Association of the Jun dimerization protein 2 gene with intracranial aneurysms in Japanese and Korean cohorts as compared to a Dutch cohort.

In a previous study a linkage region for association to IA patients was found on chromosome 14q22. In this study, we report the findings of a positional candidate gene, Jun dimerization Protein 2 (JDP2), and single nucleotide polymorphisms (SNP) of that gene that are associated with intracranial aneurysms in different ethnic populations. We screened the linkage region around chromosome 14q22 and narrowed it down to JDP2. We then genotyped case and control groups of three different ethnic populations: 403 Japanese intracranial aneurysm (IA) cases and 412 controls, 181 Korean IA cases and 181 controls, 379 Dutch cases and 642 Dutch controls. Genotyping was performed using polymerase chain reaction and direct sequencing technology. The allele distribution of three SNPs (two intronic: rs741846; P=0.0041 and rs175646; P=0.0014, and one in the untranslated region: rs8215; P=0.019) and their genotype distribution showed significant association in the Japanese IA patients. The allelic and genotypic frequency of one intronic SNP (rs175646; P=0.0135 and P=0.0137, respectively) and the genotypic frequency for the SNP in the UTR region (rs8215; P=0.049) was also significantly different between cases and controls of the Korean cohort. There was no difference in allelic or genotypic frequencies in the Dutch population. These SNPs in JDP2 are associated with intracranial aneurysms, suggesting that variation in or near JDP2 play a role in susceptibility to IAs in East Asian populations.

Participation of elderly patients in registration trials for oncology drug applications in Japan.

BACKGROUND: The objective of this study was to evaluate the age-based enrollment of cancer patients into registration trials of new drug applications or expanding the indications for use. MATERIALS AND METHODS: The data from 234 registration trials in Japan and overseas of 43 drugs, which were reviewed by the Pharmaceuticals and Medical Devices Agency and approved by the Ministry of Health, Labour and Welfare in Japan between 1999 and 2008, were retrospectively analyzed according to the age distribution of enrolled patients. The age distribution of the Japanese cancer population was derived from Cancer Statistics in Japan 2003 and Annual Report on Health, Labour and Welfare 2003-2004. RESULTS: In the Japanese cancer population, the estimated median age of cancer patients is 70 years, and 66% of cancer patients are aged 65 years or more. The estimated median age of cancer patients in all registration trials conducted in Japan was 59 years, whereas it was 55 years in the registration trials conducted overseas. The proportion of patients aged 65 years or more enrolled in registration trials conducted in Japan was 35%; this number was 28% in registration trials conducted overseas. CONCLUSION: Elderly patients are underrepresented in oncology registration trials in Japan.

[Intrathecal baclofen therapy for spastic paraparesis due to aortic dissecting aneurysm; recent progress in treatment strategy].

A 48-year-old man suffered from acute dissection of thoracic aortic aneurysm which eventually led to replacement of the ascending aorta with a tube graft. During this clinical course, circulatory failure in intercostal artery resulted in spinal cord infarction followed by moto-sensory disturbance below Th7 dermatomic area. Seven months later, spasticity with pain in both lower extremities became conspicuous that was uncontrollable by any oral medication. Eventually the patient underwent the implantation of continuous infusion pump for intrathecal baclofen therapy (ITB). The clinical condition was remarkably improved and now has been well controlled. ITB, authorized by Japanese Ministry of Health Labour and Welfare in 2006, has notable therapeutic effects on spasticity derived from any sort of central nervous disorder. More promotive enlightenment if ITB is indispensable for enhancement of its medical benefit in Japan.

Network-based gene expression analysis of intracranial aneurysm tissue reveals role of antigen presenting cells.

