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.

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.

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.

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.

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.

A gene encoding antigenic peptides of human squamous cell carcinoma recognized by cytotoxic T lymphocytes.

Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601-restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601(+) epithelial cancer patients.

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.

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.

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.

Prognostic value of an increase in fluorine-18 deoxyglucose uptake in patients with myocardial infarction: comparison with stress thallium imaging.

OBJECTIVES: This study was undertaken to evaluate the prognostic value of an increase in fluorine (F)-18 deoxyglucose uptake compared with clinical, angiographic and stress thallium findings in patients with myocardial infarction. BACKGROUND: Positron emission tomography (PET) imaging using F-18 deoxyglucose has been applied to assess tissue viability in patients with coronary artery disease. We hypothesized that patients with a myocardial segment with augmented F-18 deoxyglucose uptake are at high risk for a future cardiac event. METHODS: One hundred fifty-eight consecutive patients with myocardial infarction referred for F-18 deoxyglucose PET and stress thallium scans were studied. Follow-up was obtained in 84 patients at a mean interval of 23 months to investigate prognostic implications of radionuclide studies. RESULTS: Seventeen patients had a cardiac event during the follow-up interval. Univariate analysis showed that an increase in F-18 deoxyglucose uptake was the best predictor of a future cardiac event (p = 0.0006), followed by the number of stenosed vessels (p = 0.008). In the multivariate analysis, when an increase in F-18 deoxyglucose uptake was entered into the model, only angiographic variables had an independent prognostic value, whereas no other radionuclide variables showed significant prognostic value. Among patients who did not show redistribution, a future cardiac event was observed more often in patients with than in those without an increase in F-18 deoxyglucose uptake (p < 0.05). CONCLUSIONS: Thus, an increase in F-18 deoxyglucose uptake seemed to be the best predictor of a future cardiac event among all clinical, angiographic and radionuclide variables in this study of stable patients with myocardial infarction. Even when a stress thallium-201 scan does not show redistribution, those patients who have an increase in F-18 deoxyglucose uptake in a PET study may be at risk for a future cardiac event, and these patients may need aggressive treatment to prevent a future cardiac event.

Structural basis for the higher Ca(2+)-activation of the regulated actin-activated myosin ATPase observed with Dictyostelium/Tetrahymena actin chimeras.

Replacement of residues 228-230 or 228-232 of subdomain 4 in Dictyostelium actin with the corresponding Tetrahymena sequence (QTA to KAY replacement: half chimera-1; QTAAS to KAYKE replacement: full chimera) leads to a higher Ca(2+)-activation of the regulated acto-myosin subfragment-1 ATPase activity. The ratio of ATPase activation in the presence of tropomyosin-troponin and Ca(2+) to that without tropomyosin-troponin becomes about four times as large as the ratio for the wild-type actin. To understand the structural basis of this higher Ca(2+)-activation, we have determined the crystal structures of the 1:1 complex of Dictyostelium mutant actins (half chimera-1 and full chimera) with gelsolin segment-1 to 2.0 A and 2.4 A resolution, respectively, together with the structure of wild-type actin as a control. Although there were local changes on the surface of the subdomain 4 and the phenolic side-chain of Tyr230 displaced the side-chain of Leu236 from a non-polar pocket to a more solvent-accessible position, the structures of the actin chimeras showed that the mutations in the 228-232 region did not introduce large changes in the overall actin structure. This suggests that residues near position 230 formed part of the tropomyosin binding site on actin in actively contracting muscle. The higher Ca(2+)-activation observed with A230Y-containing mutants can be understood in terms of a three-state model for thin filament regulation in which, in the presence of both Ca(2+) and myosin heads, the local changes of actin generated by the mutation (especially its phenolic side-chain) facilitate the transition of thin filaments from a "closed" state to an "open" state. Between 394 and 469 water molecules were identified in the different structures and it was found that actin recognizes hydrated forms of the adenine base and the Ca ion in the nucleotide binding site.

