-induced malignant hyperthermia.

BACKGROUND: Malignant hyperthermia (MH) is a potentially fatal complication of general anesthesia triggered by volatile anesthetics. In animal studies, sevoflurane has been reported to be a weak triggering agent. The aim of this study was to evaluate the clinical severity of sevoflurane-induced MH compared to isoflurane. METHODS: From the Japanese MH database containing information for 520 MH cases since 1961, we analyzed 147 cases classified by the MH Clinical Grading Scale (CGS) as 'very likely' or 'almost certain', accumulated from 1990 to 2009. Sevoflurane without succinylcholine (S-SCh (-) group) was given to 48 cases, and isoflurane without succinylcholine (I-SCh (-) group) was given to 30. Variables studied were outcome, CGS score, CGS rank, the first MH sign, and time from induction to onset of MH (occurrence time). Clinical signs and maximum laboratory data from six processes of the CGS were also analyzed. Each of the Mann-Whitney U-test or the unpaired t-test was used for group comparisons. RESULTS: Mortality was 8.3% in the S-SCh (-) group and 10.0% in the I-SCh (-) group (P = 0.803). The CGS scores were 53.4 (SD, 12.2) and 52.3 (11.7) (P = 0.691), respectively. The five processes of the CGS did not differ between groups. Median occurrence times were 72.5 minutes (range, 36.3-127.5) and 65.0 minutes (30.0-131.3), respectively (P = 0.890). CONCLUSION: There were no clinically apparent differences between MH triggered by sevoflurane and isoflurane, and thus no evidence to support the postulate that sevoflurane is a weak or weaker MH triggering agent.

Epidermotropic CD8+ cytotoxic T-cell lymphoma exhibiting a transition from the indolent to the aggressive phase, accompanied by emergence of CD7+ cells and formation of neutrophilic pustules.

A 47-year-old-man presented with rashes on his trunk and limbs, and a diagnosis of parapsoriasis was made. Ten years later, the rashes had progressed gradually to form plaques and tumours. Gene rearrangement studies revealed monoclonality of the T-cell receptor ß-chain (TCR-Jß)1 gene, and results of flow cytometry and immunohistochemical examination confirmed a diagnosis of epidermotropic CD8+ cytotoxic T-cell lymphoma. The clinical course of the disease remained indolent for some time, but about 2 years later, neutrophilic pustules formed on the surface of the skin lesions, and tumours developed in the patient's testes. Using flow cytometry, emergence of CD7+ cells was found. The patient died the following year of respiratory failure due to brain herniation. On postmortem examination, CD8+ tumour cells were found in the brain. This case demonstrates an unusually protracted indolent phase in a patient with cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma; its transition into the aggressive phase was accompanied by emergence of CD7+ cells and formation of neutrophilic pustules.

Molecular characterization of a deletion in the HPRT1 gene in a patient with Lesch-Nyhan syndrome.

Lesch-Nyhan syndrome is caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT) encoded by HPRT1. About 20% of patients have a deletion of HPRT1 and large deletions of HPRT1 are not always fully characterized at the molecular level. Here, we report on a case of Lesch-Nyhan syndrome with a 33-kb deletion involving exon 1 of HPRT1. This novel mutation is caused by a nonhomologous recombination between different classes of interspersed repetitive DNA.

Analysis of human cultured myotubes responses mediated by ryanodine receptor 1.