Little is known about the pathology and pathogenesis of the rupture of intracranial aneurysms. For a better understanding of the molecular processes involved in intracranial aneurysm (IA) formation we performed a gene expression analysis comparing ruptured and unruptured aneurysm tissue to a control artery. Tissue samples of six ruptured and four unruptured aneurysms, and four cerebral arteries serving as controls, were profiled using oligonucleotide microarrays. Gene ontology classification of the differentially expressed genes was analyzed and regulatory functional networks and canonical pathways were identified with a network-based computational pathway analysis tool. Real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed as confirmation. Analysis of aneurysmal and control tissue revealed 521 differentially expressed genes. The most significantly associated gene ontology term was antigen processing (P=1.64E-16). Further network-based analysis showed the top scoring regulatory functional network to be built around overexpressed major histocompatibility class (MHC) I and II complex related genes and confirmed the canonical pathway "Antigen Presentation" to have the highest upregulation in IA tissue (P=7.3E-10). Real time RT-PCR showed significant overexpression of MHC class II genes. Immunohistochemical staining showed strong positivity for MHC II molecule specific antibody (HLA II), for CD68 (macrophages, monocytes), for CD45RO (T-cells) and HLA I antibody. Our results offer strong evidence for MHC class II gene overexpression in human IA tissue and that antigen presenting cells (macrophages, monocytes) play a key role in IA formation.

Relative contributions of myeloperoxidase and NADPH-oxidase to the early host defense against pulmonary infections with Candida albicans and Aspergillus fumigatus.

Generation of oxidative products by phagocytic cells is known to be an important host defense mechanism directed toward killing of invading microorganisms. The importance of two major oxidant-producing enzymes, myeloperoxidase (MPO) and NADPH-oxidase, in in vivo fungicidal action was directly compared in genetically engineered mice. Both MPO-deficient (MPO-/-) and NADPH-oxidase-deficient (X-linked chronic granulomatous disease [X-CGD]) mice showed increased susceptibility to pulmonary infections with Candida albicans and Aspergillus fumigatus compared with normal mice, and the X-CGD mice exhibited shorter survivals than MPO-/- mice. This increased mortality of X-CGD mice was associated with a 10- to 100-fold increased outgrowth of the fungi in their organs during the first 6 days. These results suggest that superoxide (O2-) produced by NADPH-oxidase is more important than hypochlorous acid (HOCl) produced by MPO, although both oxidative products obviously contribute to the host defense against pulmonary infection with those fungi. We also observed that MPO-/-/X-CGD double knockout mice showed comparable levels of susceptibility to the X-CGD mice against C. albicans and A. funigatus, indicating that MPO is unable to play a role in host defense in the absence of NADPH-oxidase. This strongly suggests that hydrogen peroxide, the precursor of HOCl, is solely derived from O2- produced by NADPH-oxidase.

Differential host susceptibility to pulmonary infections with bacteria and fungi in mice deficient in myeloperoxidase.

Myeloperoxidase (MPO), which is located within neutrophils capable of producing hypochlorous acid, is active in vitro against bacteria and fungi. However, MPO-deficient persons are usually healthy. To define the in vivo contribution of MPO to early host defense against pulmonary infections, MPO-deficient and control mice were intranasally infected with various fungi and bacteria, and the number of residual microorganisms in lungs was compared 48 h later. MPO-deficient mice showed severely reduced cytotoxicity to Candida albicans, Candida tropicalis, Trichosporon asahii, and Pseudomonas aeruginosa. However, the mutant mice showed a slight but significantly delayed clearance of Aspergillus fumigatus and Klebsiella pneumoniae and had comparable levels of resistance to the wild type against Candida glabrata, Cryptococcus neoformans, Staphylococcus aureus, and Streptococcus pneumoniae. These results suggest that the MPO-dependent oxidative system is important for host defense against fungi and bacteria, although the effect varies by pathogen species.

Stresgenin B, an inhibitor of heat-induced heat shock protein gene expression, produced by Streptomyces sp. AS-9.

Stresgenin B was isolated as an inhibitor of heat-induced heat shock protein (HSP) gene expression from a culture broth of Streptomyces sp. AS-9 by silica gel chromatography and HPLC. The molecular formula of the novel compound was determined as C11H13NO5 by high resolution FAB-MS analysis, and the structure was determined by UV, 1H NMR, 13C NMR, HMQC, HMBC, and NOESY spectra. Stresgenin B inhibited heat-induced luciferase reporter-gene expression directed by the human hsp70B promoter in Chinese hamster ovary (CHO) cells at concentrations lower than the concentrations for inhibition of dexamethasone-induced luciferase reporter-gene expression directed by the mouse mammary tumor virus (MMTV)-LTR promoter. The inhibition of heat-induced reporter gene expression was evident even when cells were exposed to stresgenin B only during heat stress treatment. Moreover, the compound inhibited heat-induced syntheses of hsp72/73, hsp90, and hsp110 and thereby suppressed the induction of thermotolerance. Stresgenin B showed moderate cytotoxic activities against several neoplastic cell lines and also showed antibacterial activities against Micrococcus luteus, Bacillus subtilis and Staphylococcus aureus strains.