New crystal forms and preliminary X-ray diffraction studies of mitochondrial cytochrome bc1 complex from bovine heart.

Two new crystal forms of the cytochrome bc1 complex (ubiquinol:ferricytochrome c oxidoreductase, EC. 1.10.2.2) from bovine heart mitochondria were obtained by the batch method with polyethylene glycol 4000 as the precipitant, when buffer was exchanged from Tris-HCl to potassium phosphate. The first crystal form belongs to the hexagonal space group P61 or P6(5) with unit-cell constants of a = b = 131 A and c = 720 A. The reproducibility of crystallization and the quality of this crystal form were improved by an addition of ZnCl2 to the protein solution. The second crystal form, obtained from the enzyme preparation purified by crystallization, belongs to the tetragonal space group P4(1) or P4(3) with unit-cell constants of a = b = 190 A and c = 445 A. Both crystals diffracted X-rays to 6.5 A resolution and gave sharper diffraction spots than the previous monoclinic form. X-ray intensity data to 8.0 A resolution for the hexagonal crystal and to 7.0 A resolution for the tetragonal one were collected with synchrotron radiation.

Crystallization and preliminary X-ray crystallographic studies of bovine heart mitochondrial cytochrome bc1 complex.

Cytochrome bc1 complex (ubiquinol:ferricytochrome c oxidoreductase, EC. 1.10.2.2) from bovine heart mitochondria was crystallized by a batchwise method from protein solution containing sucrose monolaurate using polyethylene glycol-4000 as a precipitant. The red parallelepiped crystals grew to a size of approximately 1 mm x 1 mm x 1 mm. The crystalline protein showed enzymic activity catalyzing electron transfer from ubiquinol-2 to cytochrome c. The subunit composition and absorption spectrum of the crystalline enzyme were identical to those reported previously for the enzyme in solution. The crystal diffracted X-rays to 7.5 A resolution. The diffraction pattern indicated a monoclinic form, space group P2(1), and unit-cell constants of a = 196 A, b = 179 A, c = 253 A and beta = 97 degrees. Most probably four functional units are present in an asymmetric unit.

An autocrine or a paracrine role of adrenomedullin in modulating cardiac fibroblast growth.

OBJECTIVE: The aim of the present study was to determine the role of adrenomedullin (AM) in cardiac fibroblasts. METHODS: The production and secretion of AM were examined in cultured neonatal rat cardiac fibroblasts, and the effects of AM on proliferation and protein synthesis of these cells were assessed by [3H]thymidine and [3H]phenylalanine incorporation, respectively. RESULTS: Cultured cardiac fibroblasts secreted AM into the medium time-dependently at a rate of 20.3 +/- 3.0 fmol/5 x 10(4) cells/48 h, mean +/- S.D. Northern blot analysis showed expression of preproAM mRNA of 1.6 kb in these cells. In addition, 10(-6) mol/l of angiotensin II (Ang II) and endothelin-1 (ET-1) significantly increased the AM secretion by 55 and 48%, respectively. Synthetic AM significantly reduced 10(-6) mol/l Ang II- or 10(-7) mol/l ET-1-stimulated [3H]thymidine and [3H]phenylalanine incorporation in a dose-dependent manner, and these effects were attenuated by a calcitonin gene-related peptide (CGRP) type 1 receptor antagonist, CGRP(8-37). Synthetic AM also had a dose-dependent stimulatory effect on cAMP accumulation in these cells, which was significantly attenuated by CGRP(8-37). A cAMP analogue, 8-bromo-cAMP, mimicked the AM effects, inhibiting the Ang II-stimulated [3H]thymidine and [3H]phenylalanine incorporation. Blockage of the effect of endogenous AM by anti-AM monoclonal antibody not only significantly reduced the basal level of intracellular cAMP, but also enhanced the [3H]thymidine and [3H]phenylalanine incorporation into the cells. CONCLUSIONS: Cultured neonatal rat cardiac fibroblasts produce and secrete AM, and the secreted AM may inhibit proliferation and protein synthesis of these cells. AM may exert these inhibitory effects partly by elevating intracellular cAMP. It is suggested that AM has an important role in modulating the growth of cardiac fibroblasts in an autocrine or a paracrine manner.