Malignant hyperthermia is a life-threatening condition caused by autosomal dominant mutations in the ryanodine receptor type 1 gene. Identifying patients predisposed to malignant hyperthermia is done through the Ca-induced Ca release test in Japan. We examined the intracellular calcium concentration in human cultured muscle cells and compared the sensitivity of myotubes to ryanodine receptor type 1 activators based on the Ca-induced Ca release rate. We assessed the utility of this method as an identifying test for predisposition to malignant hyperthermia. Muscle specimens were obtained from 34 individuals undergoing the Ca-induced Ca release test. We cultured myotubes from residual material and monitored changes in intracellular calcium concentration after exposure to the ryanodine receptor type 1 activators caffeine, halothane and 4-chloro-m-cresol by measuring fura-2 fluorescence. We determined the half maximal effective concentrations (EC50) for the test compounds in each myotube and calculated cut-off points using receiver operating characteristic curves. Seventeen patients each were classified into the accelerated and non-accelerated groups based on their Ca-induced Ca release rate. The EC50 values for caffeine, halothane and 4-chloro-m-cresol of the accelerated group were significant lower than those of the non-accelerated group (P < 0.001, P < 0.001 and P < 0.001, respectively). The calculated cut-off points of EC50 values for caffeine, halothane and 4-CmC were 3.62 mM, 2.28 mM and 197 microM, respectively. An increased sensitivity to ryanodine receptor type 1 activators was seen in myotubes in the accelerated group. This functional test on human cultured myotubes indicates that the alteration of their intracellular Ca2+ homeostasis may identify the predisposition to malignant hyperthermia.

Galanin receptor antagonist m35 but not m40 or c7 ameliorates cerulein-induced acute pancreatitis in mice.

BACKGROUND/AIMS: We compared the galanin antagonists C7, M35, M40 and galantide, for their ability to ameliorate acute pancreatitis (AP). METHODS: Galanin antagonists were co-administered with 7 hourly cerulein injections used to induce AP. Plasma amylase and lipase activities were measured as indices of AP, and pancreata were harvested at 12 h for histological examination and estimation of myeloperoxidase (MPO) activity. RESULTS: Treatment with galantide, M35 and C7 ameliorated the AP-induced plasma hyperenzymemia by 40-75%. Administration of M40 did not significantly alter plasma hyperenzymemia. Galantide, M35 and M40 significantly reduced the pancreatic MPO activity by 65-80%, whereas C7 increased MPO activity. Galantide and M35 but not C7 or M40 treatment significantly reduced the AP-induced necrosis score by 30-50% compared to the AP alone group. C7 alone increased plasma lipase activity and the pancreatic necrosis score compared with saline treatment alone, whereas the other antagonists were without effect. CONCLUSION: Galantide and M35 ameliorated the severity of AP, but M40 and C7 had mixed effects. Complex galanin pathways may be involved in cerulein-induced AP. M35 and galantide are potential therapeutic peptides for the treatment of AP and further evaluation should be considered. and IAP.

Effects of propofol on calcium homeostasis in human skeletal muscle.

Malignant hyperthermia is a pharmacogenetic skeletal muscle disorder of intracellular calcium (Ca2+) homeostasis with an autosomal dominant inheritance. The objective of this study was to investigate the safety of propofol by investigating its effects on calcium homeostasis and its effect sites in human skeletal muscles. Muscle specimens were obtained from 10 individuals with predisposition to malignant hyperthermia. In skinned fibre experiments, we measured the effects of propofol on the Ca(2+)-induced Ca2+ release and the uptake of Ca2+ into the sarcoplasmic reticulum. Ca2+ imaging in primary myotubes was employed to analyse propofol-mediated alternations in the Ca2+ regulation and propofol-induced Ca2+ responses in the presence of Ca2+ channel blocker or Ca(2+)-induced Ca2+ release inhibitor. Increased Ca2+ release from the sarcoplasmic reticulum and inhibition of Ca2+ uptake into the sarcoplasmic reticulum were not observed with 100 microM propofol. A rise of Ca2+ was not seen under 100 microM propofol and the EC50 value for propofol was 274.7 +/- 33.9 microM, which is higher than the clinical levels for anaesthesia. Propofol-induced Ca2+ responses were remarkably attenuated in the presence of Ca2+ channel blocker or Ca(2+)-induced Ca+ release inhibitor compared with the results obtained with caffeine. We conclude firstly that propofol is safe for individuals with predisposition to malignant hyperthermia when it is used within the recommended clinical dosage range, and secondly that its mode of action upon ryanodine receptors is likely to be different from that of caffeine.