Suppression of thermotolerance development through cycloheximide-induced negative control of stress protein gene expression.

Expression of a luciferase reporter gene by Chinese hamster ovary cells under the control of the human heat shock protein (hsp) 70 gene promoter was suppressed by incubation at 37 degrees C after treatment with cycloheximide (CHX) during 42 degrees C heat shock exposure. The CHX-induced suppression of hsp gene expression induced no development of thermotolerance. However, 42 degrees C heat shock treatment without CHX followed by CHX inhibition of protein synthesis during recovery incubation at 37 degrees C induced thermotolerance expression by inducing enhanced synthesis of hsps including hsp70 in subsequent heat challenge incubation at 43 degrees C. The results demonstrated a direct correlation between suppression (induction) of stress protein gene expression and non-expression (expression) of thermotolerance. Kinetic analysis showed that the CHX suppression of hsp gene induction was greater than the CHX inhibition of protein synthesis, and that it depended on the severity of heat stress: it decreased with increasing heat stress doses. Moreover, prior feeding of the proline analog L-azetidine 2-carboxylic acid abrogated the CHX-induced suppression of hsp gene expression. In addition, CHX treatment during heat cell-killing at 43 degrees C induced protection of cells. These results were well explained by the proposed model of negative or positive control of stress protein gene expression depending on the level of free hsp70, which may be modulated by both the rate of protein synthesis and the severity of heat stress.

Stress dose-dependent suppression of heat shock protein gene expression by inhibiting protein synthesis during heat shock treatment.

When a clone of Chinese hamster ovary (CHO) cells transfected with a plasmid containing a luciferase reporter gene under the control of the human heat shock protein (hsp) 70 gene promoter was treated with cycloheximide during heat exposure at 42 and 43 degrees C for 15 to 100 minutes and then incubated at 37 degrees C after removal of cycloheximide, reporter gene expression was suppressed by the protein synthesis inhibitor only at small heat shock doses (i.e., heat shock of less than 40 minutes at 42 degrees C and 15 minutes at 43 degrees C). A similar stress dose-dependent suppression of reporter gene expression by cycloheximide was also demonstrated by treatment with sodium arsenite at 37 degrees C. However, dexamethasone-dependent reporter gene expression in a different CHO clone was not inhibited after the inducer treatment for different times in the presence of cycloheximide. In addition, synthesis of most cellular proteins (except for hsp) was not affected after heat shock treatment with cycloheximide. The results suggested that the cycloheximide inhibition of gene expression is specific to hsp gene expression induced by limited stress doses. Furthermore, a prior 42 degrees C heat shock treatment for 30 minutes induced a decreased responsiveness (tolerance) to a second 42 degrees C heat treatment for hsp gene expression, but tolerance did not develop in cells exposed to the first heat shock in the presence of cycloheximide. These results confirm previous findings that induction of hsp gene expression by stress is balanced by the severity of stress and rate of protein synthesis. They also support the proposed model of autoregulation of hsp gene expression by levels of free hsp70.

Effects of light stimulation on the activity of the autonomic nerves in anesthetized rats.

The existence of the retino-hypothalamic pathway suggests that light stimulation may influence the activity of the autonomic outflows. Efferent activities of the pancreatic, hepatic, and gastric branches of the vagus nerve and those of pancreatic, hepatic, splenic, adrenal, and renal branch of the splanchnic nerve were recorded. Light stimulation with 2000 1x for 10 min to the left eye increased the splanchnic (sympathetic) outflows and suppressed the vagal outflows. The effects lasted for several hours. The minimal effective stimulation was 20 1x for 1 min or 200 1x for 0.1 min. These responses were observed in the light period as well as dark period. However, in the suprachiasmatic nucleus (SCN) lesioned rat, the changes in autonomic outflows following light stimulation were absent. The observations suggest that light stimulation modulates visceral functions through changes in the autonomic nervous system activities via the SCN.