Pentacoordination of the heme iron of Arthromyces ramosus peroxidase shown by a 1.8 A resolution crystallographic study at pH 4.5.

In the presence of ammonium sulfate the absorption spectra of a peroxidase from the fungus Arthromyces ramosus (ARP) showed that the low-spin component increased as the pH increased from 6.0 to 9.0, whereas in its absence ARP remained in the high-spin state in the pH range investigated. The crystal structure of ARP at pH 4.5 in the presence of ammonium sulfate at 1.8 A resolution showed that the electron density at the 6th position of the heme iron seen at pH 7.5 had disappeared and that the iron atom deviated markedly from the heme plane. These observations strongly suggest that under physiological conditions the heme of ARP is in the pentacoordinated high-spin state and that at a high pH the heme iron is able to bind ammonia, forming the low-spin state. The location of the water molecule at the distal side of the heme in peroxidases is also discussed.

Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma.

A specific and sensitive radioimmunoassay for human adrenomedullin has been developed and distribution and characterization of immunoreactive adrenomedullin in human tissue were investigated. The radioimmunoassay specifically recognizes its carboxyterminal region and half maximal inhibition of binding of radioiodinated adrenomedullin(40-52)NH2 was observed at 11 fmol/tube. Immunoreactive adrenomedullin was abundant in adrenal medulla (47.7 +/- 26.1 fmol/mg, mean +/- S.D.) and was ubiquitously found in all tissue examined. The mean plasma concentration of adrenomedullin in three normal individuals was 17.2 +/- 6.4 pg/ml (mean +/- S.D.). By analysis with reverse-phase high-performance liquid chromatography coupled with the radioimmunoassay, most immunoreactive adrenomedullin in the adrenal medulla, atrium and lung was found to be adrenomedullin(1-52)NH2.

Identification and hypotensive activity of proadrenomedullin N-terminal 20 peptide (PAMP).

Proadrenomedullin N-terminal 20 peptide (PAMP) is a candidate for a novel biologically active peptide processed from an adrenomedullin precursor. Using a radioimmunoassay for human PAMP, major and minor immunoreactive PAMPs were purified from porcine adrenal medulla and complete amino acid sequences were determined. The major immunoreactive peptide was PAMP itself with an amidated carboxy terminus. The minor one was determined to be PAMP[5-20]. An intravenous bolus injection of human PAMP in anesthetized rats caused a rapid and strong hypotensive effect in a dose dependent manner. The present data indicate that PAMP is an endogenous biologically active peptide which is processed from adrenomedullin precursor.

Immunoreactive adrenomedullin in human plasma.

A specific and sensitive radioimmunoassay for adrenomedullin has been developed. Half-maximal inhibition of binding of radioiodinated adrenomedullin was observed at 4 fmol/tube. The radioimmunoassay recognized the entire adrenomedullin molecule and has little crossreactivity with adrenomedullin fragment peptides. Adrenomedullin-like immunoreactivity was found to circulate in human plasma at considerable concentration (3.3 +/- 0.39 fmol/ml). The immunoreactivity of adrenomedullin was eluted at almost the same position as synthetic adrenomedullin on gel-filtration chromatography and reverse-phase high-performance liquid chromatography, suggesting that circulating adrenomedullin recognized by the present radioimmunoassay is identical or very similar to authentic adrenomedullin. Plasma immunoreactive adrenomedullin significantly increased in patients with hypertension, with a progressive rise proportionate to disease severity.