Association study of a polymorphism of the CTGF gene and susceptibility to systemic sclerosis in the Japanese population.

OBJECTIVES: To validate the association of a single nucleotide polymorphism (SNP) of the connective tissue growth factor gene (CTGF) with susceptibility to systemic sclerosis (SSc) in the Japanese population. METHODS: 395 Japanese patients with SSc, 115 patients with rheumatoid arthritis and 269 healthy Japanese volunteers were enrolled in the study. An SNP (rs6918698) at -945 bp from the start codon in the promoter region of the CTGF gene was determined by allelic discrimination with the use of a specific TaqMan probe. RESULTS: The G allele showed a significantly higher frequency in patients with SSc than in controls (p<0.001; odds ratio 1.5; 95% confidence interval 1.2 to 1.9). In particular, the clinical subsets of SSc showed a more significant association between the G allele and diffuse cutaneous SSc (p<0.001) and the presence of interstitial lung disease (p<0.001), the presence of anti-topoisomerase I antibody (p<0.001) and anti-U1RNP antibody (p = 0.010). Association analyses using the genotype of the SNP yielded results similar to those of analyses using the allele. CONCLUSIONS: This study confirms the association between an SNP in the CTGF gene and susceptibility to SSc, especially in the presence of diffuse cutaneous SSc, interstitial lung disease and anti-topoisomerase I antibody. The results strongly suggest that this SNP may be a powerful indicator of severe skin and lung involvement in patients with SSc.

Propofol-induced changes in myoplasmic calcium concentrations in cultured human skeletal muscles from RYR1 mutation carriers.

Malignant hyperthermia is a pharmacogenetic disorder caused by autosomal dominant mutations in the ryanodine receptor type 1 gene. Propofol has been reported as a safe anaesthetic for malignant hyperthermia susceptible patients but has not been tested on cultured cells from patients with the ryanodine receptor type 1 mutation. The aim of this study was to determine whether propofol could trigger abnormal calcium fluxes in human myotubes isolated from malignant hyperthermia susceptible patients harbouring the native ryanodine receptor type 1 mutation. Muscle specimens were obtained from the patients to diagnose malignant hyperthermia disposition and the calcium-induced calcium release test and molecular genetic analyses were performed. Using the calcium sensitive probe Fura 2, we determined the 340/380 nm wave-length ratios by measuring alterations in calcium homeostasis in isolated myotubes from cultured skeletal muscle specimens. Two patients, one with ryanodine receptor type 1 R2508C and one with the L4838V mutation had accelerated calcium-induced calcium release rates. The 340/380 nm ratios increased when the propofol concentration exceeded 100 microM. The half-maximal activation concentrations (EC50) for propofol from patients 1 and 2 were 181.1 and 420.5 microM, respectively. Increases in calcium concentrations in response to propofol dosage were limited to doses at least 100-fold greater than those used in clinical settings. These observations correlate well with clinical observations that propofol does not trigger malignant hyperthermia in susceptible humans.

Sphincter of Oddi function in the Australian brush-tailed possum is inhibited by intragastric ethanol.

The role of sphincter of Oddi (SO) function in alcoholic acute pancreatitis (AP) is unclear. We aimed to compare the effect of i.v. and intragastric (IG) ethanol on SO function (i.e. trans-sphincteric flow; TSF) and investigate possible neural mechanisms. The involvement of gastric mucosal damage was also investigated by pretreatment with pantoprazole. In anaesthetized Australian possums, blood pressure (BP), TSF and blood ethanol concentrations were measured after i.v. or IG ethanol. Possums were subjected to acute vagotomy, atropine, L-nitro arginine methyl ester (L-NAME) or pantoprazole pretreatment prior to IG ethanol. BP was not significantly altered by ethanol. Ethanol decreased TSF in a dose and route-dependent manner. The lowest dose of IG ethanol reduced TSF but this response was not duplicated by i.v. ethanol producing the same blood ethanol concentrations. Acute vagotomy, atropine or L-NAME pretreatment blocked the ethanol-induced decrease in TSF and simultaneously suppressed the blood ethanol concentration. Pantoprazole pretreatment reduced the TSF response and blood ethanol concentrations implicating mechanisms induced by gastric mucosal damage. We conclude that ethanol (and/or its metabolites) reduces TSF via humoral and neural mechanisms involving vagal pathways, muscarinic receptors and nitric oxide. Reduced TSF could contribute to the onset of AP.