Purification and characterization of aseanostatins: actinomycete-derived fatty acid inhibitors to myeloperoxidase release from human polymorphonuclear leukocytes.

We found inhibitors, designated aseanostatins P1 and P5, against myeloperoxidase (MPO) release from human polymorphonuclear leukocytes (PMN). Aseanostatins were extracted from an actinomycete isolated in Thailand and purified by a series of column chromatography of charcoal and silica gel, and HPLC. Physico-chemical characterization by gas liquid chromatography and GC-MS indicated that aseanostatins were fatty acids. The active forms of aseanostatins were recovered by hydrolyzing their methyl esters after HPLC. Two components P1 and P5 with the IC50 of 0.96 and 0.54 microgram/ml to the MPO release were obtained as pure forms, indicating aseanostatin P5 was higher activity than aseanostatin P1. The component P1 was identical with 12-methyltridecanoic acid and P5 was indistinguishable to 12-methyltetradecanoic acid (ante-i-15:0). Aseanostatin P5 (1 microgram/ml) did not inhibit beta-glucuronidase release, but O2- production a little. It has no effect on chemotaxis of PMN to fMet-Leu-Phe (10(-8)M), PMN adhesion or phosphorylation of a 64-kD protein in the PMN cell-lysate system.

Excision of papillary endocardial tumor.

We report the eighth case of surgical removal of a papillary endocardial tumor. The surgical approach was aided by intraoperative two-dimensional echocardiography. The tumor was attached to the chorda tendinea of the mitral valve and was successfully removed via a left atrial approach.

Characterization of a natural cointegrate of the pock-forming plasmids pSK1 and pSK2 of Streptomyces kasugaensis MB273.

Strains carrying only one species of pock-forming plasmid, designated as pSK3, were isolated from two different derivative strains of Streptomyces kasugaensis MB273 which contained three species of plasmids, pSK1, pSK2 and pSK3. Single and double digestion of pSK3 with seven restriction endonucleases yielded fragments identical with those of pSK3 and assignable to those obtained from pSK1 and pSK2. In particular, digestion with BglII alone or in combination with other restriction endonucleases afforded the same size fragments as those of pSK1 and pSK2. Strains containing pSK3 induced pocks on lawns of strains carrying pSK1 or pSK2 and resisted pock formation by the latter strains. Therefore, it was concluded that pSK3 was a pSK3 derivative with elevated pock-forming ability and represented a composite plasmid consisting of two elements, pSK1 and pSK2, without any loss of their plasmid functions. Deletion derivative plasmids constructed from the BglII fragments of pSK3 provided evidence supporting the above conclusion. Pock formation by a pSK3-containing strain against strains carrying pSK1, or pSK2 or no plasmid accompanied the transfer of pSK3 from the former to the latter. Segregation of pSK1 and pSK2 from pSK3 was observed in mycelium from pocks caused by pSK3-containing strains and on subculture of pSK3-containing strains.

Characterization and localization of plasmid functions involved in pock formation and pock resistance of plasmid pSK3 of Streptomyces kasugaensis MB273.

Deletion derivatives and recombinants of the plasmid pSK3, which is a cointegrate of pSK1 and pSK2 in Streptomyces kasugaensis, were constructed and analysed for their ability to transfer and 'pock' on strains carrying pSK1 or pSK2. Various deletions in the pSK1 and/or pSK2 regions of pSK3 were grouped into nine classes on the basis of their pock-forming ability and pock resistance. Analysis of these deletions and insertions provided tentative locations of DNA regions for two pock-resistance determinants (Por1 and Por2), two pock-forming determinants (Poc1 and Poc2) consisting of plasmid transfer and spread determinants (Tra/Spr), and two replication determinants (Rep1 and Rep2) corresponding to the pSK1 and pSK2 regions of pSK3. In particular, the Por2 function in the pSK2 region was determined to be located in a 1.35 kb segment.