Infantile hemangiopericytoma--case report and literature review.

Infantile hemangiopericytoma is a rare soft tissue neoplasm of pericytic origin and is almost always benign, despite its worrisome pathologic features. We describe a 2-month-old male infant with a soft tissue mass on his right thigh. Histologically, the lesion showed a characteristic hemangiopericytoma-like vascular pattern, multilobulation, and moderate mitotic activity. These morphologic features were prediagnosed as infantile hemangiopericytoma. However, immunohistochemical and ultrastructural studies revealed a heterogeneous cellular composition, primarily pericytes and endothelial cells, similar to that observed in infantile myofibromatosis.

Genetic polymorphisms and haplotypes of the human cardiac sodium channel alpha subunit gene (SCN5A) in Japanese and their association with arrhythmia.

Genetic variations in cardiac ion channels have been implicated not only as the causes of inherited arrhythmic syndromes, but also as genetic risk factors for some acquired arrhythmias. To elucidate the potential roles of genetic polymorphisms of the alpha subunit of the voltage-gated sodium channel type V (SCN5A) in cardiac rhythm disturbance, the entire SCN5A coding exons and their flanking introns were sequenced in 166 Japanese arrhythmic patients and 232 healthy controls. We detected 69 genetic variations, including 54 novel ones. Out of the 12 novel nonsynonymous single nucleotide polymorphisms (SNPs), p.Leu1988Arg was found at a frequency of 0.015. The other 11 SNPs were rare (0.001), with 6 found in arrhythmic patients and 5 in healthy controls. The frequency of a novel intronic SNP, c.703+130G>A, was significantly higher in the patients than in the controls, suggesting this SNP is associated with an unknown risk factor for arrhythmia. Following linkage disequilibrium analysis, the haplotype structure of SCN5A was inferred using high-frequency SNPs. The frequency of the haplotype harbouring both p.Leu1988Arg and the common SNP p.His558Arg (haplotype GG) was significantly lower in the patients than in the controls. This finding suggests that this haplotype (GG) might have been positively selected in the controls because of its protective effect against arrhythmias. This study provides fundamental information necessary to elucidate the effect of genetic variations in SCN5A on channel function and cardiac rhythm in Japanese, and probably in the Asian population.

Single nucleotide polymorphisms in the gene encoding the major histocompatibility complex class II transactivator (CIITA) in systemic lupus erythematosus.

BACKGROUND: The major histocompatibility complex (MHC) class II transactivator (CIITA) is a master switch of antigen presentation and activates expression of the MHC II gene. Insufficient up regulation of MHC class II molecules is reported to be one of the major immunological mechanisms in systemic lupus erythematosus (SLE). OBJECTIVE: To examine the association between single nucleotide polymorphisms (SNPs) in the human CIITA gene (MHC2TA) and SLE. METHODS: Promoters and coding regions of MHC2TA were evaluated for polymorphisms in 100 patients with SLE and 100 healthy donors. Eight oligonucleotide primer sets that covered the coding region and each promoter region were used for genomic analysis of SNPs. RESULTS: Allele frequencies of previously reported SNPs did not differ between healthy donors and patients with SLE. Additionally, a new polymorphism in an intronic region at nt 485 (A-->A/G) was identified, which is close to the polymorphism at nt 474 that has been associated with one of the disease causing CIITA cDNA mutations in bare lymphocyte syndrome. This SNP was found in 11% of patients with SLE and in 3% of healthy donors, suggesting it may have a role in the pathogenesis of SLE. CONCLUSIONS: A newly identified polymorphism in an intronic region at nt 485 (A-->A/G) may have an important role in the pathogenesis of SLE.